CA2162577C - Human immunoglobulin vh gene segments and dna fragments containing the same - Google Patents
Human immunoglobulin vh gene segments and dna fragments containing the same Download PDFInfo
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Abstract
Novel human immunoglobulin V H segments and DNA
fragments containing the same are disclosed. The DNA
fragment according to the present invention is the fragment having a size of about 800 kbp which is shown in Fig. 1. The human immunoglobulin V H segments according to the present invention are contained in the fragment of this DNA fragment of about 800 kbp, and there are 50 novel segments. The base sequences of these segments are shown in the Sequence Listing. The present invention also provides DNA fragments which contain two or more of these V H segments.
fragments containing the same are disclosed. The DNA
fragment according to the present invention is the fragment having a size of about 800 kbp which is shown in Fig. 1. The human immunoglobulin V H segments according to the present invention are contained in the fragment of this DNA fragment of about 800 kbp, and there are 50 novel segments. The base sequences of these segments are shown in the Sequence Listing. The present invention also provides DNA fragments which contain two or more of these V H segments.
Description
SPECIFICATION
Human Immunoglobulin VH Gene Segments and DNA Fragments Containing the Same TECHNICAL FIELD
This invention relates to novel human immunoglobulin VH gene segments and DNA fragments containing the same.
The segments and DNA fragments according to the present invention are useful for producing human antibodies using a mammalian host by a genetic engineering process.
BACKGROUND ART
Immunoglobulins are composed of the L chains and H
chains, each of which consists of a variable region (V
region) and a constant region (C regs.on) that has a structure common to immunoglobulin molecules.. What determines the antigenic specificity of an antibody is the V region. The V region of the H chain is encoded by V, D (diversity) and J (joining) genes (The gene of the H
chain is expressed by placing a suffix "H", like "VH").
One of the important reasons why the V regions of immunoglobulins are highly diverse and can provide antibodies which specifically binds to infinite number of antigens is the rearrangement of V, D and J genes. That is, there are a plurality of V genes, D genes and J
genes, respectively and they are randomly combined in somatic cells to form a gene encoding a single mRNA.
Since the combination is randomly selected, wide variety of immunoglobuiin V regions are provided.
Human Immunoglobulin VH Gene Segments and DNA Fragments Containing the Same TECHNICAL FIELD
This invention relates to novel human immunoglobulin VH gene segments and DNA fragments containing the same.
The segments and DNA fragments according to the present invention are useful for producing human antibodies using a mammalian host by a genetic engineering process.
BACKGROUND ART
Immunoglobulins are composed of the L chains and H
chains, each of which consists of a variable region (V
region) and a constant region (C regs.on) that has a structure common to immunoglobulin molecules.. What determines the antigenic specificity of an antibody is the V region. The V region of the H chain is encoded by V, D (diversity) and J (joining) genes (The gene of the H
chain is expressed by placing a suffix "H", like "VH").
One of the important reasons why the V regions of immunoglobulins are highly diverse and can provide antibodies which specifically binds to infinite number of antigens is the rearrangement of V, D and J genes. That is, there are a plurality of V genes, D genes and J
genes, respectively and they are randomly combined in somatic cells to form a gene encoding a single mRNA.
Since the combination is randomly selected, wide variety of immunoglobuiin V regions are provided.
On the other hand., antibodies currently employed for therapies of various diseases are those originated from animals other than human, such as mouse. However, if these antibodies are administered to human, since the antibodies are of exogenous origin, an immunological response occurs in the human body to present allergy and to neutralize the, antibodies. To overcome this problem, it is desired to use antibodies originated from human for the therapies 'for human. Further, if a human antibody is industrially produced using human as the host and using a human-originated antigen, a problem of immunological tolerance is brought about, so that this approach employing the known method is very difficult. Thus, the production of human immunoglobuli:ns by a genetic engineering process using an animal as a host is now being developed '(for example, Japanese Laid-open PCT
Application (Kohyo) No. 4-504365; Proc. Natl. Acad. Sci.
USA, Vol. 86, pp.5898-5902, August 1989; Proc. Natl.
Acad. Sci. USA, Vol. 87, pp.5109-5113, July 1990;
Genomics 8, 742-750 {1991)). However, in the conventional methods in which human immunoglobulin genes are expressed in host animals other than human, there is a problem that the number of human VH segments provided for the genetic recombination is very small, so that the diversity of the expressed human immunoglobulins is limited. Even if only one VH segment is recombined, the diversity of the ~immunoglobulin is assured to some degree because of the combination with D and J genes. However, as mentioned above, since the diversity of immunoglobulins is determined by the rearrangement (random combination) of V gene segments, the more the human VH segments, recombined, the higher the diversity of the immunoglobulins expressed. If the diversity of immunoglobulins is increased, not only antibodies against a number of antigens can be formed, but also the possibility of forming an antibody having a high specificity to a given antigen is promoted. Therefore, it is important for therapies and diagnoses to recombine VH segments as many as possible.
DISCLOSURE OF THE INVENTION
Accordingly, an object of the present invention is to provide a DNA fragment comprising a plurality of human immunoglobulin VH segments. Another object of the present invention is to provide a novel human immunoglobulin VH segments.
The present inventors intensively studied and succeed in determining human immunoglobulin H chain V region gene segments having a size of about 800 kb and in determining DNA sequences of 64 human VH segments contained therein.
This made it possible to provide this DNA fragment of 800 kb and various DNA fragments contained therein, thereby completing the present invention.
That is, the present invention provides a DNA
fragment having a size of about 800 kbp and having the structure shown in Fig.. 1. It should be noted that in Fig. 1, the 64 human VH segments are those having DNA
sequences shown in Sequence ID Nos. l, 2, ... 63, and 64, respectively, in the order from downstream (i.e., from the side near the Jg gene).
The present invention also provides DNA fragments containing at least two consecutive functional human VH
segments which are contained in said DNA fragment of about 800 kb according to the present invention.
The present invention further provides DNA fragments Y20, Y103, Y21, Y6, Y-24, M131, M118, M84 and 3-31, which have been deposited.
The present invention still further provides DNA
fragments consisting essentially of at least two optional DNA fragments linked in an optional order, each of which contains at least two consecutive functional human VH
segments contained in the DNA fragment of about 800 kb according to the present invention.
The present invention still further provides DNA
fragments consisting essentially of at least two DNA.
fragments selected from the group consisting of DNA
fragments Y20,~Y103, Y21, Y6, Y-24, M131, M118, M84 and 3-31 which have been deposited, which are linked in an optional order.
The present invention still further provides novel human immunoglobulin VH segments having DNA sequences shown in Sequence ID Nos. f, 7, 8, 9, 10, 11, 12, 13, 14, _5_ 15, 16, 17, 18; 19, 20, 21, 22, 23, 24, 25, 26, 27, 29, 30, 31, 32, 33, 34, 35; 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 63 and 64, respectively.
By the present invention, novel human immunoglobulin VH segments and DNA fragments containing the same were provided. The DNA fragment of about 800 kb according to the present invention contains as many. as 64 human immunoglobulin VH segments. Thus, by producing human immunoglobulins by a host animal using this DNA fragment, the diversity of the produced human immunoglobulin is largely increased, when compared with the conventional methods.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows a genetic map of the DNA fragment of about 0.8 Mb according to the present invention:
. Fig. 2 shows the results of Southern hybridization of a representative DNA inserted in YAC.
Fig..3A shows the results of Southern hybridization of the fragment digested with restriction enzymes Mlu I
and Not I.
Fig. 3B shows a physical map of a YAC clone constructed based on the results shown in Fig. 3A.
Fig. 4 shows a genetic map of YAC clone Y6.
BEST MODE FOR CARRYING OUT THE INVENTION
The present inventors prepared a library by inserting the DNA partially digested with Eco RI into YAC
~:~~2~'~'~
by the method detailed in the examples hereinbelow described, which DNA was originated from human lymphoblastoid cell line transformed by EB virus, and succeeded in determining the structure of human VH gene region having a size~of.about 800 kbp using the above-mentioned library. The structure is shown in Fig. 1. In Fig. 1, the genetic map is shown on the four thick solid lines. The right side of each solid line~is .the 3' side and the left end of the upper most solid line continues to the right end of the second solid line. In the DNA
fragment shown in Fig. 1, there exist C genes, JH genes and D genes in the order mentioned from the 3' end.
Subsequent to the D genes, there are 64 vH segments. The DNA sequences of~all of these 64 VH segments have been.
determined as described in the examples below, and Sequence ID Nos. ~1, 2, ... 63, 64 were assigned to~the 64 VH segments in the order from downstream. Among these VH
segments, the functional VH segments which are thought to encode polypeptides are indicated by solid rectangles.
On the other hand, those which have the general features of the known Vg segments but do not presently encode polypeptides because of the termination codons contained therein, that is, pseudo VH segments are indicated by hollow rectangles. Immediately below the genetic map, restriction maps by Eco RI and Hind III are shown. The restriction.sites are indicated by short perpendicular lines. The short lines to which ends circles are attached are those whose order is not determined, and the dotted boxes indicate the regions in which Eco RI sites have not been determined. In Fig. 1, the symbol which looks like "Y" indicates the sites at which two restriction sites are close. In Fig. 1, restriction sites of Mlu I are indicated by hollow triangles and restriction sites of Not I are indicated by solid triangles. The fragments inserted in the clones employed for determining the structure of the DNA fragment are shown thereunder. The structure of the 3' side farther than the 3' end shown in Fig. 1 is known and described in Ravetch, J.V. et al., (1981) Cell, Vol. 27, pp. 583-591.
Among the DNA fragments inserted in the clones shown in Fig. 1, the yeasts containing Y20, Y103, Y21, Y6 and Y24 inserted in YAC have been deposited with National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology of 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, 305, Japan, under Accession Numbers FERM BP-4272, FERM BP-4275, FERM BP-4273, FERM BP-4271 and FERM BP-4274, respectively. The E. coli cells containing M131, M118, M84 and 3-31, respectively, inserted in cosmids have been deposited with National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology of 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, 305, Japan, under Accession Numbers FERM
BP-4279, FERM BP-4278, FERM BP-4277 and FERM BP-4276, respectively.
The DNA fragment having a size of about 800 kbp shown in Fig. 1 can be prepared by linking these _g-deposited DNA fragments by known~methods. That is, a DNA
fragment A and a DNA fragment B.whose DNA sequence at its terminal region overlaps with the DNA sequence of the terminal region of DNA fragment A (i.e., the~DNA sequence of the 3' region of DNA fragment A is identical to the DNA sequence of the 5' region of DNA fragment B) can be easily ligated by a method exploiting genetic recombination in the yeast.cells. More particularly, DNA
fragments A and B are inserted in separate YAC vectors, and the resulting recombinant YAC vectors. are introduced in separate mating type yeast cells, respectively. The resulting yeast cells are then fused. By this, genetic recombination occurs in the yeast host to form a YAC
having a DNA fragment in which DNA fragment A and DNA
fragment B are ligated, Which has only one overlapping region located at the terminal regions of DNA fragments A
and B. The thus formed recombinant YAC can easily be selected using the auxotrophy encoded in the YAC as a marker. This method is well-known in the art, and is described in, for example, Japanese Laid-open PCT
Application (ICohyo) No. 4-504365; Proc. Natl. Acad. Sci.
USA, Vol. 87, pp.9913-9917, December 1990; Science Vol.
250, p.94, Proc. Natl. Acad. Sci. USA, Vol. 89, pp.5296-5300, June 1992; and Nucleic Acid Research, Vol. 20, No..
12, pp.3135-3138. Since the terminal regions of each of the deposited 8 DNA fragments overlap the respective terminal regions of the adjacent DNA fragments, they can ~~.~~~'~''~
_g-be ligated sequentially by the method described above.
Although DNA fragments 3-31, M84, M118 and M-131 are cloned in cosmid vectors,.they can be kept in an artificial.chromosome in the yeast cell by cutting the recombinant cosmid with a restriction enzyme having a restriction site only in the cosmid vector, and ligating a YAC vector to the ends of the digested recombinant cosmid vector. Further, by the above-described method, the digested recombinant vector can be ligated to a YAC
clone of other regions. It should be noted that even if the above-mentioned 9 deposited fragments are ligated, a gap of about 4 kb still remains. A DNA fragment which fills the gap can be easily prepared by the method described below. That is, as shown in Fig. 1, since the Hind IIh fragment including the region of the gap is relatively large, this Hind III fragment can be obtained by completely digesting human genome by Hind III, electrophoresing the resultant, selecting~DNA fragments having sizes of about 15 kb, detecting the desired fragment with a probe, and recovering the detected-desired fragment. The probe used here can be isolated as follows. That is, the DNA fragments located at the both ends of the gap are subcloned using a plasmid and DNA
fragments which do not contain a repetitive sequence are prepared therefrom. The thus obtained fragments are then used for screening of the library. Only those detected by the probes which are the DNA fragments at both ends of the. gap are isolated.
As.described above, the DNA.fragment of about 800 kbp shown in Fig. 1 was provided according to the present invention. The fragments consisting of the DNA region included in this DNA fragment can also be used for producing human immunoglobulin by a genetic engineering method. More particularly, to increase the diversity of human immunoglobulin produced by a genetic engineering method, it is preferred to incorporate a fragment containing human VH segments as many as possible.
However, if the fragment contains at least two human VH
segments, the diversity to some degree is given during rearrangement, so that the fragment can be employed.
Thus, DNA fragments consisting of a region containing at least two consecutive functional VH segments, which region is contained in the DNA of about 800 kb shown in Fig. l can be employed and are useful. The number of the functional VH segments contained in.such DNA fragments is at least two, and is preferably not less than 6. The more the number of the functional VH segments, the higher the diversity of the human immunoglobulin produced, so that the more pref erred . Thus , the preference i s increased when the number of the functional VH segments is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 and 33, with the order mentioned. Among these fragments, although those having large molecular weights are cloned 2~~~~'~'~
into YAC vector, small fragments having a size of about not more than 50 kb are not.necessarily cloned into YAC
vector, but can be. cloned into cosmid vectors and plasmid vectors.
Such DNA fragments can be prepared since the information disclosed.in Fig. 1 and Sequence ID Nos. 1 -64 is available: That is, for example,.a DNA fragment containing not less than two functional VH segments can be.obtained by partially digesting human genome with an appropriate restriction enzyme such as Eco RI or Hind III, separating the resulting fragments by electrophoresis, and selecting a DNA fragment containing not less than two desired functional VH segments using not~less than two probes each of which hybridizes with one of the not less than two desired functional VH
segments. Alternatively, amplification by PCR may be employed in place of the detection by the probes. In this case, since the entire DNA sequences of the functional VH segments are known, the DNA sequences of the primers which should be used are also known, so that the PCR can be carried out easily.
The present invention further provides DNA fragments consisting essentially of optional DNA fragments each of which contains not less than two functional VH segments which are ligated in optional orders. That is, by ligating a plurality of the DNA fragments each containing.
not less than two functional VH segments, the number of VH segments in the DNA fragment can be increased when compared. with the case. where only one such DNA fragment containing not less thin two VH segments is used, so that the diversity of the produced immunoglobulin can be increased accordingly. The DNA fragments are not necessarily consecutive, and optional DNA fragments may be ligated in an optional order. In cases where there is no overlapping region between two,.DNA fragments to be ligated, the above-described method for ligating the DNA
fragments having an overlapping region cannot be applied.
However, two DNA fragments having no overlapping region can also be ligated by the method as follows.
The left arm~vector region and the right arm vector region of a YAC clone containing not less than two functional VH segments are recovered by the method of Hermanson et al (1991) (Nucleic Acids. Res.,l9; 4943-4948). A plasmid (pICL) which has a sequence homologous with the ampicillin-resistant marker (AMP) in the left arm vector region of the YAC, a marker (Lys) which reverse the lysine auxotrophy to the wild type, and a multiple cloning.site immediately downstream Lys; and a plasmid (PLUS) which has a sequence homologous with YAC4 region in the right arm vector region of the YAC, the above-mentioned Lys, a kanamycin-resistant marker (KAN), and a multiple cloning site immediately downstream the KAN are linearized and then introduced into yeast cells containing YAC by a conventional method. The plasmids 2~.~~~'~'~
pICL and pLUS cause recombination in the yeast cells at an appropriate frequency, thereby being recombined with the left arm vector region and the right arm vector region of the YAC. The yeast cells carrying such a YAC
are selected by using an appropriate selection medium and the YAC in the selected yeast cells is then cut with. an appropriate restriction enzyme which has a restriction sites ~n the multiple cloning.sites of the above-mentioned plasmids: By the operation described above, DNA fragments containing the left end or the right end of the DNA fragment originated from human contained in the YAC are recovered as plasmids. After amplifying the thus obtained plasmids in E. coli by a conventional method, the recovered plasmids are digested with a restriction enzyme and then ligated by ligase. The thus ligated DNA
fragment is then ligated to the left arm vector region or the right arm vector region of the YAC and introduced into yeast cells carrying the YAC. These YAC vectors causes recombination at a certain frequency between the intrinsic left arm or.right arm vector regions and the left end or right end region of the DNA fragment originated from human. By selecting the resulting recombinant vectors, a YAC clone containing a DNA
fragment originated from human, which left end is ligated to the right end of another DNA fragment originated from human, and a YAC clone containing a DNA fragment originated from human, which right end is ligated to the left end of another DNA fragment originated from human are recovered. Since these YAC clones have the structure in which the left end or the right end of a DNA
originated from human is ligated to the right end or the left end of another DNA originated from human, they can be recombined with a YAC clone having a sequence in the ligated DNA fragments by the method described above.
Further, by optionally ligating the above-described eight actually deposited DNA fragments in an optional order, a large fragment containing a number of VH
segments can be prepared.
By the present invention, the DNA sequences of the 64 VH segments contained in the fragment of about 800 kbp shown in Fig. 1 were determined. As described in detail in the examples below, among these, 50 VH segments are novel segments which have DNA sequences that have not hitherto been known. These novel human immunoglobulin VH
segments include pseudo segments which do.not encode a polypeptide. Even a pseudo segment has an utility because it may function as a donor of gene conversion in the somatic cell level.
The fiuman.immunoglobulin VH segments and the DNA
fragments containing the same according to the present invention can be used for producing human immunoglobulins in a mammalian host as described in, for example, Japanese Laid-open PCT Application (Rohyo) No. 4-504365.
Examples The present invention will now be described in more detail by way of examples thereof. It should be noted that the present invention is not limited to the following examples.
Example 1 ~ Determination of Structure of DNA Fragment of about 8 0 0. kbp .
(1) Library Used for Screening The human YAC library screened was constructed from DNA of an Epstein-Barr virus-transformed human lymphoblastoid cell line CGM1 (T. Imai and M.V. Olson, genomics, 8, 297-303 (1990)). Eco RI partial digests of CGM1 DNA were ligated to pYAC4 vector (D. Burke and M.V.
Olson, in "Guide to Yeast Genetics and Molecular Biology"
(C.~Guthrie arid G.R. Fink, eds), p.253, Academic Press, Orlando, 1991), and introduced into AB1380 yeast host strain (D. Burke and M.V. Olson, in "Guide to Yeast Genetics and Molecular Biology" (C. Guthrie and G.R.
Fink, eds), p.253, Academic~Press, Orlando, 1991). The library consisted of 15,000 independent clones with mean YAC size of about 360 kb. The library thus contained the equivalent of approximately 1.8 haploid human genomes.
DNA rearrangement in immunoglobulin H chain (IgFi) locus was first checked by Southern hybridization.using the human D and Jg probes. The result showed that an allele kept germline configuration while the other was VDJ
rearranged.
(2) Primers~used for PCR-based Screening 21~~~ ~'~
For PCR-based screening of human VH YAC clones, oligonucleotide primers for VH-LII and VH-I families, the first and the second largest VH families, were synthesized. VH region segments of immunoglobulins contain two hypervariable regions (CDR1 and CDR2) and three framework regions (FR1, FR2 and FR3) (E.A. Rabat et al., Sequences of Proteins of Immunological Interest., Fifth edition, NIH publications, Washington D.C. (1991)).
Nucleotide sequences of the framework regions are highly conserved within the same family, suggesting the possibility of oligonucleotide synthesis is for consensus primers corresponding to the framework regions. For this purpose, nucleotide sequences of FR1, FR2 and~FR3 regions in all the known VH sequences were aligned for comparison. Nucleotide sequences corresponding to the first 8 amino acid residues of the FR1 region had extremely high conservation not only within the same family but also between VH-I and VH-III families, which enabled the synthesis of a.forward primer F-univ common for the two families as shown in Table 1. Sequences for family-specific reverse primers Were independently chosen from conserved sequences in the FR2 region so that 3'-half of .the primer sequence has 100$ identity~to known VH
segments and, in particular, 3'-most nucleotide corresponds to the first letter of the highly conserved/invariant amino acid residues. More particularly, F-univ and I-R, and F-univ and III-R were ._ used as primers for the screening. The-DNA sequences of the primers are shown in Table 1.
(3) Optimal PCR Condition Check , Analytical experiments were carried out to determine the optimal condition for specific amplification. A
reaction mixture (5 ul) was prepared in accordance with the protocol recommended by Perkin-Elmer/Cetus. Thermal cycling was performed using a DNA Thermal Cycler (Perkin-Elmer/Cetus). Reactions were carried out using 25 ng of template human DNA under various annealing temperatures (55°C, 58°C, 60°C and 62°C) and cycles (25, 30, and 35 cycles).. As a result, it was found that the reaction under high annealing temperature, namely 94°C, 1 minute - 62°C, 2 minutes - 72°C, 2 minutes, regardless of cycles, produced specific amplification in human DNA
sample~but not in.yeast strain AB1380 DNA. PCR under low annealing temperature sometimes gave false positive signals in negative control and therefore could not be used. Thus, the PCR was carried out under the above-described conditions.
(4) Polymerase Chain Reaction (PCR) PCR-based first screening was performed using synthesized oligonucleotide primers described above against seven multi-filter DNA pools each of which represents the.DNA from 1920 colonies (20 x.96-well) as described (E. D. Green and M.v. Olson, Proc. Natl. Acad.
Sci. USA, 87, 1213-1217 (1990)). Positive multi-filter 2~~2~ ~'~
pools were divided into five pools each of which consists of 384 colonies (4 x 96-well), and further screened by the same procedure. 25 ng.each of YAC pool.DNAs Were used for reaction. DNA of CGM1 whose DNA.was used to construct the YAC library, and of the yeast strain AB1380 were included during the PCR analysis as positive and negative controls, respectively. After the amplification, the entire sample was analyzed by electrophoresis in 10% polyacrylamide gels containing 15%
glycerol and visualized by ethidium bromide staining.
Application (Kohyo) No. 4-504365; Proc. Natl. Acad. Sci.
USA, Vol. 86, pp.5898-5902, August 1989; Proc. Natl.
Acad. Sci. USA, Vol. 87, pp.5109-5113, July 1990;
Genomics 8, 742-750 {1991)). However, in the conventional methods in which human immunoglobulin genes are expressed in host animals other than human, there is a problem that the number of human VH segments provided for the genetic recombination is very small, so that the diversity of the expressed human immunoglobulins is limited. Even if only one VH segment is recombined, the diversity of the ~immunoglobulin is assured to some degree because of the combination with D and J genes. However, as mentioned above, since the diversity of immunoglobulins is determined by the rearrangement (random combination) of V gene segments, the more the human VH segments, recombined, the higher the diversity of the immunoglobulins expressed. If the diversity of immunoglobulins is increased, not only antibodies against a number of antigens can be formed, but also the possibility of forming an antibody having a high specificity to a given antigen is promoted. Therefore, it is important for therapies and diagnoses to recombine VH segments as many as possible.
DISCLOSURE OF THE INVENTION
Accordingly, an object of the present invention is to provide a DNA fragment comprising a plurality of human immunoglobulin VH segments. Another object of the present invention is to provide a novel human immunoglobulin VH segments.
The present inventors intensively studied and succeed in determining human immunoglobulin H chain V region gene segments having a size of about 800 kb and in determining DNA sequences of 64 human VH segments contained therein.
This made it possible to provide this DNA fragment of 800 kb and various DNA fragments contained therein, thereby completing the present invention.
That is, the present invention provides a DNA
fragment having a size of about 800 kbp and having the structure shown in Fig.. 1. It should be noted that in Fig. 1, the 64 human VH segments are those having DNA
sequences shown in Sequence ID Nos. l, 2, ... 63, and 64, respectively, in the order from downstream (i.e., from the side near the Jg gene).
The present invention also provides DNA fragments containing at least two consecutive functional human VH
segments which are contained in said DNA fragment of about 800 kb according to the present invention.
The present invention further provides DNA fragments Y20, Y103, Y21, Y6, Y-24, M131, M118, M84 and 3-31, which have been deposited.
The present invention still further provides DNA
fragments consisting essentially of at least two optional DNA fragments linked in an optional order, each of which contains at least two consecutive functional human VH
segments contained in the DNA fragment of about 800 kb according to the present invention.
The present invention still further provides DNA
fragments consisting essentially of at least two DNA.
fragments selected from the group consisting of DNA
fragments Y20,~Y103, Y21, Y6, Y-24, M131, M118, M84 and 3-31 which have been deposited, which are linked in an optional order.
The present invention still further provides novel human immunoglobulin VH segments having DNA sequences shown in Sequence ID Nos. f, 7, 8, 9, 10, 11, 12, 13, 14, _5_ 15, 16, 17, 18; 19, 20, 21, 22, 23, 24, 25, 26, 27, 29, 30, 31, 32, 33, 34, 35; 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 63 and 64, respectively.
By the present invention, novel human immunoglobulin VH segments and DNA fragments containing the same were provided. The DNA fragment of about 800 kb according to the present invention contains as many. as 64 human immunoglobulin VH segments. Thus, by producing human immunoglobulins by a host animal using this DNA fragment, the diversity of the produced human immunoglobulin is largely increased, when compared with the conventional methods.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows a genetic map of the DNA fragment of about 0.8 Mb according to the present invention:
. Fig. 2 shows the results of Southern hybridization of a representative DNA inserted in YAC.
Fig..3A shows the results of Southern hybridization of the fragment digested with restriction enzymes Mlu I
and Not I.
Fig. 3B shows a physical map of a YAC clone constructed based on the results shown in Fig. 3A.
Fig. 4 shows a genetic map of YAC clone Y6.
BEST MODE FOR CARRYING OUT THE INVENTION
The present inventors prepared a library by inserting the DNA partially digested with Eco RI into YAC
~:~~2~'~'~
by the method detailed in the examples hereinbelow described, which DNA was originated from human lymphoblastoid cell line transformed by EB virus, and succeeded in determining the structure of human VH gene region having a size~of.about 800 kbp using the above-mentioned library. The structure is shown in Fig. 1. In Fig. 1, the genetic map is shown on the four thick solid lines. The right side of each solid line~is .the 3' side and the left end of the upper most solid line continues to the right end of the second solid line. In the DNA
fragment shown in Fig. 1, there exist C genes, JH genes and D genes in the order mentioned from the 3' end.
Subsequent to the D genes, there are 64 vH segments. The DNA sequences of~all of these 64 VH segments have been.
determined as described in the examples below, and Sequence ID Nos. ~1, 2, ... 63, 64 were assigned to~the 64 VH segments in the order from downstream. Among these VH
segments, the functional VH segments which are thought to encode polypeptides are indicated by solid rectangles.
On the other hand, those which have the general features of the known Vg segments but do not presently encode polypeptides because of the termination codons contained therein, that is, pseudo VH segments are indicated by hollow rectangles. Immediately below the genetic map, restriction maps by Eco RI and Hind III are shown. The restriction.sites are indicated by short perpendicular lines. The short lines to which ends circles are attached are those whose order is not determined, and the dotted boxes indicate the regions in which Eco RI sites have not been determined. In Fig. 1, the symbol which looks like "Y" indicates the sites at which two restriction sites are close. In Fig. 1, restriction sites of Mlu I are indicated by hollow triangles and restriction sites of Not I are indicated by solid triangles. The fragments inserted in the clones employed for determining the structure of the DNA fragment are shown thereunder. The structure of the 3' side farther than the 3' end shown in Fig. 1 is known and described in Ravetch, J.V. et al., (1981) Cell, Vol. 27, pp. 583-591.
Among the DNA fragments inserted in the clones shown in Fig. 1, the yeasts containing Y20, Y103, Y21, Y6 and Y24 inserted in YAC have been deposited with National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology of 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, 305, Japan, under Accession Numbers FERM BP-4272, FERM BP-4275, FERM BP-4273, FERM BP-4271 and FERM BP-4274, respectively. The E. coli cells containing M131, M118, M84 and 3-31, respectively, inserted in cosmids have been deposited with National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology of 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, 305, Japan, under Accession Numbers FERM
BP-4279, FERM BP-4278, FERM BP-4277 and FERM BP-4276, respectively.
The DNA fragment having a size of about 800 kbp shown in Fig. 1 can be prepared by linking these _g-deposited DNA fragments by known~methods. That is, a DNA
fragment A and a DNA fragment B.whose DNA sequence at its terminal region overlaps with the DNA sequence of the terminal region of DNA fragment A (i.e., the~DNA sequence of the 3' region of DNA fragment A is identical to the DNA sequence of the 5' region of DNA fragment B) can be easily ligated by a method exploiting genetic recombination in the yeast.cells. More particularly, DNA
fragments A and B are inserted in separate YAC vectors, and the resulting recombinant YAC vectors. are introduced in separate mating type yeast cells, respectively. The resulting yeast cells are then fused. By this, genetic recombination occurs in the yeast host to form a YAC
having a DNA fragment in which DNA fragment A and DNA
fragment B are ligated, Which has only one overlapping region located at the terminal regions of DNA fragments A
and B. The thus formed recombinant YAC can easily be selected using the auxotrophy encoded in the YAC as a marker. This method is well-known in the art, and is described in, for example, Japanese Laid-open PCT
Application (ICohyo) No. 4-504365; Proc. Natl. Acad. Sci.
USA, Vol. 87, pp.9913-9917, December 1990; Science Vol.
250, p.94, Proc. Natl. Acad. Sci. USA, Vol. 89, pp.5296-5300, June 1992; and Nucleic Acid Research, Vol. 20, No..
12, pp.3135-3138. Since the terminal regions of each of the deposited 8 DNA fragments overlap the respective terminal regions of the adjacent DNA fragments, they can ~~.~~~'~''~
_g-be ligated sequentially by the method described above.
Although DNA fragments 3-31, M84, M118 and M-131 are cloned in cosmid vectors,.they can be kept in an artificial.chromosome in the yeast cell by cutting the recombinant cosmid with a restriction enzyme having a restriction site only in the cosmid vector, and ligating a YAC vector to the ends of the digested recombinant cosmid vector. Further, by the above-described method, the digested recombinant vector can be ligated to a YAC
clone of other regions. It should be noted that even if the above-mentioned 9 deposited fragments are ligated, a gap of about 4 kb still remains. A DNA fragment which fills the gap can be easily prepared by the method described below. That is, as shown in Fig. 1, since the Hind IIh fragment including the region of the gap is relatively large, this Hind III fragment can be obtained by completely digesting human genome by Hind III, electrophoresing the resultant, selecting~DNA fragments having sizes of about 15 kb, detecting the desired fragment with a probe, and recovering the detected-desired fragment. The probe used here can be isolated as follows. That is, the DNA fragments located at the both ends of the gap are subcloned using a plasmid and DNA
fragments which do not contain a repetitive sequence are prepared therefrom. The thus obtained fragments are then used for screening of the library. Only those detected by the probes which are the DNA fragments at both ends of the. gap are isolated.
As.described above, the DNA.fragment of about 800 kbp shown in Fig. 1 was provided according to the present invention. The fragments consisting of the DNA region included in this DNA fragment can also be used for producing human immunoglobulin by a genetic engineering method. More particularly, to increase the diversity of human immunoglobulin produced by a genetic engineering method, it is preferred to incorporate a fragment containing human VH segments as many as possible.
However, if the fragment contains at least two human VH
segments, the diversity to some degree is given during rearrangement, so that the fragment can be employed.
Thus, DNA fragments consisting of a region containing at least two consecutive functional VH segments, which region is contained in the DNA of about 800 kb shown in Fig. l can be employed and are useful. The number of the functional VH segments contained in.such DNA fragments is at least two, and is preferably not less than 6. The more the number of the functional VH segments, the higher the diversity of the human immunoglobulin produced, so that the more pref erred . Thus , the preference i s increased when the number of the functional VH segments is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 and 33, with the order mentioned. Among these fragments, although those having large molecular weights are cloned 2~~~~'~'~
into YAC vector, small fragments having a size of about not more than 50 kb are not.necessarily cloned into YAC
vector, but can be. cloned into cosmid vectors and plasmid vectors.
Such DNA fragments can be prepared since the information disclosed.in Fig. 1 and Sequence ID Nos. 1 -64 is available: That is, for example,.a DNA fragment containing not less than two functional VH segments can be.obtained by partially digesting human genome with an appropriate restriction enzyme such as Eco RI or Hind III, separating the resulting fragments by electrophoresis, and selecting a DNA fragment containing not less than two desired functional VH segments using not~less than two probes each of which hybridizes with one of the not less than two desired functional VH
segments. Alternatively, amplification by PCR may be employed in place of the detection by the probes. In this case, since the entire DNA sequences of the functional VH segments are known, the DNA sequences of the primers which should be used are also known, so that the PCR can be carried out easily.
The present invention further provides DNA fragments consisting essentially of optional DNA fragments each of which contains not less than two functional VH segments which are ligated in optional orders. That is, by ligating a plurality of the DNA fragments each containing.
not less than two functional VH segments, the number of VH segments in the DNA fragment can be increased when compared. with the case. where only one such DNA fragment containing not less thin two VH segments is used, so that the diversity of the produced immunoglobulin can be increased accordingly. The DNA fragments are not necessarily consecutive, and optional DNA fragments may be ligated in an optional order. In cases where there is no overlapping region between two,.DNA fragments to be ligated, the above-described method for ligating the DNA
fragments having an overlapping region cannot be applied.
However, two DNA fragments having no overlapping region can also be ligated by the method as follows.
The left arm~vector region and the right arm vector region of a YAC clone containing not less than two functional VH segments are recovered by the method of Hermanson et al (1991) (Nucleic Acids. Res.,l9; 4943-4948). A plasmid (pICL) which has a sequence homologous with the ampicillin-resistant marker (AMP) in the left arm vector region of the YAC, a marker (Lys) which reverse the lysine auxotrophy to the wild type, and a multiple cloning.site immediately downstream Lys; and a plasmid (PLUS) which has a sequence homologous with YAC4 region in the right arm vector region of the YAC, the above-mentioned Lys, a kanamycin-resistant marker (KAN), and a multiple cloning site immediately downstream the KAN are linearized and then introduced into yeast cells containing YAC by a conventional method. The plasmids 2~.~~~'~'~
pICL and pLUS cause recombination in the yeast cells at an appropriate frequency, thereby being recombined with the left arm vector region and the right arm vector region of the YAC. The yeast cells carrying such a YAC
are selected by using an appropriate selection medium and the YAC in the selected yeast cells is then cut with. an appropriate restriction enzyme which has a restriction sites ~n the multiple cloning.sites of the above-mentioned plasmids: By the operation described above, DNA fragments containing the left end or the right end of the DNA fragment originated from human contained in the YAC are recovered as plasmids. After amplifying the thus obtained plasmids in E. coli by a conventional method, the recovered plasmids are digested with a restriction enzyme and then ligated by ligase. The thus ligated DNA
fragment is then ligated to the left arm vector region or the right arm vector region of the YAC and introduced into yeast cells carrying the YAC. These YAC vectors causes recombination at a certain frequency between the intrinsic left arm or.right arm vector regions and the left end or right end region of the DNA fragment originated from human. By selecting the resulting recombinant vectors, a YAC clone containing a DNA
fragment originated from human, which left end is ligated to the right end of another DNA fragment originated from human, and a YAC clone containing a DNA fragment originated from human, which right end is ligated to the left end of another DNA fragment originated from human are recovered. Since these YAC clones have the structure in which the left end or the right end of a DNA
originated from human is ligated to the right end or the left end of another DNA originated from human, they can be recombined with a YAC clone having a sequence in the ligated DNA fragments by the method described above.
Further, by optionally ligating the above-described eight actually deposited DNA fragments in an optional order, a large fragment containing a number of VH
segments can be prepared.
By the present invention, the DNA sequences of the 64 VH segments contained in the fragment of about 800 kbp shown in Fig. 1 were determined. As described in detail in the examples below, among these, 50 VH segments are novel segments which have DNA sequences that have not hitherto been known. These novel human immunoglobulin VH
segments include pseudo segments which do.not encode a polypeptide. Even a pseudo segment has an utility because it may function as a donor of gene conversion in the somatic cell level.
The fiuman.immunoglobulin VH segments and the DNA
fragments containing the same according to the present invention can be used for producing human immunoglobulins in a mammalian host as described in, for example, Japanese Laid-open PCT Application (Rohyo) No. 4-504365.
Examples The present invention will now be described in more detail by way of examples thereof. It should be noted that the present invention is not limited to the following examples.
Example 1 ~ Determination of Structure of DNA Fragment of about 8 0 0. kbp .
(1) Library Used for Screening The human YAC library screened was constructed from DNA of an Epstein-Barr virus-transformed human lymphoblastoid cell line CGM1 (T. Imai and M.V. Olson, genomics, 8, 297-303 (1990)). Eco RI partial digests of CGM1 DNA were ligated to pYAC4 vector (D. Burke and M.V.
Olson, in "Guide to Yeast Genetics and Molecular Biology"
(C.~Guthrie arid G.R. Fink, eds), p.253, Academic Press, Orlando, 1991), and introduced into AB1380 yeast host strain (D. Burke and M.V. Olson, in "Guide to Yeast Genetics and Molecular Biology" (C. Guthrie and G.R.
Fink, eds), p.253, Academic~Press, Orlando, 1991). The library consisted of 15,000 independent clones with mean YAC size of about 360 kb. The library thus contained the equivalent of approximately 1.8 haploid human genomes.
DNA rearrangement in immunoglobulin H chain (IgFi) locus was first checked by Southern hybridization.using the human D and Jg probes. The result showed that an allele kept germline configuration while the other was VDJ
rearranged.
(2) Primers~used for PCR-based Screening 21~~~ ~'~
For PCR-based screening of human VH YAC clones, oligonucleotide primers for VH-LII and VH-I families, the first and the second largest VH families, were synthesized. VH region segments of immunoglobulins contain two hypervariable regions (CDR1 and CDR2) and three framework regions (FR1, FR2 and FR3) (E.A. Rabat et al., Sequences of Proteins of Immunological Interest., Fifth edition, NIH publications, Washington D.C. (1991)).
Nucleotide sequences of the framework regions are highly conserved within the same family, suggesting the possibility of oligonucleotide synthesis is for consensus primers corresponding to the framework regions. For this purpose, nucleotide sequences of FR1, FR2 and~FR3 regions in all the known VH sequences were aligned for comparison. Nucleotide sequences corresponding to the first 8 amino acid residues of the FR1 region had extremely high conservation not only within the same family but also between VH-I and VH-III families, which enabled the synthesis of a.forward primer F-univ common for the two families as shown in Table 1. Sequences for family-specific reverse primers Were independently chosen from conserved sequences in the FR2 region so that 3'-half of .the primer sequence has 100$ identity~to known VH
segments and, in particular, 3'-most nucleotide corresponds to the first letter of the highly conserved/invariant amino acid residues. More particularly, F-univ and I-R, and F-univ and III-R were ._ used as primers for the screening. The-DNA sequences of the primers are shown in Table 1.
(3) Optimal PCR Condition Check , Analytical experiments were carried out to determine the optimal condition for specific amplification. A
reaction mixture (5 ul) was prepared in accordance with the protocol recommended by Perkin-Elmer/Cetus. Thermal cycling was performed using a DNA Thermal Cycler (Perkin-Elmer/Cetus). Reactions were carried out using 25 ng of template human DNA under various annealing temperatures (55°C, 58°C, 60°C and 62°C) and cycles (25, 30, and 35 cycles).. As a result, it was found that the reaction under high annealing temperature, namely 94°C, 1 minute - 62°C, 2 minutes - 72°C, 2 minutes, regardless of cycles, produced specific amplification in human DNA
sample~but not in.yeast strain AB1380 DNA. PCR under low annealing temperature sometimes gave false positive signals in negative control and therefore could not be used. Thus, the PCR was carried out under the above-described conditions.
(4) Polymerase Chain Reaction (PCR) PCR-based first screening was performed using synthesized oligonucleotide primers described above against seven multi-filter DNA pools each of which represents the.DNA from 1920 colonies (20 x.96-well) as described (E. D. Green and M.v. Olson, Proc. Natl. Acad.
Sci. USA, 87, 1213-1217 (1990)). Positive multi-filter 2~~2~ ~'~
pools were divided into five pools each of which consists of 384 colonies (4 x 96-well), and further screened by the same procedure. 25 ng.each of YAC pool.DNAs Were used for reaction. DNA of CGM1 whose DNA.was used to construct the YAC library, and of the yeast strain AB1380 were included during the PCR analysis as positive and negative controls, respectively. After the amplification, the entire sample was analyzed by electrophoresis in 10% polyacrylamide gels containing 15%
glycerol and visualized by ethidium bromide staining.
(5) Colony Hybridization After PCR-based first and second screening, the location of the positive. clone within the 384-clone array was established by conventional colony hybridization.
The nylon filters consisting of 384 YAC clones were prepared by a known method (D. Burke and M.V. Olson, in "Guide to Yeast Genetics and Molecular Biology" (C.
Guthrie and G.R. Fink, eds), p.253, Academic Press, Orlando., 1991). V266BL (Y~ Nishida, et al., Proc. Natl.
Acad. Sci. USA, 79, 3833-3837 (1992)) and VHBV (M.
Rodaira et al., J. Mol. Biol., 190, 529-541(1986)) were used for probes representative for human VH-I and VH-III
families, respectively. These probes were labeled (5 x 105 cpm) with 32P-dCTP using Oligolabeling Rit (Pharmacia) and subjected to colony hybridization according to standard procedure (D. Burke, et al., supra). After the hybridization fox 12 hours at 65°C, filters were' washed twice with 2xSSC (lxSSC is 0.15 M
NaCl-15 mM sodium citrate) for 10 minutes at room temperature, then twice with 0.2xSSC-0.1% SDS for 30 minutes at 65°C. Filters were exposed overnight and corresponding positive YAC clones were picked up for further characterization.
The nylon filters consisting of 384 YAC clones were prepared by a known method (D. Burke and M.V. Olson, in "Guide to Yeast Genetics and Molecular Biology" (C.
Guthrie and G.R. Fink, eds), p.253, Academic Press, Orlando., 1991). V266BL (Y~ Nishida, et al., Proc. Natl.
Acad. Sci. USA, 79, 3833-3837 (1992)) and VHBV (M.
Rodaira et al., J. Mol. Biol., 190, 529-541(1986)) were used for probes representative for human VH-I and VH-III
families, respectively. These probes were labeled (5 x 105 cpm) with 32P-dCTP using Oligolabeling Rit (Pharmacia) and subjected to colony hybridization according to standard procedure (D. Burke, et al., supra). After the hybridization fox 12 hours at 65°C, filters were' washed twice with 2xSSC (lxSSC is 0.15 M
NaCl-15 mM sodium citrate) for 10 minutes at room temperature, then twice with 0.2xSSC-0.1% SDS for 30 minutes at 65°C. Filters were exposed overnight and corresponding positive YAC clones were picked up for further characterization.
(6) Insert Check by Colony PCR
To test the presence of specific DNA sequence in isolated YACs, simple and rapid rescreening of colony-purified clones was carried out by using PCR without. DNA
purification (E. D. Green and M.V. Olson, Proc. Natl.
Acad. Sci. USA, 87, 1213-1217 (1990)). That is, the positive yeast clones were streaked onto AFiC plates and grown. Four each of single colonies from each clone were transferred by toothpick into 5 ~cl of PCR mixture described above. PCR and following gel electrophoresis were performed for identification of the amplified bands under.the same condition as that used for screening. In most of'the clones, all of the four colonies gave rise to specific amplification of DNA fragments.
To test the presence of specific DNA sequence in isolated YACs, simple and rapid rescreening of colony-purified clones was carried out by using PCR without. DNA
purification (E. D. Green and M.V. Olson, Proc. Natl.
Acad. Sci. USA, 87, 1213-1217 (1990)). That is, the positive yeast clones were streaked onto AFiC plates and grown. Four each of single colonies from each clone were transferred by toothpick into 5 ~cl of PCR mixture described above. PCR and following gel electrophoresis were performed for identification of the amplified bands under.the same condition as that used for screening. In most of'the clones, all of the four colonies gave rise to specific amplification of DNA fragments.
(7) Sizing of YAC Clones Using PFGE
Many researchers claimed that some YACs are clonally unstable due to intrachromosomal rearrangement during the growth in culture resulting in size variation of the human DNA insert. This artifact is considered to be often mediated by repetitive sequences or tandem repeat of homologous DNA sequences in the insert DNA. Since VH
locus contains a number of homologous DNA fragments consisting of VR gene segments and their flanking regions, such kind of rearrangement can take place at considerable frequency. An additional problem is the presence of single yeast containing more than one insert YACs. In order to exclude the artifact clones for subsequent analysis and to identify YAC clones with.
multiple insert, the sizes of the YAC clones were first determined by pulse field gel electrophoresis (PFGE).
The same four VH-positive single colonies checked by PCR
were selected from 17 colonies originating from a single well, and miniprepared from 5 ml culture in AHC medium to give Iow-gelling temperature agarose blocks by~a known method (D. Burke et al.., su ra). Appropriate sized piece of agarose block was used for 'sizing the YACs by~PFGE
with a Pulsaphor*(Pharmacia) or a Crossfield*(ATTO, Tokyo, Japan) gel electrophoresis apparatus at 60 second pulse time. Concatamerized lambda DNA was also loaded as a size standard. After the electrophoresis, DNAs were transferred to nitrocellulose filter and subjected to Southern hybridization using pBR322 plasmid as a probe.
Typical result is shown in Fig. 2. AlI of the four colonies selected from each of clones Y21, Y22 and Y24 having DNA inserts with a size of 300 kb, 330 kb and 310 kb, respectively exhibited the same size, so that they seemed to have no recombination. On the other hand, since four colonies selected from clone Y23 had DNA
*Trade-mark . . _.
inserts with different sizes, the insert of the clone Y23 looked rather unstable due to frequent recombination.
Therefore, the colony which did not cause recombination was selected for the subsequent analysis. All but 3 clones including clone Y23 of 17 VH-carrying YAC clones including the analyzed VH~displayed instability of human inserts. Subsequent analysis revealed that such recombination took place regardless of the number of VH
segments in the insert DNA, indicating some other factors might be involved in homologous recombination. From 14 stable YAC clones among the 17 YAC clones containing VH, Y20, Y103, Y21, Y6 and Y24 were selected and used for the subsequent physical mapping.
Many researchers claimed that some YACs are clonally unstable due to intrachromosomal rearrangement during the growth in culture resulting in size variation of the human DNA insert. This artifact is considered to be often mediated by repetitive sequences or tandem repeat of homologous DNA sequences in the insert DNA. Since VH
locus contains a number of homologous DNA fragments consisting of VR gene segments and their flanking regions, such kind of rearrangement can take place at considerable frequency. An additional problem is the presence of single yeast containing more than one insert YACs. In order to exclude the artifact clones for subsequent analysis and to identify YAC clones with.
multiple insert, the sizes of the YAC clones were first determined by pulse field gel electrophoresis (PFGE).
The same four VH-positive single colonies checked by PCR
were selected from 17 colonies originating from a single well, and miniprepared from 5 ml culture in AHC medium to give Iow-gelling temperature agarose blocks by~a known method (D. Burke et al.., su ra). Appropriate sized piece of agarose block was used for 'sizing the YACs by~PFGE
with a Pulsaphor*(Pharmacia) or a Crossfield*(ATTO, Tokyo, Japan) gel electrophoresis apparatus at 60 second pulse time. Concatamerized lambda DNA was also loaded as a size standard. After the electrophoresis, DNAs were transferred to nitrocellulose filter and subjected to Southern hybridization using pBR322 plasmid as a probe.
Typical result is shown in Fig. 2. AlI of the four colonies selected from each of clones Y21, Y22 and Y24 having DNA inserts with a size of 300 kb, 330 kb and 310 kb, respectively exhibited the same size, so that they seemed to have no recombination. On the other hand, since four colonies selected from clone Y23 had DNA
*Trade-mark . . _.
inserts with different sizes, the insert of the clone Y23 looked rather unstable due to frequent recombination.
Therefore, the colony which did not cause recombination was selected for the subsequent analysis. All but 3 clones including clone Y23 of 17 VH-carrying YAC clones including the analyzed VH~displayed instability of human inserts. Subsequent analysis revealed that such recombination took place regardless of the number of VH
segments in the insert DNA, indicating some other factors might be involved in homologous recombination. From 14 stable YAC clones among the 17 YAC clones containing VH, Y20, Y103, Y21, Y6 and Y24 were selected and used for the subsequent physical mapping.
(8) Physical Mapping of YAC Clones with Rare Site Endonucleases Gel blocks were prepared from the YAC clones after sizing and were used for physical map construction by PFGE. In general, detailed physical map using several enzymes might be required for long-range YAC analysis.
In this example, however, only two rare-site restriction enzymes (i.e., restriction enzymes whose restriction sites occur relatively rarely), namely Not I and Mlu _I, were used for overlapping analysis of the YAC clones mainly by the~following two reasons: 1) VH-carrying YAC
clones can be arrayed.with several other information such as comparison of the'size or the pattern of the fragments hybridized with VH probes or non-repetitive probes ~~~z~~~
isolated from VH-carrying cosmid clones, 2) it is necessary to subclone the YACs into cosmids fox detailed structural analysis including construction of physical maps using ordinary restriction enzymes.
Gel blocks digested in completion with Not I.or Mlu I were electrophoresed with a PFGE apparatus using.a pulse time of 30 to 60 seconds depending on the length of YAC. Mixtures of lambda phage DNA, its Xho I digests and Hind III digests were also used as low molecular weight size markers. Southern filters were first hybridized .
with total human large molecular. DNAs for detection of all restricted fragments. The sizes of detected bands were summed up to fit the length of undigested yAC
insert. Filters were hybridized consecutively with pBR322 DNA probes corresponding to each of the pYAC4 arms. A P'vu II and Bam HI double digest of pBR322 results in a 2.67-kb and 1.69-kb fragments which hybridize specifically to the left (trp) and the right (ura) end of YACs, respectively. Filters were also hybridized with six VH family-specific probes for the presence of VH segments in digested DNA fragments.
Origin of VH family-specific probes for VH_II, VH_IV~
VH-V and VH-VI families, respectively, are; VCE-1 (N.
Takahashi et al., Proc. Natl. Acad. Sci. USA, 81, 5194-5198 (1984)), V71-2 (R~H. Lee et al., J. Mol. Biol., 195, 761-768 (1987)), 5-1R1 (J.E. Berman~et al., EMBO J. 7, 727-1051 (1988) and 6-1R1 (J.E. Berman et.al., EMBO J. 7, =23-?27-1051 (1988)).
In order to array Not I and Mlu I fragments detected by the complete digestion experiments, hybridization experiments using partially digested YAC DNA were carried out. Analytical experiment was necessary to determine the optimal condition for partial digestion since the efficiency of the restriction enzyme reaction is highly dependent on the purity of DNA. In the DNA preparation in this example, 6-hour incubation with 1 unit of restriction enzyme was, in most cases, sufficient for complete digestion of a gel block (about 500 ng of DNA).
Partial cleavage of DNA was achieved by varying the time of digestion as follows: ~ .
1. Dialyze three gel flocks (about 50 ~1 each volume containing about 1 ~cg of DNA, stored in~0.5 M EDTA (pH
8.0)) fvr 1 hour against 50 ml of distilled water at room temperature with gentle agitation. Repeat this step for complete removal of EDTA.
2. Equilibrate the blocks with 10 ml appropriate digestion buffer at 37°C for 30 minutes.
3. Transfer each block to 250-ul reaction mixture containing 1 unit each of restriction enzyme in lx digestion buffer.
4. Incubate all three tubes for 10 minutes, 30 minutes and 1 hour at 37oC.
5. Stop the reaction by adding 100 ~cl of 0:5 M EDTA
(pH 8.0).
.,.
6. Equilibrate the blocks with appropriate gel electrophoresis buffer 2 - 3 times over a 1 hour period and immediately perform PFGE using an appropriate pulse time.
Filters were.hybridized with the above-described right- or left-end probe of YAC vector and~the size of the hybridized restriction fragments was determined by comparison with size standards (Fig. 3A). Results from complete and partial digestion experiments were combined to construct a physical map of YAC clones shown in Fig.
3B. Mapped clones were thus linked and classified into several contigs.
In this example, however, only two rare-site restriction enzymes (i.e., restriction enzymes whose restriction sites occur relatively rarely), namely Not I and Mlu _I, were used for overlapping analysis of the YAC clones mainly by the~following two reasons: 1) VH-carrying YAC
clones can be arrayed.with several other information such as comparison of the'size or the pattern of the fragments hybridized with VH probes or non-repetitive probes ~~~z~~~
isolated from VH-carrying cosmid clones, 2) it is necessary to subclone the YACs into cosmids fox detailed structural analysis including construction of physical maps using ordinary restriction enzymes.
Gel blocks digested in completion with Not I.or Mlu I were electrophoresed with a PFGE apparatus using.a pulse time of 30 to 60 seconds depending on the length of YAC. Mixtures of lambda phage DNA, its Xho I digests and Hind III digests were also used as low molecular weight size markers. Southern filters were first hybridized .
with total human large molecular. DNAs for detection of all restricted fragments. The sizes of detected bands were summed up to fit the length of undigested yAC
insert. Filters were hybridized consecutively with pBR322 DNA probes corresponding to each of the pYAC4 arms. A P'vu II and Bam HI double digest of pBR322 results in a 2.67-kb and 1.69-kb fragments which hybridize specifically to the left (trp) and the right (ura) end of YACs, respectively. Filters were also hybridized with six VH family-specific probes for the presence of VH segments in digested DNA fragments.
Origin of VH family-specific probes for VH_II, VH_IV~
VH-V and VH-VI families, respectively, are; VCE-1 (N.
Takahashi et al., Proc. Natl. Acad. Sci. USA, 81, 5194-5198 (1984)), V71-2 (R~H. Lee et al., J. Mol. Biol., 195, 761-768 (1987)), 5-1R1 (J.E. Berman~et al., EMBO J. 7, 727-1051 (1988) and 6-1R1 (J.E. Berman et.al., EMBO J. 7, =23-?27-1051 (1988)).
In order to array Not I and Mlu I fragments detected by the complete digestion experiments, hybridization experiments using partially digested YAC DNA were carried out. Analytical experiment was necessary to determine the optimal condition for partial digestion since the efficiency of the restriction enzyme reaction is highly dependent on the purity of DNA. In the DNA preparation in this example, 6-hour incubation with 1 unit of restriction enzyme was, in most cases, sufficient for complete digestion of a gel block (about 500 ng of DNA).
Partial cleavage of DNA was achieved by varying the time of digestion as follows: ~ .
1. Dialyze three gel flocks (about 50 ~1 each volume containing about 1 ~cg of DNA, stored in~0.5 M EDTA (pH
8.0)) fvr 1 hour against 50 ml of distilled water at room temperature with gentle agitation. Repeat this step for complete removal of EDTA.
2. Equilibrate the blocks with 10 ml appropriate digestion buffer at 37°C for 30 minutes.
3. Transfer each block to 250-ul reaction mixture containing 1 unit each of restriction enzyme in lx digestion buffer.
4. Incubate all three tubes for 10 minutes, 30 minutes and 1 hour at 37oC.
5. Stop the reaction by adding 100 ~cl of 0:5 M EDTA
(pH 8.0).
.,.
6. Equilibrate the blocks with appropriate gel electrophoresis buffer 2 - 3 times over a 1 hour period and immediately perform PFGE using an appropriate pulse time.
Filters were.hybridized with the above-described right- or left-end probe of YAC vector and~the size of the hybridized restriction fragments was determined by comparison with size standards (Fig. 3A). Results from complete and partial digestion experiments were combined to construct a physical map of YAC clones shown in Fig.
3B. Mapped clones were thus linked and classified into several contigs.
(9) Isolation of Insert-terminal Sequences from YACs After isolated YAC clones were classified into several contigs based on their restriction maps, insert-terminal DNA segments were isolated from both ends of each contig to synthesize oligonucleotide primers. As is often pointed out, considerable percentage (up to 30%) of YAC clones in libraries contain noncontiguous DNA
segments spliced together resulting in "chimeric clone".
Since no good strategies have been developed to exclude coligation artifact during the construction of the library, it is necessary to check this possibility with appropriate method after isolation of YAC clones. In this example, the strategy to investigate the possibility by using PCR with synthesized insert-terminal primers was taken. The reason is that the synthesized primers would be useful not only to investigate chimeric clones but also to register resulting sequences as sequence tagged sites (STS) for rescreening the YAC library by PCR. In addition, they could be used to look for overlaps between contigs~which could not be found by comparison of their restriction maps.
For isolation of insert-terminal YAC segments, several different methods can be employed including more sophisticated and rapid method by inverse PCR and the Vectorette system (J. H. Riley et al.., Nucleic Acids Res., 18,.2887-2890 (1990.)). However, in this example, a rather classical way, that is, to subclone the fragments with plasmid or lambda phage vectors was taken. High molecular weight DNA from YAC clones was digested with restriction enzymes which have recognition sites both in right- and left-arm sequences. Gel electrophoresis was performed in a 0.7~ agarose gel and Southern filter was hybridized with a 0.62-kb Hind III - Sal I fragment of pBR322 DNA (TetR) which specifically hybridizes with insert-vector boundary sequence of pYAC4 vector. The DNA
fractions of interest were recovered from the gel using DES1 paper and ligated to either EMBL4 or pUCl9 vector depending on the insert size. Isolated fragments with EMBL4 vector were subcloned into pUCl9 vector for subsequent sequencing. The chain. termination method~'with M13 forward or reverse primer was used for sequencing these plasmid clones. Sequences for insert-terminal primers were provided from the non-repetitive portion in the resulting sequence.
PCR experiments were achieved.to investigate the above-mentioned artifact using primers at the both ends of YAC-DNA against the DNA_from a human~mouse somatic , cell hybrid GM10479 line (Colier Institute) which carries human chromosome 14 alone in which the human IgH locus exists. DNA from CGM1 cells (source of YAC library) and Rag cells (mouse cell) were also used as positive and negative controls, respectively. PCR was carried out in 25- ~1 reactions according to a known method (H.S. Rim and O. Smithies, Nucleic Acids Res., 16, 8870-8903 (1988)). 200 ng each of DNA was used for the regction.
Incubations containing DNAs from GM10479, CGM1 and Rag, respectively were subjected to 35 to 40 cycles at 95°C, 1 minute - 55 to 62°C, 2 minutes = 72°C, 2 minutes according to the condition optimized by analytical experiment using CGM1 DNA. The YAC clones of which either of the two insert-terminal primers gave no specific amplification against GM10479 were concluded to be chimeric clones. Only one contig neither of which primers gave~amplified bands was turned out to cover orphan VH locus on chromosome 16.
segments spliced together resulting in "chimeric clone".
Since no good strategies have been developed to exclude coligation artifact during the construction of the library, it is necessary to check this possibility with appropriate method after isolation of YAC clones. In this example, the strategy to investigate the possibility by using PCR with synthesized insert-terminal primers was taken. The reason is that the synthesized primers would be useful not only to investigate chimeric clones but also to register resulting sequences as sequence tagged sites (STS) for rescreening the YAC library by PCR. In addition, they could be used to look for overlaps between contigs~which could not be found by comparison of their restriction maps.
For isolation of insert-terminal YAC segments, several different methods can be employed including more sophisticated and rapid method by inverse PCR and the Vectorette system (J. H. Riley et al.., Nucleic Acids Res., 18,.2887-2890 (1990.)). However, in this example, a rather classical way, that is, to subclone the fragments with plasmid or lambda phage vectors was taken. High molecular weight DNA from YAC clones was digested with restriction enzymes which have recognition sites both in right- and left-arm sequences. Gel electrophoresis was performed in a 0.7~ agarose gel and Southern filter was hybridized with a 0.62-kb Hind III - Sal I fragment of pBR322 DNA (TetR) which specifically hybridizes with insert-vector boundary sequence of pYAC4 vector. The DNA
fractions of interest were recovered from the gel using DES1 paper and ligated to either EMBL4 or pUCl9 vector depending on the insert size. Isolated fragments with EMBL4 vector were subcloned into pUCl9 vector for subsequent sequencing. The chain. termination method~'with M13 forward or reverse primer was used for sequencing these plasmid clones. Sequences for insert-terminal primers were provided from the non-repetitive portion in the resulting sequence.
PCR experiments were achieved.to investigate the above-mentioned artifact using primers at the both ends of YAC-DNA against the DNA_from a human~mouse somatic , cell hybrid GM10479 line (Colier Institute) which carries human chromosome 14 alone in which the human IgH locus exists. DNA from CGM1 cells (source of YAC library) and Rag cells (mouse cell) were also used as positive and negative controls, respectively. PCR was carried out in 25- ~1 reactions according to a known method (H.S. Rim and O. Smithies, Nucleic Acids Res., 16, 8870-8903 (1988)). 200 ng each of DNA was used for the regction.
Incubations containing DNAs from GM10479, CGM1 and Rag, respectively were subjected to 35 to 40 cycles at 95°C, 1 minute - 55 to 62°C, 2 minutes = 72°C, 2 minutes according to the condition optimized by analytical experiment using CGM1 DNA. The YAC clones of which either of the two insert-terminal primers gave no specific amplification against GM10479 were concluded to be chimeric clones. Only one contig neither of which primers gave~amplified bands was turned out to cover orphan VH locus on chromosome 16.
(10) Cosmid Subcloning and Construction of Physical Maps Isolation of~large chromosomal region using YAC
system is advantageous for the. initial step of physical mapping. However, subsequent step to analyze large DNA
fragments in YAC can be problematic since exogenous DNA
inserts cannot be. easily separated from yeast chromosomal DNA and fragments up to several hundred kb are difficult to handle without mechanical shearing. In order to map VH segments of a.large DNA fragment containing VH
segments, detailed restriction map using common 6bp-site restriction enzyme is necessary. For this purpose, YAC
clones were subcloned into cosmids. Cosmid libraries were constructed from whole YAC DNA without previous separation of cloned DNA from host chromosome. There are two major reasons for this: 1) separation of intact insert DNA and.their manipulation are difficult, 2) 4000 independent colonies are sufficient for complete coverage of ,YAC insert since the genome size of.yeast is about 1,5 x 104 bp, 1/200 of that of human.
In general, there are two major difficulties in the construction of cosmid libraries. The first is self-ligation of vector DNAs, resulting in generation of clones carrying no inserts of foreign DNA, and 'the second is insertion of more than one DNA fragments in a single vector, namely co-ligation artifact. To overcome these problems, great efforts have been made including construction of better-designed vectors with two cos sites and modified method for ligation.such as partial filling of vector and insert DNAs (J. Sambrook et al., A
Laboratory Manual, Cold Spring Harbor Laboratory Press,.
Cold Spring Harbor, N.Y. (1989)). Size fractionated insert DNA usually contains smaller DNA molecules trapped among larger molecules especially when excess amount of DNA was loaded in the preparative gel. Alkaline phosphatase treatment of insert DNA is effective in order to exclude the co-ligation between inserts but gives rise to polymerized vector DNA during the ligation step, which causes high background of empty colonies under the antibiotic selection. In this example, however, less .
than 5 gig of YAC DNA was sufficient for insert preparation and thus preparative gel electrophoresis was successful without contamination of smaller DNA
fragments. Most of the cosmid libraries were thus constructed with minimal steps in combination with alkaline phosphatase-treated cosmid vector and partially digested DNA of exact size range for cosmid~insert (from 35 kb to 45 kb).
p1 Preparation of Yeast DNA Containing YAC
Since large DNA fragments are required as starting material for preparing the DNA, extraction of DNA from yeast cells with minimal shear damage is one of the most critical steps. Obviously, the best way is to manipulate DNA in-gel because DNA is fully protected from shear damage. The present inventors found, however, that gentle extraction of DNA in liquid from.yeast- cells gives sufficient length of. DNA (>200 kb) for partial digestion and subsequent size fractionation. In addition, liquid DNA is easier to control the condition for partial ~1~~~~~
digestion than gel block DNA. With a simple and rapid (6 hours for total procedure) method described below, about 50 ~g of large size DNA. (>200 kb) can be routinely purified from 100-ml. yeast culture.
(i) Spin down~yeast cells and wash them with TE (10 mM.
Tris HC1 (pH 8.0) - 1 mM EDTA) twice.
(ii) Resuspend the cells in 20 m1 of 0.1 M EDTA (pH 7.5), 1 M sorbitol, 0.2 mg/ml of Zymolyase 100T (ICN
Cat#152270), 15 mM 2-mercaptoethanol. Incubate at 37oC
for 1 hour to form spheroplasts.
(iii) Spin down the spheroplasts and resuspend in 9' ml of 0.1 M Tris HC1 (pH 7.5), 50 mM EDTA (pH 7.5).
(iv) Add 1 ml (1/10 final volume) of 10% SDS and mix gently. Incubate at 60°C for 10 to 20 minutes.
(v) Add 1/3 volume of 4 M potassium acetate and mix gently. Leave on ice for 30 minutes.
(vi) Centrifuge at 2000xg for 30 minutes and transfer the supernatant to a new tube. Add 3 volumes of isopropanol and mix gently. Leave at room temperature for 10 to 20 minutes for precipitation of DNA.
(vii) Centrifuge again at 2000xg for 30 minutes and discard supernatant. Dissolve the pe~.let in 10 ml of water.
From~this step onwards, care should be taken not to give shear damage to the DNA.
(viii) Extract with phenol twice and with CIAA
(chloroform:isoamyl alcohol = 24:1) twice followed by ethanol.precipitation at room temperature for 1'p to 20 minute s ~.
(ix) Centrifuge at 2000xg for 30 minutes. Rins~ the pellet with 70% ethanol and dry up the pellet.
(x) Dissolve with l.ml of TE. ~
p Vector DNA Preparation Lorist 2 DNA was linearized by digestion with Hind III or Bam HI. Linearized~DNA was dephosphoryl~ted by treatment with bacterial alkaline phosphatase. Small aliquots of DNA before and after phosphatase treatment were used for test ligation for phosphatase treatment according to a known method (J. Sambrook et al.,, su ra).
~3 Insert DNA Preparation Analytical experiment of partial digestion of yeast DNA was performed according to standard procedure (J.
Sambrook et al., su ra) to determine the optimal enzyme concentration and reaction time. Preparation ofi size-fractionated DNA from the gel was achieved with~LGT
agarose and ~ agarase I. This very gentle methbd resulted in high recovery (>90%) of fractionateca DNA
without degradation. Scaled up cleavage reactiqn was done using 5 ug of DNA with optimal enzyme concentration. Digested samples were loaded in ;a preparative gel of 0.5% LGT agarose (Bio Rad preparative grade) at about 1 V/cm overnight. Linearized lambda DNA
and its Xho I-digests which give 35-kb and 15-kt~~bands were also loaded as size markers. After visualixing~the ~~.~2~7'~
=31-DNA under ultraviolet transilluminater, a small slice~of agarose containing the fraction ranging from 35 kb to 45 kb was cut out. Recovery of the DNA from the gel slice was achieved using ~ agarase I (NEB) as follows:
(i) Equilibrate the gel block with water for complete removal of gel electrophoresis buffer.
(ii) Transfer the block to a new tube and add 1/9 volume of. lOx ~ agarase I buffer.
(iii) Melt the gel at 68°C for 10 minutes. Cool to 40°C
and incubate the molten agarose at 40°C for 1 hour with optimal number of units of ~ agarase I.
(iv) Adjust the salt concentration of the solution to 0.5 M~NaCl for ethanol precipitation. Chill on ice for 10 minutes.
(v) Centrifuge at 15,OOOxg for 15 minutes to pellet any remaining undigested carbohydrates.
(vi) Transfer the DNA-containing supernatant to a new tube. Precipitate the DNA with 3 volumes of ethanol at -80°C for 10 minutes.
(vii) Centrifuge at 15,OOOxg for 15 minutes and remove the supernatant. Rinse the pellet with 70% ethanol and dry up the pellet.
(viii) Resuspend the pellet in appropriate volume of water for subsequent manipulation.
With this method,~in average 100 to 300 ng of size-fractionated DNA can be recovered.
~ Ligation, in vitro Packaging and Infection to ~ i ~~ ~2~~
This process was performed according to standard procedure ,(J. Sambrook et al., supra). By using lambda inn packaging kit (Nippon Gene) and ED8768 host strain, about 10,000 colonies were obtained from 25 ng of ligated DNA.
~.Screening of Cosmid Libraries Initial screening was carried out using Hind III-partial cosmid libraries. About .10,000 colonies (500 colonies per ~lOcm plate x20) were plated on LB plates containing 50 ~g/ml of kanamycin so that single colonies can be picked up after first screening. Colonies were then lifted from~the plates with X8.2 cm detergent-free nitrocellulose membranes (Advantec Toyo Membrane) and subjected to colony hybridization. Three different kinds of probes were used for screening, namely mixture of six VH-family specific probes to isolate VH-containing cosmid clones, YAC vector probes (TetR gene segment of pBR322, described above) for isolation of insert-terminal cosmid clones, and total human DNA for any remaining cosmid clones. In average, 50 to 100 clones from a YAC clone with approximately 300-kb insert were isolated with the probes.
~ Construction.of Cosmid Contigs DNA from cosmid clones.was isolated by the alkaline lysis method by a conventional method (J. Sambrook et al., su ra). Purified cosmid DNAs were digested with Eco RI or Hind III and subjected to agarose gel electrophoresis for restriction mapping. Overlaps between clones were easily. found by comparinglrestriction patterns among cosmid clones. Ordered cosmid clones were then cleaved with Eco. RI or Hind III and loaded in a 0.7~
agarose gel. Southern filters were hybridized with six VH-family specific probes for identification of location and number of VH segments in cosmid clones. Filters were washed three times for 30 minutes under standard conditions (at 50°C in lxSSC, 0.1% SDS) followed by stringent conditions (at 65°C in O.IxSSC, 0.1~ SDS).
Location of VH segments were determined by comparison between hybridization pattern of cosmids and their physical maps.
Theoretically, approximately 50 independent cosmid clones (about 7 fold of the whole YAC insert) would be sufficient to cover the whole YAC insert of 300 kb in length. However, the distribution of cosmid clones were uneven and there still remained a few gaps. The clones corresponding to the gaps could not be isolated even after screening of Sau 3AI partial. library or chromosomal walking by using the probes isolated from the edge of each contig. Regions not present in the cosmid libraries were subcloned with phage or plasmid vectors by isolation' of DNA fragments of required size from YAC DNA as shown in Fig. 4. After the complete physical map was constructed, the present inventors found out that this was not due to the nonrandom distribution of restriction sites within the YAC insert. The presence of some classes of sequences such as pahindromic.or tandem repeat DNA might make these regions unclonable or under-represented by using cosmid system. The complete physical map of the 0.8-Mb region constructed in this example is shown in Fig. 1 as mentioned above. The distance.from JH of each VH segment shown in Fig. 1 and the sizes of Eco RI and Hind III fragments are shown in Table 3.
Example 2 Construction of Cosmid Clones A cosmid library was constructed from human high molecular DNAs as follows:
3-31: High molecular DNAs obtained ,from human placenta were partially digested with Tag ~I and the resultant was subjected to electrophoresis on 0.5~ agarose gel. The 35 - 45-kb bands were recovered by using DEAF paper. The recovered DNAs were treated with alkaline phosphatase and the resultant was ligated to cosmid vector pJBB which.had been completely digested with a.restriction enzyme Cla I.
The ligation product was subjected to in vitro packaging and the resultant was infected to host E. coli 490A, followed by the screening by the conventional colony hybridization to obtain the clone.
M131, M84 and M118: These fragments were obtained by the same method as for 3-31 except that the DNA used was human pro B cell line FLEB14-14, the vector and the host E.- coli used were Lorist 2 and ED8767, respectively, the combination of restriction enzymes employed was Xba I and Hind III, and the edges of the fragments were modified by the partial repairing. The partial repairing was carried out according.to a known method (J. Sambrook, E.F.
Fritsch and T. Maniatis, 1989, Molecular Cloning; a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
Example 3 Sequencing Analysis of VH Segments Instead of sequencing subcloned VH-containing DNA
fragments using vector primers, VH family-specific oligonucleotide primers were synthesized. As mentioned above, nucleotide sequences of FR regions of VH.segments are highly conserved within the same family, so the present inventors selected consensus sequences from the conserved portions and synthesized family-specific oligonucleotide primers for sequence analysis. For this purpose, automated fluorescence-based sequencing system Model 373A developed by Applied Biosystems was employed.
Dye-Deoxy*terminator sequencing kit supplied from the same company using fluorescent-dye labeled dideoxy nucleotides was suitable for our purpose~since synthesized VH-specific primers could be directly used without fluorescence-label.
(I) Subcloning of VH-containing Restriction Fragments In order to use VH-family specific primers for sequencing, it is essential to subclone VH-containing DNA
fragments so that each plasmid contains only one VH
*Trade-mark kb was cut out. Recovery of th 76432-1 ca o2i62s~~ Zooo-io-2o segment. Several other 6bp-site enzymes than Eco RI and Hind III were used to isolate single VH-carrying DNA
fragments. Plasmid DNA of the subcloned fragments was isolated by alkaline lysis method followed by ult=acentrifugation twice to obtain high~quality DNA
samples for accurate sequences.
(2) Oligonucleotide Synthesis for Sequencing To select consensus sequences for VH family-specific oligonucleotide primer synthesis, nucleotide sequences of framework regions and axon-intron boundaries of the known VH segments were aligned by family. Attention was paid so that 3'-half of them have 100% identities to reference sequences and 3'-most nucleotide corresponds to the first or the second letter of highly conserved/iwariant amino acid residues. Nineteen additional primers were designed for five VH families as shown in Table 1 (described below).
(3) Sequencing Reaction and Gel Electrophoresis The sequencing reaction was performed by PCR using Dye-Deoxy terminator sequencing kit (ABI) according to manufacturer's instruction. Gel electrophoresis and detection of signals were done in the sequencing apparatus according to the users manual of the system.
In average, sequences of over 350 bases were obtained from each reaction.
(4) Evaluation of Synthesized VH Family-specific Primers The primers F-univ and I-R were first' chosen to *Trade-t~k 21~2~'~~
sequence VH-I segments. As shown in Table 2, they annealed 11 of 12 VH_I segments analyzed. It i~s to be noted that all of 6 functional VH-I segments could be sequenced with these two primers. Two more primers, I-NF1 and I-NR1 were designed for V1-14P and V1-27P
segments. These two primers were also used for. some other VH segments to verify their sequences obtained by first two primers (Table 2).
Eight primers were designed and used for sequencing VH-III family segments. The first sequencing reaction of each VH segment was performed with F-univ and III-R
primers. They annealed more. than $0$ of the VH_III
.segments analyzed (25/30 for F-univ and 24/30 for III=
R)(Table 2). Importantly, again, all the functional VH-. III segments with one exception could be sequenced with this combination of primers, suggesting that they would be good for most of VH-III cDNA. Based on the nucleotide sequences obtained from first experiment, six additional primers (III-F3, III-R3, III-F4, III-R4, III-NF1 and III-F2) were designed and appropriate combination among them were used for further analysis. Among these, III-R3 and III-F4 were used to determine the sequence of 5' regulatory region and 3' flankii-ig region, respectively.
V3-29P and v3-32P were pseudogenes with extensive divergence in their sequences and thus all but III-NF1 failed to anneal these two VH segments. Sequences of V3-25P, V3-44P and V3-63P were determined using M13 ~~.~~~'~'~
vector primers from their internal restriction~sites.
Five each of synthesized primers were used to determine the sequences of VH segments belonging to VH-III VH-IV and VH-V families. Since VH segments belonging to each of these three families are highly homologous with each other, it was thought that four each of the primers are.enough for most. of the VH segments belonging to these smaller VH families. In fact,~all four VH-II
family-specific primers annealed three VH-II segments (V2-5, V2-lOP and V2-26). In brief, in total 11 primers (F-univ and I-R for VH-I; II-R1, II-F2 and II-R2 for VH-II% F-univ and IiI-R for VH-III% IV-R1, IV-F2 and IV-R2 for VH-IV% V-R2 and V-R3 for VH~V) would be sufficient for sequencing most of the VH segments belonging to five VH families. The II-F1, III-NF2 and IV-F1 primers contain intron sequences and thus cannot be used for cDNA
sequencing.
By this procedure, the DNA sequences of the 64 VH
segments were determined and they are shown in Sequence ID Nos. 1 - 64 as mentioned above. The distance of each VH segment from JH and the sizes of Eco RI and Hind III
fragments are summarized in Table 3.
(5) Transcriptional Polarities of VH Segments The strategy for sequencing VH segments with family-specific primers was not suitable for determination of transcriptional polarities of the VH
segments because it did not require restriction map of ..._ single VH-containing subcloned fragments. The. present inventors could not determine orientations of all the VH
segments within this region for that reason. The present inventcsrs found, however, that 8 regions containing 21'VH
segments-were already isolated in cosmid or phage clones since sequences.between corresponding VH segments as well as their restriction maps were identical with each other.
As the relative orders of these 21 VH segments within these clones are identical to those in the 0.8-Mb region, it was concluded that the orientation of these 21 VH
segments are the same as those of the JH segments.
- ~~.~"~~7'~
Table 1 V" family-specific primers~used for screening and sequencing family name sequence(5'to 3' ) X location direction I, 13, F univ AGGTGCAGCTGGTGCAGTCTG 1-8 ~ forward V - .
I I R ~CCAGGGGCCTGTCGCACCCA 36-42 reverse -I N F TGGGGCCTCAGTGAAGGTCTCCTG 14-22 forward I N R GATCC(A/G)TCCCATCCACTCAAG45-51 reverse II II F 1 TGTCTTCTCCACAGGGGTCTT intron-(-2)forward -II F 2 GGGAAGGCCCTGGAGTGGCT 42-48 forward -IL R 1 GTGCAGGTCAGCGTGAGGGT 17-23 reverse -II R 2 TGGTTTTTG.GAGGTGTCCTTGG 70-77 reverse -III III R CACTCCAGCCCCTTCCCTGGAGC 40-47 reverse -III F 3 GTGAGGTTCAGCTGGTGGAGT (-1)-7 forward -III R 3 AGCTGAACCTCACACTGGAC (-3)-4 reverse -111 F 4 AAGGGCCGATTCACCATCT 64-70 forward -II< R 4 TTGTCTCTGGAGATGGTGAA 68-73 reverse -II< N F TGAGACTCTCCTGTGCAGCCTCTG 18-26 forward Ill N F TCT(T/C)TGTGTTTGCAGGTGT intron-(-3)forward IV F 1 TCTGTTCACAGGGGTCCTGTC ntron-(-1) forward - i N F 2 TCCGGCAGCCCCCAGGGAA 37-43 forward -Iv R 1 GCAGGTGAGGGACAGGGT 17-22 reverse -IV R 2 CAGGGAGAACTGGTTCTTGGA 74-80 reverse -V V R 1 CCCGGGCATCTGGCGCACCCA 36-42 reverse -V R 2 GCTGCTCCACTGCAGGTAGGC 78-82R reverse -V R 3 CTTCAGGCTGCTCCACTGCAG 74-83 reverse -X Locations of the primers are indicated as amino acid residue number according to Kabat et al. Bases with redundancy are shown in the parentheses. Directions relative to coding sequence are also shown.
~~.~w~~~
Table 2 List of useful primers for sequencing V" clones VH-, primers VH-,m primers VH_", primers V" segments univ R NFI NRI univ R F3 R3 F4 R4 NF1 NF2 FI Rl F2 R2 V" I 1-2 + +
1-3 + +
1-8 + + .
1-12P + +
I-14P - + +
1-17P + ~ + + +
1-18 + + +
1-24P + + + +
1-27P + - + +
1-40P + +
1-45 + +
1=46 +. +
VH DI 3-6P - + - + - + + +
3-7 + + + +
3-9 + + +
3-11 + + + +
3-13 + - + + + +
3-15 + + + +
3-16P + + + +
3-19P + + +
3-20 + + + + +
3-21 + + + + +
3-22P + + +
3-23 + + + : + +
3-29P _ _ _ _ _ ~..
3-30 + + + + +
3-32P _ _ _ _ _ _ + _ 3-33 + + + + +
3-35 + +
3-36P - + +
3-37P + - +
3-38P + +
3-41P + +
3-42P. + - + +
3-43 ~ + + .
3-47P + +
3-48 + +
3-49 + +
3-50P + +
3-52P~ + +
3-53 + +
3-54P + + +
3-64 + +
yH Iv 4-4 + + + +
+ + - +
4-34 + + + +
Table 3 V " kb from . Fragment size(kb) 1H EcoRI Hind I~
6-1 75 0. 9 25 1-2 125 7. 2 12. 5 1-3 150 , 3.4 l.'7 4-4 160 5.1 8. 0 2-5 175 5. 4 16. 0 3-6P 185 11. 8 16. 0 3-7 190 ~ 2.2 5.0 1-8 215 3. 8 2. 0 3-9 230 2.6 5.4 2-lOP 235 13.5 18.5 3-11 245 1. 6 18. 5 1=12P 250 4.5 2.8 3-13 260 1.7 5.8 1-14P 275 2.9 13.0 3-15 280 4.8 13.0 3-16P 290 5.4 1.8 1-17P 295 5.4+1.6 10.2 1-18 315 3. 4 8. 8 3-19P 330 4.3 ~ 14.7 3-20 345 11.8 12.8 3-21 360 2. 2 6. 8 3-22P 385 5.7 7.0 3-23 395 2.0 5.7 1-24P 410 3.0 5.2 3-25P 420 10.0 7.3 2-26 430 8.1 6.6 1-27P 450 8.3 11.3 4-28 455 8.3 5.4 3-29P 460 3.5 5.8 3-30 470 9. 8 6, 8 4-31 475 10. 3 13. 0 3-32P 485 13.3 5.6 3-33 490 13.3 6.8 4-34 505 11.5 16.2 3-35 520 5.3 3.2 3-36P 525 5.3 5.7 3-37P 540 7.5 13.2 3-38P 545 8.0 15.4 4-39 555 7.0 15.4 __ -Table 3 (continued) V " kb from Fragment size(kb) JH EcoRI Hind IB
1-40P 560 1.4 3.2 3-41P 580 4.4 11.9 3-42P 590 - 3:0 3.8 3-43 600 6.5 8.1 3-44P 610 8.8 17.0 1-45 635 10.7 2.7 1-46 640 2.0 4.6 3-47P 650 2.7 10.5 3-48 670 2.7 3.9 3-49 690 1.6 16.5 3-50P 695 10.0 16.5 5-51 710 8. 0 11.
3-52P 715 4.0 11.0 3-53 725 ~ 8.3 6.3 3-54P 730 6.4- 15.4 4-55P 735 3.9 15.4 1-56P 740 3.4 15.4 3-57P 745 9.7 6.6 1-58P 750 8.3 17.5 4-59 755 8. 3 17.
3-60P 760 0.8+3.0 17.5 4-61 770 8. 1 9. 0 3-62P 'l75 4..6 9.0 3-63P 780 8.9 6.2 3-64 790 4.4 >7.4 ~,1~~~'~
SEQUENCE ID N0. : 1 .
SEQUENCE LENGTH . 1 4 2 9 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA .
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE: hucian lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Ser Val Ser Phe Leu Ile Phe Leu Pro Val Leu Gly Leu Pro Trp Gly Val Leu Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp 2~ ~~~'~'~
Ser Val Ser Ser Asn Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp.Tyr Ala Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro 80 85 ' 90 95 GAC ACA TCC AAG AAC CAG TTC TCC CTG~CAG CTG AAC TCT GTG ACT CCC ?52 Asp Thr Ser Lys Asn Gln Phe Ser Leu GIn Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Vat Tyr Tyr Cys Ala Arg TTCATTCCTT TTTACTCTTT TTTTCTTTTT A1'TCATCTGC CTGAATTATT TCAAAAGATC 1344 SEQUENCE ID HO.: 2 SEQUENCE LENGTH . 5 1 2 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : l inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
~TGAGAGCTCC GTTCCTCACC ATG GAC TGG ACC TGG AGG ATC CTC TTC TTG GTG 53 Met Asp Trp,Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr GTCCAGTCCA GGGAGATCTC ~CACAG 162 ATCCACTTCT GA
GTGTTCTCTC GCC
CAC
TCC
Gly Ala His Ser GlnVal GlnLeuVal GlnSerGly AlaGluValLysLys ProGlyAla SerVal LysValSer CysLysAla SerGlyTyrThrPhe ThrGlyTyr TyrMet HisTrpVal ArgGlnAla.ProGlyGlnGlyLeu GluTrpMet 55 . 60 65 GlyTrp IleAsnPro AsnSerGly GlyThrAsnTyrAla GlnLysPhe GlnGly ArgValThr MetThrArg AspThrSerIleSer ThrAlaTyr 85 90 ~ 95 ~1.~25 ~ "~
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg GCAG
SEQUENCE ID NO.: 3 SEQUENCE LENGTH . 4 9 6 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Asp Tcp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr GCT ATG CAT TGG GTG CGC CAG GCC CCC GGA CAA AGG CTT GAG TGG.ATG 300 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met Gly Trp Ser Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Glu Phe Gln Gly Arg Val Thr Ile Thr.Arg Asp Thr Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Met Ala Val Tyr Tyr Cys Ala Arg SEQUENCE ID N0. : 4 SEQUENCE LENGTH . 6 5 0 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Lys His Leu Trp CTC CTG
CTG GTG
GCA GCT
T GTGAGTGTCT
CAAGGCTGCA
Phe Phe Leu Leu Leu Val Ala Ala Pro Arg _ 10 15 CCTCTGATCC
CAGGGCTCAC
TGTGGGTCTC
CT TCC GTG
CAG
GTG
CAC
CTG
CAG
GAG
TCG
GGC
CCA
GG
Trp Val u r Gln Va1 y Pro y Leu Le Se Gln Leu Gln Gl Val Glu Ser Gl TCG CTC ACT GGT
Lys Pro GluThr Leu Ser Thr Cys ValSer Gly Ser Ser Leu Thr Gly AGT TGG CAG GGG
Ile Ser TyrTyr Trp Ser Ile Arg ProAla Lys Gly Ser Trp Gln Gly 50 55 ~ 60 TGG TAT GGG AAC
Leu Glu IleGly Arg Ile Thr Ser SerThr Tyr Asn Trp Tyr Gly Asn CTC ACC GTA TCC
Pro Ser LysSer Arg.Val Met Ser AspThr Lys Asn Leu Thr Val Ser TCC TCT GCC ACG
GIn Phe LeuLys Leu Ser Val Thr AlaAsp Ala Vat Ser Ser Ala Thr 100 105 Iln Tyr Tyr Cys Ala Arg lI5 SEQUENCE ID NO.: 5 2~~~5~~
SEQUENCE LENGTH . 6 1 3 SEQUENCE TYPE : nucleic acid STRANDEDNESS : doubl.e TOPOLOGY : 1 inear .
MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Asp Thr Leu Cys Ser Thr.Leu Leu Leu Leu Thr Ile Pro Ser Trp Val Leu Ser Gln Ile AAA
Thr LeuLysGlu SerGlyProThr .LeuValLysPro ThrGlnThr Leu Thr LeuThrCys ThrPheSerGly PheSerLeuSer ThrSerGly Val Gly ValGlyTrp IleArgGlnPro ProGlyLysAla LeuGluTrp Leu Ala Leu. Ile Tyr Trp Asn Asp Asp Lys Arg Tyr Ser Pro Ser Leu Lys 2~.~~~'~"~
Ser Arg Leu Thr IIe Thr Lys Asp Thr Ser Lys Asn Gln Val VaI Leu Thr Met Thr~Asn Met Asp Pro Val Asp Thr Ala Thr Tyr~Tyr Cys Ala His Arg SEQUENCE ID NO.: 6 SEQUENCE LENGTH . 5 9 4 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Glu Leu Gly Leu Arg Tcp Val Phe Leu Ala Ala Ile Leu Lys Gly Val GlnCys GluMetGln LeuValGlu SerGlyAlaAsn LeuThrLysPro GlyCys Pro Asp SerProVal GlnProLeuAsp SerProSerVal AlaIle AlaArgThr GlySerPro ArgLeuGlnGly ArgValCysSer C
GlySer GlnLeuLeu ValValVal YalValProCys ThrThrGlnThr Leu ArgAlaAsp SerProPhe ProGluThrIle ProLysThrHis CysIle CysLys ThrAsp ly GlnArgMet GlnLeuHisMet G
100~ 105 TACGGTAAGG ACACAAACCT
ThrLeu Glu T AACCAGGGAA GTGCTGACCC
T ACAAGGG
SEQUENCE ID NO.: 7 SEQUENCE LENGTH . 8 7 7 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double T OPOLOGY : 1 i near MOLECULE TYPE : Genomic DNA
ORIGINAL
SOURCE
: Homo sapiens IMMEDIATE line SOURCE: CGMI
human lymphoblastoid cell SEQUENCE
DESCRIPTION
GAA TTC CTT ATT GAA
G
Met Glu Leu Gly Leu Ser Trp Val~ ValAla Leu Phe Leu Ile Glu GTC
Gly GlnCys Val CAG GGC TTG CCT
Glu Val Leu Val Glu Ser Gly Gly ValGln GlyGly Gln Gly Leu Pro AGA GGA TTC AGT
Ser Leu Leu Ser Cys Ala Ala Ser ThrPhe SerTyr Arg Gly Phe Ser 40 45 . 50 AGC GGG AAG GAG
Trp Met Trp Val Arg Gln Ala Pro GlyLeu TrpVal Ser Gly Lys Glu ATA AAA TAC GAC
Ala Asn Lys Gln Asp Gly Ser Glu TyrVal SerVal Ile Lys Tyr Asp CGA AAC GCC TCA
Lys Gly Phe Thr Ile Ser Arg Asp LysAsn LeuTyr Arg Asn Ala Ser ATG GAC ACG GCT TAT
Leu Gln Asn Ser Leu Arg Ala Glu Val TyrCys Met Asp Thr Ala Tyr ~~~~5"~'~
w Ala Arg S EQUENCE I D N0. : 8 SEQUENCE LENGTH . 5 6 4 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Ser Ala His Ser Gln Val Gln Leu Val Gl.n Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser ~ 30 35 40 Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asp Ile Asn Trp Val 45 50 ~ 55 Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met Gly Trp Met Asn Pro AAC AGT GGT AAC ACA GGC TAT GCA CAG AAG TTC CAG GGC AGA GTC ACC .407 Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe Gln Gly Arg.Val Thr 75 ~ 80 85 Met Thr Arg Asn Thr Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Ser 90 ~ 95 100 Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg SEQUENCE ID N0. : 9 SEQUENCE LENGTH . 6 4 0 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : t inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Glu Leu Gly Leu Ser Trp Ile Phe Leu Leu Ala IIeI
AAA ATGAGTGGAT
G
GTGATTCATG
GAGAAATAGA
GAGATTGAGT
.
Leu Lys TTGGTGTCT TG G GTC TG
GA
Gl y l n s Va Gl Cy GAG
Glu Val Leu ValGluSerGly GlyGlyLeuVal ln ProGly Arg Gln G
20 25 ~ ~ 3Q 35 AGA
Ser Leu Leu SerCysAlaAla SerGlyPheThr PheAspAsp Tyr Arg CAC
Ala Met Trp ValArgGlnAla ProGlyLysGly L.euGluTrp Val His ATT
Ser Gly Ser TrpAsnSerGly SerIleGlyTyr AlaAspSer Val Ile CGA.
Lys Gly Phe ThrIleSerArg AspAsnAlaLys AsnSerLeu Tyr Arg ATG
Leu Gln Asn SerLeuArgAla GluAspThrAla LeuTyrTyr Cys Met GCA AAA CAGTGAGG GGAAGTCAGC. GAGAGCCCAG 617 GAT ACAAAAACCT
A
CA
Ala Lys Asp GACAGGAGGG
GCC
SEQUENCE ID NO.: 1 0 SEQUENCE LENGTH . 6 3 0 y .
SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear .
MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met GIu Leu Tyr Ser Thr Leu Leu Leu Leu Thr Val Pro 1 5 m Ser Trp Val Leu Ser Gln Val 15 ~n AAA
ThrLeu LysGluSer GIyProAla LeuValLysProThr GlnThrLeu MetLeu ThrCysThr PheSerGly PheSerLeuSerThr SerGlyMet 40 45 . 50 GlyVal Gly Ile CysGlnPro SerAlaLysA1aLeu GluTrpLeu Ala His Ile Tyr Asn Asp Asn Lys Tyr Tyr~Ser Pro Ser Leu Lys Ser Arg Leu Ile Ile Ser Lys Asp Thr Ser Lys Asn Glu Val Val Leu ACA GTG ATC AAC ATG .GAC ATT GTG GAC ACA GCC ACA CAT TACTGTGCAA 505 Thr Val Ile Asn Met Asp Ile Val Asp Thr Ala Thr His 100 , 105 110 SEQUENCE ID NO.: 1 1 SEQUENCE LENGTH . 7 1 5 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa .iens IMMEDIATE SOURCE : human lymphoblastoidline CGMI
cell SEQUENCE DESCRIPTION
CACCCCAGGC~TTTACACTTT ATGCTTCCGG GTGTGGAATT GTGAGCGGAT60 CTCGTATGTT
ATGATTACGC
TCGAATTCCC
TTT GGG TTC
Met Glu Phe Gly Leu Ser Trp Val Phe GAGAACTAGA GACATTGAGT
Leu Val Ala Ile Ile Lys GTGGCAGTTT
CTG GTG GAG~TCT GGG GGA GGC
'~~.~~~7'~
Gly Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly LeuVal LysProGlyGly SerLeuArgLeu SerCysAlaAla SerGly 30 35 40 ~ 45 PheThr PheSerAspTyr TyrMetSerTrp IleArgGlnAla ProGly LysGly Leu.GluTrpVal SerTyrIleSer SerSerGlySer ThrIle TyrTyr AlaAspSerVal LysGlyArgPhe ThrIleSerArg AspAsn 80 ~ ~
Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg SEQUENCE ID NO.: 1 2 SEQUENCE LENGTH . 6 6 0 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens ~1~~~ ~'~
-so-IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION .
CATCTCCTC ATG GNC TGG ACC TAC AAG ATC CTC TTC .TTG GTG GCA GCA GCC 171 Met Xaa Trp Thr Tyr Lys Ile Leu Phe Leu Val Ala Ala Ala Thr Gly Ala His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys 25 30 ~ 35 ValSer CysLysAla SerGlyTyr ThrPheThrTyr CysTyrLeuHis TrpVal GlnAla ProGlyGln GlyLeuGluTrp ThrGlyPhe LeuPhe GluArgPhe PheIleGln HisLeuPheCys LysGlnIleSer GlyIle ValGlu IleIleLeu ThrAsnLeuThr GlnAsnPheLeu 85 . 90 95 TGA ATC AAT CTT TGT AAA CAT .CAA TTT CTG AAT CAA TGT TGT NAA TAT 568 -sl-Ile Asn Leu Cys Lys His Gln Phe Leu Asn Gln Cys Cys Xaa Tyr 100 105 1.10 TTC AGA ACA CAA GCA CAA NTT CAC ATT TNA ACT CTA CfiT TNA TCT CTA 616 Phe Arg Thr Gln Ala Gln Xaa His Ile Xaa Thr Leu Leu Xaa Ser Leu Phe Lys Xaa Tyr Gln Lys Xaa Ser Ser Xaa Ala Cys Asn Val SEQUENCE ID NO.: 1 3 SEQUENCE LENGTH . 8 1 9 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
GCCCCAGCCT TGGGATTCCC~AAGTGTTTGT ATTCAGTGAT CAGGACTGAA CACACAGGAC 300 Met Glu Leu Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Glu Gly Val Gln Cys Glu Val His Leu Val Glu Ser Gly Gly Gly Leu Val Gln CCT GGG GGG GCC~CTG AGA CTC TCC TGT GCA GCC TCT GGA TTC ACC TTC 553 Pro Gly Gly Ala Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr Asp Met His Trp Val Arg Gln Ala Thr Gly Lys Gly Leu 50 55 ~ 60 Glu Trp Val Ser Ala Asn Gly Thr Ala Gly Asp Thr Tyr Tyr Pro Gly TCC GTG AAG GGG CGA TTC ACC ATC TCC AGA GAA ~AAT GCC AAG AAC TCC 697 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr 100 105 . 110 Tyr Cys Ala Arg SEQUENCE ID NO. : 1 4 SEQUENCE LENGTH . 8 1 6 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
~~.~2~ ~~.
ORIGINAL SOURCE : Homo Sapiens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
AGCAGTCAGA GATCTGAGGA CATAGATGTG TACTACTGTG CGANACACAC AGTGTGACAN 540' GAGTCGTACC CNGGATCCCT GNTTGGCCTG AGNATA . 816 SEQUENCE ID NO.: 1 5 SEQUENCE LENGTH . 5 3 5 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : l inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo Sapiens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Glu Phe Gly Leu Ser Trp Ile Phe Leu Pro Ala Ile Leu Lys GTC
CAG
TGT
GAG
GTG
CAG
CTG
GTG
GAG
TCT
GGG
GGA
GCC
TTG
Gly Yal Gln Cys.
Glu Val Gln Leu Val Glu Ser Gly Gly Ala Leu TGT
ValLysPro GlyGly SerLeuArgLeu Ser AlaAla SerGlyPhe Cys 35 . 40 45 CGC .
ThrPheSer AswAla TrpMetSerTrp Val GlnAla ProGlyLys Arg AAA .
GlyLeuGlu TrpVal GlyArg.IleLys Ser ThrAsp GlyGlyThr Lys 65 70 i5 Thr Asp Tyr Ala A,la Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu GAC ACA GCC GTG TAT TAC TGT ACC ACA GA CACAGTGAGG GGAGGTCAGT 497.
Asp Thr Ala Val Tyr Tyr Cys Thr Thr ~~~~'~
SEQUENCE ID NO.: 1 6 SEQUENCE LENGTH . 5 4 2 SEQUENCE TYPE : nucleic acid STRANDEDNES$ : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Glu Phe Gly Leu Ser Trp Yal Phe Leu Ala Gly Ile Leu Lys Gly Val Gln Cys Glu Val Gln Leu Val G!u Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser GGA TTC ACC TTC.AGT AAC AGT GAC ATG AAC TGG GCC CGC AAG GCT CCA 310 Gly Phe Thr Phe Ser Asn Ser Asp Met Asn Trp Ala Arg Lys Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly Val Ser Trp Asn Gly Ser Arg i E
Thr His Tyr~Val Asp Val Arg Arg Ile Ile Ser Arg Ser. Lys Phe Asp g0 g5 90 CTG CTG AAC GAG' Asn Ser Arg Asn Ser Tyr Gln Lys Arg Arg Arg Ala Leu Leu Asn Glu TAC GTG TCCTGTGAGG
GGACACAAGT
Asp Met Ala Val~ Cys Arg Tyr Tyr Val GCGAGCCCAG ACACAAACCT
CCTGCAGGAA CACTGGGCG
SEQUENCE ID NO.:
SEQUENCE LENGTH .
SEQUENCE TYPE : nucleicacid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : GenomicDNA
ORIGINAL SOURCE :
Homo Sapiens IMMEDIATE SOURCE lymphoblastoid line CGMI
: human cell SEQUENCE DESCRIPTION
C CACAGCTCCT ACC
Met Asp Cys Thr Trp Gly Ile Leu Phe Leu Val Ala Ser Xaa Thr ' Asp Val His Ser Gln Val Gln Leu Leu Gln Pro Gly ~~~.~~5'~
Ala GluValLys LysProAlaSer SerValLysVal SerTrpPro Gly Phe GlnIleHis LeuHisGlnIle LeuTyrThrVal GlyAlaThr Gly Pro TrpThrArg Ala TrpLeu GlyCysIleAsn ProTyrAsn Asp Asn ThrHisTyr AlaGlnLysPhe ArgGlyArgVal ThrIleThr Ser Asp ArgSerVal SerThrAlaTyr MetGluLeuSer SerLeuArg Ser .
AAACCCACAT
Glu AspMetVal ValTyrSerCys ValArg AAGATTATTA GATTAACGAT TTTCTTAGA ~ 591 SEQUENCE ID NO.: 1.8 SEQUENCE LENGTH . 5 3 9 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : I inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
_6g_ Met Asp Trp . 1 TGG
AGC
ATC
CTT
TTC
TTG
GTG
GCA
GCA
CCA
ACA
G GTAACGGACT
Thr Trp Ser Ile Leu Phe Leu Val Ala Ala Pro Thr AGGGCTGAGA
GAGAAACCAG
TGTCCTCTCC.ACAG 211 GT
GCC
CAC
TCC
CAG
GTT
CAG
CTG
GTG
CAG
TCT
GGA
Gly Ala His Ser G ln Val Gln Leu Val Gln Ser Gly GAG GCC AAG~ GCT
Ala Val Lys Lys Pro Gly Ser Val Val Ser Cys Lys Glu Ala Lys Ala GGT TAT AGC GCC
Ser Tyr Thr Phe Thr Ser Gly Ile Trp Val Arg Gln Gly Tyr Ser Ala GGA ATG ATC GGT
Pro Gln G1y Leu Glu Trp Gly Trp Ser Ala Tyr Asn Gly Met Ile Gly ACA CTC AGA ACA
Asn Asn Tyr Ala Gin Lys Gln Gly Val Thr Met Thr Thr Leu Arg Thr GAC TCC ACG AGC ACA GCC ATG GAG AGG AGC.CTG AGA 451 ACA TAC CTG TCT
Asp Ser Thr Ser Thr Ala Met Glu Arg Ser Leu Arg Thr Tyr Leu Ser GAC GA
ACG
GCC
GTG
TAT
TAC
TGT
Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg ~~.~~~ 1 6 CCTGAGGGTT TCAGAAACCC CAGGGAGGAG GCAGCT ~ 539 SEQUENCE ID NO.: 1 9 SEQUENCE LENGTH . 7 2 7 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : I inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE~DESCRIPTION
SEQUENCE ID NO.: 2 0 SEQUENCE LENGTH . 5 1 4 SEQUENCE TYPE : nucleic acid STRANDEDNESS
: double TOPOLOGY: 1 inear MOLECULETYPE : Genomic DNA
ORIGINALSOURCE : Homo sapiens IMMEDIATE lineCGMI
SOURCE
: human lymphoblastoid cell SEQUENCE. DESCRIPTION
ATG GTT TTC
GAG CTT GTT
TTT GCT ATT
GGG
CTG
AG
Met Glu Phe Gly Leu r Trp a Ile Se Val Phe Leu Val Al 1 5 .
i0 AAA GTGAGTGAAC
ACGAGTGAGA
Leu Lys ATTTGTGTGG CAG
CAGTTTCTGA GT
GTC
Gly Val TGT GAG GGA GTA
Gln Glu Val Gln Leu Val Ser Gly~GlyGly Val Arg Pro Cys Glu Val GGG TGT TCT ACC
Gly Ser Leu Arg Leu Ser Ala Ala Gly Phe Phe Asp Gly Cys Ser Thr TAT CGC CCA GGG
Asp Gly Met Ser Trp Val Gln Ala Gly Lys Leu Glu Tyr Arg Pro Gly GTC AAT AGC TAT
Trp Ser Gly Ile Asn Trp Gly Gly Thr Gly Ala Asp Val Asn Ser Tyr GTG ATC GAC AAG
Ser Lys Gly Arg Phe Thr Ser Arg Asn Ala Asn Ser Val Ile Asp Lys Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr CAC TGT GCG AGA GA CACAGTGAGG GGAAGCCAGT GAGAGCCCAG ACACAAACGT 505.
His Cys Ala Arg SEQUENCE ID N0. : 2 1 SEQUENCE LENGTH . 5 1 9 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 i near MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
AGGATTCACC ATG GAA CTG GGG CTC CGC TGG GTT TTC CTT GTT GCT AT'T 49 Met Glu Leu Gly Leu Arg Trp Val Phe Leu Val Ala Ile Leu Glu GAAACGGTGG ATGTGTGTGA CAGTTTCTGA CCAATGTCTC.TCTGTTTGCA G GT GTC 162 Gly Yal Gln Cys Glu Val Gln Leu Val Glu Ser Gly.Gly Gly Leu Val Lys Pro ~~~25'~'~
GlyGly SerLeuArg LeuSerCysAla AlaSerGlyPhe ThrPheSer 35 40 ~ 45 SerTyr SerMetAsn TrpValArgGln AlaProGlyLys GlyLeuGlu TrpVal SerSerIle SerSerSerSer SerTyrIleTyr TyrAlaAsp SerVal LysGlyArg PheThrIleSer Arg~AspAsnAla LysAsnSer CTGTAT CTGCAAATG AACAGCCTGAGA GCCGAGGACACG GCT~GTGTAT 450 LeuTyr LeuGlnMet ~AsnSerLeuArg AlaGluAspThr AlaValTyr GGAAGTCAGT
GTGAGCCCAG
TyrCys AlaArg GTCCC
SEQUENCE ID NO.: 2 2 SEQUENCE LENGTH . 6 0 6 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
2~.6~~'~'~
CACC ATG~GAG
TCA TGG CTG AGC
TGG GTT TTT
Met Glu Ser Trp Leu Ser Trp Val Phe TTA AAA G GTAATTCATT
GAGAACTATT GAAATTGAGT
Leu Ala Ala Ile Leu Lys 15 . .
GAAACAGTGG ATACGTGTGG
CAG TGT
GAG GTG
CAT CTG
GTG GAG
TCT GGG
GGA
Gly Val n Cys Glu Val Gl His Leu Val Glu Ser Gly Gly.
GGG CTC TCT
Ala Leu ValGln Pro Gly Ser Leu Arg Ser Cys Ala Ala Gly Leu Ser TAC GGG CCC
Gly Phe ThrPhe Ser Tyr Tyr Met Ser Val Arg Gln Ala Tyr Gly Pro 45 50 55 ~ 60 TGG AGA GGT
GIy Lys GlyLeu Glu Val Gly Phe Ile Asn Lys Ala Asn Trp Arg Gly GGG ACA ACAGAA TAG ACG TCT GTG AAA AGA TTC ACA ATC~TCA463 ACC GGC
~Gly Thr ThrGlu Thr Thr Ser Val Lys Arg Phe Thr Ile Gly Ser . 80 85 90 AGC CAA AAA
Arg Asp AspSer Lys Ile.Thr Tyr Leu Met Lys Ser Leu Ser Gln Lys ACC GAG GACACG GCC T.AT TAC TGT GA CACAGTGAGG 556 GTG TCC AGA
Thr Glu AspThr Ala Tyr Tyr Cys Ser Val Arg ACACAAACCT
CCCTGCAGGG
SEQUENCE ID NO.: 2 3 SEQUENCE LENGTH . 5 1 4 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : l inear MOLECULE TYPE : Genomic DNA
ORIGINAL. SOURCE : Homo sa iens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Lys GTAATTCATG GTGAATGGAT
AAGAGTGAGA
I Lys I
a ATACGTGTGG~CAGTTTCTGA CAG
CCAGGGTTTC GT
Gl y GTC CAGTGTGAG GTGCAGCTG TTGGAGTCTGGG~GGA GGCTTGGTA CAG 208 Val GlnCysGIu ValGlnLeu LeuGluSerGlyGly GlyLeuVal Gln Pro Gly.GlySer LeuArgLeu SerCysAlaAlaSer GlyPheThr Phe 35 ~ 40 . 45 Ser SerTyrAla MetSerTrp ValArgGlnAlaPro GlyLysGly Leu 50 55 ~ 60 Glu TrpValSer AlaIleSer GlySerGlyGlySer ThrTyrTyr Ala .
AspSer Va LysGly ArgPheThr~Ile SerArgAspAsn SerLysAsn l ThrLeu TyrLeuGln MetAsnSerLeu ArgAlaGluAsp ThrAlaVal . 100 105 110 GGAAGTCATT
GTGAGCCCAG
TyrTyr CysAlaLys SEQUENCE ID N0. : 2 4 SEQUENCE LENGTH . 6 0 0 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE : human lymphoblastoid cell tine CGMI
SEQUENCE DESCRIPTION
Met Asp Cys Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr r,..
Gly Thr His Ala Gln Val Gln CTG GTA CAG GGG GAGGTG.AAG AAGCCTGGGGCC TCAGTGAAG 266 TCT GCT
Leu Yal Gln Gly GluValLys LysProGlyAla SerValLys Ser Ala ~ 30 35 AAG TCC
Val Ser Cys Val GlyTyrThr LeuThrGluLeu SerMetHis Lys Ser CAG CCT
Trp Val Arg Ala GlyLysGly LeuGluTrpMet GlyGlyPhe Gln Pro 55 60 65. 70 GAT GAA
Asp Pro Glu Gly ThrIleTyr AlaGinLysPhe GlnGlyArg Asp Glu ACC GAC
Val Thr Met Glu ThrSerThr AspThrAlaTyr MetGluLeu Thr Asp 90 95 ~ 100 AGA GAG
Ser Ser Leu Ser AspThrAla ValTyrTyrCys AlaThr Arg Glu AAAC CCTGAGAGC TCAGAAACCC
GGAG GGATTTATG AG
SEQUENCE ID : 2 NO: 5 SEQUENCE LENGTH
. 6 5 5 SEQUENCE TYPE: nucleicacid STRANDEDNESS
: double TOPOLOGY :
1 inear MOLECULE TYPEGenomicDNA
:
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
GAGGAGCCCA GCCCCCGAAT TCCCAGGTGT TTTCATCTGG TGATCAGCAC CGAACA.CAGA 120 Trp Ser Leu Cys Ala Gly Phe Ser Leu Leu Leu Phe Asn AACAGTGGAT ACGTGTGGCA GTTTCTGACC GGGGTGTCTC TGTGTTTGCA G.GTA TCC 282 Yal Ser AGT GTG AGA.TGC AGC TGG TGG AGT CTG GGG GAG GCT TGC AAA AGC CTG 330 Ser Yal Arg Cys Ser Trp Trp Ser Leu Gly Glu Ala Cys Lys Ser Leu ArgGly ProArgAspSer ProValGlnPro LeuAsnSer ProSerVal AlaThr Thr ThrVal SerAlaArgLeu GlnGlyMet GlyTrpSer TrpPhe AspLysLeuIle LeuMetGlyVal AlaHisThr Ser Thr 65~ 70 75 ProVal ArgThrAspSer IleProProGlu IleThrPro ArgThrHis PheIIe ~CysLys Thr Ala LysPro ArgThrArg ProSerIle Ser Val Pro Glu SEQUENCE ID NO.: 2 6 SEQUENCE LENGTH . 5 4 6 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Asp Thr Leu Cys Tyr Thr Leu Leu Leu Leu 1 5 . 10 Thr Thr Pro Ser Trp Val Leu Ser Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu ACC CTC ACG CTG ACC TGC ACC GTC TCT GGG~TTC TCA CTC AGC AAT GCT 259 21625~'~
ThrLeu TheLeuThr CysThrValSer GlyPheSerLeu SerAsnAla 40 45 ~ 50 ArgMet GlyValSer TrpIIeArgGln ProProGlyLys AlaLeuGlu TrpLeu AlaHisIle PheSer~AsnAsp GluLysSerTyr SerThrSer LeuLys SerArgLeu ThrIleSerLys AspThrSerLys SerGlnVal ValLeu ThrMetThr AsnMetAspPro ValAspThrAla ThrTyrTyr CACAGAGACA ACAAGAACCT
CAGCCCAGGA
Cys Ala Arg Ile SEQUENCE ID NO.: 2 7 SEQUENCE LENGTH . 5 8 7 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : I inear MOLECULE TYPE : Genomic DNA
ORIGINAL.SOURCE : Homo sapiens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
~c,~~w~~~
Met Tyr Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly Val His Ser Gln Val Gln CTG GTG CAG~TCT GGG CCT GAG GTG AAG AAG CCT GGA GCC TCA TTG AAG 262 Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr~Ser Tyr Ala Ile Ser 40 45 ~ 50 TrpVal GlnAla HisGlyGlnGly LeuGluGluMet GlyTrpIle AsnThr AsnThrGly AsnLeuThrTyr AlaGlnGlyPhe ThrGlyArg PheVal PheSerMet AspThrSerVal SerMetAlaTyr LeuHisIle 90 ~ 95 100 SerSer LeuLysAla GluAspThrCys LysArg CCTGAGAGAA CCAGAAATCC CTGAGCTGAG GCAGTGACAG
TGAGGGAGGA
GGCAGCTGTG
SEQUENCE ID NO.: 2 8 SEQUENCE LENGTH
. 6 2 4 SEQUENCE TYPE : acid nucleic STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : DNA
Genomic ORIGINAL SOURCE
: Homo sapiens IMMEDIATE SOURCE: lymphoblastoid lineCGMI
human cell SEQUENCE DESCRIPTION
TACAGAGGCA CTTAGGCACC
TTCCTTAAAT GAAATACTTT
GTGCAAGAAC AAA CTG TTC
Met His Trp Phe Leu Lys Leu Phe GCT CCC T GTGAGTGTCT
Leu Leu Val Ala Arg Ala Pro CCAGGGCTCA AG
GG
Trp Val CAG CTG GAG GGC AAG
Leu Ser Gln Val Gln Ser ProGlyLeu Val Pro Gln Leu Glu Gly Lys TCC CTC TGC GTC ATC
Ser Asp Thr Leu Thr Ala SerGlyTyr Ser Ser Ser Leu Cys Val Ile AGT AGT AAC.TGG TGG CGG CCCGCAGGG AAG CTG 427 TGG GGC ATC CAG GGA
Ser Ser Asn Trp Trp Arg ProProGly Lys Leu -Trp Gly Ile Gln Gly 50 55 60 . 65 TAC ATC TAT GGG AAC
Glu Trp Ile Gly Tyr Ser SerThrTyr Tyr Pro Tyr Ile Tyr Gly Asn Ser Leu Lys Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln 85 90 . 95 Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Val Asp Thr Ala Val Tyr 100 ~ 105 110 TAC TGT~GCG AGA AA CACAGTGAGG GGAGGTGAGT GTGAGCCCAG ACACAAACC 624 Tyr Cys Ala Arg SEQUENCE ID N0. : 2 9 SEQUENCE LENGTH . 3 0 4 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo Sapiens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
SEQUENCE ID NO.: 3 0 SEQUENCE LENGTH . 5 1 2 SEQUENCETYPE ,: nucleic acid STRANDEDNESS
: double TOPOLOGY: 1 inear.
MOLECULETYPE : Genomic DNA .
ORIGINALSOURCE : Homo sapiens IMMEDIATE lineCGMI
SOURCE:
human.lymphoblastoid cell SEQUENCEDESCRIPTION
ATG CTC
GAG GTT
TTT GCT
GGG CTT
CTG TTA
AGC
TGG
GTT
TT
Met Glu Phe Gly Leu r Trp e Se Val Ph Leu Val Ala Leu Leu G GTGATTCATG GTGAGTGAA
GAGAAATAGA
GAGA
A rg G
Gly Val TGT GAG GGA GTC
Gln Gln Val Gln Leu Val Ser Gly Gly Val Gln Pro Cys Glu Gly Val AGG TGT TCT ACC
Gly Ser Leu Arg Leu.Ser Ala Ala Gly Phe Phe Ser Arg Cys Ser Thr TAT CGC CCA GGG
Ser Gly Met His Trp Val Gln Ala Gly Lys Leu Glu Tyr Arg Pro Gly TGG GCA GTT ATA TCA TAT GGA AGT AAA TAC GCA .GAC 353 GTG GAT AAT TAT
Trp Ala Val Ile Ser Tyr Gly Ser Ly5 Tyr Ala Asp Val Asp Asn Tyr GTG ATC GAC AAG
Ser Lys Gly Arg Phe Thr Ser Arg Asn Ser Asn Thr Val Ile Asp Lys Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg .115 CCCTGCAGG ~ 512 SEQUENCE ID NO.: 3 1 SEQUENCE LENGTH . 6 3 1 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : linear MOLECULE TYPE : Genomic DNA.
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Lys His Leu Trp Phe~Phe Leu Leu Leu Val Ala Ala Pro Arg Trp Val w LeuPro GlnValGln LeuGln GluSer GlyProGlyLeu ValLysPro SerGln ThrLeuSer LeuThr CysThr ValSerGlyGly SerIleSer SerGly GlyTyrTyr TrpSer TrpIle ArgGlnHisPro GIyLysGly 50 55 ~ 60 65 LeuGlu TrpIleGly TyrIle TyrTyr SerGlySerThr TyrTyrAsn ProSer LeuLysSer ArgVal ThrIle SerValAspThr SerLysAsn GlnPhe SerLeuLys LeuSer SerVal ThrAlaAlaAsp ThrAlaVal GGAGGTGAGT
GTGAGCCCAG
TyrTyr CysAlaArg SEQUENCE ID NO.: 3 2.
SEQUENCE LENGTH . 3 4 1 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens ~,.~-_86_ IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
CTCTGTATCT GCAAATGAAC ACTCAGAGAG CTGAGGACGT~GGCCGTGTAT GGCTATACAT I80 SEQUENCE ID NO.: 3 3 SEQUENCE LENGTH . 5 8 3 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE ~: Homo sapiens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val GCT CTT TTA AGA G GTGATTCATT GGAGAAATAG AGAGACTGAG.TGTGAGTGAA 105 Ala Leu Leu Arg Gly Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser.Cys Ala Ala Ser Gly Phe ,' 1~~~ ~ '~
35 40 45 .
ThrPheSerSer TyrGlyMet HisTrp ValArgGln AlaProGly Lys GlyLeuGluTrp ValAlaVal IleTrp TyrAspGly SerAsnLys Tyr TyrAlaAspSer AlaLysGly ArgPhe ThrIleSer ArgAspAsn Ser ThrAsnThrLeu PheLeuGln MetAsn SerLeuArg AlaGluAsp Thr ACAGTGAGGG
AlaValTyrTyr CysAlaArg CACAAACCTC CCTGCAGGAA G CAGGGGGGGC TCAGGAGCCA
CGCTGGCGG AAATCAGCTG
CTGATCAGAG TCAGCCCT ~ 583 SEQUENCE ID NO.: 3 4 SEQUENCE LENGTH .: 6 8 7 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : l inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
CAAATCCTCA CTTAGGCACC CACAGGAAAT GACTACACAT ~TTCCTTAAAT TCAGGGTCCA 120 Met Lys His Leu Trp Phe.Phe Leu Leu Leu Yal Ala Ala Pro Arg Trp Val Leu Ser Gln Val Gln Leu Gln Gln Trp Gly Ala GGA CTG TTG AAG CCT TCG GAG ACC CTG TCC CTC ACC TGC GCT.GTC TAT 385 Gly Leu Leu Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val~Tyr C
GlyGly SerPheSer GlyTyrTyrTrp SerTrpIleArg GlnProPro GlyLys GlyLeuG1-uTrpIleCly~GluIleAsnHisSer GlySerThr G
AsnTyr AsnProSer LeuLysSerArg ValThrIleSer ValAspThr SerLys AsnGlnPhe SerLeuLysLeu SerSerValThr AlaAlaAsp CACAGTGAGG
GGAGGTGAGT
ThrAla ValTyrTyr CysAlaArg ~~~~~'~~
GTGAGCCCAG ACAAAAACCT CCCTGCAGGT AGGCAGAGGG GGCGGGCGCA GGTACTGCTC. 683 SEQUENCE ID NO.: 3 5 SEQUENCE LENGTH . 7 0 0 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : I inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Glu.Phe Gly Leu Ser Trp Val Phe Leu. Ala Ala Ile Leu Lys Gly Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala.Ser Gly Phe Thr '..-i ~A
PheSer AsnSerAsp MetAsnTrpVal GlnAlaPro LysGly His Gly 50 ~ 55 60 CTGGAG TGGGTA.TCG GGTGTTAGTTGG GGCAGTAGG CACTAT 477 AAT ACG
LeuGlu TrpValSer GlyValSerTrp GlySerArg HisTyr Asn Thr GCAGAC TCTGTGAAG GGCCGATTCATC TCC.AGAGAC TCCAGG 525 ATC AAT
AlaAsp SerValLys GlyArgPheIle SerArgAsp SerArg Ile Asn CTG GAC
AsnThr LeuTyrLeu GlnThrAsnSer ArgAlaGlu ThrAla Leu Asp CACTGTGAGA
GGTCGGAAGT
GTGAGC
ValTyr TyrCysVal Arg ACACAAACCT CCTGCAGGAA AGGGGGCGCT CAGGACCCAC
CGTTGGGGGA
AATCAGCTGC
SEQUENCE ID NO.: 3 6 SEQUENCE LENGTH . 8 0 6 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
CCCCAGAGCT TGCTATAGAG GAGGAGACAT CCACATAGGG.CCCTCNCTTG TCCTGATGAA 120 ...
ATTTGGTGGT CAGGTCTCTG AACACAGAGG ACTCACT ATG GAG TTT GGG CTG AGC . 235 Met Glu Phe Gly Leu Ser 1 5 ~
GAGAACTAGA
TrpGly PheHisVal AlaAsnVal Lys GATATTGAGT GAAACAGTGG ATATGTGTGG
GTGAGTGGAC CAGGT
ACAAGTGAGA
CCAGGGTGTC GT GAG TG 394.
TGTGTGTGTT GTC GTG ~
TGCAG CAG CAC
TGT CTG
G
Gly Glu al Val Val Gln His Cys Leu V
GGT
GluSer LeuGlyGly LeuLeu ProGly Pro AspPhe Leu Gly TTA
LeuGln ProLeuAsp SerProLeu ValPro LeuGly ThrGly Leu 40 ~ 45 50 TGG
AlaGly SerIleArg LeuLeuGly LysGly SerArg SerHis Leu Trp CAG
Val VatVal ValAlaGln AlaMet ThrLeu Arg Val Gln AAT
AspSer ProSer.ProGluMetMet ProArg HisCys IleCys Lys Asn TGT
Thr Ala Ser Glu Pro Arg Ile Gly Leu Cys Ile Thr Val Val a'~r TCCTGTTTTG GGGACACCAA TTTTGGAGTT TGCAGTATCC TTGAGTCCAG TACGTTCATG. 800 GTGGCA ~ 806 SEQUENCE ID NO.: 3 7 SEQUENCE LENGTH . 5 0 0 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met.Leu Phe Gly Leu Ser Trp Pro Phe Arg Phe Thr Ile Leu AGG G GTGACACGTG AAGCACTACA GATATTGCTC'GTGAGTGGAT ATTAGAGAAA 105 Arg Gly Val Gln Tyr Glu Yal Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Leu Trp Trp Val Leu Arg Leu Ser Cys Ala Ala Cys Gly Phe Ile Leu Arg Ser Asn Trp Ser His Arg Ala Ser Arg Lys Gly Leu Ala Trp Asn Asp Met Val Ser Tyr Ile Ser Ala Ser Gly Gly Ser Leu Tyr Tyr Ala Asp Thr Glu Gly Ile His His Leu Arg Gln Trp Gln Glu His Ala Val 85 90 ~ 95 Leu Ala Asn Glu Gln Ser Glu Arg Gly Leu Gly Cys Val Glu Arg 100 105 ' 110 SEQUENCE ID NO.: 3 8 SEQUENCE LENGTH . 5 0 7 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION ' ' Met Gln Phe Val Leu Ser Trp Val Phe Leu Val Gly Ile Leu Lys Gly Val .._ _g4_ Gln Cys. Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Arg.Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn Glu Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Arg AAG GGC AGA TTC ACC ATC TCC AGA~GAC AAT TCC AAG AAC ACG CTG.TAT 402 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Gly Thr Ala Ala Tyr Tyr Cys Ala Arg _ SEQUENCE ID NO.: 3 9 SEQUENCE LENGTH . 8 0 0 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Lys His Leu Trp Phe Phe Leu Leu Leu Vat Ala Ala Pro Arg 10~ 15 Trp Bal Leu Ser Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val. Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Ser Ile.Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg SEQUENCE ID NO.: 4 0 SEQUENCE LENGTH . 9 7 0 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : I inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
CACAACCTCC ATGAAAAACA ACATAGAAAT TTCTCAAAGA.ACTAAAATTA GAATTACCAT 60 GATGGGATGG ATCATCACCT ACACTGGGAA CCCAACATAT ACCAACGGCT TCACAGGACG . 360 CTAGATACAT .GTGAGCCCAT TTCCTGGTCT TTGCTTAACT GACAAGCTCT CATCAGTGCA 780 SEQUENCE ID N0. : 4 1 SEQUENCE LENGTH . 8 1 9 SEQUENCE~TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : linear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
GATTCACC ATG GAG TCA TGG CTG AGC TGG GTT TTT CTT GCC GCT~ATT TTA 230 Met Glu Ser Trp Leu Ser Trp Val Phe Leu Ala Ala Ile Leu 1 5 ~ ~ 10 Lys Gly Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Yal Gln Pro w ~ ~ 6 2 5'~'~
GlyGlySer LeuArgLeu SerCysAla AlaSerGlyPhe SerPheSer SerTyrGly MetSerTrp ValArgGln AlaProGlyLys GlyLeuGlu 50 ~ 55 60 65 ValAla HisIleTrp AsnAspGly SerGlnLysTyr TyrAlaAsp 70 75 . . 80 SerValLys~GlyArgPhe ThrIleSer GluThrIleLeu ArgAlaCys 85 90 ~ 95 Ser Ile Cys Lys Trp Thr Val Lys Leu Arg Thr Arg Pro Cys Ile Thr Val Pro SEQUENCE ID N0. : 4 2 SEQUENCE L$NGTH . 4 7 1 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE :Homo sapiens ~~~~w~~~
IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
SEQUENCE ID N0. : 4 3 SEQUENCE LENGTH . 8 7 0 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo s_apiens IMMEDIATE~SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ana Ile Leu Lys .,_ Gly Val Gln Cys Glu Val Gln LeuVal GluSerGly GlyVaIVal ValGinProGlyGly SerLeuArg LeuSer CysAlaAla SerGlyPhe ThrPheAspAspTyr ThrMetHis T Val ArgGlnAla ProGlyLys GlyLeuGluTrpYal SerLeuIle rp SerTrp AspGlyGly SerThrTyr TyrAlaAspSerVal LysGlyArg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Lys Asp GCCTGTGTCA CCT ~ 870 SEQUENCE ID N0. : 4 4 SEQUENCE LENGTH . 5 2 9 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE : human lymphoblastoidline CGMI
cell SEQUENCE DESCRIPTION
TCCCAGGTGT
GGG CTG AGC TGG GCT TTC CTT GTT
Met Leu Phe Gly Leu Ser Trp Ala Phe Leu Val GATATTGTTT GTGAGTGGAT
Thr Ile Leu Arg TGCTGACCAG
GAG TCT TTT
Gly Val Gln Tyr Glu Val Gln Leu Val Phe Phe Phe Phe Glu Ser Phe TTA CAA GCT
Phe His Phe Leu Ala Asn Ile His Gly Asn Asn Gly Leu Leu Gln Ala CAA TTT TAA
Phe Leu Pro Thr Leu Tyr Arg His His Ser Pro Cys Leu Gln Phe CAT CTT AGG
.._ Gly Phe Pro Glu Glu Cys Cys His His Leu Ser Cys Ser Phe Arg Lys Asn Ala Pro Ser Thr His Leu His Leu Ser Ala Cys Ile Ser Ile Cys Leu Gly Arg Ser Gln Gln Pro Xaa Glu His Ser Pro His Pro Thr ATG CTG CTC GAG GGG GTG ~ 529 Met Leu Leu Glu Gly Val 110 ~ 115 SEQUENCE ID NO.: 4 5 SEQUENCE LENGTH . 7 4 8 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo Sapiens .
IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
CAGGATCAGG GCTTGAGTCA TCAGCATCTC ACTCTTGCAA~AGNCTGATGT GTCGTTTGTC 60 Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr ~~ ~~~'~7 Asp Ala Tyr.Ser Gln Met Gln Leu Val Gln Ser Gly GCTGAG GTG.AAG ACTGGGTCCTCA GTG GTTTCC TGCAAGGCT 446 AAG AAG
AlaGlu ValLysLys ThrGlySerSer ValLysYalSer CysLysAla 30 35 40 .
SerGly TyrThrPhe ThrTyrArgTyr LeuHisTrpVal ArgGlnAla ProGly GlnAlaLeu GluTrpMetGly TrpIleThePro PheAsnGly AsnThr AsnTyrAla GlnLysPheGln AspArgValThr IleThrArg GACAGG TCTATGAGC ACAGCCTACATG~GAGCTGAGCAGC CTGAGATCT 638 AspArg SerMetSer ThrAlaTyrMet GluLeuSerSer LeuArgSer CACAGTGTGA
AAACCCACAT
Glu~Asp ThrAlaMet TyrTyrCysAla Arg TCAGAAACCC GGTTACAG
CAAGGAGGAG
SEQUENCE ID NO:: 4 6 SEQUENCE LENGTH . 7 9 9 SEQUENCE TYPE : nucleic acid .._ STRANDEDNESS : double TOPOLOGY : linear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo Sapiens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
ACC ATG GAC TGG ACC TGG~AGG GTC TTC~TGC~TTG CTG GCT GTA GCT CCA G 289 Met Asp Trp Thr Trp Arg Val Phe Cys Leu Leu Ala Val Ala Pro Gly Ala His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met .... r ~;
ACC AGG GAC ACG ACG AGC GTC TAC GAG CTG AGC AGC CTG
A
Thr Arg Asp Thr Thr Ser Val Tyr Glu Leu Ser Ser Ser Thr Met eu L
ACG TAT GCG
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg lI0 115 SEQUENCE ID NO.: 4 7 SEQUENCE LENGTH . 6 2 7 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Arg Glu Phe Val Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Cys Val 2~s~~~~r GlnCys GluAspGlnLeu ValGluSer GlyGlyGlyLeu ValGlnPro GlyGly SerLeuA.rgPro SerCysAla AlaSerGlyPhe AlaPheSer AGC~TAT GTTCTGCACTGG GTTCGCCGG GCTCCAGGGAAG GGTCCGGAG 483 SerTyr ValLeu~HisTrp ValArgArg AlaProGlyLys GlyProGlu TGGGTA TCAGCTATTGGT ACTGGTGGT GATACATACTAT GCAGACTCC .531 TrpVal SerAlaIleGly ThrGlyGly AspThrTyrTyr AlaAspSer Val Met Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr Leu Lys Thr Ala Leu Arg Thr Trp Leu Cys Ile Met SEQUENCE ID NO.: 4 8 SEQUENCE LENGTH .- 7 4 3 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Glu Leu Gly Leu Cys Trp Val Phe Leu Val Ala Ile Leu Glu GAGATTTAGT
GTGTGTGGAT
CCTTGGTGT TCTTTGTTTG
Gly ValGlnCys Glu ValGlnLeu ValGluSerGlyGly GlyLeuYalGln ProGlyGiy Ser LeuArgLeu SerCysAlaAlaSer GlyPheThrPhe SerSerTyr Ser MetAsnTrp ValArgGlnAlaPro GlyLysGlyLeu GluTrpVal Ser TyrIleSer SerSerSerSerThr IleTyrTyrAla AspSerVal Lys GlyArgPhe ThrIleSerArgAsp AsnAlaLysAsn SerLeuTyr Leu GlnMetAsn SerLeuArgAlaGlu AspThrAlaVal TyrTyrCys CACAGTGAGG CCCTGCAGGG
GGAGGTCAGT
GTGACAC
Ala Arg SEQUENCE ID NO.: 4 9 SEQUENCE LENGTH . 7 6 3 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 i near MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
GAGTTTCCAC TTGGTGATCA GCACTGAACA CAGACCACCA~ACC ATG GAG TTT GGG 235 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly.Leu Val Gln Pro Gly Arg Ser Leu Arg ~ ~. ~ 2 5'~'~
LeuSer CysThrAla SerGlyPheThr PheGlyAspTyr AlaMetSer TrpPhe ArgGlnAla ProGlyLysGly LeuGluTrpVal GlyPheIle ArgSer LysAlaTyr GlyGlyThrThr GluTyrThrAla SerValLys Gly~Arg PheThrIle SerArgAspGly SerLysSerIle .AlaTyrLeu GlnMet AsnSerLeu LysThrGluAsp ThrAlaValTyr TyrCysThr CACAGTGNGG CCCTGCAGGC
GGAGGTCAAT
GTGAGCCCAG
A
rg CCACCAGGGG
GCGCTAGG
SEQUENCE ID NO.: 5 0 SEQUENCE LENGTH . 2 8 3 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Glu Leu Gly Leu Ser Trp Val Ser Leu Val Ile Ile ..._ 1 ~ 5 10 Leu Lys AAACAGTGAT TCTGTGTGGC AGGTTCTGAC~TCAGATGTCC TCTGTGCTTG TAG GT GTC 163 Gly Val Cys Gly Val Gln Met Val Glu Ser Trp Gly Glu Leu Ala Gln Xaa Glu Cys Ala Asp Ser Ala Val His Pro Leu Asn Pro~Pro Ser Val GCT ACT AGA TCA GCT GAA TCT GCC ~ 283 Ala Thr Arg Ser Ala Glu Ser Ala SEQUENCE ID NO.: 5 1 SEQUENCE LENGTH . 7 0 0 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Gly Ser ..._ ~1~~ ~~
ACC GCCATC GCCCTC~CTC GCTGTTCTC CAAG GTCAGTCCTG 223 CTC CTG
Thr AlaIle AlaLeuLeu AlaValLeu Gln Leu Leu A GTGGAAAGGA CAAATTTTGT
G T GAG
GTG
CAG
CTG
GTG
CAG
TCT
GGA
GCA
G ly al ys Ser lu ln V C G Val Leu G Val Gln Ser Gly Ala TCT TCT
Glu ValLysLys ProGlyGlu LeuLysIle SerCys Lys Gly Ser Ser TGG CCC
Gly TyrSerPhe ThrSerTyr IleGlyTrp ValArg Gln Met Trp Pro GGG GAT
Gly LysGlyLeu_GluTrpMet IleIleTyr ProGly Asp Ser Gly Asp ACC AGATACAGC CCGTCCTTC.CAAGGCCAGGTC ACCATC TCA GCC 526 GAC
Thr ArgTyrSer ProSerPhe GlyGlnYal ThrIl~e Ser Ala Gln Asp 0 $5 90 CTG TCG
Lys SerIleSer ThrAlaTyr GlnTrpSer SerLeu Lys Ala Leu Ser GCG CACAGTGAG
Asp ThrAlaMet TyrTyrCys Arg Ala CTAAAACCCT AGACTCACTC
CCACACCGCA
TCTCT
-." , SEQUENCE ID NO.: 5 2 SEQUENCE LENGTH . 7 6 7 SEQUENCE TYPE : nucleic acid STRAND):DNESS : doub 1 a TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens .
IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Glu Ser Gly Leu Ser Tcp Val Phe Leu Val Ala Ile Leu Lys Gly Val Gln Cys Glu Val Gln Leu Val Glu Ser GGG TGA GGC TTG GTA CAG CCT GGA GGG TCC CTG AGA CTC~TCC TGT GCA 435 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala . 35 40 Ala Ser Gly Phe The Phe Ser Ser Ser Trp Met His Trp Val Cys Gln GCT CCG GAG~AAG GGG CTG GAG TGG GTG GCC GAC ATA AAG TGT GAC GGA 531 -..
AlaPro GluLys Leu GluTrpValAla AspIleLysCys AspGly Gly AGTGAG AAATAC GTA.GACTCTGTGAAG GGCCGATTGACC ATCTCC 579 TAT
SerGlu LysTyr Val AspSerValLys GlyArgLeuThr IleSer Tyr AAG
ArgAsp AsnAla Asn SerLeuTyrLeu GlnValAsnSer LeuArg Lys ACC CACAGTGAGG
AlaGlu AspMet Val TyrTyrCysVal Arg Thr G ACACAAACCT CACAA
A ACAGGGGCAG
C
SEQUENC$ ID NO.: 5 3 SEQUENCE LENGTH . 7 2 4 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Glu Phe Trp Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys CCAATGTCTC.TGTGTTTGCA G ~GT GTC CAG TGT GAG GTG CAC CTG GTG GAG 213 Gly Val GIn Cys Glu Val Gln Leu Val Glu GGG
SerGlyGlyGly LeuIleG~InProGlyGlySerLeu Ai-gLeuSer Cys 30 35 ~ 40 AlaAlaSerGly PheThrVal SerSerAsnTyrMet SerTrpYal Arg GlnAlaProGly LysGlyLeu GluTrpYalSerVal IleTyrSer Gly GlySerThrTyr TyrAlaAsp SerValLysGlyArg PheThrIle Ser 75 80 g5 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg GCC GAG GAC ACG GCC GTG.TAT TAC TGT GCG AGA GA CACAGTGAGG 498 Ala Glu Asp Thr Ala Val Tyr Tyr Cys A~la Arg 110 . 115 SEQUENCE ID NO.: 5 4 SEQUENCE LENGTH . 7 0 6 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
CCCCATAGCT.CTGGGAGAGA AGCGCCAGCC CTGGGATTCC CAGGGGTTTC CATTTGGTGA 120 TCAGGACTAA AGACAGAGGA CCCACC ATG GAG CTT GGG CTG AGC TGG GTT TTC 173 .
Met Glu Leu .Gly Leu Ser Trp Val Phe .1 5 Thr Val Thr Vat Leu Lys Gly Val Gln Cys Glu Val Gln Leu Val Glu Ser Glu Glu Asn Gln Arg Gln Leu Gly Gly Ser Leu Arg Leu Ser Cys Ala Asp Ser Gly Leu Thr Phe Ser Ser Tyr Met Ser Ser Asp Ser Gln Ala Pro Gly Lys Gly Leu Glu Val Val Asp Ile Asp Arg Ser Gin Leu Cys Tyr Ala Gln Ser.Yal Lys Ser Arg Phe Thr Ile Ser Lys Glu Asn Ala AAG AAC TCA CTC TGT TTG CAA ATG~AAC AGT CTG AGA GCA GAG GGC ACG 569 Lys Asn Ser Leu Cys Leu Gln Met Asn Ser Leu Arg Ala Glu Gly Thr 95 . 100 ~ 105 GCC GTG.TAT TAC TGT ATG TGA GT CACCAGGTAA GAAGACATCA GTGTGATCAC 622 Ala Val Tyr Tyr Cys Met SEQUENCE ID NO:: 5 5 SEQUENCE LENGTH . 8 0 0 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Lys His I
Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg ,.
Trp Val Leu Ser Gln Val Gln Leu Gln Glu Ser Gly Pro GGA CTGGTG CCTTCGGAG.ACCCTGTCC.CTC GCT GTC 444 AAG ATC TCT
TGC
Gly LeuValLys ProSerGlu LeuSerLeu Ile Ala ValSer Thr Cys AAG GTC
Gly AspSerIle SerSerGly Trp Ile Trp Arg GlnPro Asn Val ATT CAT
Pro GlyLysGly LeuGluTrp GlyGluIle His Ser GlySer Ile His AAG ATG
Thr TyrTyrAsn ProSerLeu SerArgIle Thr Ser ValAsp Lys Met CTG GTG
Thr SerLysAsn GlnPheTyr LysLeuSer Ser Thr AlaAla Leu Val GAC ACGGCCGTG TATTACTGT AGATACACA~GTG GGAGGTGAGT 685 GCG AGG
Asp ThrAtaVal TyrTyrCys Arg Thr Val Ala Tyr Arg GTGAGCCCAG CACAAACCT AGGCAGAGGG GGNGGGCACA
A CCCTACAGAT GGTGCTGCTC
GGGGGCGCGC AGCAG
GANGNCACAG
SEQUENCE ID NO.: 5 6 SEQUENCE LENGTH : 4 2 9 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double -,.
TOPOLOGY : linear MOLECULE TYPE :. Genomic DNA
ORIGINAL SOURCE : Homo sa iens , IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Asp Trp Thr Trp Ser Ile Leu Phe Leu Val Ala Ala Ala Thr Gly Ala His Ser Arg Val Gln Leu Val Gln Ser Giy Pro Glu Val Lys Gln Pro Gly Ala Ser Ala Lys Val Ser Cys Lys Val Ser Gly Thr Val Ile Thr Tyr Gly ATG AAT TGG ATA CGA. CAG ACC CCA GGA CAG GGG CTT GAG TGG ATG GGA 422 Met Asn Trp Ile Arg Gln Thr Pro Gly Gln Gly Leu Glu Trp Met Gly Trp Ile SEQUENCE ID N0. : 5 7 SEQUENCE LENGTH . 4 6 2 ~~~2~'~'~
-lI9-SEQUENCE TYPE : nucleic acid STRANDEDNESS :double TOPOLOGY : I inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
GGTGTGGGTG GAAACAGTGA.GTAGTCAAGT GGGAGTTCTC AGAGTTACTC TCCATGAGTA 360 ACCTGAAAGT CCAAGGACAA GGCTGTGTAT TACTGTGAGG GA ~ 462 SEQUENCE ID NO.: 5 8 SEQUENCE LENGTH . 6 2 9 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Asp Trp Ile Trp Arg Ile Leu ._ GCAAGGAGAT
GCCAAGTCCC
Phe LeuValGly AlaAlaIle GTCCAGTCAA GT
GGTGGCTTTC
ATCCACTCCT
Gly Ala HisSerGln MetGlnLeu ValGlnSerGlyProGlu ValLysLys Pro GlyThrSer ValLysVaI .SerCysLysAlaSerGly PheThrPhe Thr SerSerAla ValGlnTrp ValArgGlnAlaArgGly GlnArgLeu Glu TrpIleGly TrpIleVal ValGlySerGlyAsnThr AsnTyrAla Gln LysPheGln GluArg.ValThrIleThrArgAspMet SerThrSer Thr AlaTyrMet GluLeuSer SerLeuArgSerGluAsp ThrAlaVal CACAGTGTGA
AAACCCACAT
Tyr Tyr~CysAla Ala C
2 ~. 62 ~? ?
SEQUENCE ID NO.: 5 9 SEQUENCE LENGTH . 6 2 2 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa i.ens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE.DESCRIPTION
GCAAATCCTC
AGCTCACATG
CAT CTG TGG
Met Lys His Leu Trp Phe Phe 1 ~ 5 CAGGGATCCA
Leu Leu Leu Val Ala Ala Pro Arg CTGTGGGTCT
TCG GGC CCA G GTG
Trp Val Leu Ser Gln Val Gln Leu Gln Glu Ser ly Leu Gly Pro G Val GTC TCT GGT
Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Gly Ser Val Ser Gly CCC CCA GGG
Val Ser Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Lys Gly Pro Pro Gly AGC ACC AAC
Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Tyr Asn Ser Thr Asn __ ~2~~2~'~'~
70 , 75 CCCTCCCTC. AAG CGA GTC ATA GTA GAC ACG AAG AAC 518 AGT ACC TCA TCC
ProSerLeu Lys Arg Val Ile Val Asp Thr Lys Asn Ser Thr Ser Ser CAGTTC.TCC CTG CTG AGC GTG GCT GCG GAC GCC GTG 566.
AAG TCT ACC ACG
GlnPheSer Leu Leu Ser Val Ala Ala Asp Ala Val Lys Ser Thr Thr TATTACTGT GCG GA CACAGTGAGG GTGAGCCCAGACAAAAACC
TyrTyrCys Ala Arg SEQUENCE ID NO.: 6 0 SEQUENCE LENGTH . 5 8 8 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : linear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE: human lymphoblastoidline CGMI
cell SEQUENCE DESCRIPTION
ATGAACACTG
TTC CTT GTT G
Met Glu Ser Gly Leu Ser Trp Ile Ala Val Leu Lys Phe Leu Val TGACTGGACA
CCGTGTTTGC TGA
Gly Cys Pro Val GGT GCA GCT GGT GGA GTC TGG GGG .AAA GAC TGG GGG 266 AGG CTT AGT GTC
Gly Ala Ala Gly Gly Val Trp Gly Lys Asp Trp Gly Arg Leu Ser Val CTC ATT
Ser GluThrLeu LeuCysSerLeu Trp HisLeu Gln Leu Cys Ile AAA
Tyr AlaLeuGly ProProGlySer Arg Gly.PheGlyValGly Leu Lys AGT TATTAGTAC AAGTGGTGATAC CGT CTACAC AGACTC.TGT GAA 410 ' ACT
Ser Tyr Tyr LysTrp Tyr Arg LeuHis ArgLeuCys Glu .
Thr TGC
Gly LeuIleHis HisLeu Arg Gln ProGlu PheThrVal Ser Cys AAT CAT
Ala GluGln ProGluSerrg Arg GlyCys ValLeuLeu Cys Asn A His Glu Arg TGGGACAACC AGGGAAAGCC TGGGAC 5gg S EQUENCE I D N0. : 6 1 SEQUENCE LENGTH . 1 2 1 2 SEQUENCE TYPE : nucleic acid S TRANDEDNESS : doub 1 a TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
~~.~5'~
CCTCCTTTTT CACCTCTCCG TACAAAGGCA CC ~ CACAT GCAAATCCTT ACTTAAGCAC 60 TCTGAGAGCC TGGACCTCCT GTGCAAGAAC ATG.AAA CAC CTG TGG TTC TTC CTC 174 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg TATGGGAGGT GCCTCTGATC CCAGGGC'fCA CTGTGGGTCT CTCTGTTCAC AG GG GTC 283 Trp Val Leu Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro 25 sn SerGlu ThrLeuSer LeuThrCysThr ValSerGly GlySerVal Ser SerGly SerTyrTyr TrpSerTrpIle ArgGlnPro ProGlyLys Gly CTGGAG TGGATTGGG TATATCTATTAC AGTGGG~AGC ACCAACTAC AAC 475 LeuGlu TrpIleGly TyrIleTyrTyr SerGlySer ThrAsnTyr Asn CCCTCC CTCAAGAGT CGAGTCACCATA TCA.GTAGAC ACGTCCAAG AAC 523 ProSer LeuLysSer ArgYalThrIle SerYalAsp ThrSerLys Asn .
. 85 90 95 GlnPhe SerLeuLys LeuSerSerVal ThrAlaAla AspThrla Val A
CACAGTGAGG
GGAGGTGAGT
Tyr Tyr Cys Ala Arg TATCATCAAC TGAATTGTAC CCTCTTTGAA ATTCATATGA,TGAAACCTTA AATTCAATGG 1038 SEQUENCE ID NO.: 6 2 SEQUENCE LENGTH . 5 6 0 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Asn Leu Arg ~~.62~'~~'~
Gly Yal Gln Cys Glu Val Gln Leu VaI Glu Ser Gly Glu Gly LeuVal.GlnProGlyGly SerLeuArgLeu SerCysAla AlaSer Gly PheThrPhe SerSerSer AlaMetHisTrp ValArgGln AlaPro Arg LysGlyLeu TrpVal SerValIleSer ThrSerGly AspThr Val LeuTyrThr AspSerVal LysGlyArgPhe ThrIleSer ArgAsp g5 90 Asn AlaGlnAsn SerLeuSer LeuGlnMetAsn SerLeuArg AlaGlu 95 100 105 .
CGCAG AG
Gly ThrValVal TyrTyrCys ValLys CAAACCTCCT TGGGAC
GCAGGGTACC
SEQUENCE ID NO.: 6 3 SEQUENCE LENGTH . 5 1 5 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : linear MOLECULE TYPE': Genomic DNA
2~~~ ~ '~
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
CTT GTT TTA
Met Gly Phe Glu Leu Thr Arg Ile Phe Leu Val AlaIle Leu ATAAGTGAGA
Lys TGTGTATTTT GTT
Gly Val CAG TGT GAG GTG GAG CTG ATA,GAG TCC ATA GAG 211 GGC CTG AGA CAA CTT
Gln Cys Glu Val Glu Leu Ile Glu Ser Ile Glu Gly Leu Arg Gln Leu GGG AAG TTC C1'G AGA CTC TCC TGT GTA GCC TCT TTCAGT 259 GGA TTC ACC
Gly Lys Phe Leu Arg Leu Ser Cys Val Ala Ser PheSer Gly Phe Thr 35 40 . 45 GGG AAG GGG
Ser Tyr Met Ser Trp Val~Asn Glu Thr Leu Gly LeuGlu Lys Gly 50: 55 60 ATA TAC CAT
Gly Val Ile Asp Yal Lys Tyr Asp Gly Ser Gln AlaAsp Ile Tyr His AAT GCT AAG
Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp AsnSer Asn Ala Lys CCG TAT CTC CAA ACG AAC AGT CTG AGA GCT GAG ATGCAT
Pro Tyr Leu Gln Thr Asn Ser Leu Arg Ala Glu MetHis Asp Met Thr ,_ GGC TGT ACA TAA GG ~TTCCAAGTGA GGAAA1~ CG.GTGTGAGTCC AGACCAAAAT 505 Gly Cys Thr SEQUENCE ID NO.: 6 4 SEQUENCE LENGTH . 6 4 9 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo Sapiens IMMEDIATE SOURCE: human lymphoblastoidline CGMI
cell SEQUENCE DESCRIPTION
AGCT~CTGGGA GAGGAGCCCC CCCCCTGGGA TTTTCATTTGGTGATCAGCA60 TTCCCAGGTG
GGG CTG GTT
Met Thr Glu Phe Gly Leu Ser Trp Phe Leu Val AGGAAATAGA
Val Ala Ile Phe Lys ' CAGTTTCTGA
CTG GTG GAG GGA
G1y Va1 Gln Cys Glu Val Gln Leu Ser Gly Gly Leu Val Glu Gly CTC TCC TGT TCT
Val Gln Pro Gly Gly Ser Leu Arg Ala Ala Gly Phe Leu Ser Cys Ser 35 ~ 40 45 TGG GTC CGC CCA
Thr Phe Ser Ser.Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val Ser Ala Ile Ser Ser Asn Gly Gly Ser Thr Tyr TAT GCA AAC TCT.GTG AAG GGC AGA TTC ACC ATC TCC AGA GAC AAT TCC 465 Tyr Ala Asn Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Gly Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys Ala Arg CTGATCAGCG TCAACCGCAG GGG
system is advantageous for the. initial step of physical mapping. However, subsequent step to analyze large DNA
fragments in YAC can be problematic since exogenous DNA
inserts cannot be. easily separated from yeast chromosomal DNA and fragments up to several hundred kb are difficult to handle without mechanical shearing. In order to map VH segments of a.large DNA fragment containing VH
segments, detailed restriction map using common 6bp-site restriction enzyme is necessary. For this purpose, YAC
clones were subcloned into cosmids. Cosmid libraries were constructed from whole YAC DNA without previous separation of cloned DNA from host chromosome. There are two major reasons for this: 1) separation of intact insert DNA and.their manipulation are difficult, 2) 4000 independent colonies are sufficient for complete coverage of ,YAC insert since the genome size of.yeast is about 1,5 x 104 bp, 1/200 of that of human.
In general, there are two major difficulties in the construction of cosmid libraries. The first is self-ligation of vector DNAs, resulting in generation of clones carrying no inserts of foreign DNA, and 'the second is insertion of more than one DNA fragments in a single vector, namely co-ligation artifact. To overcome these problems, great efforts have been made including construction of better-designed vectors with two cos sites and modified method for ligation.such as partial filling of vector and insert DNAs (J. Sambrook et al., A
Laboratory Manual, Cold Spring Harbor Laboratory Press,.
Cold Spring Harbor, N.Y. (1989)). Size fractionated insert DNA usually contains smaller DNA molecules trapped among larger molecules especially when excess amount of DNA was loaded in the preparative gel. Alkaline phosphatase treatment of insert DNA is effective in order to exclude the co-ligation between inserts but gives rise to polymerized vector DNA during the ligation step, which causes high background of empty colonies under the antibiotic selection. In this example, however, less .
than 5 gig of YAC DNA was sufficient for insert preparation and thus preparative gel electrophoresis was successful without contamination of smaller DNA
fragments. Most of the cosmid libraries were thus constructed with minimal steps in combination with alkaline phosphatase-treated cosmid vector and partially digested DNA of exact size range for cosmid~insert (from 35 kb to 45 kb).
p1 Preparation of Yeast DNA Containing YAC
Since large DNA fragments are required as starting material for preparing the DNA, extraction of DNA from yeast cells with minimal shear damage is one of the most critical steps. Obviously, the best way is to manipulate DNA in-gel because DNA is fully protected from shear damage. The present inventors found, however, that gentle extraction of DNA in liquid from.yeast- cells gives sufficient length of. DNA (>200 kb) for partial digestion and subsequent size fractionation. In addition, liquid DNA is easier to control the condition for partial ~1~~~~~
digestion than gel block DNA. With a simple and rapid (6 hours for total procedure) method described below, about 50 ~g of large size DNA. (>200 kb) can be routinely purified from 100-ml. yeast culture.
(i) Spin down~yeast cells and wash them with TE (10 mM.
Tris HC1 (pH 8.0) - 1 mM EDTA) twice.
(ii) Resuspend the cells in 20 m1 of 0.1 M EDTA (pH 7.5), 1 M sorbitol, 0.2 mg/ml of Zymolyase 100T (ICN
Cat#152270), 15 mM 2-mercaptoethanol. Incubate at 37oC
for 1 hour to form spheroplasts.
(iii) Spin down the spheroplasts and resuspend in 9' ml of 0.1 M Tris HC1 (pH 7.5), 50 mM EDTA (pH 7.5).
(iv) Add 1 ml (1/10 final volume) of 10% SDS and mix gently. Incubate at 60°C for 10 to 20 minutes.
(v) Add 1/3 volume of 4 M potassium acetate and mix gently. Leave on ice for 30 minutes.
(vi) Centrifuge at 2000xg for 30 minutes and transfer the supernatant to a new tube. Add 3 volumes of isopropanol and mix gently. Leave at room temperature for 10 to 20 minutes for precipitation of DNA.
(vii) Centrifuge again at 2000xg for 30 minutes and discard supernatant. Dissolve the pe~.let in 10 ml of water.
From~this step onwards, care should be taken not to give shear damage to the DNA.
(viii) Extract with phenol twice and with CIAA
(chloroform:isoamyl alcohol = 24:1) twice followed by ethanol.precipitation at room temperature for 1'p to 20 minute s ~.
(ix) Centrifuge at 2000xg for 30 minutes. Rins~ the pellet with 70% ethanol and dry up the pellet.
(x) Dissolve with l.ml of TE. ~
p Vector DNA Preparation Lorist 2 DNA was linearized by digestion with Hind III or Bam HI. Linearized~DNA was dephosphoryl~ted by treatment with bacterial alkaline phosphatase. Small aliquots of DNA before and after phosphatase treatment were used for test ligation for phosphatase treatment according to a known method (J. Sambrook et al.,, su ra).
~3 Insert DNA Preparation Analytical experiment of partial digestion of yeast DNA was performed according to standard procedure (J.
Sambrook et al., su ra) to determine the optimal enzyme concentration and reaction time. Preparation ofi size-fractionated DNA from the gel was achieved with~LGT
agarose and ~ agarase I. This very gentle methbd resulted in high recovery (>90%) of fractionateca DNA
without degradation. Scaled up cleavage reactiqn was done using 5 ug of DNA with optimal enzyme concentration. Digested samples were loaded in ;a preparative gel of 0.5% LGT agarose (Bio Rad preparative grade) at about 1 V/cm overnight. Linearized lambda DNA
and its Xho I-digests which give 35-kb and 15-kt~~bands were also loaded as size markers. After visualixing~the ~~.~2~7'~
=31-DNA under ultraviolet transilluminater, a small slice~of agarose containing the fraction ranging from 35 kb to 45 kb was cut out. Recovery of the DNA from the gel slice was achieved using ~ agarase I (NEB) as follows:
(i) Equilibrate the gel block with water for complete removal of gel electrophoresis buffer.
(ii) Transfer the block to a new tube and add 1/9 volume of. lOx ~ agarase I buffer.
(iii) Melt the gel at 68°C for 10 minutes. Cool to 40°C
and incubate the molten agarose at 40°C for 1 hour with optimal number of units of ~ agarase I.
(iv) Adjust the salt concentration of the solution to 0.5 M~NaCl for ethanol precipitation. Chill on ice for 10 minutes.
(v) Centrifuge at 15,OOOxg for 15 minutes to pellet any remaining undigested carbohydrates.
(vi) Transfer the DNA-containing supernatant to a new tube. Precipitate the DNA with 3 volumes of ethanol at -80°C for 10 minutes.
(vii) Centrifuge at 15,OOOxg for 15 minutes and remove the supernatant. Rinse the pellet with 70% ethanol and dry up the pellet.
(viii) Resuspend the pellet in appropriate volume of water for subsequent manipulation.
With this method,~in average 100 to 300 ng of size-fractionated DNA can be recovered.
~ Ligation, in vitro Packaging and Infection to ~ i ~~ ~2~~
This process was performed according to standard procedure ,(J. Sambrook et al., supra). By using lambda inn packaging kit (Nippon Gene) and ED8768 host strain, about 10,000 colonies were obtained from 25 ng of ligated DNA.
~.Screening of Cosmid Libraries Initial screening was carried out using Hind III-partial cosmid libraries. About .10,000 colonies (500 colonies per ~lOcm plate x20) were plated on LB plates containing 50 ~g/ml of kanamycin so that single colonies can be picked up after first screening. Colonies were then lifted from~the plates with X8.2 cm detergent-free nitrocellulose membranes (Advantec Toyo Membrane) and subjected to colony hybridization. Three different kinds of probes were used for screening, namely mixture of six VH-family specific probes to isolate VH-containing cosmid clones, YAC vector probes (TetR gene segment of pBR322, described above) for isolation of insert-terminal cosmid clones, and total human DNA for any remaining cosmid clones. In average, 50 to 100 clones from a YAC clone with approximately 300-kb insert were isolated with the probes.
~ Construction.of Cosmid Contigs DNA from cosmid clones.was isolated by the alkaline lysis method by a conventional method (J. Sambrook et al., su ra). Purified cosmid DNAs were digested with Eco RI or Hind III and subjected to agarose gel electrophoresis for restriction mapping. Overlaps between clones were easily. found by comparinglrestriction patterns among cosmid clones. Ordered cosmid clones were then cleaved with Eco. RI or Hind III and loaded in a 0.7~
agarose gel. Southern filters were hybridized with six VH-family specific probes for identification of location and number of VH segments in cosmid clones. Filters were washed three times for 30 minutes under standard conditions (at 50°C in lxSSC, 0.1% SDS) followed by stringent conditions (at 65°C in O.IxSSC, 0.1~ SDS).
Location of VH segments were determined by comparison between hybridization pattern of cosmids and their physical maps.
Theoretically, approximately 50 independent cosmid clones (about 7 fold of the whole YAC insert) would be sufficient to cover the whole YAC insert of 300 kb in length. However, the distribution of cosmid clones were uneven and there still remained a few gaps. The clones corresponding to the gaps could not be isolated even after screening of Sau 3AI partial. library or chromosomal walking by using the probes isolated from the edge of each contig. Regions not present in the cosmid libraries were subcloned with phage or plasmid vectors by isolation' of DNA fragments of required size from YAC DNA as shown in Fig. 4. After the complete physical map was constructed, the present inventors found out that this was not due to the nonrandom distribution of restriction sites within the YAC insert. The presence of some classes of sequences such as pahindromic.or tandem repeat DNA might make these regions unclonable or under-represented by using cosmid system. The complete physical map of the 0.8-Mb region constructed in this example is shown in Fig. 1 as mentioned above. The distance.from JH of each VH segment shown in Fig. 1 and the sizes of Eco RI and Hind III fragments are shown in Table 3.
Example 2 Construction of Cosmid Clones A cosmid library was constructed from human high molecular DNAs as follows:
3-31: High molecular DNAs obtained ,from human placenta were partially digested with Tag ~I and the resultant was subjected to electrophoresis on 0.5~ agarose gel. The 35 - 45-kb bands were recovered by using DEAF paper. The recovered DNAs were treated with alkaline phosphatase and the resultant was ligated to cosmid vector pJBB which.had been completely digested with a.restriction enzyme Cla I.
The ligation product was subjected to in vitro packaging and the resultant was infected to host E. coli 490A, followed by the screening by the conventional colony hybridization to obtain the clone.
M131, M84 and M118: These fragments were obtained by the same method as for 3-31 except that the DNA used was human pro B cell line FLEB14-14, the vector and the host E.- coli used were Lorist 2 and ED8767, respectively, the combination of restriction enzymes employed was Xba I and Hind III, and the edges of the fragments were modified by the partial repairing. The partial repairing was carried out according.to a known method (J. Sambrook, E.F.
Fritsch and T. Maniatis, 1989, Molecular Cloning; a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
Example 3 Sequencing Analysis of VH Segments Instead of sequencing subcloned VH-containing DNA
fragments using vector primers, VH family-specific oligonucleotide primers were synthesized. As mentioned above, nucleotide sequences of FR regions of VH.segments are highly conserved within the same family, so the present inventors selected consensus sequences from the conserved portions and synthesized family-specific oligonucleotide primers for sequence analysis. For this purpose, automated fluorescence-based sequencing system Model 373A developed by Applied Biosystems was employed.
Dye-Deoxy*terminator sequencing kit supplied from the same company using fluorescent-dye labeled dideoxy nucleotides was suitable for our purpose~since synthesized VH-specific primers could be directly used without fluorescence-label.
(I) Subcloning of VH-containing Restriction Fragments In order to use VH-family specific primers for sequencing, it is essential to subclone VH-containing DNA
fragments so that each plasmid contains only one VH
*Trade-mark kb was cut out. Recovery of th 76432-1 ca o2i62s~~ Zooo-io-2o segment. Several other 6bp-site enzymes than Eco RI and Hind III were used to isolate single VH-carrying DNA
fragments. Plasmid DNA of the subcloned fragments was isolated by alkaline lysis method followed by ult=acentrifugation twice to obtain high~quality DNA
samples for accurate sequences.
(2) Oligonucleotide Synthesis for Sequencing To select consensus sequences for VH family-specific oligonucleotide primer synthesis, nucleotide sequences of framework regions and axon-intron boundaries of the known VH segments were aligned by family. Attention was paid so that 3'-half of them have 100% identities to reference sequences and 3'-most nucleotide corresponds to the first or the second letter of highly conserved/iwariant amino acid residues. Nineteen additional primers were designed for five VH families as shown in Table 1 (described below).
(3) Sequencing Reaction and Gel Electrophoresis The sequencing reaction was performed by PCR using Dye-Deoxy terminator sequencing kit (ABI) according to manufacturer's instruction. Gel electrophoresis and detection of signals were done in the sequencing apparatus according to the users manual of the system.
In average, sequences of over 350 bases were obtained from each reaction.
(4) Evaluation of Synthesized VH Family-specific Primers The primers F-univ and I-R were first' chosen to *Trade-t~k 21~2~'~~
sequence VH-I segments. As shown in Table 2, they annealed 11 of 12 VH_I segments analyzed. It i~s to be noted that all of 6 functional VH-I segments could be sequenced with these two primers. Two more primers, I-NF1 and I-NR1 were designed for V1-14P and V1-27P
segments. These two primers were also used for. some other VH segments to verify their sequences obtained by first two primers (Table 2).
Eight primers were designed and used for sequencing VH-III family segments. The first sequencing reaction of each VH segment was performed with F-univ and III-R
primers. They annealed more. than $0$ of the VH_III
.segments analyzed (25/30 for F-univ and 24/30 for III=
R)(Table 2). Importantly, again, all the functional VH-. III segments with one exception could be sequenced with this combination of primers, suggesting that they would be good for most of VH-III cDNA. Based on the nucleotide sequences obtained from first experiment, six additional primers (III-F3, III-R3, III-F4, III-R4, III-NF1 and III-F2) were designed and appropriate combination among them were used for further analysis. Among these, III-R3 and III-F4 were used to determine the sequence of 5' regulatory region and 3' flankii-ig region, respectively.
V3-29P and v3-32P were pseudogenes with extensive divergence in their sequences and thus all but III-NF1 failed to anneal these two VH segments. Sequences of V3-25P, V3-44P and V3-63P were determined using M13 ~~.~~~'~'~
vector primers from their internal restriction~sites.
Five each of synthesized primers were used to determine the sequences of VH segments belonging to VH-III VH-IV and VH-V families. Since VH segments belonging to each of these three families are highly homologous with each other, it was thought that four each of the primers are.enough for most. of the VH segments belonging to these smaller VH families. In fact,~all four VH-II
family-specific primers annealed three VH-II segments (V2-5, V2-lOP and V2-26). In brief, in total 11 primers (F-univ and I-R for VH-I; II-R1, II-F2 and II-R2 for VH-II% F-univ and IiI-R for VH-III% IV-R1, IV-F2 and IV-R2 for VH-IV% V-R2 and V-R3 for VH~V) would be sufficient for sequencing most of the VH segments belonging to five VH families. The II-F1, III-NF2 and IV-F1 primers contain intron sequences and thus cannot be used for cDNA
sequencing.
By this procedure, the DNA sequences of the 64 VH
segments were determined and they are shown in Sequence ID Nos. 1 - 64 as mentioned above. The distance of each VH segment from JH and the sizes of Eco RI and Hind III
fragments are summarized in Table 3.
(5) Transcriptional Polarities of VH Segments The strategy for sequencing VH segments with family-specific primers was not suitable for determination of transcriptional polarities of the VH
segments because it did not require restriction map of ..._ single VH-containing subcloned fragments. The. present inventors could not determine orientations of all the VH
segments within this region for that reason. The present inventcsrs found, however, that 8 regions containing 21'VH
segments-were already isolated in cosmid or phage clones since sequences.between corresponding VH segments as well as their restriction maps were identical with each other.
As the relative orders of these 21 VH segments within these clones are identical to those in the 0.8-Mb region, it was concluded that the orientation of these 21 VH
segments are the same as those of the JH segments.
- ~~.~"~~7'~
Table 1 V" family-specific primers~used for screening and sequencing family name sequence(5'to 3' ) X location direction I, 13, F univ AGGTGCAGCTGGTGCAGTCTG 1-8 ~ forward V - .
I I R ~CCAGGGGCCTGTCGCACCCA 36-42 reverse -I N F TGGGGCCTCAGTGAAGGTCTCCTG 14-22 forward I N R GATCC(A/G)TCCCATCCACTCAAG45-51 reverse II II F 1 TGTCTTCTCCACAGGGGTCTT intron-(-2)forward -II F 2 GGGAAGGCCCTGGAGTGGCT 42-48 forward -IL R 1 GTGCAGGTCAGCGTGAGGGT 17-23 reverse -II R 2 TGGTTTTTG.GAGGTGTCCTTGG 70-77 reverse -III III R CACTCCAGCCCCTTCCCTGGAGC 40-47 reverse -III F 3 GTGAGGTTCAGCTGGTGGAGT (-1)-7 forward -III R 3 AGCTGAACCTCACACTGGAC (-3)-4 reverse -111 F 4 AAGGGCCGATTCACCATCT 64-70 forward -II< R 4 TTGTCTCTGGAGATGGTGAA 68-73 reverse -II< N F TGAGACTCTCCTGTGCAGCCTCTG 18-26 forward Ill N F TCT(T/C)TGTGTTTGCAGGTGT intron-(-3)forward IV F 1 TCTGTTCACAGGGGTCCTGTC ntron-(-1) forward - i N F 2 TCCGGCAGCCCCCAGGGAA 37-43 forward -Iv R 1 GCAGGTGAGGGACAGGGT 17-22 reverse -IV R 2 CAGGGAGAACTGGTTCTTGGA 74-80 reverse -V V R 1 CCCGGGCATCTGGCGCACCCA 36-42 reverse -V R 2 GCTGCTCCACTGCAGGTAGGC 78-82R reverse -V R 3 CTTCAGGCTGCTCCACTGCAG 74-83 reverse -X Locations of the primers are indicated as amino acid residue number according to Kabat et al. Bases with redundancy are shown in the parentheses. Directions relative to coding sequence are also shown.
~~.~w~~~
Table 2 List of useful primers for sequencing V" clones VH-, primers VH-,m primers VH_", primers V" segments univ R NFI NRI univ R F3 R3 F4 R4 NF1 NF2 FI Rl F2 R2 V" I 1-2 + +
1-3 + +
1-8 + + .
1-12P + +
I-14P - + +
1-17P + ~ + + +
1-18 + + +
1-24P + + + +
1-27P + - + +
1-40P + +
1-45 + +
1=46 +. +
VH DI 3-6P - + - + - + + +
3-7 + + + +
3-9 + + +
3-11 + + + +
3-13 + - + + + +
3-15 + + + +
3-16P + + + +
3-19P + + +
3-20 + + + + +
3-21 + + + + +
3-22P + + +
3-23 + + + : + +
3-29P _ _ _ _ _ ~..
3-30 + + + + +
3-32P _ _ _ _ _ _ + _ 3-33 + + + + +
3-35 + +
3-36P - + +
3-37P + - +
3-38P + +
3-41P + +
3-42P. + - + +
3-43 ~ + + .
3-47P + +
3-48 + +
3-49 + +
3-50P + +
3-52P~ + +
3-53 + +
3-54P + + +
3-64 + +
yH Iv 4-4 + + + +
+ + - +
4-34 + + + +
Table 3 V " kb from . Fragment size(kb) 1H EcoRI Hind I~
6-1 75 0. 9 25 1-2 125 7. 2 12. 5 1-3 150 , 3.4 l.'7 4-4 160 5.1 8. 0 2-5 175 5. 4 16. 0 3-6P 185 11. 8 16. 0 3-7 190 ~ 2.2 5.0 1-8 215 3. 8 2. 0 3-9 230 2.6 5.4 2-lOP 235 13.5 18.5 3-11 245 1. 6 18. 5 1=12P 250 4.5 2.8 3-13 260 1.7 5.8 1-14P 275 2.9 13.0 3-15 280 4.8 13.0 3-16P 290 5.4 1.8 1-17P 295 5.4+1.6 10.2 1-18 315 3. 4 8. 8 3-19P 330 4.3 ~ 14.7 3-20 345 11.8 12.8 3-21 360 2. 2 6. 8 3-22P 385 5.7 7.0 3-23 395 2.0 5.7 1-24P 410 3.0 5.2 3-25P 420 10.0 7.3 2-26 430 8.1 6.6 1-27P 450 8.3 11.3 4-28 455 8.3 5.4 3-29P 460 3.5 5.8 3-30 470 9. 8 6, 8 4-31 475 10. 3 13. 0 3-32P 485 13.3 5.6 3-33 490 13.3 6.8 4-34 505 11.5 16.2 3-35 520 5.3 3.2 3-36P 525 5.3 5.7 3-37P 540 7.5 13.2 3-38P 545 8.0 15.4 4-39 555 7.0 15.4 __ -Table 3 (continued) V " kb from Fragment size(kb) JH EcoRI Hind IB
1-40P 560 1.4 3.2 3-41P 580 4.4 11.9 3-42P 590 - 3:0 3.8 3-43 600 6.5 8.1 3-44P 610 8.8 17.0 1-45 635 10.7 2.7 1-46 640 2.0 4.6 3-47P 650 2.7 10.5 3-48 670 2.7 3.9 3-49 690 1.6 16.5 3-50P 695 10.0 16.5 5-51 710 8. 0 11.
3-52P 715 4.0 11.0 3-53 725 ~ 8.3 6.3 3-54P 730 6.4- 15.4 4-55P 735 3.9 15.4 1-56P 740 3.4 15.4 3-57P 745 9.7 6.6 1-58P 750 8.3 17.5 4-59 755 8. 3 17.
3-60P 760 0.8+3.0 17.5 4-61 770 8. 1 9. 0 3-62P 'l75 4..6 9.0 3-63P 780 8.9 6.2 3-64 790 4.4 >7.4 ~,1~~~'~
SEQUENCE ID N0. : 1 .
SEQUENCE LENGTH . 1 4 2 9 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA .
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE: hucian lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Ser Val Ser Phe Leu Ile Phe Leu Pro Val Leu Gly Leu Pro Trp Gly Val Leu Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp 2~ ~~~'~'~
Ser Val Ser Ser Asn Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp.Tyr Ala Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro 80 85 ' 90 95 GAC ACA TCC AAG AAC CAG TTC TCC CTG~CAG CTG AAC TCT GTG ACT CCC ?52 Asp Thr Ser Lys Asn Gln Phe Ser Leu GIn Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Vat Tyr Tyr Cys Ala Arg TTCATTCCTT TTTACTCTTT TTTTCTTTTT A1'TCATCTGC CTGAATTATT TCAAAAGATC 1344 SEQUENCE ID HO.: 2 SEQUENCE LENGTH . 5 1 2 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : l inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
~TGAGAGCTCC GTTCCTCACC ATG GAC TGG ACC TGG AGG ATC CTC TTC TTG GTG 53 Met Asp Trp,Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr GTCCAGTCCA GGGAGATCTC ~CACAG 162 ATCCACTTCT GA
GTGTTCTCTC GCC
CAC
TCC
Gly Ala His Ser GlnVal GlnLeuVal GlnSerGly AlaGluValLysLys ProGlyAla SerVal LysValSer CysLysAla SerGlyTyrThrPhe ThrGlyTyr TyrMet HisTrpVal ArgGlnAla.ProGlyGlnGlyLeu GluTrpMet 55 . 60 65 GlyTrp IleAsnPro AsnSerGly GlyThrAsnTyrAla GlnLysPhe GlnGly ArgValThr MetThrArg AspThrSerIleSer ThrAlaTyr 85 90 ~ 95 ~1.~25 ~ "~
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg GCAG
SEQUENCE ID NO.: 3 SEQUENCE LENGTH . 4 9 6 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Asp Tcp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr GCT ATG CAT TGG GTG CGC CAG GCC CCC GGA CAA AGG CTT GAG TGG.ATG 300 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met Gly Trp Ser Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Glu Phe Gln Gly Arg Val Thr Ile Thr.Arg Asp Thr Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Met Ala Val Tyr Tyr Cys Ala Arg SEQUENCE ID N0. : 4 SEQUENCE LENGTH . 6 5 0 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Lys His Leu Trp CTC CTG
CTG GTG
GCA GCT
T GTGAGTGTCT
CAAGGCTGCA
Phe Phe Leu Leu Leu Val Ala Ala Pro Arg _ 10 15 CCTCTGATCC
CAGGGCTCAC
TGTGGGTCTC
CT TCC GTG
CAG
GTG
CAC
CTG
CAG
GAG
TCG
GGC
CCA
GG
Trp Val u r Gln Va1 y Pro y Leu Le Se Gln Leu Gln Gl Val Glu Ser Gl TCG CTC ACT GGT
Lys Pro GluThr Leu Ser Thr Cys ValSer Gly Ser Ser Leu Thr Gly AGT TGG CAG GGG
Ile Ser TyrTyr Trp Ser Ile Arg ProAla Lys Gly Ser Trp Gln Gly 50 55 ~ 60 TGG TAT GGG AAC
Leu Glu IleGly Arg Ile Thr Ser SerThr Tyr Asn Trp Tyr Gly Asn CTC ACC GTA TCC
Pro Ser LysSer Arg.Val Met Ser AspThr Lys Asn Leu Thr Val Ser TCC TCT GCC ACG
GIn Phe LeuLys Leu Ser Val Thr AlaAsp Ala Vat Ser Ser Ala Thr 100 105 Iln Tyr Tyr Cys Ala Arg lI5 SEQUENCE ID NO.: 5 2~~~5~~
SEQUENCE LENGTH . 6 1 3 SEQUENCE TYPE : nucleic acid STRANDEDNESS : doubl.e TOPOLOGY : 1 inear .
MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Asp Thr Leu Cys Ser Thr.Leu Leu Leu Leu Thr Ile Pro Ser Trp Val Leu Ser Gln Ile AAA
Thr LeuLysGlu SerGlyProThr .LeuValLysPro ThrGlnThr Leu Thr LeuThrCys ThrPheSerGly PheSerLeuSer ThrSerGly Val Gly ValGlyTrp IleArgGlnPro ProGlyLysAla LeuGluTrp Leu Ala Leu. Ile Tyr Trp Asn Asp Asp Lys Arg Tyr Ser Pro Ser Leu Lys 2~.~~~'~"~
Ser Arg Leu Thr IIe Thr Lys Asp Thr Ser Lys Asn Gln Val VaI Leu Thr Met Thr~Asn Met Asp Pro Val Asp Thr Ala Thr Tyr~Tyr Cys Ala His Arg SEQUENCE ID NO.: 6 SEQUENCE LENGTH . 5 9 4 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Glu Leu Gly Leu Arg Tcp Val Phe Leu Ala Ala Ile Leu Lys Gly Val GlnCys GluMetGln LeuValGlu SerGlyAlaAsn LeuThrLysPro GlyCys Pro Asp SerProVal GlnProLeuAsp SerProSerVal AlaIle AlaArgThr GlySerPro ArgLeuGlnGly ArgValCysSer C
GlySer GlnLeuLeu ValValVal YalValProCys ThrThrGlnThr Leu ArgAlaAsp SerProPhe ProGluThrIle ProLysThrHis CysIle CysLys ThrAsp ly GlnArgMet GlnLeuHisMet G
100~ 105 TACGGTAAGG ACACAAACCT
ThrLeu Glu T AACCAGGGAA GTGCTGACCC
T ACAAGGG
SEQUENCE ID NO.: 7 SEQUENCE LENGTH . 8 7 7 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double T OPOLOGY : 1 i near MOLECULE TYPE : Genomic DNA
ORIGINAL
SOURCE
: Homo sapiens IMMEDIATE line SOURCE: CGMI
human lymphoblastoid cell SEQUENCE
DESCRIPTION
GAA TTC CTT ATT GAA
G
Met Glu Leu Gly Leu Ser Trp Val~ ValAla Leu Phe Leu Ile Glu GTC
Gly GlnCys Val CAG GGC TTG CCT
Glu Val Leu Val Glu Ser Gly Gly ValGln GlyGly Gln Gly Leu Pro AGA GGA TTC AGT
Ser Leu Leu Ser Cys Ala Ala Ser ThrPhe SerTyr Arg Gly Phe Ser 40 45 . 50 AGC GGG AAG GAG
Trp Met Trp Val Arg Gln Ala Pro GlyLeu TrpVal Ser Gly Lys Glu ATA AAA TAC GAC
Ala Asn Lys Gln Asp Gly Ser Glu TyrVal SerVal Ile Lys Tyr Asp CGA AAC GCC TCA
Lys Gly Phe Thr Ile Ser Arg Asp LysAsn LeuTyr Arg Asn Ala Ser ATG GAC ACG GCT TAT
Leu Gln Asn Ser Leu Arg Ala Glu Val TyrCys Met Asp Thr Ala Tyr ~~~~5"~'~
w Ala Arg S EQUENCE I D N0. : 8 SEQUENCE LENGTH . 5 6 4 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Ser Ala His Ser Gln Val Gln Leu Val Gl.n Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser ~ 30 35 40 Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asp Ile Asn Trp Val 45 50 ~ 55 Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met Gly Trp Met Asn Pro AAC AGT GGT AAC ACA GGC TAT GCA CAG AAG TTC CAG GGC AGA GTC ACC .407 Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe Gln Gly Arg.Val Thr 75 ~ 80 85 Met Thr Arg Asn Thr Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Ser 90 ~ 95 100 Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg SEQUENCE ID N0. : 9 SEQUENCE LENGTH . 6 4 0 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : t inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Glu Leu Gly Leu Ser Trp Ile Phe Leu Leu Ala IIeI
AAA ATGAGTGGAT
G
GTGATTCATG
GAGAAATAGA
GAGATTGAGT
.
Leu Lys TTGGTGTCT TG G GTC TG
GA
Gl y l n s Va Gl Cy GAG
Glu Val Leu ValGluSerGly GlyGlyLeuVal ln ProGly Arg Gln G
20 25 ~ ~ 3Q 35 AGA
Ser Leu Leu SerCysAlaAla SerGlyPheThr PheAspAsp Tyr Arg CAC
Ala Met Trp ValArgGlnAla ProGlyLysGly L.euGluTrp Val His ATT
Ser Gly Ser TrpAsnSerGly SerIleGlyTyr AlaAspSer Val Ile CGA.
Lys Gly Phe ThrIleSerArg AspAsnAlaLys AsnSerLeu Tyr Arg ATG
Leu Gln Asn SerLeuArgAla GluAspThrAla LeuTyrTyr Cys Met GCA AAA CAGTGAGG GGAAGTCAGC. GAGAGCCCAG 617 GAT ACAAAAACCT
A
CA
Ala Lys Asp GACAGGAGGG
GCC
SEQUENCE ID NO.: 1 0 SEQUENCE LENGTH . 6 3 0 y .
SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear .
MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met GIu Leu Tyr Ser Thr Leu Leu Leu Leu Thr Val Pro 1 5 m Ser Trp Val Leu Ser Gln Val 15 ~n AAA
ThrLeu LysGluSer GIyProAla LeuValLysProThr GlnThrLeu MetLeu ThrCysThr PheSerGly PheSerLeuSerThr SerGlyMet 40 45 . 50 GlyVal Gly Ile CysGlnPro SerAlaLysA1aLeu GluTrpLeu Ala His Ile Tyr Asn Asp Asn Lys Tyr Tyr~Ser Pro Ser Leu Lys Ser Arg Leu Ile Ile Ser Lys Asp Thr Ser Lys Asn Glu Val Val Leu ACA GTG ATC AAC ATG .GAC ATT GTG GAC ACA GCC ACA CAT TACTGTGCAA 505 Thr Val Ile Asn Met Asp Ile Val Asp Thr Ala Thr His 100 , 105 110 SEQUENCE ID NO.: 1 1 SEQUENCE LENGTH . 7 1 5 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa .iens IMMEDIATE SOURCE : human lymphoblastoidline CGMI
cell SEQUENCE DESCRIPTION
CACCCCAGGC~TTTACACTTT ATGCTTCCGG GTGTGGAATT GTGAGCGGAT60 CTCGTATGTT
ATGATTACGC
TCGAATTCCC
TTT GGG TTC
Met Glu Phe Gly Leu Ser Trp Val Phe GAGAACTAGA GACATTGAGT
Leu Val Ala Ile Ile Lys GTGGCAGTTT
CTG GTG GAG~TCT GGG GGA GGC
'~~.~~~7'~
Gly Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly LeuVal LysProGlyGly SerLeuArgLeu SerCysAlaAla SerGly 30 35 40 ~ 45 PheThr PheSerAspTyr TyrMetSerTrp IleArgGlnAla ProGly LysGly Leu.GluTrpVal SerTyrIleSer SerSerGlySer ThrIle TyrTyr AlaAspSerVal LysGlyArgPhe ThrIleSerArg AspAsn 80 ~ ~
Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg SEQUENCE ID NO.: 1 2 SEQUENCE LENGTH . 6 6 0 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens ~1~~~ ~'~
-so-IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION .
CATCTCCTC ATG GNC TGG ACC TAC AAG ATC CTC TTC .TTG GTG GCA GCA GCC 171 Met Xaa Trp Thr Tyr Lys Ile Leu Phe Leu Val Ala Ala Ala Thr Gly Ala His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys 25 30 ~ 35 ValSer CysLysAla SerGlyTyr ThrPheThrTyr CysTyrLeuHis TrpVal GlnAla ProGlyGln GlyLeuGluTrp ThrGlyPhe LeuPhe GluArgPhe PheIleGln HisLeuPheCys LysGlnIleSer GlyIle ValGlu IleIleLeu ThrAsnLeuThr GlnAsnPheLeu 85 . 90 95 TGA ATC AAT CTT TGT AAA CAT .CAA TTT CTG AAT CAA TGT TGT NAA TAT 568 -sl-Ile Asn Leu Cys Lys His Gln Phe Leu Asn Gln Cys Cys Xaa Tyr 100 105 1.10 TTC AGA ACA CAA GCA CAA NTT CAC ATT TNA ACT CTA CfiT TNA TCT CTA 616 Phe Arg Thr Gln Ala Gln Xaa His Ile Xaa Thr Leu Leu Xaa Ser Leu Phe Lys Xaa Tyr Gln Lys Xaa Ser Ser Xaa Ala Cys Asn Val SEQUENCE ID NO.: 1 3 SEQUENCE LENGTH . 8 1 9 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
GCCCCAGCCT TGGGATTCCC~AAGTGTTTGT ATTCAGTGAT CAGGACTGAA CACACAGGAC 300 Met Glu Leu Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Glu Gly Val Gln Cys Glu Val His Leu Val Glu Ser Gly Gly Gly Leu Val Gln CCT GGG GGG GCC~CTG AGA CTC TCC TGT GCA GCC TCT GGA TTC ACC TTC 553 Pro Gly Gly Ala Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr Asp Met His Trp Val Arg Gln Ala Thr Gly Lys Gly Leu 50 55 ~ 60 Glu Trp Val Ser Ala Asn Gly Thr Ala Gly Asp Thr Tyr Tyr Pro Gly TCC GTG AAG GGG CGA TTC ACC ATC TCC AGA GAA ~AAT GCC AAG AAC TCC 697 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr 100 105 . 110 Tyr Cys Ala Arg SEQUENCE ID NO. : 1 4 SEQUENCE LENGTH . 8 1 6 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
~~.~2~ ~~.
ORIGINAL SOURCE : Homo Sapiens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
AGCAGTCAGA GATCTGAGGA CATAGATGTG TACTACTGTG CGANACACAC AGTGTGACAN 540' GAGTCGTACC CNGGATCCCT GNTTGGCCTG AGNATA . 816 SEQUENCE ID NO.: 1 5 SEQUENCE LENGTH . 5 3 5 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : l inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo Sapiens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Glu Phe Gly Leu Ser Trp Ile Phe Leu Pro Ala Ile Leu Lys GTC
CAG
TGT
GAG
GTG
CAG
CTG
GTG
GAG
TCT
GGG
GGA
GCC
TTG
Gly Yal Gln Cys.
Glu Val Gln Leu Val Glu Ser Gly Gly Ala Leu TGT
ValLysPro GlyGly SerLeuArgLeu Ser AlaAla SerGlyPhe Cys 35 . 40 45 CGC .
ThrPheSer AswAla TrpMetSerTrp Val GlnAla ProGlyLys Arg AAA .
GlyLeuGlu TrpVal GlyArg.IleLys Ser ThrAsp GlyGlyThr Lys 65 70 i5 Thr Asp Tyr Ala A,la Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu GAC ACA GCC GTG TAT TAC TGT ACC ACA GA CACAGTGAGG GGAGGTCAGT 497.
Asp Thr Ala Val Tyr Tyr Cys Thr Thr ~~~~'~
SEQUENCE ID NO.: 1 6 SEQUENCE LENGTH . 5 4 2 SEQUENCE TYPE : nucleic acid STRANDEDNES$ : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Glu Phe Gly Leu Ser Trp Yal Phe Leu Ala Gly Ile Leu Lys Gly Val Gln Cys Glu Val Gln Leu Val G!u Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser GGA TTC ACC TTC.AGT AAC AGT GAC ATG AAC TGG GCC CGC AAG GCT CCA 310 Gly Phe Thr Phe Ser Asn Ser Asp Met Asn Trp Ala Arg Lys Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly Val Ser Trp Asn Gly Ser Arg i E
Thr His Tyr~Val Asp Val Arg Arg Ile Ile Ser Arg Ser. Lys Phe Asp g0 g5 90 CTG CTG AAC GAG' Asn Ser Arg Asn Ser Tyr Gln Lys Arg Arg Arg Ala Leu Leu Asn Glu TAC GTG TCCTGTGAGG
GGACACAAGT
Asp Met Ala Val~ Cys Arg Tyr Tyr Val GCGAGCCCAG ACACAAACCT
CCTGCAGGAA CACTGGGCG
SEQUENCE ID NO.:
SEQUENCE LENGTH .
SEQUENCE TYPE : nucleicacid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : GenomicDNA
ORIGINAL SOURCE :
Homo Sapiens IMMEDIATE SOURCE lymphoblastoid line CGMI
: human cell SEQUENCE DESCRIPTION
C CACAGCTCCT ACC
Met Asp Cys Thr Trp Gly Ile Leu Phe Leu Val Ala Ser Xaa Thr ' Asp Val His Ser Gln Val Gln Leu Leu Gln Pro Gly ~~~.~~5'~
Ala GluValLys LysProAlaSer SerValLysVal SerTrpPro Gly Phe GlnIleHis LeuHisGlnIle LeuTyrThrVal GlyAlaThr Gly Pro TrpThrArg Ala TrpLeu GlyCysIleAsn ProTyrAsn Asp Asn ThrHisTyr AlaGlnLysPhe ArgGlyArgVal ThrIleThr Ser Asp ArgSerVal SerThrAlaTyr MetGluLeuSer SerLeuArg Ser .
AAACCCACAT
Glu AspMetVal ValTyrSerCys ValArg AAGATTATTA GATTAACGAT TTTCTTAGA ~ 591 SEQUENCE ID NO.: 1.8 SEQUENCE LENGTH . 5 3 9 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : I inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
_6g_ Met Asp Trp . 1 TGG
AGC
ATC
CTT
TTC
TTG
GTG
GCA
GCA
CCA
ACA
G GTAACGGACT
Thr Trp Ser Ile Leu Phe Leu Val Ala Ala Pro Thr AGGGCTGAGA
GAGAAACCAG
TGTCCTCTCC.ACAG 211 GT
GCC
CAC
TCC
CAG
GTT
CAG
CTG
GTG
CAG
TCT
GGA
Gly Ala His Ser G ln Val Gln Leu Val Gln Ser Gly GAG GCC AAG~ GCT
Ala Val Lys Lys Pro Gly Ser Val Val Ser Cys Lys Glu Ala Lys Ala GGT TAT AGC GCC
Ser Tyr Thr Phe Thr Ser Gly Ile Trp Val Arg Gln Gly Tyr Ser Ala GGA ATG ATC GGT
Pro Gln G1y Leu Glu Trp Gly Trp Ser Ala Tyr Asn Gly Met Ile Gly ACA CTC AGA ACA
Asn Asn Tyr Ala Gin Lys Gln Gly Val Thr Met Thr Thr Leu Arg Thr GAC TCC ACG AGC ACA GCC ATG GAG AGG AGC.CTG AGA 451 ACA TAC CTG TCT
Asp Ser Thr Ser Thr Ala Met Glu Arg Ser Leu Arg Thr Tyr Leu Ser GAC GA
ACG
GCC
GTG
TAT
TAC
TGT
Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg ~~.~~~ 1 6 CCTGAGGGTT TCAGAAACCC CAGGGAGGAG GCAGCT ~ 539 SEQUENCE ID NO.: 1 9 SEQUENCE LENGTH . 7 2 7 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : I inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE~DESCRIPTION
SEQUENCE ID NO.: 2 0 SEQUENCE LENGTH . 5 1 4 SEQUENCE TYPE : nucleic acid STRANDEDNESS
: double TOPOLOGY: 1 inear MOLECULETYPE : Genomic DNA
ORIGINALSOURCE : Homo sapiens IMMEDIATE lineCGMI
SOURCE
: human lymphoblastoid cell SEQUENCE. DESCRIPTION
ATG GTT TTC
GAG CTT GTT
TTT GCT ATT
GGG
CTG
AG
Met Glu Phe Gly Leu r Trp a Ile Se Val Phe Leu Val Al 1 5 .
i0 AAA GTGAGTGAAC
ACGAGTGAGA
Leu Lys ATTTGTGTGG CAG
CAGTTTCTGA GT
GTC
Gly Val TGT GAG GGA GTA
Gln Glu Val Gln Leu Val Ser Gly~GlyGly Val Arg Pro Cys Glu Val GGG TGT TCT ACC
Gly Ser Leu Arg Leu Ser Ala Ala Gly Phe Phe Asp Gly Cys Ser Thr TAT CGC CCA GGG
Asp Gly Met Ser Trp Val Gln Ala Gly Lys Leu Glu Tyr Arg Pro Gly GTC AAT AGC TAT
Trp Ser Gly Ile Asn Trp Gly Gly Thr Gly Ala Asp Val Asn Ser Tyr GTG ATC GAC AAG
Ser Lys Gly Arg Phe Thr Ser Arg Asn Ala Asn Ser Val Ile Asp Lys Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr CAC TGT GCG AGA GA CACAGTGAGG GGAAGCCAGT GAGAGCCCAG ACACAAACGT 505.
His Cys Ala Arg SEQUENCE ID N0. : 2 1 SEQUENCE LENGTH . 5 1 9 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 i near MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
AGGATTCACC ATG GAA CTG GGG CTC CGC TGG GTT TTC CTT GTT GCT AT'T 49 Met Glu Leu Gly Leu Arg Trp Val Phe Leu Val Ala Ile Leu Glu GAAACGGTGG ATGTGTGTGA CAGTTTCTGA CCAATGTCTC.TCTGTTTGCA G GT GTC 162 Gly Yal Gln Cys Glu Val Gln Leu Val Glu Ser Gly.Gly Gly Leu Val Lys Pro ~~~25'~'~
GlyGly SerLeuArg LeuSerCysAla AlaSerGlyPhe ThrPheSer 35 40 ~ 45 SerTyr SerMetAsn TrpValArgGln AlaProGlyLys GlyLeuGlu TrpVal SerSerIle SerSerSerSer SerTyrIleTyr TyrAlaAsp SerVal LysGlyArg PheThrIleSer Arg~AspAsnAla LysAsnSer CTGTAT CTGCAAATG AACAGCCTGAGA GCCGAGGACACG GCT~GTGTAT 450 LeuTyr LeuGlnMet ~AsnSerLeuArg AlaGluAspThr AlaValTyr GGAAGTCAGT
GTGAGCCCAG
TyrCys AlaArg GTCCC
SEQUENCE ID NO.: 2 2 SEQUENCE LENGTH . 6 0 6 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
2~.6~~'~'~
CACC ATG~GAG
TCA TGG CTG AGC
TGG GTT TTT
Met Glu Ser Trp Leu Ser Trp Val Phe TTA AAA G GTAATTCATT
GAGAACTATT GAAATTGAGT
Leu Ala Ala Ile Leu Lys 15 . .
GAAACAGTGG ATACGTGTGG
CAG TGT
GAG GTG
CAT CTG
GTG GAG
TCT GGG
GGA
Gly Val n Cys Glu Val Gl His Leu Val Glu Ser Gly Gly.
GGG CTC TCT
Ala Leu ValGln Pro Gly Ser Leu Arg Ser Cys Ala Ala Gly Leu Ser TAC GGG CCC
Gly Phe ThrPhe Ser Tyr Tyr Met Ser Val Arg Gln Ala Tyr Gly Pro 45 50 55 ~ 60 TGG AGA GGT
GIy Lys GlyLeu Glu Val Gly Phe Ile Asn Lys Ala Asn Trp Arg Gly GGG ACA ACAGAA TAG ACG TCT GTG AAA AGA TTC ACA ATC~TCA463 ACC GGC
~Gly Thr ThrGlu Thr Thr Ser Val Lys Arg Phe Thr Ile Gly Ser . 80 85 90 AGC CAA AAA
Arg Asp AspSer Lys Ile.Thr Tyr Leu Met Lys Ser Leu Ser Gln Lys ACC GAG GACACG GCC T.AT TAC TGT GA CACAGTGAGG 556 GTG TCC AGA
Thr Glu AspThr Ala Tyr Tyr Cys Ser Val Arg ACACAAACCT
CCCTGCAGGG
SEQUENCE ID NO.: 2 3 SEQUENCE LENGTH . 5 1 4 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : l inear MOLECULE TYPE : Genomic DNA
ORIGINAL. SOURCE : Homo sa iens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Lys GTAATTCATG GTGAATGGAT
AAGAGTGAGA
I Lys I
a ATACGTGTGG~CAGTTTCTGA CAG
CCAGGGTTTC GT
Gl y GTC CAGTGTGAG GTGCAGCTG TTGGAGTCTGGG~GGA GGCTTGGTA CAG 208 Val GlnCysGIu ValGlnLeu LeuGluSerGlyGly GlyLeuVal Gln Pro Gly.GlySer LeuArgLeu SerCysAlaAlaSer GlyPheThr Phe 35 ~ 40 . 45 Ser SerTyrAla MetSerTrp ValArgGlnAlaPro GlyLysGly Leu 50 55 ~ 60 Glu TrpValSer AlaIleSer GlySerGlyGlySer ThrTyrTyr Ala .
AspSer Va LysGly ArgPheThr~Ile SerArgAspAsn SerLysAsn l ThrLeu TyrLeuGln MetAsnSerLeu ArgAlaGluAsp ThrAlaVal . 100 105 110 GGAAGTCATT
GTGAGCCCAG
TyrTyr CysAlaLys SEQUENCE ID N0. : 2 4 SEQUENCE LENGTH . 6 0 0 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE : human lymphoblastoid cell tine CGMI
SEQUENCE DESCRIPTION
Met Asp Cys Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr r,..
Gly Thr His Ala Gln Val Gln CTG GTA CAG GGG GAGGTG.AAG AAGCCTGGGGCC TCAGTGAAG 266 TCT GCT
Leu Yal Gln Gly GluValLys LysProGlyAla SerValLys Ser Ala ~ 30 35 AAG TCC
Val Ser Cys Val GlyTyrThr LeuThrGluLeu SerMetHis Lys Ser CAG CCT
Trp Val Arg Ala GlyLysGly LeuGluTrpMet GlyGlyPhe Gln Pro 55 60 65. 70 GAT GAA
Asp Pro Glu Gly ThrIleTyr AlaGinLysPhe GlnGlyArg Asp Glu ACC GAC
Val Thr Met Glu ThrSerThr AspThrAlaTyr MetGluLeu Thr Asp 90 95 ~ 100 AGA GAG
Ser Ser Leu Ser AspThrAla ValTyrTyrCys AlaThr Arg Glu AAAC CCTGAGAGC TCAGAAACCC
GGAG GGATTTATG AG
SEQUENCE ID : 2 NO: 5 SEQUENCE LENGTH
. 6 5 5 SEQUENCE TYPE: nucleicacid STRANDEDNESS
: double TOPOLOGY :
1 inear MOLECULE TYPEGenomicDNA
:
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
GAGGAGCCCA GCCCCCGAAT TCCCAGGTGT TTTCATCTGG TGATCAGCAC CGAACA.CAGA 120 Trp Ser Leu Cys Ala Gly Phe Ser Leu Leu Leu Phe Asn AACAGTGGAT ACGTGTGGCA GTTTCTGACC GGGGTGTCTC TGTGTTTGCA G.GTA TCC 282 Yal Ser AGT GTG AGA.TGC AGC TGG TGG AGT CTG GGG GAG GCT TGC AAA AGC CTG 330 Ser Yal Arg Cys Ser Trp Trp Ser Leu Gly Glu Ala Cys Lys Ser Leu ArgGly ProArgAspSer ProValGlnPro LeuAsnSer ProSerVal AlaThr Thr ThrVal SerAlaArgLeu GlnGlyMet GlyTrpSer TrpPhe AspLysLeuIle LeuMetGlyVal AlaHisThr Ser Thr 65~ 70 75 ProVal ArgThrAspSer IleProProGlu IleThrPro ArgThrHis PheIIe ~CysLys Thr Ala LysPro ArgThrArg ProSerIle Ser Val Pro Glu SEQUENCE ID NO.: 2 6 SEQUENCE LENGTH . 5 4 6 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Asp Thr Leu Cys Tyr Thr Leu Leu Leu Leu 1 5 . 10 Thr Thr Pro Ser Trp Val Leu Ser Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu ACC CTC ACG CTG ACC TGC ACC GTC TCT GGG~TTC TCA CTC AGC AAT GCT 259 21625~'~
ThrLeu TheLeuThr CysThrValSer GlyPheSerLeu SerAsnAla 40 45 ~ 50 ArgMet GlyValSer TrpIIeArgGln ProProGlyLys AlaLeuGlu TrpLeu AlaHisIle PheSer~AsnAsp GluLysSerTyr SerThrSer LeuLys SerArgLeu ThrIleSerLys AspThrSerLys SerGlnVal ValLeu ThrMetThr AsnMetAspPro ValAspThrAla ThrTyrTyr CACAGAGACA ACAAGAACCT
CAGCCCAGGA
Cys Ala Arg Ile SEQUENCE ID NO.: 2 7 SEQUENCE LENGTH . 5 8 7 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : I inear MOLECULE TYPE : Genomic DNA
ORIGINAL.SOURCE : Homo sapiens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
~c,~~w~~~
Met Tyr Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly Val His Ser Gln Val Gln CTG GTG CAG~TCT GGG CCT GAG GTG AAG AAG CCT GGA GCC TCA TTG AAG 262 Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr~Ser Tyr Ala Ile Ser 40 45 ~ 50 TrpVal GlnAla HisGlyGlnGly LeuGluGluMet GlyTrpIle AsnThr AsnThrGly AsnLeuThrTyr AlaGlnGlyPhe ThrGlyArg PheVal PheSerMet AspThrSerVal SerMetAlaTyr LeuHisIle 90 ~ 95 100 SerSer LeuLysAla GluAspThrCys LysArg CCTGAGAGAA CCAGAAATCC CTGAGCTGAG GCAGTGACAG
TGAGGGAGGA
GGCAGCTGTG
SEQUENCE ID NO.: 2 8 SEQUENCE LENGTH
. 6 2 4 SEQUENCE TYPE : acid nucleic STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : DNA
Genomic ORIGINAL SOURCE
: Homo sapiens IMMEDIATE SOURCE: lymphoblastoid lineCGMI
human cell SEQUENCE DESCRIPTION
TACAGAGGCA CTTAGGCACC
TTCCTTAAAT GAAATACTTT
GTGCAAGAAC AAA CTG TTC
Met His Trp Phe Leu Lys Leu Phe GCT CCC T GTGAGTGTCT
Leu Leu Val Ala Arg Ala Pro CCAGGGCTCA AG
GG
Trp Val CAG CTG GAG GGC AAG
Leu Ser Gln Val Gln Ser ProGlyLeu Val Pro Gln Leu Glu Gly Lys TCC CTC TGC GTC ATC
Ser Asp Thr Leu Thr Ala SerGlyTyr Ser Ser Ser Leu Cys Val Ile AGT AGT AAC.TGG TGG CGG CCCGCAGGG AAG CTG 427 TGG GGC ATC CAG GGA
Ser Ser Asn Trp Trp Arg ProProGly Lys Leu -Trp Gly Ile Gln Gly 50 55 60 . 65 TAC ATC TAT GGG AAC
Glu Trp Ile Gly Tyr Ser SerThrTyr Tyr Pro Tyr Ile Tyr Gly Asn Ser Leu Lys Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln 85 90 . 95 Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Val Asp Thr Ala Val Tyr 100 ~ 105 110 TAC TGT~GCG AGA AA CACAGTGAGG GGAGGTGAGT GTGAGCCCAG ACACAAACC 624 Tyr Cys Ala Arg SEQUENCE ID N0. : 2 9 SEQUENCE LENGTH . 3 0 4 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo Sapiens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
SEQUENCE ID NO.: 3 0 SEQUENCE LENGTH . 5 1 2 SEQUENCETYPE ,: nucleic acid STRANDEDNESS
: double TOPOLOGY: 1 inear.
MOLECULETYPE : Genomic DNA .
ORIGINALSOURCE : Homo sapiens IMMEDIATE lineCGMI
SOURCE:
human.lymphoblastoid cell SEQUENCEDESCRIPTION
ATG CTC
GAG GTT
TTT GCT
GGG CTT
CTG TTA
AGC
TGG
GTT
TT
Met Glu Phe Gly Leu r Trp e Se Val Ph Leu Val Ala Leu Leu G GTGATTCATG GTGAGTGAA
GAGAAATAGA
GAGA
A rg G
Gly Val TGT GAG GGA GTC
Gln Gln Val Gln Leu Val Ser Gly Gly Val Gln Pro Cys Glu Gly Val AGG TGT TCT ACC
Gly Ser Leu Arg Leu.Ser Ala Ala Gly Phe Phe Ser Arg Cys Ser Thr TAT CGC CCA GGG
Ser Gly Met His Trp Val Gln Ala Gly Lys Leu Glu Tyr Arg Pro Gly TGG GCA GTT ATA TCA TAT GGA AGT AAA TAC GCA .GAC 353 GTG GAT AAT TAT
Trp Ala Val Ile Ser Tyr Gly Ser Ly5 Tyr Ala Asp Val Asp Asn Tyr GTG ATC GAC AAG
Ser Lys Gly Arg Phe Thr Ser Arg Asn Ser Asn Thr Val Ile Asp Lys Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg .115 CCCTGCAGG ~ 512 SEQUENCE ID NO.: 3 1 SEQUENCE LENGTH . 6 3 1 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : linear MOLECULE TYPE : Genomic DNA.
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Lys His Leu Trp Phe~Phe Leu Leu Leu Val Ala Ala Pro Arg Trp Val w LeuPro GlnValGln LeuGln GluSer GlyProGlyLeu ValLysPro SerGln ThrLeuSer LeuThr CysThr ValSerGlyGly SerIleSer SerGly GlyTyrTyr TrpSer TrpIle ArgGlnHisPro GIyLysGly 50 55 ~ 60 65 LeuGlu TrpIleGly TyrIle TyrTyr SerGlySerThr TyrTyrAsn ProSer LeuLysSer ArgVal ThrIle SerValAspThr SerLysAsn GlnPhe SerLeuLys LeuSer SerVal ThrAlaAlaAsp ThrAlaVal GGAGGTGAGT
GTGAGCCCAG
TyrTyr CysAlaArg SEQUENCE ID NO.: 3 2.
SEQUENCE LENGTH . 3 4 1 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens ~,.~-_86_ IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
CTCTGTATCT GCAAATGAAC ACTCAGAGAG CTGAGGACGT~GGCCGTGTAT GGCTATACAT I80 SEQUENCE ID NO.: 3 3 SEQUENCE LENGTH . 5 8 3 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE ~: Homo sapiens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val GCT CTT TTA AGA G GTGATTCATT GGAGAAATAG AGAGACTGAG.TGTGAGTGAA 105 Ala Leu Leu Arg Gly Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser.Cys Ala Ala Ser Gly Phe ,' 1~~~ ~ '~
35 40 45 .
ThrPheSerSer TyrGlyMet HisTrp ValArgGln AlaProGly Lys GlyLeuGluTrp ValAlaVal IleTrp TyrAspGly SerAsnLys Tyr TyrAlaAspSer AlaLysGly ArgPhe ThrIleSer ArgAspAsn Ser ThrAsnThrLeu PheLeuGln MetAsn SerLeuArg AlaGluAsp Thr ACAGTGAGGG
AlaValTyrTyr CysAlaArg CACAAACCTC CCTGCAGGAA G CAGGGGGGGC TCAGGAGCCA
CGCTGGCGG AAATCAGCTG
CTGATCAGAG TCAGCCCT ~ 583 SEQUENCE ID NO.: 3 4 SEQUENCE LENGTH .: 6 8 7 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : l inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
CAAATCCTCA CTTAGGCACC CACAGGAAAT GACTACACAT ~TTCCTTAAAT TCAGGGTCCA 120 Met Lys His Leu Trp Phe.Phe Leu Leu Leu Yal Ala Ala Pro Arg Trp Val Leu Ser Gln Val Gln Leu Gln Gln Trp Gly Ala GGA CTG TTG AAG CCT TCG GAG ACC CTG TCC CTC ACC TGC GCT.GTC TAT 385 Gly Leu Leu Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val~Tyr C
GlyGly SerPheSer GlyTyrTyrTrp SerTrpIleArg GlnProPro GlyLys GlyLeuG1-uTrpIleCly~GluIleAsnHisSer GlySerThr G
AsnTyr AsnProSer LeuLysSerArg ValThrIleSer ValAspThr SerLys AsnGlnPhe SerLeuLysLeu SerSerValThr AlaAlaAsp CACAGTGAGG
GGAGGTGAGT
ThrAla ValTyrTyr CysAlaArg ~~~~~'~~
GTGAGCCCAG ACAAAAACCT CCCTGCAGGT AGGCAGAGGG GGCGGGCGCA GGTACTGCTC. 683 SEQUENCE ID NO.: 3 5 SEQUENCE LENGTH . 7 0 0 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : I inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Glu.Phe Gly Leu Ser Trp Val Phe Leu. Ala Ala Ile Leu Lys Gly Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala.Ser Gly Phe Thr '..-i ~A
PheSer AsnSerAsp MetAsnTrpVal GlnAlaPro LysGly His Gly 50 ~ 55 60 CTGGAG TGGGTA.TCG GGTGTTAGTTGG GGCAGTAGG CACTAT 477 AAT ACG
LeuGlu TrpValSer GlyValSerTrp GlySerArg HisTyr Asn Thr GCAGAC TCTGTGAAG GGCCGATTCATC TCC.AGAGAC TCCAGG 525 ATC AAT
AlaAsp SerValLys GlyArgPheIle SerArgAsp SerArg Ile Asn CTG GAC
AsnThr LeuTyrLeu GlnThrAsnSer ArgAlaGlu ThrAla Leu Asp CACTGTGAGA
GGTCGGAAGT
GTGAGC
ValTyr TyrCysVal Arg ACACAAACCT CCTGCAGGAA AGGGGGCGCT CAGGACCCAC
CGTTGGGGGA
AATCAGCTGC
SEQUENCE ID NO.: 3 6 SEQUENCE LENGTH . 8 0 6 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
CCCCAGAGCT TGCTATAGAG GAGGAGACAT CCACATAGGG.CCCTCNCTTG TCCTGATGAA 120 ...
ATTTGGTGGT CAGGTCTCTG AACACAGAGG ACTCACT ATG GAG TTT GGG CTG AGC . 235 Met Glu Phe Gly Leu Ser 1 5 ~
GAGAACTAGA
TrpGly PheHisVal AlaAsnVal Lys GATATTGAGT GAAACAGTGG ATATGTGTGG
GTGAGTGGAC CAGGT
ACAAGTGAGA
CCAGGGTGTC GT GAG TG 394.
TGTGTGTGTT GTC GTG ~
TGCAG CAG CAC
TGT CTG
G
Gly Glu al Val Val Gln His Cys Leu V
GGT
GluSer LeuGlyGly LeuLeu ProGly Pro AspPhe Leu Gly TTA
LeuGln ProLeuAsp SerProLeu ValPro LeuGly ThrGly Leu 40 ~ 45 50 TGG
AlaGly SerIleArg LeuLeuGly LysGly SerArg SerHis Leu Trp CAG
Val VatVal ValAlaGln AlaMet ThrLeu Arg Val Gln AAT
AspSer ProSer.ProGluMetMet ProArg HisCys IleCys Lys Asn TGT
Thr Ala Ser Glu Pro Arg Ile Gly Leu Cys Ile Thr Val Val a'~r TCCTGTTTTG GGGACACCAA TTTTGGAGTT TGCAGTATCC TTGAGTCCAG TACGTTCATG. 800 GTGGCA ~ 806 SEQUENCE ID NO.: 3 7 SEQUENCE LENGTH . 5 0 0 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met.Leu Phe Gly Leu Ser Trp Pro Phe Arg Phe Thr Ile Leu AGG G GTGACACGTG AAGCACTACA GATATTGCTC'GTGAGTGGAT ATTAGAGAAA 105 Arg Gly Val Gln Tyr Glu Yal Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Leu Trp Trp Val Leu Arg Leu Ser Cys Ala Ala Cys Gly Phe Ile Leu Arg Ser Asn Trp Ser His Arg Ala Ser Arg Lys Gly Leu Ala Trp Asn Asp Met Val Ser Tyr Ile Ser Ala Ser Gly Gly Ser Leu Tyr Tyr Ala Asp Thr Glu Gly Ile His His Leu Arg Gln Trp Gln Glu His Ala Val 85 90 ~ 95 Leu Ala Asn Glu Gln Ser Glu Arg Gly Leu Gly Cys Val Glu Arg 100 105 ' 110 SEQUENCE ID NO.: 3 8 SEQUENCE LENGTH . 5 0 7 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION ' ' Met Gln Phe Val Leu Ser Trp Val Phe Leu Val Gly Ile Leu Lys Gly Val .._ _g4_ Gln Cys. Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Arg.Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn Glu Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Arg AAG GGC AGA TTC ACC ATC TCC AGA~GAC AAT TCC AAG AAC ACG CTG.TAT 402 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Gly Thr Ala Ala Tyr Tyr Cys Ala Arg _ SEQUENCE ID NO.: 3 9 SEQUENCE LENGTH . 8 0 0 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Lys His Leu Trp Phe Phe Leu Leu Leu Vat Ala Ala Pro Arg 10~ 15 Trp Bal Leu Ser Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val. Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Ser Ile.Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg SEQUENCE ID NO.: 4 0 SEQUENCE LENGTH . 9 7 0 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : I inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
CACAACCTCC ATGAAAAACA ACATAGAAAT TTCTCAAAGA.ACTAAAATTA GAATTACCAT 60 GATGGGATGG ATCATCACCT ACACTGGGAA CCCAACATAT ACCAACGGCT TCACAGGACG . 360 CTAGATACAT .GTGAGCCCAT TTCCTGGTCT TTGCTTAACT GACAAGCTCT CATCAGTGCA 780 SEQUENCE ID N0. : 4 1 SEQUENCE LENGTH . 8 1 9 SEQUENCE~TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : linear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
GATTCACC ATG GAG TCA TGG CTG AGC TGG GTT TTT CTT GCC GCT~ATT TTA 230 Met Glu Ser Trp Leu Ser Trp Val Phe Leu Ala Ala Ile Leu 1 5 ~ ~ 10 Lys Gly Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Yal Gln Pro w ~ ~ 6 2 5'~'~
GlyGlySer LeuArgLeu SerCysAla AlaSerGlyPhe SerPheSer SerTyrGly MetSerTrp ValArgGln AlaProGlyLys GlyLeuGlu 50 ~ 55 60 65 ValAla HisIleTrp AsnAspGly SerGlnLysTyr TyrAlaAsp 70 75 . . 80 SerValLys~GlyArgPhe ThrIleSer GluThrIleLeu ArgAlaCys 85 90 ~ 95 Ser Ile Cys Lys Trp Thr Val Lys Leu Arg Thr Arg Pro Cys Ile Thr Val Pro SEQUENCE ID N0. : 4 2 SEQUENCE L$NGTH . 4 7 1 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE :Homo sapiens ~~~~w~~~
IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
SEQUENCE ID N0. : 4 3 SEQUENCE LENGTH . 8 7 0 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo s_apiens IMMEDIATE~SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ana Ile Leu Lys .,_ Gly Val Gln Cys Glu Val Gln LeuVal GluSerGly GlyVaIVal ValGinProGlyGly SerLeuArg LeuSer CysAlaAla SerGlyPhe ThrPheAspAspTyr ThrMetHis T Val ArgGlnAla ProGlyLys GlyLeuGluTrpYal SerLeuIle rp SerTrp AspGlyGly SerThrTyr TyrAlaAspSerVal LysGlyArg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Lys Asp GCCTGTGTCA CCT ~ 870 SEQUENCE ID N0. : 4 4 SEQUENCE LENGTH . 5 2 9 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE : human lymphoblastoidline CGMI
cell SEQUENCE DESCRIPTION
TCCCAGGTGT
GGG CTG AGC TGG GCT TTC CTT GTT
Met Leu Phe Gly Leu Ser Trp Ala Phe Leu Val GATATTGTTT GTGAGTGGAT
Thr Ile Leu Arg TGCTGACCAG
GAG TCT TTT
Gly Val Gln Tyr Glu Val Gln Leu Val Phe Phe Phe Phe Glu Ser Phe TTA CAA GCT
Phe His Phe Leu Ala Asn Ile His Gly Asn Asn Gly Leu Leu Gln Ala CAA TTT TAA
Phe Leu Pro Thr Leu Tyr Arg His His Ser Pro Cys Leu Gln Phe CAT CTT AGG
.._ Gly Phe Pro Glu Glu Cys Cys His His Leu Ser Cys Ser Phe Arg Lys Asn Ala Pro Ser Thr His Leu His Leu Ser Ala Cys Ile Ser Ile Cys Leu Gly Arg Ser Gln Gln Pro Xaa Glu His Ser Pro His Pro Thr ATG CTG CTC GAG GGG GTG ~ 529 Met Leu Leu Glu Gly Val 110 ~ 115 SEQUENCE ID NO.: 4 5 SEQUENCE LENGTH . 7 4 8 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo Sapiens .
IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
CAGGATCAGG GCTTGAGTCA TCAGCATCTC ACTCTTGCAA~AGNCTGATGT GTCGTTTGTC 60 Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr ~~ ~~~'~7 Asp Ala Tyr.Ser Gln Met Gln Leu Val Gln Ser Gly GCTGAG GTG.AAG ACTGGGTCCTCA GTG GTTTCC TGCAAGGCT 446 AAG AAG
AlaGlu ValLysLys ThrGlySerSer ValLysYalSer CysLysAla 30 35 40 .
SerGly TyrThrPhe ThrTyrArgTyr LeuHisTrpVal ArgGlnAla ProGly GlnAlaLeu GluTrpMetGly TrpIleThePro PheAsnGly AsnThr AsnTyrAla GlnLysPheGln AspArgValThr IleThrArg GACAGG TCTATGAGC ACAGCCTACATG~GAGCTGAGCAGC CTGAGATCT 638 AspArg SerMetSer ThrAlaTyrMet GluLeuSerSer LeuArgSer CACAGTGTGA
AAACCCACAT
Glu~Asp ThrAlaMet TyrTyrCysAla Arg TCAGAAACCC GGTTACAG
CAAGGAGGAG
SEQUENCE ID NO:: 4 6 SEQUENCE LENGTH . 7 9 9 SEQUENCE TYPE : nucleic acid .._ STRANDEDNESS : double TOPOLOGY : linear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo Sapiens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
ACC ATG GAC TGG ACC TGG~AGG GTC TTC~TGC~TTG CTG GCT GTA GCT CCA G 289 Met Asp Trp Thr Trp Arg Val Phe Cys Leu Leu Ala Val Ala Pro Gly Ala His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met .... r ~;
ACC AGG GAC ACG ACG AGC GTC TAC GAG CTG AGC AGC CTG
A
Thr Arg Asp Thr Thr Ser Val Tyr Glu Leu Ser Ser Ser Thr Met eu L
ACG TAT GCG
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg lI0 115 SEQUENCE ID NO.: 4 7 SEQUENCE LENGTH . 6 2 7 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Arg Glu Phe Val Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Cys Val 2~s~~~~r GlnCys GluAspGlnLeu ValGluSer GlyGlyGlyLeu ValGlnPro GlyGly SerLeuA.rgPro SerCysAla AlaSerGlyPhe AlaPheSer AGC~TAT GTTCTGCACTGG GTTCGCCGG GCTCCAGGGAAG GGTCCGGAG 483 SerTyr ValLeu~HisTrp ValArgArg AlaProGlyLys GlyProGlu TGGGTA TCAGCTATTGGT ACTGGTGGT GATACATACTAT GCAGACTCC .531 TrpVal SerAlaIleGly ThrGlyGly AspThrTyrTyr AlaAspSer Val Met Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr Leu Lys Thr Ala Leu Arg Thr Trp Leu Cys Ile Met SEQUENCE ID NO.: 4 8 SEQUENCE LENGTH .- 7 4 3 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Glu Leu Gly Leu Cys Trp Val Phe Leu Val Ala Ile Leu Glu GAGATTTAGT
GTGTGTGGAT
CCTTGGTGT TCTTTGTTTG
Gly ValGlnCys Glu ValGlnLeu ValGluSerGlyGly GlyLeuYalGln ProGlyGiy Ser LeuArgLeu SerCysAlaAlaSer GlyPheThrPhe SerSerTyr Ser MetAsnTrp ValArgGlnAlaPro GlyLysGlyLeu GluTrpVal Ser TyrIleSer SerSerSerSerThr IleTyrTyrAla AspSerVal Lys GlyArgPhe ThrIleSerArgAsp AsnAlaLysAsn SerLeuTyr Leu GlnMetAsn SerLeuArgAlaGlu AspThrAlaVal TyrTyrCys CACAGTGAGG CCCTGCAGGG
GGAGGTCAGT
GTGACAC
Ala Arg SEQUENCE ID NO.: 4 9 SEQUENCE LENGTH . 7 6 3 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 i near MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
GAGTTTCCAC TTGGTGATCA GCACTGAACA CAGACCACCA~ACC ATG GAG TTT GGG 235 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly.Leu Val Gln Pro Gly Arg Ser Leu Arg ~ ~. ~ 2 5'~'~
LeuSer CysThrAla SerGlyPheThr PheGlyAspTyr AlaMetSer TrpPhe ArgGlnAla ProGlyLysGly LeuGluTrpVal GlyPheIle ArgSer LysAlaTyr GlyGlyThrThr GluTyrThrAla SerValLys Gly~Arg PheThrIle SerArgAspGly SerLysSerIle .AlaTyrLeu GlnMet AsnSerLeu LysThrGluAsp ThrAlaValTyr TyrCysThr CACAGTGNGG CCCTGCAGGC
GGAGGTCAAT
GTGAGCCCAG
A
rg CCACCAGGGG
GCGCTAGG
SEQUENCE ID NO.: 5 0 SEQUENCE LENGTH . 2 8 3 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Glu Leu Gly Leu Ser Trp Val Ser Leu Val Ile Ile ..._ 1 ~ 5 10 Leu Lys AAACAGTGAT TCTGTGTGGC AGGTTCTGAC~TCAGATGTCC TCTGTGCTTG TAG GT GTC 163 Gly Val Cys Gly Val Gln Met Val Glu Ser Trp Gly Glu Leu Ala Gln Xaa Glu Cys Ala Asp Ser Ala Val His Pro Leu Asn Pro~Pro Ser Val GCT ACT AGA TCA GCT GAA TCT GCC ~ 283 Ala Thr Arg Ser Ala Glu Ser Ala SEQUENCE ID NO.: 5 1 SEQUENCE LENGTH . 7 0 0 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Gly Ser ..._ ~1~~ ~~
ACC GCCATC GCCCTC~CTC GCTGTTCTC CAAG GTCAGTCCTG 223 CTC CTG
Thr AlaIle AlaLeuLeu AlaValLeu Gln Leu Leu A GTGGAAAGGA CAAATTTTGT
G T GAG
GTG
CAG
CTG
GTG
CAG
TCT
GGA
GCA
G ly al ys Ser lu ln V C G Val Leu G Val Gln Ser Gly Ala TCT TCT
Glu ValLysLys ProGlyGlu LeuLysIle SerCys Lys Gly Ser Ser TGG CCC
Gly TyrSerPhe ThrSerTyr IleGlyTrp ValArg Gln Met Trp Pro GGG GAT
Gly LysGlyLeu_GluTrpMet IleIleTyr ProGly Asp Ser Gly Asp ACC AGATACAGC CCGTCCTTC.CAAGGCCAGGTC ACCATC TCA GCC 526 GAC
Thr ArgTyrSer ProSerPhe GlyGlnYal ThrIl~e Ser Ala Gln Asp 0 $5 90 CTG TCG
Lys SerIleSer ThrAlaTyr GlnTrpSer SerLeu Lys Ala Leu Ser GCG CACAGTGAG
Asp ThrAlaMet TyrTyrCys Arg Ala CTAAAACCCT AGACTCACTC
CCACACCGCA
TCTCT
-." , SEQUENCE ID NO.: 5 2 SEQUENCE LENGTH . 7 6 7 SEQUENCE TYPE : nucleic acid STRAND):DNESS : doub 1 a TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens .
IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Glu Ser Gly Leu Ser Tcp Val Phe Leu Val Ala Ile Leu Lys Gly Val Gln Cys Glu Val Gln Leu Val Glu Ser GGG TGA GGC TTG GTA CAG CCT GGA GGG TCC CTG AGA CTC~TCC TGT GCA 435 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala . 35 40 Ala Ser Gly Phe The Phe Ser Ser Ser Trp Met His Trp Val Cys Gln GCT CCG GAG~AAG GGG CTG GAG TGG GTG GCC GAC ATA AAG TGT GAC GGA 531 -..
AlaPro GluLys Leu GluTrpValAla AspIleLysCys AspGly Gly AGTGAG AAATAC GTA.GACTCTGTGAAG GGCCGATTGACC ATCTCC 579 TAT
SerGlu LysTyr Val AspSerValLys GlyArgLeuThr IleSer Tyr AAG
ArgAsp AsnAla Asn SerLeuTyrLeu GlnValAsnSer LeuArg Lys ACC CACAGTGAGG
AlaGlu AspMet Val TyrTyrCysVal Arg Thr G ACACAAACCT CACAA
A ACAGGGGCAG
C
SEQUENC$ ID NO.: 5 3 SEQUENCE LENGTH . 7 2 4 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Glu Phe Trp Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys CCAATGTCTC.TGTGTTTGCA G ~GT GTC CAG TGT GAG GTG CAC CTG GTG GAG 213 Gly Val GIn Cys Glu Val Gln Leu Val Glu GGG
SerGlyGlyGly LeuIleG~InProGlyGlySerLeu Ai-gLeuSer Cys 30 35 ~ 40 AlaAlaSerGly PheThrVal SerSerAsnTyrMet SerTrpYal Arg GlnAlaProGly LysGlyLeu GluTrpYalSerVal IleTyrSer Gly GlySerThrTyr TyrAlaAsp SerValLysGlyArg PheThrIle Ser 75 80 g5 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg GCC GAG GAC ACG GCC GTG.TAT TAC TGT GCG AGA GA CACAGTGAGG 498 Ala Glu Asp Thr Ala Val Tyr Tyr Cys A~la Arg 110 . 115 SEQUENCE ID NO.: 5 4 SEQUENCE LENGTH . 7 0 6 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
CCCCATAGCT.CTGGGAGAGA AGCGCCAGCC CTGGGATTCC CAGGGGTTTC CATTTGGTGA 120 TCAGGACTAA AGACAGAGGA CCCACC ATG GAG CTT GGG CTG AGC TGG GTT TTC 173 .
Met Glu Leu .Gly Leu Ser Trp Val Phe .1 5 Thr Val Thr Vat Leu Lys Gly Val Gln Cys Glu Val Gln Leu Val Glu Ser Glu Glu Asn Gln Arg Gln Leu Gly Gly Ser Leu Arg Leu Ser Cys Ala Asp Ser Gly Leu Thr Phe Ser Ser Tyr Met Ser Ser Asp Ser Gln Ala Pro Gly Lys Gly Leu Glu Val Val Asp Ile Asp Arg Ser Gin Leu Cys Tyr Ala Gln Ser.Yal Lys Ser Arg Phe Thr Ile Ser Lys Glu Asn Ala AAG AAC TCA CTC TGT TTG CAA ATG~AAC AGT CTG AGA GCA GAG GGC ACG 569 Lys Asn Ser Leu Cys Leu Gln Met Asn Ser Leu Arg Ala Glu Gly Thr 95 . 100 ~ 105 GCC GTG.TAT TAC TGT ATG TGA GT CACCAGGTAA GAAGACATCA GTGTGATCAC 622 Ala Val Tyr Tyr Cys Met SEQUENCE ID NO:: 5 5 SEQUENCE LENGTH . 8 0 0 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Lys His I
Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg ,.
Trp Val Leu Ser Gln Val Gln Leu Gln Glu Ser Gly Pro GGA CTGGTG CCTTCGGAG.ACCCTGTCC.CTC GCT GTC 444 AAG ATC TCT
TGC
Gly LeuValLys ProSerGlu LeuSerLeu Ile Ala ValSer Thr Cys AAG GTC
Gly AspSerIle SerSerGly Trp Ile Trp Arg GlnPro Asn Val ATT CAT
Pro GlyLysGly LeuGluTrp GlyGluIle His Ser GlySer Ile His AAG ATG
Thr TyrTyrAsn ProSerLeu SerArgIle Thr Ser ValAsp Lys Met CTG GTG
Thr SerLysAsn GlnPheTyr LysLeuSer Ser Thr AlaAla Leu Val GAC ACGGCCGTG TATTACTGT AGATACACA~GTG GGAGGTGAGT 685 GCG AGG
Asp ThrAtaVal TyrTyrCys Arg Thr Val Ala Tyr Arg GTGAGCCCAG CACAAACCT AGGCAGAGGG GGNGGGCACA
A CCCTACAGAT GGTGCTGCTC
GGGGGCGCGC AGCAG
GANGNCACAG
SEQUENCE ID NO.: 5 6 SEQUENCE LENGTH : 4 2 9 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double -,.
TOPOLOGY : linear MOLECULE TYPE :. Genomic DNA
ORIGINAL SOURCE : Homo sa iens , IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Asp Trp Thr Trp Ser Ile Leu Phe Leu Val Ala Ala Ala Thr Gly Ala His Ser Arg Val Gln Leu Val Gln Ser Giy Pro Glu Val Lys Gln Pro Gly Ala Ser Ala Lys Val Ser Cys Lys Val Ser Gly Thr Val Ile Thr Tyr Gly ATG AAT TGG ATA CGA. CAG ACC CCA GGA CAG GGG CTT GAG TGG ATG GGA 422 Met Asn Trp Ile Arg Gln Thr Pro Gly Gln Gly Leu Glu Trp Met Gly Trp Ile SEQUENCE ID N0. : 5 7 SEQUENCE LENGTH . 4 6 2 ~~~2~'~'~
-lI9-SEQUENCE TYPE : nucleic acid STRANDEDNESS :double TOPOLOGY : I inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
GGTGTGGGTG GAAACAGTGA.GTAGTCAAGT GGGAGTTCTC AGAGTTACTC TCCATGAGTA 360 ACCTGAAAGT CCAAGGACAA GGCTGTGTAT TACTGTGAGG GA ~ 462 SEQUENCE ID NO.: 5 8 SEQUENCE LENGTH . 6 2 9 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sapiens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Asp Trp Ile Trp Arg Ile Leu ._ GCAAGGAGAT
GCCAAGTCCC
Phe LeuValGly AlaAlaIle GTCCAGTCAA GT
GGTGGCTTTC
ATCCACTCCT
Gly Ala HisSerGln MetGlnLeu ValGlnSerGlyProGlu ValLysLys Pro GlyThrSer ValLysVaI .SerCysLysAlaSerGly PheThrPhe Thr SerSerAla ValGlnTrp ValArgGlnAlaArgGly GlnArgLeu Glu TrpIleGly TrpIleVal ValGlySerGlyAsnThr AsnTyrAla Gln LysPheGln GluArg.ValThrIleThrArgAspMet SerThrSer Thr AlaTyrMet GluLeuSer SerLeuArgSerGluAsp ThrAlaVal CACAGTGTGA
AAACCCACAT
Tyr Tyr~CysAla Ala C
2 ~. 62 ~? ?
SEQUENCE ID NO.: 5 9 SEQUENCE LENGTH . 6 2 2 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa i.ens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE.DESCRIPTION
GCAAATCCTC
AGCTCACATG
CAT CTG TGG
Met Lys His Leu Trp Phe Phe 1 ~ 5 CAGGGATCCA
Leu Leu Leu Val Ala Ala Pro Arg CTGTGGGTCT
TCG GGC CCA G GTG
Trp Val Leu Ser Gln Val Gln Leu Gln Glu Ser ly Leu Gly Pro G Val GTC TCT GGT
Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Gly Ser Val Ser Gly CCC CCA GGG
Val Ser Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Lys Gly Pro Pro Gly AGC ACC AAC
Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Tyr Asn Ser Thr Asn __ ~2~~2~'~'~
70 , 75 CCCTCCCTC. AAG CGA GTC ATA GTA GAC ACG AAG AAC 518 AGT ACC TCA TCC
ProSerLeu Lys Arg Val Ile Val Asp Thr Lys Asn Ser Thr Ser Ser CAGTTC.TCC CTG CTG AGC GTG GCT GCG GAC GCC GTG 566.
AAG TCT ACC ACG
GlnPheSer Leu Leu Ser Val Ala Ala Asp Ala Val Lys Ser Thr Thr TATTACTGT GCG GA CACAGTGAGG GTGAGCCCAGACAAAAACC
TyrTyrCys Ala Arg SEQUENCE ID NO.: 6 0 SEQUENCE LENGTH . 5 8 8 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : linear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE: human lymphoblastoidline CGMI
cell SEQUENCE DESCRIPTION
ATGAACACTG
TTC CTT GTT G
Met Glu Ser Gly Leu Ser Trp Ile Ala Val Leu Lys Phe Leu Val TGACTGGACA
CCGTGTTTGC TGA
Gly Cys Pro Val GGT GCA GCT GGT GGA GTC TGG GGG .AAA GAC TGG GGG 266 AGG CTT AGT GTC
Gly Ala Ala Gly Gly Val Trp Gly Lys Asp Trp Gly Arg Leu Ser Val CTC ATT
Ser GluThrLeu LeuCysSerLeu Trp HisLeu Gln Leu Cys Ile AAA
Tyr AlaLeuGly ProProGlySer Arg Gly.PheGlyValGly Leu Lys AGT TATTAGTAC AAGTGGTGATAC CGT CTACAC AGACTC.TGT GAA 410 ' ACT
Ser Tyr Tyr LysTrp Tyr Arg LeuHis ArgLeuCys Glu .
Thr TGC
Gly LeuIleHis HisLeu Arg Gln ProGlu PheThrVal Ser Cys AAT CAT
Ala GluGln ProGluSerrg Arg GlyCys ValLeuLeu Cys Asn A His Glu Arg TGGGACAACC AGGGAAAGCC TGGGAC 5gg S EQUENCE I D N0. : 6 1 SEQUENCE LENGTH . 1 2 1 2 SEQUENCE TYPE : nucleic acid S TRANDEDNESS : doub 1 a TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
~~.~5'~
CCTCCTTTTT CACCTCTCCG TACAAAGGCA CC ~ CACAT GCAAATCCTT ACTTAAGCAC 60 TCTGAGAGCC TGGACCTCCT GTGCAAGAAC ATG.AAA CAC CTG TGG TTC TTC CTC 174 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg TATGGGAGGT GCCTCTGATC CCAGGGC'fCA CTGTGGGTCT CTCTGTTCAC AG GG GTC 283 Trp Val Leu Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro 25 sn SerGlu ThrLeuSer LeuThrCysThr ValSerGly GlySerVal Ser SerGly SerTyrTyr TrpSerTrpIle ArgGlnPro ProGlyLys Gly CTGGAG TGGATTGGG TATATCTATTAC AGTGGG~AGC ACCAACTAC AAC 475 LeuGlu TrpIleGly TyrIleTyrTyr SerGlySer ThrAsnTyr Asn CCCTCC CTCAAGAGT CGAGTCACCATA TCA.GTAGAC ACGTCCAAG AAC 523 ProSer LeuLysSer ArgYalThrIle SerYalAsp ThrSerLys Asn .
. 85 90 95 GlnPhe SerLeuLys LeuSerSerVal ThrAlaAla AspThrla Val A
CACAGTGAGG
GGAGGTGAGT
Tyr Tyr Cys Ala Arg TATCATCAAC TGAATTGTAC CCTCTTTGAA ATTCATATGA,TGAAACCTTA AATTCAATGG 1038 SEQUENCE ID NO.: 6 2 SEQUENCE LENGTH . 5 6 0 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE : human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Asn Leu Arg ~~.62~'~~'~
Gly Yal Gln Cys Glu Val Gln Leu VaI Glu Ser Gly Glu Gly LeuVal.GlnProGlyGly SerLeuArgLeu SerCysAla AlaSer Gly PheThrPhe SerSerSer AlaMetHisTrp ValArgGln AlaPro Arg LysGlyLeu TrpVal SerValIleSer ThrSerGly AspThr Val LeuTyrThr AspSerVal LysGlyArgPhe ThrIleSer ArgAsp g5 90 Asn AlaGlnAsn SerLeuSer LeuGlnMetAsn SerLeuArg AlaGlu 95 100 105 .
CGCAG AG
Gly ThrValVal TyrTyrCys ValLys CAAACCTCCT TGGGAC
GCAGGGTACC
SEQUENCE ID NO.: 6 3 SEQUENCE LENGTH . 5 1 5 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : linear MOLECULE TYPE': Genomic DNA
2~~~ ~ '~
ORIGINAL SOURCE : Homo sa iens IMMEDIATE SOURCE: human lymphoblastoid cell line CGMI
SEQUENCE DESCRIPTION
CTT GTT TTA
Met Gly Phe Glu Leu Thr Arg Ile Phe Leu Val AlaIle Leu ATAAGTGAGA
Lys TGTGTATTTT GTT
Gly Val CAG TGT GAG GTG GAG CTG ATA,GAG TCC ATA GAG 211 GGC CTG AGA CAA CTT
Gln Cys Glu Val Glu Leu Ile Glu Ser Ile Glu Gly Leu Arg Gln Leu GGG AAG TTC C1'G AGA CTC TCC TGT GTA GCC TCT TTCAGT 259 GGA TTC ACC
Gly Lys Phe Leu Arg Leu Ser Cys Val Ala Ser PheSer Gly Phe Thr 35 40 . 45 GGG AAG GGG
Ser Tyr Met Ser Trp Val~Asn Glu Thr Leu Gly LeuGlu Lys Gly 50: 55 60 ATA TAC CAT
Gly Val Ile Asp Yal Lys Tyr Asp Gly Ser Gln AlaAsp Ile Tyr His AAT GCT AAG
Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp AsnSer Asn Ala Lys CCG TAT CTC CAA ACG AAC AGT CTG AGA GCT GAG ATGCAT
Pro Tyr Leu Gln Thr Asn Ser Leu Arg Ala Glu MetHis Asp Met Thr ,_ GGC TGT ACA TAA GG ~TTCCAAGTGA GGAAA1~ CG.GTGTGAGTCC AGACCAAAAT 505 Gly Cys Thr SEQUENCE ID NO.: 6 4 SEQUENCE LENGTH . 6 4 9 SEQUENCE TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : 1 inear MOLECULE TYPE : Genomic DNA
ORIGINAL SOURCE : Homo Sapiens IMMEDIATE SOURCE: human lymphoblastoidline CGMI
cell SEQUENCE DESCRIPTION
AGCT~CTGGGA GAGGAGCCCC CCCCCTGGGA TTTTCATTTGGTGATCAGCA60 TTCCCAGGTG
GGG CTG GTT
Met Thr Glu Phe Gly Leu Ser Trp Phe Leu Val AGGAAATAGA
Val Ala Ile Phe Lys ' CAGTTTCTGA
CTG GTG GAG GGA
G1y Va1 Gln Cys Glu Val Gln Leu Ser Gly Gly Leu Val Glu Gly CTC TCC TGT TCT
Val Gln Pro Gly Gly Ser Leu Arg Ala Ala Gly Phe Leu Ser Cys Ser 35 ~ 40 45 TGG GTC CGC CCA
Thr Phe Ser Ser.Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val Ser Ala Ile Ser Ser Asn Gly Gly Ser Thr Tyr TAT GCA AAC TCT.GTG AAG GGC AGA TTC ACC ATC TCC AGA GAC AAT TCC 465 Tyr Ala Asn Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Gly Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys Ala Arg CTGATCAGCG TCAACCGCAG GGG
Claims (10)
1.. An isolated polynucleotide comprising the following nucleic acid sequences of (a) through (f) in the 5' to 3' order:
(a) a nucleic acid sequence of a portion of a human genome inserted in:
(i) yeast artificial chromosome clone Y6 which is isolable from a transformant identified by the international deposit number FERM BP-4271; or (ii) yeast artificial chromosome clone Y24 which is isolable from a transformant identified by the international deposit number FERM BP-4274;
(b) a nucleic acid sequence of a portion of a human genome inserted in yeast artificial chromosome clone Y21 which is isolable from a transformant identified by the international deposit number FERM BP-4273, wherein the nucleic acid sequence lacks the 5' terminal sequence of the portion of a human genome so isolable from the clone Y21 that duplicates the 3' terminal sequence of the nucleic acid sequence (a);
(c) a nucleic acid sequence of a portion of a human genome inserted in cosmid vector clone M118 which is isolable from a transformant identified by the international deposit number FERM BP-4278, wherein the nucleic acid sequence lacks the 5' terminal sequence of the portion of a human genome so isolable from the clone M118 that duplicates the 3' terminal sequence of the nucleic acid sequence (b);
(d) a nucleic acid sequence of a portion of a human genome inserted in cosmid vector clone M84 which is isolable from a transformant identified by the international deposit number FERM BP-4277, wherein the nucleic acid sequence lacks the 5' terminal sequence of the portion of a human genome so isolable from the clone M84 that duplicates the 3' terminal sequence of the nucleic acid sequence (c);
(e) a nucleic acid sequence of a portion of a human genome inserted in cosmid vector clone M131 which is isolable from a transformant identified by the international deposit number FERM BP-4279, wherein the nucleic acid sequence lacks the 5' terminal sequence of the portion of a human genome so isolable from the clone M131 that duplicates the 3' terminal sequence of the nucleic acid sequence (d); and (f) a nucleic acid sequence of a portion of a human genome inserted in cosmid vector clone 3-31 which is isolable from a transformant identified by the international deposit number FERM BP-4276, wherein nucleic acid sequence lacks the 5' terminal sequence of the portion of a human genome so isolable from the clone 3-31 that duplicates the 3' terminal sequence of the nucleic acid sequence (e), wherein each of the portions of a human genome inserted in the clone Y6, Y24, Y21, M118, M84, M131 and 3-31 respectively is in a relative position in a human genome as shown in Figure 1.
(a) a nucleic acid sequence of a portion of a human genome inserted in:
(i) yeast artificial chromosome clone Y6 which is isolable from a transformant identified by the international deposit number FERM BP-4271; or (ii) yeast artificial chromosome clone Y24 which is isolable from a transformant identified by the international deposit number FERM BP-4274;
(b) a nucleic acid sequence of a portion of a human genome inserted in yeast artificial chromosome clone Y21 which is isolable from a transformant identified by the international deposit number FERM BP-4273, wherein the nucleic acid sequence lacks the 5' terminal sequence of the portion of a human genome so isolable from the clone Y21 that duplicates the 3' terminal sequence of the nucleic acid sequence (a);
(c) a nucleic acid sequence of a portion of a human genome inserted in cosmid vector clone M118 which is isolable from a transformant identified by the international deposit number FERM BP-4278, wherein the nucleic acid sequence lacks the 5' terminal sequence of the portion of a human genome so isolable from the clone M118 that duplicates the 3' terminal sequence of the nucleic acid sequence (b);
(d) a nucleic acid sequence of a portion of a human genome inserted in cosmid vector clone M84 which is isolable from a transformant identified by the international deposit number FERM BP-4277, wherein the nucleic acid sequence lacks the 5' terminal sequence of the portion of a human genome so isolable from the clone M84 that duplicates the 3' terminal sequence of the nucleic acid sequence (c);
(e) a nucleic acid sequence of a portion of a human genome inserted in cosmid vector clone M131 which is isolable from a transformant identified by the international deposit number FERM BP-4279, wherein the nucleic acid sequence lacks the 5' terminal sequence of the portion of a human genome so isolable from the clone M131 that duplicates the 3' terminal sequence of the nucleic acid sequence (d); and (f) a nucleic acid sequence of a portion of a human genome inserted in cosmid vector clone 3-31 which is isolable from a transformant identified by the international deposit number FERM BP-4276, wherein nucleic acid sequence lacks the 5' terminal sequence of the portion of a human genome so isolable from the clone 3-31 that duplicates the 3' terminal sequence of the nucleic acid sequence (e), wherein each of the portions of a human genome inserted in the clone Y6, Y24, Y21, M118, M84, M131 and 3-31 respectively is in a relative position in a human genome as shown in Figure 1.
2. The polynucleotide of claim 1, which has the restriction pattern and organization shown in Figure 1.
3. The polynucleotide of claim 1 wherein the nucleic acid sequence of a portion of a human genome is inserted in the clone Y6 comprises the nucleic acid sequences of SEQ ID NOS:
32 through 64;
the nucleic acid sequence of a portion of a human genome inserted in the clone Y24 comprises the nucleic acid sequences of SEQ ID NOS: 32 through 64;
the nucleic acid sequence of a portion of a human genome inserted in the clone Y21 comprises the nucleic acid sequences of SEQ ID NOS: 15 through 34;
the nucleic acid sequence of a portion of a human genome inserted in the clone M118 comprises the nucleic acid sequences of SEQ ID NOS: 14 and 15;
the nucleic acid sequence of a portion of a human genome inserted in the clone M84 comprises the nucleic acid sequences of SEQ ID NOS : 9 through 13;
the nucleic acid sequence of a portion of a human genome inserted in the clone M131 comprises the nucleic acid sequences of SEQ ID NOS : 8 and 9; and the nucleic acid sequence of a portion of a human genome inserted in the clone 3-31 comprises the nucleic acid sequences of SEQ ID NOS: 6 through 8.
32 through 64;
the nucleic acid sequence of a portion of a human genome inserted in the clone Y24 comprises the nucleic acid sequences of SEQ ID NOS: 32 through 64;
the nucleic acid sequence of a portion of a human genome inserted in the clone Y21 comprises the nucleic acid sequences of SEQ ID NOS: 15 through 34;
the nucleic acid sequence of a portion of a human genome inserted in the clone M118 comprises the nucleic acid sequences of SEQ ID NOS: 14 and 15;
the nucleic acid sequence of a portion of a human genome inserted in the clone M84 comprises the nucleic acid sequences of SEQ ID NOS : 9 through 13;
the nucleic acid sequence of a portion of a human genome inserted in the clone M131 comprises the nucleic acid sequences of SEQ ID NOS : 8 and 9; and the nucleic acid sequence of a portion of a human genome inserted in the clone 3-31 comprises the nucleic acid sequences of SEQ ID NOS: 6 through 8.
4. An isolated polynucleotide of a portion of a human genome comprising the following nucleic acid sequences (1) through (28) in the 5' to 3' order : (1) SEQ ID NO : 64 ; (2) SEQ ID NO: 61; (3) SEQ ID NO: 59; (4) SEQ ID NO: 53; (5) SEQ
ID NO: 51; (6) SEQ ID NO: 49; (7) SEQ ID NO: 48; (8) SEQ ID
NO: 46; (9) SEQ ID NO: 45; (10) SEQ ID NO; 43; (11) SEQ ID
NO: 39; (12) SEQ ID NO: 35; (13) SEQ ID NO: 34; (14) SEQ ID
NO: 33; (15) SEQ ID NO: 31; (16) SEQ ID NO: 30; (17) SEQ ID
NO: 28; (18) SEQ ID NO: 26; (19) SEQ ID NO: 23; (20) SEQ ID
NO: 21; (21) SEQ ID NO: 20; (22) SEQ ID NO: 18; (23) SEQ ID
NO: 15; (24) SEQ ID NO: 13; (25) SEQ ID NO: 11; (26) SEQ ID
NO: 9; (27) SEQ ID NO: 8; and (28) SEQ ID NO: 7;
wherein an intervening nucleic acid sequence appears between each of the adjacent nucleic acid sequences (1) through (28), the intervening nucleic acid sequence being that found in:
a) a yeast artificial chromosome clone which is isolable from a transformant identified by the international deposit number selected from the group consisting of FERM BP-4271, FERM BP-4273, and FERM BP-4274; or b) a cosmid vector clone which is isolable from a transformant identified by the international deposit number selected from the group consisting of FERM BP-42 76, FERM BP-4277, FERM BP-4278, and FERM BP-4279.
ID NO: 51; (6) SEQ ID NO: 49; (7) SEQ ID NO: 48; (8) SEQ ID
NO: 46; (9) SEQ ID NO: 45; (10) SEQ ID NO; 43; (11) SEQ ID
NO: 39; (12) SEQ ID NO: 35; (13) SEQ ID NO: 34; (14) SEQ ID
NO: 33; (15) SEQ ID NO: 31; (16) SEQ ID NO: 30; (17) SEQ ID
NO: 28; (18) SEQ ID NO: 26; (19) SEQ ID NO: 23; (20) SEQ ID
NO: 21; (21) SEQ ID NO: 20; (22) SEQ ID NO: 18; (23) SEQ ID
NO: 15; (24) SEQ ID NO: 13; (25) SEQ ID NO: 11; (26) SEQ ID
NO: 9; (27) SEQ ID NO: 8; and (28) SEQ ID NO: 7;
wherein an intervening nucleic acid sequence appears between each of the adjacent nucleic acid sequences (1) through (28), the intervening nucleic acid sequence being that found in:
a) a yeast artificial chromosome clone which is isolable from a transformant identified by the international deposit number selected from the group consisting of FERM BP-4271, FERM BP-4273, and FERM BP-4274; or b) a cosmid vector clone which is isolable from a transformant identified by the international deposit number selected from the group consisting of FERM BP-42 76, FERM BP-4277, FERM BP-4278, and FERM BP-4279.
5. An isolated polynucleotide consisting of a nucleic acid sequence of a portion of a human genome inserted in a clone selected from the group consisting of:
(a) yeast artificial chromosome clone Y6 which is isolable from a transformant identified by the international deposit number FERM BP-4271;
(b) yeast artificial chromosome clone Y24 which is isolable from a transformant identified by the international deposit number FERM BP-4274;
(c) yeast artificial chromosome clone Y21 which is isolable from a transformant identified by the international deposit number FERM BP-4273;
(d) cosmid vector clone M118 which is isolable from a transformant identified by the international deposit number FERM BP-4278;
(e) cosmid vector clone M84 which is isolable from a transformant identified by the international deposit number FERM BP-4277;
(f) cosmid vector clone M131 which is isolable from a transformant identified by the international deposit number FERM BP-4279; and (g) cosmid vector clone 3-31 which is isolable from a transformant identified by an international deposit number FERM BP-4276, wherein each of the portions of a human genome inserted in the clone Y6, Y24, Y21, M118, M84, M131 and 3-31 respectively is in a relative position in a human genome as shown in Figure 1, or a fragment of the polynucleotide, which comprises one or more functional human V H gene.
(a) yeast artificial chromosome clone Y6 which is isolable from a transformant identified by the international deposit number FERM BP-4271;
(b) yeast artificial chromosome clone Y24 which is isolable from a transformant identified by the international deposit number FERM BP-4274;
(c) yeast artificial chromosome clone Y21 which is isolable from a transformant identified by the international deposit number FERM BP-4273;
(d) cosmid vector clone M118 which is isolable from a transformant identified by the international deposit number FERM BP-4278;
(e) cosmid vector clone M84 which is isolable from a transformant identified by the international deposit number FERM BP-4277;
(f) cosmid vector clone M131 which is isolable from a transformant identified by the international deposit number FERM BP-4279; and (g) cosmid vector clone 3-31 which is isolable from a transformant identified by an international deposit number FERM BP-4276, wherein each of the portions of a human genome inserted in the clone Y6, Y24, Y21, M118, M84, M131 and 3-31 respectively is in a relative position in a human genome as shown in Figure 1, or a fragment of the polynucleotide, which comprises one or more functional human V H gene.
6. An isolated polynucleotide consisting of a nucleic acid sequence of a portion of a human genome inserted in a clone selected from the group consisting of:
(a) yeast artificial chromosome clone Y6 which is isolable from a transformant identified by the international deposit number FERM BP-4271;
(b) yeast artificial chromosome clone Y24 which is isolable from a transformant identified by the international deposit number FERM BP-4274;
(c) yeast artificial chromosome clone Y21 which is isolable from a transformant identified by the international deposit number FERM BP-4273;
(d) cosmid vector clone M118 which is isolable from a transformant identified by the international deposit number FERM BP-4278;
(e) cosmid vector clone M84 which is isolable from a transformant identified by the international deposit number FERM BP-4277;
(f) cosmid vector clone M131 which is isolable from a transformant identified by the international deposit number FERM BP-4279; and (g) cosmid vector clone 3-31 which is isolable from a transformant identified by the international deposit number FERM BP-4276, wherein each of said portions of a human genome inserted in the clone Y6, Y24, Y21, M118, M84, M131 and 3-31 respectively is in a relative position in a human genome as shown in Figure 1, wherein the nucleic acid sequence of a portion of a human genome inserted in the clone Y6 comprises the nucleic acid sequences derived from SEQ ID NOS: 32 through 64, wherein the nucleic acid sequence of a portion of a human genome inserted in the clone Y24 comprises the nucleic acid sequences derived from SEQ ID NOS: 32 through 64, wherein the nucleic acid sequence of a portion of a human genome inserted in the clone Y21 comprises the nucleic acid sequences derived from SEQ ID NOS: 15 through 34, wherein the nucleic acid sequence of a portion of a human genome inserted in the clone M118 comprises the nucleic acid sequences derived from SEQ ID NOS: 14 and 15, wherein the nucleic acid sequence of a portion of a human genome inserted in the clone M84 comprises the nucleic acid sequences derived from SEQ ID NOS: 9 through 13, wherein the nucleic acid sequence of a human genome inserted in the clone M131 comprises the nucleic acid sequences derived from SEQ ID NOS: 8 and 9, and wherein the nucleic acid sequence of a portion of a human genome inserted in the clone 3-31 comprises the nucleic acid sequences derived from SEQ ID NOS: 6 through 8, or a fragment of the polynucleotide, which comprises one or more functional human V H gene.
(a) yeast artificial chromosome clone Y6 which is isolable from a transformant identified by the international deposit number FERM BP-4271;
(b) yeast artificial chromosome clone Y24 which is isolable from a transformant identified by the international deposit number FERM BP-4274;
(c) yeast artificial chromosome clone Y21 which is isolable from a transformant identified by the international deposit number FERM BP-4273;
(d) cosmid vector clone M118 which is isolable from a transformant identified by the international deposit number FERM BP-4278;
(e) cosmid vector clone M84 which is isolable from a transformant identified by the international deposit number FERM BP-4277;
(f) cosmid vector clone M131 which is isolable from a transformant identified by the international deposit number FERM BP-4279; and (g) cosmid vector clone 3-31 which is isolable from a transformant identified by the international deposit number FERM BP-4276, wherein each of said portions of a human genome inserted in the clone Y6, Y24, Y21, M118, M84, M131 and 3-31 respectively is in a relative position in a human genome as shown in Figure 1, wherein the nucleic acid sequence of a portion of a human genome inserted in the clone Y6 comprises the nucleic acid sequences derived from SEQ ID NOS: 32 through 64, wherein the nucleic acid sequence of a portion of a human genome inserted in the clone Y24 comprises the nucleic acid sequences derived from SEQ ID NOS: 32 through 64, wherein the nucleic acid sequence of a portion of a human genome inserted in the clone Y21 comprises the nucleic acid sequences derived from SEQ ID NOS: 15 through 34, wherein the nucleic acid sequence of a portion of a human genome inserted in the clone M118 comprises the nucleic acid sequences derived from SEQ ID NOS: 14 and 15, wherein the nucleic acid sequence of a portion of a human genome inserted in the clone M84 comprises the nucleic acid sequences derived from SEQ ID NOS: 9 through 13, wherein the nucleic acid sequence of a human genome inserted in the clone M131 comprises the nucleic acid sequences derived from SEQ ID NOS: 8 and 9, and wherein the nucleic acid sequence of a portion of a human genome inserted in the clone 3-31 comprises the nucleic acid sequences derived from SEQ ID NOS: 6 through 8, or a fragment of the polynucleotide, which comprises one or more functional human V H gene.
7. A yeast artificial chromosome or cosmid vector comprising the polynucleotide of claim 5 or 6.
8. A yeast artificial chromosome selected from the group consisting of:
(a) a yeast artificial chromosome clone Y6 which is isolable from a transformant identified by an international deposit number FERM BP-4271;
(b) a yeast artificial chromosome clone Y24 which is isolable from a transformant identified by an international deposit number FERM BP-4274; and (c) a yeast artificial chromosome clone Y21 which is isolable from a transformant identified by an international deposit number FERM BP-4273.
(a) a yeast artificial chromosome clone Y6 which is isolable from a transformant identified by an international deposit number FERM BP-4271;
(b) a yeast artificial chromosome clone Y24 which is isolable from a transformant identified by an international deposit number FERM BP-4274; and (c) a yeast artificial chromosome clone Y21 which is isolable from a transformant identified by an international deposit number FERM BP-4273.
9. A cosmid vector selected from the group consisting of:
(a) a cosmid vector clone M118 which is isolable from a transformant identified by an international deposit number FERM BP-4278;
(b) a cosmid vector clone M84 which is isolable from a transformant identified by an international deposit number FERM BP-4277;
(c) a cosmid vector clone M131 which is isolable from a transformant identified by an international deposit number FERM BP-4279; and (d) a cosmid vector clone 3-31 which is isolable from a transformant identified by an international deposit number FERM BP-4276.
(a) a cosmid vector clone M118 which is isolable from a transformant identified by an international deposit number FERM BP-4278;
(b) a cosmid vector clone M84 which is isolable from a transformant identified by an international deposit number FERM BP-4277;
(c) a cosmid vector clone M131 which is isolable from a transformant identified by an international deposit number FERM BP-4279; and (d) a cosmid vector clone 3-31 which is isolable from a transformant identified by an international deposit number FERM BP-4276.
10. An isolated cell transformed by either one selected from the group consisting of:
(i) the polynucleotide of any one of claims 1 through 6; and (ii) the yeast artificial chromosome or cosmid vector of any one of claims 7 through 9.
(i) the polynucleotide of any one of claims 1 through 6; and (ii) the yeast artificial chromosome or cosmid vector of any one of claims 7 through 9.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/JP1993/000603 WO1994026895A1 (en) | 1993-05-10 | 1993-05-10 | Human immunoglobulin vh gene and dna fragment containing the same |
| CA002162577A CA2162577C (en) | 1993-05-10 | 1993-05-10 | Human immunoglobulin vh gene segments and dna fragments containing the same |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/JP1993/000603 WO1994026895A1 (en) | 1993-05-10 | 1993-05-10 | Human immunoglobulin vh gene and dna fragment containing the same |
| CA002162577A CA2162577C (en) | 1993-05-10 | 1993-05-10 | Human immunoglobulin vh gene segments and dna fragments containing the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2162577A1 CA2162577A1 (en) | 1994-11-24 |
| CA2162577C true CA2162577C (en) | 2005-06-14 |
Family
ID=34682997
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002162577A Expired - Fee Related CA2162577C (en) | 1993-05-10 | 1993-05-10 | Human immunoglobulin vh gene segments and dna fragments containing the same |
Country Status (2)
| Country | Link |
|---|---|
| CA (1) | CA2162577C (en) |
| WO (1) | WO1994026895A1 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6787638B1 (en) * | 1998-12-02 | 2004-09-07 | Applied Molecular Evolution, Inc. | Tumor specific human monoclonal antibodies and methods of use |
| JP2001206899A (en) * | 1999-11-18 | 2001-07-31 | Japan Tobacco Inc | HUMAN MONOCLONAL ANTIBODY AGAINST TGF-beta II TYPE RECEPTOR AND MEDICINAL USE THEREOF |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AT396939B (en) * | 1990-05-29 | 1993-12-27 | Alois Dipl Ing Dr Jungbauer | COMPLEX VIRAL ANTIQUE OF HIV-1 BINDING RECOMBINANT PROTEIN |
-
1993
- 1993-05-10 WO PCT/JP1993/000603 patent/WO1994026895A1/en active Application Filing
- 1993-05-10 CA CA002162577A patent/CA2162577C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| WO1994026895A1 (en) | 1994-11-24 |
| CA2162577A1 (en) | 1994-11-24 |
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