JP2006262911A - Human immunoglobulin vh gene and dna fragment containing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide DNA fragments containing a plurality of human immunoglobulin V<SB>H</SB>genes or a novel human immunoglobulin V<SB>H</SB>gene, furthermore, vectors containing the gene or transformed cells containing the vector. <P>SOLUTION: Disclosed are a novel human immunoglobulin V<SB>H</SB>gene and DNA fragments containing the gene, wherein the DNA fragment has a specific nucleic acid sequence of about 800 kbp. The human immunoglobulin V<SB>H</SB>gene is contained in the fragment of 800kbp and there are novel fifty genes. A DNA fragment containing two or more V<SB>H</SB>genes is also provided. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention relates to a novel human immunoglobulin _VH gene and a DNA fragment containing the same, a vector containing them, and a transformed cell containing the vector. The gene and DNA fragment of the present invention are useful for producing a human antibody by genetic engineering using a mammalian host.

An immunoglobulin is composed of an L chain and an H chain, and each chain is composed of a variable region (V region) having a different structure for each immunoglobulin molecule and a constant region (C region) having a common structure between immunoglobulin molecules. It is the V region that determines the antigen specificity of an antibody. The V region of the H chain is recoded by the genes V, D (diversity), and J (joining) (the H chain gene is indicated with a suffix _H like V _H ). One of the important reasons that the immunoglobulin V region is very diverse and can provide antibodies that can react specifically to a myriad of antigens is the rearrangement of V, D, and J. . That is, there are a plurality of V, D, and J genes, and they are randomly combined in somatic cells to form a single mRNA encoding gene. An immunoglobulin V region is provided.

  On the other hand, antibodies currently used for treatment of various diseases are antibodies derived from animals other than humans such as mice. However, when this is administered to humans, since the antibody is of a heterologous animal origin, an immune response to the administered antibody may occur in the human body, exhibiting allergic symptoms, etc. This causes problems such as neutralization and inactivation. In order to avoid this problem, it is desirable to administer baboon-derived antibodies for human therapy. In addition, it is difficult to industrially produce human antibodies from human-derived antigens using a human as a host, even if existing methods are used. Therefore, research on the production of human immunoglobulins by genetic engineering using an animal as a host is proceeding (for example, Japanese Patent Application Publication No. 4-504365: Proc. Natl. Acad. Sci. USA, V01.86, 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 method of expressing human immunoglobulin genes in host animals other than humans, the number of human _VH genes subjected to genetic recombination is very small, and the diversity of expressed human immunoglobulins is limited. There is a problem of being. The H chain is a D. cerevisiae even if only one V _H gene is incorporated. The combination of J genes ensures a certain degree of immunoglobulin diversity. However, as described above, basically, the rearrangement (random combination) of the V genes determines the diversity of immunoglobulins. Therefore, the greater the number of human _VH genes incorporated, the greater the variety of immunoglobulins that are expressed. Increases nature. As the diversity of immunoglobulins increases, not only can a large number of antigens be handled, but the possibility of producing antibodies with a very high affinity for a single antigen is increased. It is important when trying to use it.
Ravetch, JV. et al, (1981) Cell, Vol. 27, pp.583-591

Accordingly, an object of the present invention is to provide a DNA fragment comprising a plurality of human immunoglobulin _VH genes. Another object of the present invention is to provide a novel human immunoglobulin _VH gene. A further object of the present invention is to provide a vector containing the DNA fragment or the gene, and a transformed cell containing the vector.

According to the present invention, a novel human immunoglobulin _VH gene and a DNA fragment containing the same are provided. The DNA fragment of about 800 kb of the present invention contains as many as 64 human immunoglobulin _VH genes, and if human immunoglobulins are genetically engineered to be produced in a host animal using this, it will be compared with the conventional one. The diversity of human immunoglobulin produced is greatly increased.

As a result of intensive studies, the inventors of the present application succeeded in determining the structure of a human H chain V region gene group covering about 800 kb, and determining the base sequences of 64 human V _H genes contained therein. Succeeded in doing. As a result, the 800 kb DNA fragment and various DNA fragments contained therein can be provided, thereby completing the present invention.

  That is, the present invention includes a DNA sequence derived from a human genome obtained from a yeast artificial chromosome clone Y6, Y24, Y21, M118, M84, M131, 3-31, Y103, or Y20 obtained from the deposited transformant. Is provided.

  In a preferred embodiment, the human genome-derived DNA sequence obtained from the clone Y6 contains the DNA sequence set forth in SEQ ID NO: 32 to SEQ ID NO: 64, and the human genome-derived DNA sequence obtained from the clone Y24 is SEQ ID NO: 32 Thru | or the DNA sequence derived from the human genome obtained from this clone M118 including the DNA sequence represented by sequence number 15 thru | or SEQ ID NO: 34 The DNA sequence of SEQ ID NO: 14 and SEQ ID NO: 15 is included, the human genome-derived DNA sequence obtained from the clone M84 includes the DNA sequence of SEQ ID NO: 9 to SEQ ID NO: 13, The DNA sequence derived from the human genome obtained from the clone M131 contains the DNA sequence set forth in SEQ ID NO: 9 to SEQ ID NO: 13, The human genome-derived DNA sequence obtained from clone 3-31 includes the DNA sequence described in SEQ ID NO: 6 to SEQ ID NO: 8, and the human genome-derived DNA sequence obtained from clone Y103 is SEQ ID NO: 1 to SEQ ID NO: 5. And the DNA sequence derived from the human genome obtained from the clone Y20 includes the DNA sequences described in SEQ ID NO: 1 to SEQ ID NO: 4.

The present invention also includes a DNA sequence derived from a human genome obtained from a yeast artificial chromosome clone Y6, Y24, Y21, M118, M84, M131, 3-31, Y103, or Y20 obtained from the deposited transformant. Provided is a DNA comprising at least (1) any two functional _VH genes; and (2) any two other two functional _VH genes different from the gene of (1).

  In a preferred embodiment, the human genome-derived DNA sequence obtained from the clone Y6 contains the DNA sequence set forth in SEQ ID NO: 32 to SEQ ID NO: 64, and the human genome-derived DNA sequence obtained from the clone Y24 is SEQ ID NO: 32 Thru | or the DNA sequence derived from the human genome obtained from this clone M118 including the DNA sequence represented by sequence number 15 thru | or SEQ ID NO: 34 The DNA sequence of SEQ ID NO: 14 and SEQ ID NO: 15 is included, the human genome-derived DNA sequence obtained from the clone M84 includes the DNA sequence of SEQ ID NO: 9 to SEQ ID NO: 13, The DNA sequence derived from the human genome obtained from the clone M131 contains the DNA sequence set forth in SEQ ID NO: 9 to SEQ ID NO: 13, The human genome-derived DNA sequence obtained from clone 3-31 includes the DNA sequence described in SEQ ID NO: 6 to SEQ ID NO: 8, and the human genome-derived DNA sequence obtained from clone Y103 is SEQ ID NO: 1 to SEQ ID NO: 5. And the DNA sequence derived from the human genome obtained from the clone Y20 includes the DNA sequences described in SEQ ID NO: 1 to SEQ ID NO: 4.

The present invention also provides DNA for producing a non-human mammal that produces an immunoglobulin heavy chain comprising a variable region derived from a human _VH gene, wherein the DNA is any one of the above DNA comprising the DNA of the above is provided.

The present invention also provides the following DNA sequences (a) to (f):
(A) a human genome-derived DNA sequence obtained from either yeast artificial chromosome clone Y6 or Y24 obtained from the deposited transformant; (b) obtained from yeast artificial chromosome clone Y21 obtained from the deposited transformant. (C) DNA sequence derived from human genome obtained from cosmid vector clone M118 obtained from the deposited transformant; (d) Obtained from cosmid vector clone M84 obtained from the deposited transformant A human genome-derived DNA sequence; (e) a human genome-derived DNA sequence obtained from the deposited cosmid vector clone M131; and (f) a cosmid vector clone 3-derived from the deposited transformant. A DNA sequence derived from the human genome obtained from 31 in the order of 5 'to 3' Providing a vector with that DNA.

  In a preferred embodiment, there is provided a vector characterized in that the DNA is a DNA having a restriction enzyme recognition site pattern as shown in FIG.

In another preferred embodiment, the DNA has the following features (1) to (7):
(1) The human genome-derived DNA sequence obtained from the clone Y6 includes the DNA sequence described in SEQ ID NO: 32 to SEQ ID NO: 64; (2) the human genome-derived DNA sequence obtained from the clone Y24 is SEQ ID NO: (3) the DNA sequence derived from the human genome obtained from the clone Y21 comprises the DNA sequence described in SEQ ID NO: 15 to SEQ ID NO: 34; (4) the DNA sequence described in SEQ ID NO: 15 to SEQ ID NO: 34; The human genome-derived DNA sequence obtained from clone M118 includes the DNA sequences set forth in SEQ ID NO: 14 and SEQ ID NO: 15; (5) the human genome-derived DNA sequence obtained from clone M84 is SEQ ID NO: 9 to SEQ ID NO: (6) the DNA sequence derived from the human genome obtained from the clone M131 is the DNA described in SEQ ID NO: 8 and SEQ ID NO: 9; And (7) a vector characterized in that the DNA sequence derived from the human genome obtained from the clone 3-31 is a DNA comprising the DNA sequence set forth in SEQ ID NO: 6 to SEQ ID NO: 8. I will provide a.

  The present invention also relates to (a) a DNA sequence derived from a human genome obtained from yeast artificial chromosome clone Y103 obtained from the deposited transformant; and (b) a yeast artificial chromosome clone Y20 obtained from the deposited transformant. A vector having a DNA comprising a human genome-derived DNA sequence obtained from 5 'to 3' in this order is provided.

  In a preferred embodiment, there is provided a vector characterized in that the DNA is a DNA having a restriction enzyme recognition site pattern as shown in FIG.

In another preferred embodiment, the DNA has the following features (1) and (2):
(1) The human genome-derived DNA sequence obtained from the clone Y103 includes the DNA sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 5; and (2) the human genome-derived DNA sequence obtained from the clone Y20 is a sequence Comprising the DNA sequence set forth in No. 1 to SEQ ID No. 4;
Provided is a vector characterized by being a DNA having

  The present invention also includes SEQ ID NOs: 64, 61, 59, 53, 51, 49, 48, 46, 45, 43, 39, 35, 34, 33, 31, 30, 28, 26, 23, 21, 20, Provided is a vector having a DNA comprising the DNA sequence described in 18, 15, 13, 11, 9, 8, 7 in the order of 5 ′ to 3 ′.

  The present invention also provides a vector having a DNA comprising the DNA sequence set forth in SEQ ID NOs: 5, 4, 3, 2, 1 in 5 ′ to 3 ′ order.

  The present invention also provides a vector comprising a human genome-derived DNA sequence obtained from a yeast artificial chromosome Y6, Y24, Y21, Y103 or Y20 obtained from the deposited transformant.

  In a preferred embodiment, the vector is a yeast artificial chromosome Y6, Y24, Y21, Y103, or Y20 obtained from the deposited transformant.

  The present invention also provides a vector comprising a human genome-derived DNA sequence obtained from a cosmid vector clone M118, M84, M131, or 3-31 obtained from the deposited transformant.

  In a preferred embodiment, the vector is characterized in that it is a cosmid vector clone M118, M84, M131 or 3-31 obtained from the deposited transformant.

  The present invention also provides a cell transformed with any of the DNAs described above or a part of the DNAs, or any of the vectors described above.

  In a preferred embodiment, the cell has an international deposit number of FERM BP-4271, FERM BP-4272, FERM BP-4273, FERM BP-4274, FERM BP-4275, FERM BP-4276, FERM BP-4277, FERM BP-4278 Or a transformed cell identified by FERM BP-4279.

The inventors of the present invention use a library in which DNA of a human lymphoblastoid cell line transformed with EB virus is partially digested with Eco RI and incorporated into YAC by the method described in detail in the following Examples. The structure of the human _VH gene cluster region spanning about 800 kbp was successfully determined. This is shown in FIG. In FIG. 1, the gene map is shown on four thick solid lines. Each solid line is 3 'on the right and the left end of the top solid line is connected to the right end of the second solid line. The DNA fragment shown in FIG. 1 has a C gene from the 3 ′ side, then a J _H gene, and then a D gene, followed by 64 V _H genes. The base sequences of these 64 _VH genes are all determined as shown in the following examples, and are shown in the order of SEQ ID NOs: 1, 2,... 63, 64 from the downstream side.

Of these V _H genes, functional believed to encode a polypeptide (functional) of V _H is indicated by a rectangular black. On the other hand, although it has the general characteristics of the _VH gene known so far, it contains a stop codon and does not currently encode a polypeptide, ie, pseudo V _{The H} gene is indicated by a white rectangle. A cutting map by Eco RI and Hind III is shown directly under this gene map. Restriction enzyme cleavage sites are indicated by short vertical lines. Those with a circle at the end of the short line are those in which the order is not determined, and the region indicated by a box with dotted points is a region where the Eco RI cutting site has not been determined. In FIG. 1, what looks like “Y” indicates that the two restriction enzyme sites are in close proximity. Further, in FIG. 1, the Mul I cleavage site is indicated by a white triangle, and the Not I cleavage site is indicated by a black triangle. The insert in each clone used to determine this structure is further shown below. The structure on the 3 ′ side of FIG. 1 is known, and Ravetch, JV. et al, (1981) Cell, Vol. 27, pp.583-591.

  Among the inserted DNA fragments in the clone shown in FIG. 1, yeasts each containing Y20, Y103, Y21, Y6, and Y24 inserted in YAC have been deposited at the Biotechnology Institute of Technology, and the deposits are made. The numbers are FERM-BP4272, FERM-BP4275, FERM-BP4273, FERM-BP4271 and FERM-BP4274, respectively. In addition, E. coli containing M131, M118, M84 and 3-31 inserted in the cosmid has also been deposited with the Biotechnology Institute of Technology. FERM-BP4276.

  The DNA fragment of about 800 kbp shown in FIG. 1 can be produced by connecting these deposited DNA fragments by a known method. That is, a DNA fragment A and a DNA fragment B in which the terminal nucleotide sequence overlaps with this DNA fragment A (that is, the nucleotide sequences of the 3 ′ region of DNA fragment A and the 5 ′ region of DNA fragment B are identical) are yeast It can be easily ligated by a method utilizing genetic recombination. That is, by inserting DNA fragments A and B into separate YAC vectors, placing the recombinant YAC into different mating type yeasts, and fusing them with each other, genetic recombination occurs in the yeast host. A YAC having a DNA fragment in which the DNA fragment A and the DNA fragment B are connected by sharing only one unrecognized overlapping portion is obtained.

  Such a recombinant YAC can be easily selected using the auxotrophy encoded in the YAC vector used as a marker. This method is a conventional method. For example, Japanese Patent Application Publication 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. 12, 3135-3138.

The eight DNA fragments deposited have overlapping ends and adjacent ends, and can be sequentially connected by this method. Although 3-31, M84, M118, and M-131 have been cloned into cosmid vectors, the cosmid clones are cleaved with a restriction enzyme that has only a recognition site in the vector, and then YAC vectors are inserted at both ends. By connecting, it can be maintained as an artificial chromosome in yeast. Furthermore, by the above-mentioned method, it can be connected to YAC clones in other regions. Even if the nine fragments deposited above are joined together, a gap of about 4 kb exists between Y103 and 3-31, but the DNA fragment that fills this gap can also be easily prepared by the method shown below. it can. That is, as shown in FIG. 1, since the Hin d III fragment of this part is relatively large, first, the human genome is completely digested with Hin d III, and this is separated by electrophoresis, and then has a molecular weight of about 15 kb. It can be obtained by selecting one, detecting it with a probe, and collecting it. The probe used here can be isolated as follows.

  That is, DNA fragments at both ends of the gap are subcloned using a plasmid, DNA fragments not containing repetitive sequences are prepared from them, and these are used for library screening. Isolate only those with signals detected at both ends of the probe.

As described above, according to the present invention, the DNA fragment of about 800 kbp shown in FIG. 1 was provided. In order to produce a human immunoglobulin by genetic engineering, a fragment consisting of a DNA region contained in this DNA fragment was also provided. Can be used. That is, in order to increase the diversity of human immunoglobulins produced by genetic engineering, it is preferable to incorporate a fragment containing as many human _VH genes as possible, but at least two human _VH genes should be included. For example, it can be used because it gives a certain degree of diversity during reconstruction. Therefore, a DNA fragment comprising a region containing at least two consecutive functional _VH genes contained in the approximately 800 kb DNA fragment shown in FIG. 1 can also be used.

The number of functional _VH genes contained in such a DNA fragment is at least 2, but preferably 6 or more, 7, 8, 9, 10, 11, 12 13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 The number of individuals, 30, 31, 32, and 33 increases, and the diversity of human immunoglobulins produced increases, which is preferable. Such fragments have a large molecular weight and are cloned with a YAC vector. However, relatively small fragments with a molecular weight of about 50 kb or less are not necessarily cloned with a YAC vector, and are cloned with a cosmid vector or a plasmid vector. You can also

Such a DNA fragment can be produced since the information shown in FIG. 1 and SEQ ID NOs: 1 to 64 is known. That is, for example, the human genome was partially digested with appropriate restriction enzymes such as Eco RI and Hin d III, The resulting fragments were separated by electrophoresis, respectively the desired two or more functional V _H Haburidaizu A DNA fragment containing two or more functional _VH genes can be obtained using two or more probes. Alternatively, PCR amplification can be used instead of probe detection. Also in this case, since all the base sequences of functional _VH genes are known, the base sequences of the primers to be used are also known and can be easily performed.

The present invention further provides such a DNA fragment in which DNA fragments containing two or more functional _VH genes are arbitrarily linked. That is, by joining a plurality of such DNA fragments, the number of _VH genes in the DNA fragments can be increased compared to the case where they are used alone, thereby reducing the diversity of human immunoglobulins produced thereby. Can be increased. The DNA fragments do not necessarily have to be continuous, and may be obtained by joining arbitrary DNA fragments in an arbitrary order. When DNA fragments to be linked do not have overlapping portions, the above-described method for linking DNA fragments having overlapping portions cannot be applied, but it is also possible to join DNA fragments without overlapping portions. It can be done as follows.

The left arm vector part and the right arm vector part of a YAC clone containing two or more functional _VH genes are recovered using the method of Hermanson et al. (1991) (Nucleic Acids. Res., 19; 4943-4948). In this case, it has a homologous sequence of the ampicillin resistance marker part (AMP) of the YAC left arm vector part and a marker (Lys) that restores the lysine requirement to the original requirement, and has a multiple cloning site immediately downstream of Lys. A plasmid (pLUS) having a homologous sequence of the YAC4 portion of the YAC right arm vector portion and the above-mentioned Lys and kanamycin resistance marker (KAN) and having a multiple cloning site immediately downstream of KAN It introduce | transduces into a yeast with YAC later using a conventional method. pICL and pLUS recombine in yeast at an appropriate frequency and are incorporated into the left and right arm vector portions of YAC. These yeasts retaining YAC are selected with an appropriate selective medium, and cleaved with an appropriate one of the same restriction enzymes as the restriction enzyme recognition site contained in the multiple cloning site.

By this method, a DNA fragment containing the left end or right end of the cocoon-derived DNA fragment contained in the YAC is recovered as a plasmid. This plasmid is increased in E. coli using a conventional method, and then both recovered plasmids are cleaved with a restriction enzyme and connected with ligase. Such a connected DNA fragment is further connected to the vector portion of the left arm or the right arm of YAC, and introduced into the yeast holding YAC. These YAC vectors recombine at a certain frequency in the left arm and right arm vector portions of the original YAC and the left end or right end fragment of the human-derived DNA fragment. If this is selected with a suitable marker, a fragment of the right end of another human origin DNA is collected at the left end of human origin DNA or a YAC clone in which the left end of another human origin DNA is connected to the right end of human origin DNA is recovered. Since these YAC clones have a structure in which the right end and left end DNA fragments of different human-derived DNAs are connected to the left end and right end of human-derived DNA, respectively, the YAC clone having this connected sequence and the method described above Can be recombined.
In addition, a large fragment containing a large number of _VH genes can be produced by arbitrarily combining the eight DNA fragments actually deposited.

Further, according to the present invention, the base sequences of 64 _VH genes contained in the fragment of about 800 kbp shown in FIG. 1 were determined. As will be described in detail in the Examples below, 50 of these were novel genes having base sequences that have not been known so far. Among these novel human immunoglobulin _VH genes, pseudogenes that do not encode polypeptides are also included, but even pseudogenes may function as donors for gene conversion at the somatic level. There is utility because there is.

The human immunoglobulin _VH gene of the present invention and a DNA fragment containing the same are used for producing rabbit immunoglobulin in a mammalian host as described in, for example, Japanese Patent Application Publication No. 4-504365. Can do.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

Example 1 Structure determination of DNA fragment of about 800 kb (1) Library used for screening The human YAC library used for screening was human lymphoblastoid cell line CGM1 (T. pylori) transformed with Epstein-Barr virus. Imaiand MV0lson, genomics, 8, 297-303 (1990)). An Eco RI partial digest of CGM1 DNA was obtained using the pYAC4 vector (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, Orland, 1991), AB1380 yeast host strain (D. Burke and M.V. 0lson, in “Guide to Yeast Genetics and Molecular Biology” (C. guthrie and G. R. Fink, eds), p. .253, Academic Press, Orland, 1991). The library consisted of 15000 independent clones with an average size of the inserted DNA fragment of about 360 kb. This library therefore represents approximately 1.8 times the human haploid genome. DNA reconstitution at the immunoglobulin H (IgH) locus was first rechecked by Southern hybridization using human D and _JH probes. As a result, one of the alleles retained the germline composition, while the other was reconstituted by V _H , D, J _H.

(2) Primers used for PCR-based screening Oligonucleotide primers for V _H-III and V _H-I families, the largest and second largest V _H families, for PCR-based screening of human V _H YAC clones Was synthesized. The V _H region of an immunoglobulin has two hypervariable regions (CDR1 and CDR2) and three framework regions (FR1, FR2 and FR3) (EA. Kabat et al., Sequences of Proteins of immunological interest, Fifth edition, NIH publications, Washington DC (1991)). The nucleotide sequence of the framework part is very highly conserved within the same family. Therefore, it is suggested to synthesize a common oligonucleotide primer corresponding to the framework part. For this purpose, the base sequences of the FR1, FR2 and FR3 regions of all known _VH sequences were aligned and compared. The nucleotide sequences corresponding to the first 8 amino acid residues of FR1 were very similar not only within the same family but also between the V _H-I and V _H-III families. This made it possible to synthesize F-univ, a forward primer shown in Table 1, common to these two families. The family-specific reverse primer has the same sequence as the known _VH gene in the 3 ′ half of the primer sequence, and the most 3 ′ terminal nucleotide is common among many _VH fragments. Each sequence was selected independently from the highly conserved sequences in the FR2 region so as to correspond to the amino acid residues. Specifically, F-univ and IR, and F-univ and III-R were used as screening primers. The base sequence of each primer is shown in Table 1.

(3) Examination of optimum PCR conditions Preliminary experiments were conducted to determine the optimum conditions for specific amplification. A reaction solution (5 μl) was prepared according to the protocol of Perkin-Elmer / Cetus. The reaction was performed using DNA Thermal Cycler (Perkin-Elmer / Cetus), 25 ng template human DNA, various annealing temperatures (55 ° C., 58 ° C., 60 ° C. and 62 ° C.) and cycle numbers (25, 30 and 35). Cycle). As a result, a high annealing temperature, that is, a temperature condition of 94 ° C for 1 minute-62 ° C for 2 minutes-72 ° C for 2 minutes, specifically amplifies a human DNA sample, but does not amplify yeast strain AB1380 DNA regardless of the number of cycles. I found. At lower annealing temperatures, it was not possible to employ a false control signal in the negative control (-control) because it sometimes appears. Therefore, the following PCR was performed under the conditions described above.

(4) Polymerase chain reaction (PCR)
The first PCR-based screening consists of 7 multi-filter DNA pools (ED Green and MV. Olson, Proc. Natl. Acad. Sci. USA, each corresponding to DNA from 1920 colonies (20 × 96 wells). 87, 1213-1217 (1990)) using the above synthetic oligonucleotide primer. The positive multifilter pool was divided into 5 pools each consisting of 384 colonies (4 × 96 wells) and further screened by the same procedure. Each 25 ng of YAC pool DNA was used in the reaction. CGM1 DNA and yeast strain AB380 DNA used to construct the YAC library were added during PCR analysis to serve as positive (+) and negative control (-control), respectively. After amplification, all samples were electrophoresed in a 10% polyacrylamide gel containing 15% glycerol and analyzed by ethidium bromide staining.

(5) Colony hybridization After the first and second screening based on PCR, the position of the positive clone in 384 clones was determined by conventional colony hybridization. A nylon filter composed of 384 YAC clone was prepared by a known method (D. Burke and MV Olson, in “Guide to Yeast Genetics and Molecular Biology” (C. guthrie and GR Fink, eds), p. .253, Academic Press, Orland, 1991). V _266BL (Y. Nishida, et al., Proc. Natl. Acad. Sci. USA, 79, 3833-3837 (1982)) and V _HBV (M. kodaira et al., J. Mol. Biol., 190, 529-541 (1986)) were used as probes for the human _VH-I and _VH-III families, respectively. These probes were labeled with ³² P-dCTP (5 × 10 ⁵ cpm) using an oligo labeling kit (Pharmacia) and subjected to colony hybridization based on a conventional method (D. Burke et al., Supra). After hybridizing at 65 ° C. for 12 hours, the filter was washed twice with 2 × SSC (1 × SSC is 0.15 M NaCl—15 mM sodium citrate) for 10 minutes at room temperature, and then with 0.2 × SSC—0.1% SDS at 65 ° C. For 30 minutes. The filter was exposed to X-ray film overnight and the corresponding positive YAC clone was picked up for further analysis.

(6) Insertion check by colony PCR Simple and rapid re-screening of colony-purified clones using PCR without purifying DNA to determine whether a specific DNA sequence is present in an isolated YAC (ED. Green and MV. Olson, Proc. Natl. Acad. Sci. USA, 87, 1213-1217 (1990)). That is, positive yeast clones were removed and cultured on AHC plates. Four single colonies from each clone were transferred with a toothpick to the 5 μl PCR mix described above. Amplified bands were identified by PCR and subsequent gel electrophoresis under the same conditions used in the screening. In most clones, all four colonies showed specific amplification of the DNA fragment.

(7) Determining the size of YAC clones using PFGE Researchers have found that some YACs are unstable due to rearrangement between chromosomes during culture, resulting in variations in the size of human-derived DNA inserts. Has been a problem between. This artifact is often thought to be mediated by repetitive sequences or tandem repeats of homologous DNA sequences in the inserted DNA. Such rearrangements can occur with considerable frequency because the _VH locus contains a number of homologous DNA fragments consisting of the _VH gene and its surrounding region. More problematically, a single yeast may have more than one YAC. In order to remove the artifact clones from the subsequent analysis and identify YAC clones with multiple inserted DNAs, the size of the YAC clones was first determined by pulse field gel electrophoresis (PFGE).

Four colonies were selected from 17 single-well colonies positive for _VH rechecked by PCR, prepared in a small amount from a 5 ml culture in AHC medium, and subjected to a known method. Based on (D. Burke et al., Supra), blocks were prepared with low melting point agarose. The size of YAC was determined by PFGE using an agarose block fragment of an appropriate size. PFGE was performed using a gel electrophoresis apparatus of Pulsaphor (manufactured by Pharmacia) or Crossfield (manufactured by ATTO (Tokyo)) with a pulse time of 60 seconds. Concatemerized lambda DNA was added as a molecular weight marker. After the electrophoresis, the DNA was transferred to a nitrocellulose filter, and Southern hybridization was performed using the pBR322 plasmid as a probe.

A typical result is shown in FIG. Colonies selected from clones Y21, Y22, and Y24 with insert DNA sizes of 300 kb, 330 kb, and 310 kb, respectively, did not appear to have recombination because they exhibited the same size of four colonies. . On the other hand, the inserted DNA of clone Y23 appeared to be quite unstable due to recombination due to the size difference between the four colonies. Therefore, colonies that had not undergone recombination were selected for the following analysis. Of the 17 YAC clones containing _VH analyzed, the human insert was very unstable except for 3 containing clone Y23. Later analysis showed that such recombination occurred regardless of the number of _VH genes in the inserted DNA. This indicates that some other factor may be involved in homologous recombination. Of the 14 stable YAC clones out of 17 YAC clones containing _VH , Y20, Y103, Y21, Y6 and Y24 were selected and used for the following physical map construction.

(8) Physical map construction of YAC clone by rare site restriction enzyme A gel block of the YAC clone after size determination was prepared and used for physical map construction by PFGE. In order to analyze long YACs, it is usually necessary to use several enzymes. However, in this example, only two rare site restriction enzymes (restriction enzymes with relatively rare cleavage sites), namely Not I and Mlu I, were used for the overlap analysis of YAC clones. This is mainly due to the following two reasons. 1) YAC clones with _VH can be ordered by several other types of information such as the size or pattern of fragments that hybridize with _VH probes or non-repetitive probes isolated from cosmid clones with _VH. it can. 2) YAC needs to be subcloned into a cosmid for the construction of a detailed physical map using common restriction enzymes.

Gel blocks completely digested with Not I and Mlu I were electrophoresed using a PFGE apparatus with a pulse time of 30 to 60 seconds depending on the length of YAC. A mixture of lambda phage DNA, other Xho I degradation products and Hin d III degradation products was also used as a low molecular weight marker. In order to detect all restriction enzyme fragments, a Southern filter was first hybridized with human polymeric DNA. From the total size of the detected bands, the length of undigested YAC was calculated. Filters were subsequently hybridized with pBR322 DNA probes corresponding to each of the pYAC4 arms. Double degradation of pBR322 with Pvu II and Bam HI resulted in fragments of 2.67 kb and 1.96 kb that hybridized specifically to the left end (trp side) and right end (ura side) of the YAC vector, respectively. . The filter was also hybridized with six _VH family specific probes to see if the _VH gene was present in the cleaved DNA fragment. The origin of V _H- specific probes for V _H-II , V _H-IV , V _H-V and V _H-VI is V _CE-1 (N. Takahashi et al., Proc. Natl. Acad. Sci. USA, 81, 5194-5198 (1984)), V _71-2 (KH 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 (JE Berman et al., EMBO J. 7, 727-1051 (1988)).

Hybridization experiments using partially degraded YAC DNA were performed to align Not I and Mlu I fragments detected by complete degradation experiments. Preliminary tests were needed to determine the optimal conditions for partial degradation. This is because the efficiency of the restriction enzyme reaction largely depends on the purity of DNA. In most cases, the prepared DNA in this example was reacted with 1 unit of restriction enzyme for 6 hours for complete degradation of the gel block (˜500 ng DNA). Partial cleavage of DNA was achieved by changing the reaction time as follows.

1. Three gel blocks (each having a volume of about 50 μl, containing about 1 μg of DNA and stored in 0.5 M EDTA, pH 8.0) against 50 ml of distilled water at room temperature under gentle agitation Dialyze for 1 hour. This operation is repeated to completely remove EDTA.
2. The block is equilibrated at 37 ° C. for 30 minutes in 10 ml enzyme optimal buffer.
3. Transfer each block into 250 μl of reaction solution containing 1 unit of restriction enzyme each in 1 × reaction buffer.
4. React the three tubes at 37 ° C. for 10 minutes, 30 minutes and 1 hour, respectively.
5. The reaction is stopped by adding 100 μl of 0.5 M EDTA (pH 8.0).
6). The gel block is equilibrated 2-3 times with an appropriate gel electrophoresis buffer for 1 hour at a time, and PFGE is performed immediately using an appropriate pulse time.

  The size of the hybridized restriction enzyme fragment was determined by hybridizing the filter with the left end probe and the right end probe of the YAC vector and comparing with the molecular weight marker (FIG. 3A). The physical map of the YAC clone shown in FIG. 3B was constructed by combining the results of complete and partial degradation experiments. Mapped clones were combined in this way and classified into several island regions.

(9) Isolation of inserted DNA terminal sequence from YAC After the isolated clones are classified into several regions based on the restriction enzyme map, the inserted DNA end sequence is isolated from both ends of each region to obtain oligonucleotides. Primers were synthesized. As is often pointed out, a significant percentage (about 30%) of YAC clones in the library contain noncontiguous DNA regions called chimeric clones to which multiple DNA fragments have been ligated. Since an effective method for eliminating such artifact DNA during library construction has not been developed, it is necessary to check this possibility by an appropriate method after isolating the YAC clone. In this example, a method of examining this possibility using PCR using a synthetic inserted DNA terminal primer was employed. This is because synthetic primers are not only useful for examining chimeric clones, but also register the resulting sequence as the site (STS) from which the sequence for rescreening the YAC library by PCR was determined. Because it is useful. It can also be used to look for overlaps between contigs that cannot be found by comparing restriction enzyme maps.

More sophisticated and rapid method using reverse PCR and vectorette system (JH. Riley et al., Nucleic Acids Res., 18, 2887-1890 (1990)) to isolate the inserted DNA unterminated YAC portion There are several different methods including: However, in this example, a fairly traditional method of subcloning the fragment with a plasmid or lambda phage vector was employed. High molecular weight DNA from the YAC clone was digested with a restriction enzyme having a recognition site in both the right arm sequence and the left arm sequence. Gel electrophoresis in 0.7% agarose and a 0.62 kb Hin d III- Sal I fragment and Southern of pBR322 DNA (Tet ^R ) that specifically hybridizes with the insert-vector border sequence of the pYAC4 vector The filter was hybridized. The DNA fragment in question was recovered using DE81 paper and ligated with EMBL4 or pUC19 vector depending on the size of the insert. The isolated fragment ligated to the EMBL4 vector was then subcloned into the pUC19 vector for sequencing. In order to determine the base sequences of these plasmid clones, a chain termination method using M13 forward or reverse primers was used. The sequence for the inserted DNA unterminated primer was obtained from a portion other than the repetitive sequence in the obtained sequence.

The above-mentioned artifacts were examined by PCR experiments using primers at both ends of YAC-DNA against DNA of human mouse somatic cell hybrid cell GM10479 strain (Coriel Institute) having only human chromosome 14 where the human IgH locus is present. did. DNA from CGM1 cells (origin of YAC library) and DNA from Rag cells (mouse cells) were used as + and − controls, respectively. PCR was carried out in a 25 μl reaction solution based on a known method (HS. Kim and O. Smithies, Nucleic Acids res., 16, 88870-8903 (1988)). Each 200 ng of DNA was subjected to the reaction. PCR was performed on GM10479, CGM1, and Rag DNA in 35 to 40 cycles of 95 ° C. for 1 minute—55 to 62 ° C. for 2 minutes—72 ° C. for 2 minutes under the best condition by a split prayer experiment using CGM1 DNA. It was concluded that chimera clones did not specifically amplify DNA in GM10479 when using either of the two insert DNA end primers. Only one contig in which none of the primers gave an amplification band was found to cover the _VH locus translocated on chromosome 16 (orphon).

(10) Construction of cosmid subcloning and physical map Isolation of a large chromosomal region using the YAC system is advantageous for the first stage of physical map construction. However, exogenous DNA cannot be easily separated from yeast chromosomal DNA, and fragments reaching several hundred kb are difficult to handle without mechanical shearing, so large DNA fragments in YAC are analyzed in more detail. There are various problems involved. In order to map the _VH gene of a long DNA fragment containing the _VH gene, a detailed restriction enzyme map using a general 6 bp site recognition restriction enzyme is required. For this purpose, YAC clones were subcloned into cosmids.
The cosmid library was constructed from total yeast DNA containing YAC without separating the previously cloned DNA from YAC. There are two main reasons for this. 1) It is difficult to separate a completely inserted DNA portion and to manipulate it. 2) Since the genome size of yeast is about 1.5 × 10 ⁴ bp and 1/2000 of human, 4000 independent colonies are sufficient to completely cover the YAC insert DNA.

In general, there are two major difficulties in building a cosmid library. The first difficulty is self-ligation of vector DNA, which generates clones into which no foreign DNA is inserted. The second difficulty is the insertion of two or more DNA fragments into a single vector, ie, a discontinuous ligation artifact. To overcome these problems, a great deal of effort is involved, including the construction of better designed vectors with two cos sites and modified ligation methods such as partial filling of the vector and the inserted DNA. (J. Sambrook et al., A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold spring harbor, NY (1989)).

  Size-fractionated DNA usually contains smaller DNA molecules in larger fractions of DNA molecules, especially when an excess of DNA is run on a separation gel. In order to eliminate discontinuous ligation between DNAs, it is effective to treat DNA fragments with alkaline phosphatase. However, this causes polymerization of the vector DNA during ligation, which selects colonies that do not contain insert DNA when selected by antibiotics. However, in this example, since YAC DNA of less than 5 μg is sufficient, the DNA could be separated by electrophoresis without contamination of small DNA fragments in the target fraction. Most cosmid libraries were thus constructed using a combination of alkaline phosphatase-treated cosmid vectors and partially degraded DNA prepared in the 35 kb to 45 kb size range.

[1] Preparation of yeast DNA containing YAC Since a long DNA fragment is required as a starting material to prepare DNA, extracting DNA from yeast cells with minimal shear damage is one of the most important steps. It is. Obviously, the best way is to handle the DNA in a gel. This is because DNA is completely protected from shear damage. However, the inventors have found that gentle extraction of DNA in liquid from yeast cells yields DNA (> 200 kb) long enough for partial degradation and subsequent size fractionation. It was. Furthermore, the solution-like DNA is easier to control the partial digestion conditions than the DNA filled in the gel block. About 50 μg of large size DNA (> 200 kb) can be routinely purified from 100 ml of yeast culture by the following simple and rapid method (6 hours for all operations).

(I) Spin down yeast cells and wash twice with TE (10 mM Tris HCl (pH 8.0) -1 mM EDTA).
(Ii) Cells were resuspended in 20 ml 0.1 M EDTA (pH 7.5), 1 M sorbitol, 0.2 mg / ml Zymolase 100T (ICN catalog # 152270), 15 mM 2-mercaptoethanol, 1 hour at 37 ° C. Incubate and spheroplast.
(Iii) Spin down the spheroplasts and resuspend in 9 ml 0.1 M Tris HCl (pH 7.5), 50 mM EDTA (pH 7.5).
(Iv) Add 1 ml of 10% SDS (final concentration 1/10) and gently stir. Incubate at 60 ° C. for 10-20 minutes.
(V) Add 1/3 volume of 4M potassium acetate, mix gently and leave on ice for 30 minutes.
(Vi) Centrifuge at 2000 xg for 30 minutes and transfer the supernatant to a new tube. Add 3 times the amount of isopropanol and stir gently. Allow the DNA to wash for 10-20 minutes at room temperature.
(Vii) Centrifuge again at 2000 xg for 30 minutes and discard the supernatant. Dissolve the pellet in 10 ml of water.

In subsequent steps, care should be taken not to damage the DNA.
(Viii) Extract twice with phenol and twice with CIAA (chloroform: isoamyl alcohol = 24: 1), and wash with ethanol at room temperature for 10 to 20 minutes.
(Ix) Centrifuge at 2000xg for 30 minutes. Rinse the pellet with 70% ethanol and dry completely.
(X) Dissolve in 1 ml TE.

[2] Preparation of vector DNA
Lorist 2 DNA was linearized by digestion with Hin d III or Bam HI and dephosphorylated by treatment with bacterial alkaline phosphatase. A small amount of DNA before and after phosphatase treatment was used for test ligation to check for phosphatase treatment based on known methods (J. Sambrook et al., Supra).

[3] Preparation of Inserted DNA Preliminary experiments for partial degradation of yeast DNA were performed based on a conventional method (J. Sambrook et al., Supra), and the optimum enzyme concentration and reaction time were determined. The preparation of size-fractionated DNA from the gel was accomplished using LGT agarose and β agarase I. By this very gentle method, it was possible to recover the fractionated DNA in high yield (> 90%) without shearing. The scaled-up cleavage reaction was performed at an optimal enzyme concentration using 5 μg of DNA. The decomposed sample was run on 0.5% LGT agarose (Biorad, preparative grade) and electrophoresed overnight under the condition of about 1 V / cm. The linearized λDNA and its Xho I digest giving 35 kb and 15 kb bands also migrated as size markers. After the DNA was visualized under an ultraviolet transilluminator, a small piece of agarose containing fragments ranging from 35 kb to 45 kb was cut out. For recovery of DNA from the gel slice, β-agarase I (NEB) was used according to the following method.

(i) Equilibrate gel block with distilled water to completely remove gel electrophoresis buffer.
(ii) Transfer the block to a new tube and add 1/9 volume of 10 × β agarase I buffer.
(iii) The gel is dissolved at 68 ° C. for 10 minutes. After cooling to 40 ° C., the molten agarose is incubated with an optimal enzyme unit amount of β-agarase I at 40 ° C. for 1 hour.
(iv) The salt concentration of the solution is adjusted to 0.5 M NaCl for ethanol precipitation and cooled on ice for 10 minutes.
(v) Centrifuge at 15000 xg for 15 minutes to pellet residual undigested carbohydrate.
(vi) Transfer the DNA-containing supernatant to a new tube. The DNA is washed with 3 volumes of ethanol at −80 ° C. for 10 minutes.
(vii) Centrifuge at 15000xg for 15 minutes and remove supernatant. Rinse the pellet with 70% ethanol and dry the pellet.
(viii) Resuspend the pellet in an appropriate amount of distilled water for the next operation.
By this method, 100-300 ng average size fractionated DNA can be recovered.

[4] Ligation, in vitro packaging, and infection of E. coli This procedure was performed by conventional methods (J. Sambrook et al., Supra). About 10000 colonies were obtained from 25 ng of ligated DNA by using λ inpackaging kit (Nippon Gene Co., Ltd.) and ED8768 host strain.

[5] Screening of cosmid library The initial screening was performed using a cosmid library of partially digested DNA by Hin d III. Approximately 10,000 colonies (500 colonies × 20 per 10 cm diameter plate) were plated on LB plates containing 50 μg / ml kanamycin so that a single colony could be picked up after the initial screening. Next, the colony was taken with a nitrocellulose membrane (Advantech Toyo Membrane Co., Ltd.) having a diameter of 8.2 cm and containing no surfactant, and subjected to colony hybridization. Three different types of probes: a mixture of six _VH family-specific probes for isolating _VH- containing cosmid clones, a YAC vector probe (pBR322) for isolating inserted DNA endless cosmid clones The Tet ^R portion of, as described above), and total human DNA for any remaining cosmid clones were used for screening. On average, 50 to 100 clones corresponding to approximately 300 kb of inserted DNA were obtained with these probes from one YAC clone.

[6] Construction of cosmid contig DNA was isolated from cosmid clones by the alkaline lysis method (J. Sambrook et al., Supra). Purified cosmid DNA was digested with Eco RI or Hin d III and subjected to agarose gel electrophoresis for restriction enzyme map construction. Duplication between clones could be easily found by comparing restriction enzyme patterns between cosmid clones. Was cut with Eco RI or Hin dIII is then cosmid clones to determine the order, it was run on 0.7% agarose gel. To identify the location and number of _VH genes in the cosmid clone, Southern filters were hybridized with six _VH -family specific probes. Filters were washed three times for 30 minutes under standard conditions (50 ° C., 1 × SSC, in 0.1% SDS) and then washed under more stringent conditions (65 ° C., 0.1 × SSC, in 0.1% SDS). The location of the _VH gene was determined by comparing cosmid hybridization patterns and their physical maps.

Theoretically, about 50 independent cosmid clones (about 7 times the total YAC insert DNA) are sufficient to cover the 300 kb long total YAC insert DNA. However, cosmid clone variability was heterogeneous and some gaps still remained. Clones corresponding to these gaps could not be isolated by chromosome walking by using a probe isolated from the Sau 3AI partial degradation library or the respective contig. Regions not present in the cosmid library were recovered by isolating a DNA fragment of the required size from YAC DNA and subcloning into a phage or plasmid as shown in FIG. After constructing a complete physical map, the inventors have found that such a heterogeneous distribution of cosmid clones is not due to a block of restriction enzyme sites in the YAC insert DNA. The reason why these regions cannot be cloned in the cosmid system or are less likely to appear in reality may be due to sequences such as palindromic or tandem repeat DNA. The 0.8 Mb region constructed in this example is shown in FIG. 1 as described above. Further, it summarizes the magnitude of the distance as well as Eco RI and Hin d III fragment from J _H of the V _H gene represented in Figure 1 in Tables 3 and 4.

Example 2 Preparation of Cosmid Clone Preparation of a cosmid library from human high molecular DNA was performed as follows.
3-31: High molecular DNA obtained from human placenta was partially digested with restriction enzyme Taq I, and after electrophoresis on 0.5% agarose gel, a 35-45 kb fraction was recovered using DEAE paper. The recovered DNA was treated with alkaline phosphatase and then ligated to a cosmid vector pJB8 that had been completely digested with the restriction enzyme ClaI . The ligation product was packaged in vitro, infected with host E. coli 490A, and then clones were obtained by screening by colony hybridization in the usual manner.

M131, M84, M118: Basically the same method as 3-31, except that the DNA used was human pro B cell line FLEB14-14, the vector and host E. coli were Lorist2, ED8767, and the restriction enzyme combination was Xba I. Using Hind III, stumps were made using the partial repair method. The partial modification method is performed by a known method (J. Sambrook, EF Fritsch and T. Maniatis, 1989, Molecular Cloning; a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). It was.

Example 3 Determination of _VH gene base sequence Instead of sequencing the subcloned _VH- containing fragment using a vector primer, a _VH family-specific oligonucleotide primer was synthesized. As described above, the base sequence of the FR region of the _VH gene is very similar within the same family. Therefore, the present inventors selected a consensus sequence from this highly conserved portion, and synthesized a family-specific oligonucleotide primer for sequence analysis. For this purpose, an automatic fluorescence sequencing system model 373A developed by Applied Biosystems was used. A dye-deoxy terminator sequencing kit supplied by the company using fluorescently labeled dideoxynucleotides was suitable for this purpose. This is because the synthetic V _H -specific primer could be used directly without fluorescent labeling.

(1) Subcloning of _VH- containing restriction enzyme fragments In order to use _VH family-specific primers for base sequencing, _VH- containing DNA fragments can be subcloned so that each plasmid contains only one _VH gene. is necessary. Several 6-base site recognition restriction enzymes other than Eco RI and Hind III were used to isolate DNA fragments containing a single _VH . Plasmid DNA of the subcloned fragment was isolated by the alkaline lysis method and then ultracentrifuged twice to obtain a high purity DNA sample for accurate sequencing.

(2) Oligonucleotide synthesis for nucleotide sequence determination In order to select a consensus sequence for _VH family-specific oligonucleotide primer synthesis, the nucleotide region of the known _VH gene framework region and exon-intron boundary is selected. It arranged for every family. The 3 ′ half is 100% identical to the standard sequence, and the most 3 ′ unterminated nucleotide corresponds to the first or second base of a highly conserved (universal) amino acid residue Careful to do. For the 5 _VH families, an additional 19 primers were designed as described in Table 1 (described below).

(3) Sequencing reaction and gel electrophoresis The sequencing reaction was performed by PCR using a dye-deoxy terminator sequencing kit (ABI) based on the manufacturer's instructions. In the sequencing apparatus, gel electrophoresis and signal detection were performed according to the user manual of the apparatus. On average, sequences of more than 350 bases could be determined from each reaction.

(4) Evaluation of synthetic _VH family-specific primers Primers F-univ and IR were first chosen to determine the sequence of the _VH-I gene. As shown in Table 2, they annealed with 11 of the 12 _VH-I genes analyzed. All six functional _VH-I genes could be sequenced with these two primers. I-NF1 and I-NR1 were designed for two genes, V1-14P and V1-27P. These two primers were also used for other _VH genes to confirm the sequence determined using the first two primers (Table 2).

Eight primers were designed and used for sequencing of _VH-III family genes. Initial sequencing reactions for each _VH gene were performed using F-univ and III-R primers. These annealed to over 80% of the analyzed _VH-III genes (F-univ 25/30, III-R 24/30) (Table 2). Importantly, and with one exception, all functional V _H-III genes could be sequenced with this primer combination. This suggests that they are effective for most _VH-III cDNAs.

Based on the nucleotide sequence determined in the first experiment, six additional primers (III-F3, III-R3, III-F4, III-R4, III-NFl and III-F2) were designed and their appropriate The combination was used for further analysis. Of these, III-R3 and III-F4 were used to determine the sequences of the 5 ′ regulatory region and the 3 ′ peripheral region, respectively. V3-29P and V3-32P are pseudogenes that are highly misaligned, and therefore all but III-NFl did not anneal to these two _VH genes. V3-25P, V3-44P and V3-63P were determined from the restriction enzyme recognition site inside the gene using M13 vector.

In order to determine the sequences of the _VH genes belonging to the _VH-II , _VH-IV and _VH-V families, 5 synthetic primers were used respectively. Since the V _H genes belonging to these three families are very homologous to each other, most of the V _H genes belonging to these small V _H families were considered to be sequenceable with 4 primers each. In fact, all four _VH-II family-specific primers annealed with three _VH-II genes (V2-5, V2-10P and V2-26).

In summary, F-univ and I-R for a total of 11 primers _{(V H-I;} for _{V H-III; V II-} R1 for _{H-II, II-F2} and II-R2 F-univ and III-R; _V IV-R1 for _{H-IV, IV-F2} and IV-R2, _{V V-R2} and V-R3 for _H-V) is five _{V H} families It is sufficient to sequence most of the _VH genes to which it belongs. II-F1, III-NF2 and IV-F1 primers have intron sequences and therefore cannot be used for cDNA sequencing.

By this method, the base sequences of 64 _VH genes were determined and are shown in SEQ ID NOs: 1 to 64 as described above. Tables 3 and 4 summarize the distance of each _VH gene from _JH and the sizes of the Eco RI and Hind III fragments.

(5) _VH gene transcription direction The method of determining the base sequence of a _VH gene using a family-specific primer is not suitable for determining the transcriptional direction of a _VH gene. This is because this method does not require a restriction map of the subcloned fragment containing a single _VH . For this reason, the present inventors have not been able to determine the directionality of all _VH genes. However, 8 regions containing 21 _VH genes have already been isolated with cosmids or phages. This is because the sequences between corresponding _VH genes and their restriction enzyme maps are identical to each other. Since the relative order of these 21 V _H genes in these clones is the same as the relative order in the 0.8 Mb region, the transcription direction of these 21 V _H genes is the J _H gene. It was concluded that the direction of transcription was the same.

It is a genetic map of the DNA fragment of about 0.8 Mb of this invention. It is a figure which shows the result of the Southern hybridization of typical YAC insertion DNA. A Southern hybridization peptidase fragment was digested with restriction enzymes Mlu I and Not I - is a diagram showing the results of Chillon. B is a diagram showing a physical map of a YAC clone constructed based on the result of A. FIG. It is a gene map of YAC clone Y6.

Claims (35)

Any of the following clones (a) to (i):
(A) a yeast artificial chromosome clone Y6 obtained from a transformant identified by International Deposit Number FERM BP-4271;
(B) a yeast artificial chromosome clone Y24 obtained from a transformant identified by International Deposit Number FERM BP-4274;
(C) a yeast artificial chromosome clone Y21 obtained from the transformant identified by International Deposit Number FERM BP-4273;
(D) a cosmid vector clone M118 obtained from the transformant identified by International Deposit Number FERM BP-4278;
(E) Cosmid vector clone M84 obtained from the transformant identified by International Deposit Number FERM BP-4277;
(F) Cosmid vector clone M131 obtained from the transformant identified by International Deposit Number FERM BP-4279;
(G) Cosmid vector clone 3-31 obtained from a transformant identified by International Deposit Number FERM BP-4276;
(H) a yeast artificial chromosome clone Y103 obtained from a transformant identified by international deposit number FERM BP-4275; or (i) a yeast artificial chromosome obtained from a transformant identified by international deposit number FERM BP-4272. Clone Y20
DNA comprising a human genome-derived DNA sequence obtained from (wherein said clones Y6, Y24, Y21, M118, M84, M131, 3-31, Y103 and Y20 Each has a relative position as shown in FIG. 1 in the human genome.) The DNA according to claim 1, wherein the human genome-derived DNA sequence obtained from the clone Y6 comprises the DNA sequence set forth in SEQ ID NO: 32 to SEQ ID NO: 64. The DNA according to claim 1, wherein the DNA sequence derived from the human Y genome obtained from the clone Y24 comprises the DNA sequence set forth in SEQ ID NO: 32 to SEQ ID NO: 64. The DNA according to claim 1, wherein the DNA sequence derived from the human genome obtained from the clone Y21 comprises the DNA sequence set forth in SEQ ID NO: 15 to SEQ ID NO: 34. The DNA according to claim 1, wherein the DNA sequence derived from the human genome obtained from the clone M118 comprises the DNA sequences shown in SEQ ID NO: 14 and SEQ ID NO: 15. The DNA according to claim 1, wherein the DNA sequence derived from the human genome obtained from the clone M84 comprises the DNA sequence shown in SEQ ID NO: 9 to SEQ ID NO: 13. The DNA according to claim 1, wherein the DNA sequence derived from the human genome obtained from the clone M131 comprises the DNA sequence shown in SEQ ID NO: 9 to SEQ ID NO: 13. The DNA according to claim 1, wherein the DNA sequence derived from the human genome obtained from the clone 3-31 comprises the DNA sequence set forth in SEQ ID NO: 6 to SEQ ID NO: 8. The DNA according to claim 1, wherein the DNA sequence derived from the human genome obtained from the clone Y103 comprises the DNA sequence described in SEQ ID NO: 1 to SEQ ID NO: 5. The DNA according to claim 1, wherein the DNA sequence derived from the human genome obtained from the clone Y20 comprises the DNA sequence described in SEQ ID NO: 1 to SEQ ID NO: 4. Any of the following clones (a) to (i):
(A) a yeast artificial chromosome clone Y6 obtained from a transformant identified by International Deposit Number FERM BP-4271;
(B) a yeast artificial chromosome clone Y24 obtained from a transformant identified by International Deposit Number FERM BP-4274;
(C) a yeast artificial chromosome clone Y21 obtained from the transformant identified by International Deposit Number FERM BP-4273;
(D) a cosmid vector clone M118 obtained from the transformant identified by International Deposit Number FERM BP-4278;
(E) Cosmid vector clone M84 obtained from the transformant identified by International Deposit Number FERM BP-4277;
(F) Cosmid vector clone M131 obtained from the transformant identified by International Deposit Number FERM BP-4279;
(G) Cosmid vector clone 3-31 obtained from a transformant identified by International Deposit Number FERM BP-4276;
(H) a yeast artificial chromosome clone Y103 obtained from a transformant identified by international deposit number FERM BP-4275; or (i) a yeast artificial chromosome obtained from a transformant identified by international deposit number FERM BP-4272. Clone Y20
At least the following genes (1) and (2) contained in the human genome-derived DNA sequence obtained from:
(1) any two functional V _H genes; and (2) DNA comprising any two other two functional V _H genes different from the gene of (1) above, wherein the clone Each of the DNA sequences derived from the human genome obtained from each of Y6, Y24, Y21, M118, M84, M131, 3-31, Y103 and Y20 has a relative position as shown in FIG. 1 in the human genome. ). The DNA according to claim 11, wherein the human genome-derived DNA sequence obtained from the clone Y6 comprises the DNA sequence set forth in SEQ ID NO: 32 to SEQ ID NO: 64. The DNA according to claim 11, wherein the human genome-derived DNA sequence obtained from the clone Y24 comprises a DNA sequence represented by SEQ ID NO: 32 to SEQ ID NO: 64. The DNA according to claim 11, wherein the human genome-derived DNA sequence obtained from the clone Y21 comprises the DNA sequence set forth in SEQ ID NO: 15 to SEQ ID NO: 34. The DNA according to claim 11, wherein the DNA sequence derived from the human genome obtained from the clone M118 comprises the DNA sequences shown in SEQ ID NO: 14 and SEQ ID NO: 15. The DNA according to claim 11, wherein the DNA sequence derived from the human genome obtained from the clone M84 comprises the DNA sequence set forth in SEQ ID NO: 9 to SEQ ID NO: 13. The DNA according to claim 11, wherein the DNA sequence derived from the human genome obtained from the clone M131 comprises the DNA sequence set forth in SEQ ID NO: 9 to SEQ ID NO: 13. The DNA according to claim 11, wherein the DNA sequence derived from the human genome obtained from the clone 3-31 comprises the DNA sequence set forth in SEQ ID NO: 6 to SEQ ID NO: 8. The DNA according to claim 11, wherein the DNA sequence derived from the human genome obtained from the clone Y103 comprises the DNA sequence described in SEQ ID NO: 1 to SEQ ID NO: 5. The DNA according to claim 11, wherein the human genome-derived DNA sequence obtained from the clone Y20 comprises the DNA sequence described in SEQ ID NO: 1 to SEQ ID NO: 4. 21. A DNA for producing a non-human mammal producing an immunoglobulin heavy chain comprising a variable region derived from a human _VH gene, wherein the DNA is any one of claims 1 to 20. DNA comprising the DNA of The following DNA sequences (a) to (f):
(A) (i) or (ii) below:
(i) a yeast artificial chromosome clone Y6 obtained from a transformant identified by international deposit number FERM BP-4271, or
(ii) a human genome-derived DNA sequence obtained from any of the yeast artificial chromosome clone Y24 obtained from the transformant identified by International Deposit Number FERM BP-4274;
(B) a DNA sequence derived from the human genome obtained from the yeast artificial chromosome clone Y21 obtained from the transformant identified by the international deposit number FERM BP-4273 (here, the DNA sequence derived from the human genome obtained from the clone Y21 is: 5 'terminal sequence of the DNA sequence, which is a DNA sequence excluding a sequence overlapping with the 3' terminal sequence of the DNA sequence of (a).
(C) a DNA sequence derived from the human genome obtained from the cosmid vector clone M118 obtained from the transformant identified by the international deposit number FERM BP-4278 (here, the DNA sequence derived from the human genome obtained from the clone M118 A DNA sequence excluding a sequence that is the 5 ′ end sequence of the DNA sequence and overlaps with the 3 ′ end sequence of the DNA sequence of (b));
(D) a DNA sequence derived from a human genome obtained from a cosmid vector clone M84 obtained from a transformant identified by the international deposit number FERM BP-4277 (here, the DNA sequence derived from the human genome obtained from the clone M84 is 5 'terminal sequence of the DNA sequence, which is a DNA sequence excluding a sequence overlapping with the 3' terminal sequence of the DNA sequence of (c) above);
(E) a human genome-derived DNA sequence obtained from the cosmid vector clone M131 obtained from the transformant identified by the international deposit number FERM BP-4279 (here, the human genome-derived DNA sequence obtained from the clone M131 (5) the sequence of the 5 ′ end of the DNA sequence, excluding the sequence that overlaps the 3 ′ end sequence of the DNA sequence of (d) above)); DNA sequence derived from human genome obtained from cosmid vector clone 3-31 obtained from the transformant to be identified (here, the DNA sequence derived from human genome obtained from clone 3-31 is the 5 ′ end sequence of the DNA sequence) And a DNA sequence excluding a sequence overlapping with the 3 ′ terminal sequence of the DNA sequence of (e).
A vector comprising DNA comprising 5 ′ to 3 ′ in order, wherein each of the DNA sequences derived from the human genome obtained from each of the clones Y6, Y24, Y21, M118, M84, M131 and 3-31 Has a relative position as shown in FIG. 1 in the human genome). The vector according to claim 22, wherein the DNA is a DNA having a restriction enzyme recognition site pattern as shown in FIG. The DNA has the following features (1) to (7):
(1) The DNA sequence derived from the human genome obtained from the clone Y6 includes the DNA sequence set forth in SEQ ID NO: 32 to SEQ ID NO: 64;
(2) The DNA sequence derived from the human genome obtained from the clone Y24 includes the DNA sequence set forth in SEQ ID NO: 32 to SEQ ID NO: 64;
(3) The DNA sequence derived from the human genome obtained from the clone Y21 includes the DNA sequence set forth in SEQ ID NO: 15 to SEQ ID NO: 34;
(4) The DNA sequence derived from the human genome obtained from the clone M118 includes the DNA sequences set forth in SEQ ID NO: 14 and SEQ ID NO: 15;
(5) The DNA sequence derived from the human genome obtained from the clone M84 includes the DNA sequence set forth in SEQ ID NO: 9 to SEQ ID NO: 13;
(6) The human genome-derived DNA sequence obtained from the clone M131 includes the DNA sequences set forth in SEQ ID NO: 8 and SEQ ID NO: 9; and (7) the human genome-derived DNA sequence obtained from the clone 3-31 24. The vector according to claim 22 or 23, wherein the vector comprises the DNA sequence described in SEQ ID NO: 6 to SEQ ID NO: 8. DNA sequences of (a) and (b) below:
(A) a DNA sequence derived from a human genome obtained from a yeast artificial chromosome clone Y103 obtained from a transformant identified by International Deposit Number FERM BP-4275; and (b) a trait identified by International Deposit Number FERM BP-4272 DNA sequence derived from the human genome obtained from the yeast artificial chromosome clone Y20 obtained from the transformant (where the DNA sequence derived from the human genome obtained from the clone M20 is the 5 ′ end sequence of the DNA sequence, The DNA sequence excluding the sequence overlapping with the 3 ′ end sequence of the DNA sequence of
Vector having DNA comprising 5 ′ to 3 ′ in order (wherein each of the DNA sequences derived from the human genome obtained from each of the clones Y103 and Y20 is relative to the human genome as shown in FIG. Have a typical position.) The vector according to claim 25, wherein the DNA is a DNA having a restriction enzyme recognition site pattern as shown in FIG. The DNA has the following features (1) and (2):
(1) The human genome-derived DNA sequence obtained from the clone Y103 includes the DNA sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 5; and (2) the human genome-derived DNA sequence obtained from the clone Y20 is a sequence Comprising the DNA sequence set forth in No. 1 to SEQ ID No. 4;
27. The vector according to claim 25 or claim 26, wherein the vector is a DNA having. The following DNA sequences (1) to (28):
(1) the DNA sequence set forth in SEQ ID NO: 64;
(2) the DNA sequence set forth in SEQ ID NO: 61;
(3) the DNA sequence set forth in SEQ ID NO: 59;
(4) the DNA sequence set forth in SEQ ID NO: 53;
(5) the DNA sequence set forth in SEQ ID NO: 51;
(6) the DNA sequence set forth in SEQ ID NO: 49;
(7) the DNA sequence set forth in SEQ ID NO: 48;
(8) the DNA sequence set forth in SEQ ID NO: 46;
(9) the DNA sequence set forth in SEQ ID NO: 45;
(10) the DNA sequence set forth in SEQ ID NO: 43;
(11) the DNA sequence set forth in SEQ ID NO: 39;
(12) the DNA sequence set forth in SEQ ID NO: 35;
(13) the DNA sequence set forth in SEQ ID NO: 34;
(14) the DNA sequence set forth in SEQ ID NO: 33;
(15) the DNA sequence set forth in SEQ ID NO: 31;
(16) the DNA sequence set forth in SEQ ID NO: 30;
(17) the DNA sequence set forth in SEQ ID NO: 28;
(18) the DNA sequence set forth in SEQ ID NO: 26;
(19) the DNA sequence set forth in SEQ ID NO: 23;
(20) the DNA sequence set forth in SEQ ID NO: 21;
(21) the DNA sequence set forth in SEQ ID NO: 20;
(22) the DNA sequence set forth in SEQ ID NO: 18;
(23) the DNA sequence set forth in SEQ ID NO: 15;
(24) the DNA sequence set forth in SEQ ID NO: 13;
(25) the DNA sequence set forth in SEQ ID NO: 11;
(26) the DNA sequence set forth in SEQ ID NO: 9;
(27) a DNA sequence set forth in SEQ ID NO: 8; and (28) a vector comprising DNA comprising the DNA sequence set forth in SEQ ID NO: 7 in the order of 5 ′ to 3 ′ (wherein the DNA is In each of the two DNA sequences adjacent to each other of (1) to (28), either one of the following clones (a) or (b):
(A) a yeast artificial chromosome clone obtained from a transformant identified by either international deposit number FERM BP-4271, FERM BP-4273 or FERM BP-4274; or (b) international deposit number FERM BP-4276, It has a part of the human genome-derived DNA sequence obtained from a cosmid vector clone obtained from a transformant identified by either FERM BP-4277, FERM BP-4278 or FERM BP-4279). The following DNA sequences (1) to (5):
(1) the DNA sequence set forth in SEQ ID NO: 5;
(2) the DNA sequence set forth in SEQ ID NO: 4;
(3) the DNA sequence set forth in SEQ ID NO: 3;
(4) a DNA sequence set forth in SEQ ID NO: 2; and (5) a vector having DNA comprising the DNA sequence set forth in SEQ ID NO: 1 in the order of 5 ′ to 3 ′ (wherein the DNA is The yeast artificial chromosome obtained from the transformant identified by either the international deposit number FERM BP-4272 or FERM BP-4275, between each two adjacent DNA sequences of (1) to (5) Having a portion of the human genome-derived DNA sequence obtained from the clone). Any of the following clones (a) to (e):
(A) Yeast artificial chromosome Y6 obtained from the transformant identified by International Deposit Number FERM BP-4271;
(B) Yeast artificial chromosome Y24 obtained from the transformant identified by International Deposit Number FERM BP-4274;
(C) Yeast artificial chromosome Y21 obtained from the transformant identified by international deposit number FERM BP-4273;
(D) Yeast artificial chromosome Y103 obtained from the transformant identified by international deposit number FERM BP-4275; or (e) Yeast artificial chromosome Y20 obtained from the transformant identified by international deposit number FERM BP-4272.
A vector comprising a human genome-derived DNA sequence obtained from The vector is a clone of any one of the following (a) to (e):
(A) Yeast artificial chromosome Y6 obtained from the transformant identified by International Deposit Number FERM BP-4271;
(B) Yeast artificial chromosome Y24 obtained from the transformant identified by International Deposit Number FERM BP-4274;
(C) Yeast artificial chromosome Y21 obtained from the transformant identified by international deposit number FERM BP-4273;
(D) Yeast artificial chromosome Y103 obtained from the transformant identified by international deposit number FERM BP-4275; or (e) Yeast artificial chromosome Y20 obtained from the transformant identified by international deposit number FERM BP-4272.
The vector according to claim 30, wherein Any of the following clones (a) to (d):
(A) a cosmid vector clone M118 obtained from a transformant identified by international deposit number FERM BP-4278;
(B) Cosmid vector clone M84 obtained from the transformant identified by International Deposit Number FERM BP-4277;
(C) Cosmid vector clone M131 obtained from a transformant identified by international deposit number FERM BP-4279; or (d) Cosmid vector clone 3 obtained from a transformant identified by international deposit number FERM BP-4276. -31;
A vector comprising a human genome-derived DNA sequence obtained from The clone is any one of the following clones (a) to (d):
(A) a cosmid vector clone M118 obtained from a transformant identified by international deposit number FERM BP-4278;
(B) Cosmid vector clone M84 obtained from the transformant identified by International Deposit Number FERM BP-4277;
(C) Cosmid vector clone M131 obtained from a transformant identified by international deposit number FERM BP-4279; or (d) Cosmid vector clone 3 obtained from a transformant identified by international deposit number FERM BP-4276. -31;
The vector according to claim 32, wherein A cell transformed with any one of the DNA according to any one of claims 1 to 21 or a part of the DNA, or the vector according to any one of claims 22 to 33. The cell is any one of the following (a) to (i):
(A) a transformed cell identified by international deposit number FERM BP-4271;
(B) a transformed cell identified by International Deposit Number FERM BP-4272;
(C) a transformed cell identified by International Deposit Number FERM BP-4273;
(D) a transformed cell identified by International Deposit Number FERM BP-4274;
(E) a transformed cell identified by International Deposit Number FERM BP-4275;
(F) a transformed cell identified by International Deposit Number FERM BP-4276;
(G) a transformed cell identified by International Deposit Number FERM BP-4277;
35. The cell according to claim 34, which is (h) a transformed cell identified by international deposit number FERM BP-4278; or (i) a transformed cell identified by international deposit number FERM BP-4279. .

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