AU745420B2 - Human catalytic telomerase sub-unit and its diagnostic and therapeutic use - Google Patents

Description

WO 98/59040 PCT/EP98/03468 -1- Catalytic subunit of human telomerase and its diagnostic and therapeutic use Structure and function of the chromosome ends The genetic material of eukaryotic cells is distributed on Linear chromosomes. The ends of these hereditary units are termed telomeres, derived from the Greek words telos (end) and meros (part or segment). Most telomeres consist of repeats of short sequences which are mainly constructed from thymine and guanine (Zakian, 1995). The telomere sequences of related organisms are often similar and these sequences are even conserved between species which are more phylogenetically remote. It is a remarkable fact that the telomeres are constructed from the sequence TTAGGG in all the vertebrates which have so far been examined (Meyne et al., 1989).

The telomeres exert a variety of important functions. They prevent the fusion of chromosomes (McClintock, 1941) and consequently the formation of dicentric hereditary units. Chromosomes of this nature, possessing two centromeres, can lead to the development of cancer due to loss of heterozygosity or the duplication or loss of genes.

In addition, telomeres serve the purpose of distinguishing intact hereditary units from damaged hereditary units. Thus, yeast cells ceased dividing when they harboured a chromosome which lacked a telomere (Sandell and Zakian, 1993).

Telomeres carry out another important task in association with DNA replication in eukaryotic cells. In contrast to the circular genomes of prokaryotes, the Linear chromosomes of eukaryotes cannot be completely replicated by the DNA polymerase complex. RNA primers are required for initiating DNA replication. After the RNA primers have been eliminated and the Okazaki fragments have been extended and then ligated, the newly synthesized DNA strand lacks the 5' end because the RNA primer at that point cannot be replaced with DNA.

For this reason, without special protective mechanisms, the chromosomes would shrink with every cell division ("end-replication problem", Harley et al., 1990). The non-coding telomere -o l2'\.NT O 11 sequences probably represent a buffer zone for preventing the loss of genes (Sandell and Zakian, 1993).

Over and above this, telomeres also play an important role in regulating cell ageing (Olovnikov, 1973). Human somatic cells exhibit a limited capacity to replicate in culture; after a certain time they become senescent. In this condition, the cells no longer divide even after being stimulated with growth factors; however, they do not die but remain metabolically active (Goldstein, 1990). Various observations provide support for the hypothesis that a cell determines from the length of its telomeres how often it can still divide (Allsopp et al., 1992).

In summary, the telomeres consequently possess central functions in the ageing of cells and in the stabilization of the genetic material and prevention of cancer.

The enzyme telomerase synthesizes the telomeres As described above, organisms possessing Linear chromosomes are only able to replicate their genomes incompletely in the absence of a special protective mechanism. Most eukaryotes use a special enzyme, i.e. telomerase, to regenerate the telomere sequences.

Telomerase is expressed constitutively in the single-cell organisms which have so far been examined. By contrast, in humans, telomerase activity was only detected in germ cells and tumour cells whereas neighbouring somatic tissue did not contain any telomerase (Kim et al., 1994).

Telomerase in ciliates Like the telomeres, telomerase was identified for the first time in the ciliate Tetrahymena thermophila. Telomerase activity was detected by extending the single-stranded oligonucleotide d(TTGGGG) 4 in the presence of dTTP and dGTP (Greider and Blackburn, 1985). In this reaction, the Tetrahymena telomere sequence TTGGGG was added repeatedly to the primer. Even when an oligonucleotide having the irregular telomere sequence of Saccharomyces cerevisiae, T(G)1- 3 was offered as the starting material, the telomerase -3extended the primer with the telomere sequence of Tetrahymena (Greider and Blackburn, 1985). From these results, it was concluded that the telomerase itself carries the template for the sequence of the telomeres.

Once the existence of an RNA component in the telomerase had initially been demonstrated (Greider and Blackburn, 1987), the gene for the RNA subunit of the telomerase was cloned a short while later (Greider and Blackburn, 1989). This RNA contains a region which is complementary to the Tetrahymena telomere sequence (termed "complementary region" below). The activity of the telomerase depended on the RNA component, as was demonstrated by digesting the RNA, leading in turn to subsequent loss of activity. If the complementary region of the telomerase RNA was mutated, the corresponding mutations were incorporated in vivo into the Tetrahymena telomeres (Yu et al., 1990). Telomerase consequently belongs to the class of RNA-dependent DNA polymerases.

The first protein subunits of the Tetrahymena telomerase, i.e. p80 and p95, were identified in 1995 (Collins et al., 1995). The observation that p95 anchors the enzyme to the DNA and binds the RNA component led to the following model: the telomerase RNA anneals by its complementary region to the single-stranded 3' overhang. The 3' overhang is extended by incorporating the corresponding nucleotides in the direction. The de novo synthesis of telomeres probably involves an elongation step and a translocation step. Once a telomere sequence has been synthesized, the telomerase presumably moves along the DNA until it is once again in a position to be able to add a complete telomere sequence. This model does not have to be generally valid since great differences exist between the telomerases of different species with regard to the number of nucleotides which the enzyme adds before it dissociates from the telomere (Prowse et al., 1993).

In addition to this, telomerase subunits from other organisms have also recently been identified. Two protein subunits, i.e. p123 and p43, which do not exhibit any homology with the Tetrahymena telomerase proteins, have been found in the ciliate Euplotes aediculatus.

The telomerase subunit p123 exhibits a basic domain at its N terminus and a domain for a reverse transcriptase (RT) at the C terminus, suggesting this protein has a catalytic function, -4- (Lingner et al., 1997). Furthermore, p123 has been reported to share significant homology with the Saccharomyces cerevisiae protein Est2 which was found by Lundblad (Lingner et al., 1997).

Whereas p80 and p95 have not hitherto been demonstrated to possess any function which is essential for telomerase activity, the potential catalytic telomerase subunits p123/est2p have been unambiguously shown to have a key function: mutation of the active centre of the est2p RT led to significant truncation of the telomeres in yeast cells (Lingner et al., 1997).

Telomerase components from mammalian cells The RNA components of the telomerases of various organisms, inter alia of Saccharomyces cerevisiae, mice and humans (Singer and Gottschling, 1994; Blasco et al., 1996; Feng et al., 1995), have by now been cloned. All the telomerase RNAs known to date comprise a region which is complementary to the telomere sequence of a particular organism. However, the primary sequence of the human telomerase RNA (hTR) does not display any similarity to the RNA components of the ciliates or of Saccharomyces cerevisiae. On the other hand, regions exist which are conserved between human and murine telomerase RNA (Feng et al., 1995).

The isolation of a human telomerase-associated protein (hTPl) has recently been described (Hamngton et al., 1997). On the basis of its homology with the Tetrahymena telomerase subunit, the corresponding gene was found in an EST data base which is not available to the general public (Harrington et al., 1997). hTPl is composed of 2627 amino acids and, in the N-terminus, exhibits three domains which possess at most 46% homology with p 8 0 16 repeats of the amino acids tryptophan and asparagine, which presumably mediate a protein/protein interaction, were shown to be present, as an additional structural element, in the C-terminal region.

Activation of the telomerase in human tumours In humans, it was originally only possible to demonstrate telomerase activity in germ line cells and not in normal somatic cells (Hastie et al., 1990; Kim et al., 1994). After a more sensitive detection method had been developed (Kim et al., 1994) a low level of telomerase activity was also detected in hematopoietic cells (Broccoli et al., 1995; Counter et al., 1995; Hiyama et al., 1995). However, these cells nevertheless exhibited a reduction in the telomeres (Vaziri et al., 1994; Counter et al., 1995). It has still not been clarified whether the quantity of enzyme in these cells is insufficient to compensate for the telomere loss or whether the measured telomerase activity stems from a subpopulation, e.g. of incompletely differentiated CD34 38 precursor cells (Hiyama et al., 1995). In order to clarify this point, it would be necessary to detect the telomerase activity which was present in a single cell.

Interestingly enough, however, significant telomerase activity has been detected in a large number of the tumour tissues which have been tested to date (1734/2031, 85%; Shay, 1997), whereas no activity has been found in normal somatic tissue (1/196, Shay, 1997). In addition, a variety of investigations demonstrated that the telomeres continued to shrink in senescent cells which were transformed with viral oncoproteins and that it was only possible to find telomerase in the subpopulation which survived the growth crisis (Counter et al., 1992). The telomeres were also stable in these immortalized cells (Counter et al., 1992).

Similar findings derived from investigations in mice (Blasco et al., 1996) support the assumption that reactivation of the telomerase is a late event in tumorigenesis.

Based on these results, a "telomerase hypothesis" was developed which links the loss of telomere sequences and cell ageing to telomerase activity and the genesis of cancer. In longlived species such as humans, the shrinking of the telomeres can be regarded as a tumour suppression mechanism. Differentiated cells, which do not contain any telomerase, cease dividing when the telomeres have reached a particular length. If such a cell mutates, a tumour can only develop from it if the cell is able to extend its telomeres. Otherwise, the cell would continue to lose telomere sequences until its chromosomes became unstable and it finally died. Reactivation of the telomerase is presumably the main mechanism which tumour cells deploy in order to stabilize their telomeres.

-6- It follows from these observations and ideas that it should be possible to develop a therapy for tumours based on inhibiting telomerase activity. Conventional cancer therapies using cytostatic agents or short-wave irradiation damage all the dividing cells in the body in addition to damaging the tumour cells. However, since it is only germ line cells which contain significant telomerase activity, apart from tumour cells, telomerase inhibitors would attack the tumour cells more specifically and consequently evoke fewer undesirable side effects. Since telomerase activity has been detected in all the tumour tissues tested to date, it would be possible to employ these therapeutic agents against all types of cancer. The effect of telomerase inhibitors would then set in when the telomers of the cells had shortened to such an extent that the genome had become unstable. Since tumour cells usually exhibit shorter telomeres than do normal somatic cells, it would be cancer cells which would first of all be eliminated by telomerase inhibitors. By contrast, cells possessing long telomeres, such as the germ cells, would not be damaged until a much later stage. Telomerase inhibitors consequently represent an approach which points the way forward for cancer therapy.

However, it will only be possible to provide unambiguous answers to questions regarding the nature and the points of attack of physiological telomerase inhibitors when the protein structures of the enzyme, together with their functions, have also been identified and a deeper understanding of the various telomere-binding proteins has been obtained.

The invention relates to the catalytically active human telomerase subunit (phTC), where appropriate in purified form, to active moieties of the protein, to modulators, in particular agonists of the protein, to substances which imitate the function of the protein and to combinations of these components.

The invention furthermore relates to: The nucleic acid sequence which encodes the human protein phTC, specifically: the genomic sequence of the hTC gene, the cDNA sequence of the hTC gene, the DNA sequence of hTC variants the sequence of the mRNA which is transcribed from the hTC gene, parts of the abovementioned sequences, including the DNA sequence (SEQ ID No. 1) of hTC which is shown in Fig. 1.

The nucleic acid sequences which encode hTC-homologous proteins in other mammals, specifically: the genomic sequences of hTC-homologous genes, the cDNA sequences of hTC-homologous genes, the sequences of the mRNAs which are transcribed from hTC-homologous genes, parts of the abovementioned sequences.

Nucleic acid sequences which, in humans and other mammals, encode proteins which are related to the phTC protein, specifically: the genomic sequences ofhTC-related genes in humans and other mammals, the cDNA sequences of hTC-related genes in humans and other mammals, the sequences of the mRNAs which are transcribed from hTC-related genes in humans and other mammals, parts of the abovementioned sequences.

The above-described phTC protein, which is isolated from mammalian cells (cf.

Fig. 2 and SEQ ID No. 2).

The phTC protein which is labelled with a detection reagent, with the detection reagent preferably being an enzyme, a radioactively labelled element or a fluorescent chemical.

An antibody which is directed against the phTC protein.

i I According to a preferred embodiment, this antibody is a polyclonal antibody.

According to another preferred embodiment, this antibody is a monoclonal antibody.

Antibodies of this nature can be produced, for example, by injecting a host, which is substantially immunocompetent, with a quantity of a phTC polypeptide, or a fragment thereof, which is effective for producing the antibody, and by subsequently isolating this antibody.

In addition, an immortalized cell line which produces monoclonal antibodies can be obtained in a manner known per se.

Where appropriate, the antibodies can be labelled with a detection reagent.

Fragments which possess the desired specific binding properties can also be employed instead of the complete antibody.

Preferred examples of such a detection reagent are enzymes, radioactively labelled elements, fluorescent chemicals or biotin.

Oligonucleotides in purified form which have a sequence which is identical or exactly complementary to a contiguous sequence, of from 10 to 500 nucleotides in length, of the above-described genomic DNA, cDNA or mRNA.

An oligonucleotide of this nature can, in particular, be an oligodeoxy-ribonucleotide or an oligoribonucleotide or a peptide nucleic acid (PNA).

Preference is given to oligonucleotides which inhibit, repress or block the activity of the telomerase when they bind to the hTC mRNA.

L- -9- A DNA sequence, or a degenerate variation of this sequence, which encodes the phTC protein, or a fragment of this protein, where appropriate comprising the DNA sequence in Figure 1 a, or a DNA sequence which hybridizes with the previously cited DNA sequence under standard hybridization conditions.

A recombinant DNA molecule which comprises a DNA sequence, or a degenerate variation of this sequence, which encodes phTC or a fragment of phTC, with the latter sequence preferably comprising the DNA sequence in Figure 1 a, or which comprises a DNA sequence which hybridizes with the previously cited DNA sequence under standard hybridization conditions.

In the abovementioned recombinant DNA molecule, the described DNA is preferably linked to an expression control sequence.

Examples of expression control sequences which are particularly preferred are the early or late promoter of the SV40 virus or adenovirus, the lac system, the trp system, the TAG system, the TRC system, the main operator and promoter regions of phage X, the control regions of the fd coat protein, the 3-phosphoglycerate kinase promoter, the acid phosphatase promoter and the yeast a-mating factor promoter.

A single-cell host which has been transformed with the above-described recombinant DNA molecule which comprises the DNA sequence, or a degenerate variation of this sequence, which encodes the phTC protein or a part of this protein. In this recombinant DNA molecule, the said DNA sequence is linked to an expression control sequence.

Preferred examples of the single-cell host are: E coli, Pseudomonas, Bacillus, Streptomyces, yeasts, CHO, RI1, B-W, L-M, COS 1, COS 7, BSC1, BSC40 and BMT 10 cells, plant cells, insect cells and mammalian cells in cell culture.

A recombinant virus which is transformed with one of the previously described DNA molecules or a derivative or fragment of this molecule.

A method for inhibiting telomerase activity in human cells, preferably neoplastic cells, in which an exogenous polynucleotide which consists of a transcription unit is transferred into the cells. This transcription unit comprises a polynucleotide sequence of at least 29 consecutive nucleotides, which sequence is substantially identical or substantially complementary to the hTC RNA sequence and is linked to a heterologous transcription-regulating sequence which controls the transcription of the linked polynucleotide in the said cells.

Preferably, the abovementioned heterologous transcription-regulating sequence comprises a promoter which is constitutively active in human cells.

Alternatively, the heterologous transcription-regulating sequence can comprise a promoter which can be induced or repressed in human cells by adding a regulatory substance. Examples of such promoters are inducible and repressible tetracyclinedependent promoters, heat shock promoters and metal ion-dependent promoters.

The abovementioned exogenous polynucleotide can, for example, be a viral genome containing a transcription unit from the human hTC DNA component.

Particularly preferably, the said transcription unit produces antisense RNA which is substantially complementary to the human hTC RNA component.

Particular preference is also given to the exogenous polynucleotide being able to comprise the sequence in Fig. la.

A polynucleotide for the genetic therapy of a human disease. This polynucleotide consists of a transcription unit which comprises a polynucleotide sequence of at least 9 consecutive nucleotides, which sequence is substantially identical or substantially -11complementary to the hTC RNA sequence and is linked to a heterologous transcription-regulating sequence which controls the transcription of the linked polynucleotide in said cells.

A method for detecting telomerase-associated conditions in a patient, which method comprises the following steps: A. Detecting the phTC protein in body fluids or cell samples in order to obtain a diagnostic value; B. Comparing the diagnostic value with standard values for the phTC protein in standardized normal cells or body fluids of the same type as the test sample; C. Detecting diagnostic values which are higher or lower than the standard comparative values and which indicate a telomerase-associated condition, which condition in turn indicates a pathogenic condition.

This method is preferably employed for detecting a neoplastic disease in a patient.

The method then comprises the following steps: A. Detecting the phTC protein in cell samples in order to obtain a diagnostic value; B. Comparing the diagnostic value with standard values for the phTC protein in non-neoplastic cells of the same type as the test sample; C. Diagnostic values which are clearly higher than standard comparative values indicate a neoplastic condition.

A method for determining the presence of the phTC protein in a cell or cell sample, which method is based on amplifying an hTC polynucleotide, or hybridizing an hTC polynucleotide, a primer or an hTC-complementary sequence with an hTC polynucleotide.

A test kit for detecting phTC in cell samples and body fluids, with it being possible, for example, for labelled, immunochemically-reactive components to be: polyclonal -12antibodies against phTC, monoclonal antibodies against phTC, fragments of these antibodies or a mixture of these components.

A method for preventing and/or treating cell disturbance or destruction and/or malfunction and/or other symptoms in humans, which method is based on administering a therapeutically effective quantity of catalytically active human telomerase, its functional equivalents or its catalytically active fragments. It is also possible to conceive of using a substance which promotes the production and/or activity of phTC; a substance which can imitate the activity of phTC; a substance which can inhibit the production and/or activity of phTC, or a mixture of these substances. A specific binding partner can also be employed.

The method is preferably employed for preventing or treating ageing or cancer diseases.

Substances which are able to affect the activity of phTC, i.e. inhibit or promote, are here termed modulators. Such modulators can be found, in a manner known per se, by testing their effect on telomerase activity in a telomerase assay. Examples of telomerase assays are given in Example Modulators of phTC are of interest for treating diseases which are connected with telomerase. The prevention or treatment of ageing processes or of cancer diseases may, in particular, be mentioned in this context.

An antisense nucleic acid against the hTC mRNA, which nucleic acid comprises a nucleotide sequence which hybridizes with the said mRNA, with the antisense nucleic acid being an RNA or a DNA.

Preferably, the antisense nucleic acid binds to the start codon of the particular mRNA.

-13- A recombinant DNA molecule which contains a DNA sequence from which an antisense ribonucleic acid against the hTC mRNA is produced during transcription.

This said antisense ribonucleic acid comprises a nucleic acid sequence which can hybridize to the said hTC mRNA.

A DNA molecule of this nature can be used to prepare a cell line having a reduced expression of phTC by transfecting a phTC-producig cell line with this recombinant DNA molecule.

A ribozyme which cleaves the hTC mRNA.

This ribozyme is preferably a Tetrahymena-type ribozyme or a hammerhead-type ribozyme.

A recombinant DNA molecule which contains a DNA sequence whose transcription leads to the production of a ribozyme of this nature.

This recombinant DNA molecule can be used to transfect a phTC-producing cell line.

A combination which consists of a pair of human hTC polynucleotide PCR primers, with the primers preferably consisting of sequences which correspond to the sequence of the human hTC mRNA or which are complementary to this sequence.

A combination which comprises a polynucleotide hybridization probe for the human hTC gene, with the probe preferably comprising at least 29 consecutive nucleotides which correspond to the sequence of the human hTC gene or which are complementary to this sequence.

Animal models which can be used to investigate telomerase/telomere regulation in vivo. Thus, tumour development and ageing can, for example, be directly investigated using knockout animals or transgenic animals.

Le A 32 486-Foreign countries -14- In the case of proteins or peptides, functional equivalents are those compounds which, while being distinguishable with regard to amino acid sequence, essentially have the same functions.

Known examples of these compounds are isoenzymes or so-called microheterogeneities in proteins.

In the case of the oligonucleic or polynucleic acids, functional equivalents are to be understood as being those compounds which differ in nucleotide sequence but which encode the same protein. The existence of such compounds may be attributed, for example, to the fact that the genetic code is degenerate.

9.

In accordance with the description herein before, in one aspect the invention as claimed herein after provides functional equivalents, variants and catalytically active fragments of oo9...

the catalytically active human telomerase subunit in isolated or purified form, comprising the amino acid sequence depicted in Fig. 2, characterized in that they comprise an amino *r 9 S•acid sequence encoded by the DNA sequence depicted in Fig. 1 with a deletion of 182 bp in length extending from nucleotide 2345 to 2526, the DNA sequence depicted in Fig. 1 with a deletion of 36 bp in length extending from nucleotide 2184 to 2219, the DNA sequence depicted in Fig. 1 with a deletion of 36 bp in length extending from nucleotide 2184 to 2219 and a deletion of 182 bp in length extending from nucleotide 2345 to 2526 or the DNA sequence depicted in Fig. 14.

9 9.

9 •9 In addition, the invention as claimed provides nucleic acid sequences in isolated or purified form, encoding functional equivalents, variants and catalytically active fragments of the catalytically active human telomerase subunit comprising the amino acid sequence depicted in Fig. 2, characterized in that they comprise the DNA sequence depicted in Fig. 1 with a deletion of 182 bp in length extending from nucleotide 2345 to 2526, the DNA sequence depicted in Fig. 1 with a deletion of 36 bp in length extending from nucleotide 2184 to 2219, the DNA sequence depicted in Fig. 1 with a deletion of 36 bp in length extending from P:\WPDOCS\CRN\ShcllycSpc\7462436.spe.doc-29/01/02 -14anucleotide 2184 to 2219 and a deletion of 182 bp in length extending from nucleotide 2345 to 2526 or the DNA sequence depicted in Fig. 14.

Further, the invention as claimed provides: i) antisense nucleic acids in isolated or purified form which bind to the nucleic acid sequence according to the previous paragraph herein; (ii) antibodies against telomerase according to the fourth paragraph of page 14 herein before, where appropriate labelled with one or more labels; (iii) use of nucleic acid sequences according to the previous paragraph herein for preparing telomerase; (iv) use of antibodies according to item (ii) herein before for diagnosis; use of antibodies according to item (ii) herein before for preparing medicaments; (vi) vector comprising a nucleic acid sequence, in particular DNA, according to the previous paragraph herein; (vii) microrganisms harbouring the vector according to item (vi) herein before; (viii) screening assay for identifying modulators of human telomerase comprising the telomerase according to the fourth 15 paragraph of page 14 herein before; and (ix) process for preparing the telomerase according to the fourth paragraph of page 14 herein before, characterized in that the microorganism according to item (vii) herein before is cultured and the telomerase is isolated.

Explanation of the figures: e• 20 Fig. 1: cDNA sequence of the catalytic subunit of human telomerase (hTC) (SEQ ID No. 1).

Fig. 2: Amino acid sequence which is deduced from the hTC DNA sequence depicted in Fig. 1 (SEQ ID No. 2).

The DNA sequence depicted in Fig. 1 can be completely translated from Position 64 to Position 3461 into an amino acid sequence. The amino acid residues are depicted in accordance with their single-letter code.

Fig. 3: Ethidium bromide-stained agarose gel containing AA281296 DNA which has been treated in different ways.

P:\WPDOCS\CRN\Shel leySpc\7462436.spe.doc-29/01/02 -14b- The figure shows an ethidium bromide-stained 0.8% agarose gel. Two different DNA size standards are loaded in lanes 1 and 8, with the DNA fragment lengths 3, 2, 0.5 and 0.4 kb being pointed out. The AA281296 DNA in pT7T3D was digested with a restriction enzyme Eco RI/Not I (lane Pst I (lane 6) and Xho 1 (lane 7).

Undigested AA281296 DNA in pT7T3D was loaded onto lane 2. 1/10 of a PCR a a a.

Z

~~Illj -i mixture (1 minute 94 0 C, 2 minutes at 60 0 C, 3 minutes at 72 0 C) with the hTC cDNA in pT7T3D and primers 1 GAGTGTGTACGTC- GTCGAGCTGCTCAGGTC and 4 [lane 4] and, especially, with primers 6 GCTCGTAGTTGAGCACGCTGAACAGTG and 7 GCCAAGTTCCTGCACTGGCTGATGAG [lane 5] was applied to lanes 4 and Fig. 4: Detail from a comparison of the protein sequences of the Euplotes p123 (p123) and human (phTC) catalytic telomerase subunits.

The conditions (ktuple, gap penalty and gap length penalty) are listed for the Lipman-Pearson protein comparison, using the Lasergene program software (Dnastar, Inc.), which is depicted in this figure. The amino acid residues are depicted in accordance with their single-letter code. The amino acids which are identical between Euplotes aediculatus p123 and the identified EST+, are also highlighted using the corresponding letter from the single-letter code. Amino acids which are not identical but whose function is similar or comparable are marked by a Fig. 5: Part of a comparison of the protein sequences of the catalytic telomerase subunits of Euplotes p 12 3 (p123), and yeast (est2p).

The condition (Ktuple, gap penalty and gap length penalty) are listed for the Lipman-Pearson protein comparison using Lasergene program software (Dnastar, Inc.) which is dipicted in this figure. The amino acid residues are shown in accordance with their single letter code. The amino acids which are identical between Euplotes aediculatus p123 and yeast est2p are likewise given prominence by the corresponding letter from the single-letter code. Amino acids which are not identical, but which are similar or comparable in function, are marked with a Fig. 6: Detail from a comparison of the protein sequences of the yeast (est2p) and human (phTC) catalytic telomerase subunits.

:,L1LLL---~ -16- The conditions (ktuple, gap penalty and gap length penalty) are listed for the Lipman-Pearson protein comparison, using the Lasergene program software (Dnastar, Inc.), which is depicted in this figure. The amino acid residues are depicted in accordance with their single-letter code. The amino acids which are identical between yeast est2p and the identified EST+I are also highlighted using the corresponding letter from the single-letter code. Amino acids which are not identical but whose function is similar or comparable are marked by a Fig. 7: Detail from a comparison of the protein sequences of the Euplotes p123 (p123), yeast (est2p) and human (phTC) catalytic telomerase subunits. The comparison, depicted in Fig. 5, between Euplotes p123 (p123), yeast (est2p) and humans (phTC) was carried out using the Clustal Method subprogram of the Lasergene program software (Dnastar, Inc.) under standard conditions. The amino acid residues are depicted in accordance with their single-letter code. The amino acids which are identical between yeast est2p, Euplotes aediculatus p123 and the identified EST+I are also highlighted using the corresponding letter from the single-letter code. In addition, the regions which are identical between all three proteins are marked by a light grey bar above the protein sequence.

Fig. 8: Generated DNA sequence from Example 6 (RACE round 1) (SEQ ID No. 3).

Fig. 9: Generated DNA sequence from Example 6 (RACE round 2) (SEQ ID No. 4).

Fig. 10: Generated DNA sequence from Example 6 (RACE round 3) (SEQ ID No. Fig. 11: Generated DNA sequence from Example 8 (RACE round 3) (SEQ ID No. 6).

Fig. 12: Outline of the cloning of the complete hTC cDNA sequence. The positions of the start and stop codons are marked by arrows. The black regions of the rectangles symbolize protein-encoding sequence sections, whereas the pale grey regions symbolize and untranslated cDNA regions and/or denote intronsequences.

-17- The dark grey blocks in the rectangle for the full-length cDNA either denote the telomerase-specific motif or the seven reverse transcriptase motifs (numbers 1-7).

The DNA fragments which are required for preparing the complete hTC cDNA are likewise depicted as rectangles and are marked in accordance with their origin. All the rectangles are arranged in their positions relative to each other. The origin of the DNA fragment which is denoted by rectangle AA261296 is described in Example 2. The relative position of the 182 bp deletion in this fragment (compare Example 2) is shown by a gap in the rectangle. The origin of the DNA fragments which are denoted by the rectangles RACE 1, RACE 2 and RACE 3 is described in Example 6. The origin of the DNA fragment which is denoted by the fragment rectangle is described in Example 7. The origin of the DNA fragment which is denoted by the lambda 12 rectangle is described in Example 9. The 3' part in the lambda 12 DNA fragment which encodes a cDNA which is not connected to hTC (compare Example 9) is not depicted in this figure. The complete hTC-cDNA sequence was joined together at the 5' and 3' splice sites using the lambda 12 and DNA fragments shown in this figure (compare Example These splice sites were identified in a variety of fragments (RACE 1, RACE 3, lambda 12 and Fig. 13: Detailed sections from a comparison of the protein sequences of the catalytic telomerase subunits of Euplotes and man (hTC).

The figure shows sections from a comparison of the protein sequences of the catalytic telomerase subunits of Euplotes and man (hTC). Attention is drawn to the reverse transcriptase motifs in the boxed-in areas. The figures under the boxes refer to the respective amino acid positions in Fig. 2. The amino acid residues are shown in accordance with their single-letter code. Identical amino acids are printed in bold. In the consensus sequence for the reverse transcriptase (RT consensus) motif, h denotes a hydrophobic amino acid and p denotes a polar amino acid. If these groups of amino acids are retained in the Euplotes and hTC amino acid sequences, p and/or h is/are then printed in bold. Very highly conserved amino acids are underlaid in grey. In RT3, the boxed-in area is extended in order to cover -18additional homologous amino acids. The telomerase-specific motif is described in Example 9.

Fig. 14: Generated DNA sequence from Example 11 version) (SEQ ID No. The region which is not homologous with the DNA sequence depicted in Fig. 1 is made to stand out in bold.

Fig. 15: hTC expression in cancer cell lines and normal human tissue. Fig. A: Approximately 2 Lg of poly-A RNA from different human cell lines were immobilized on the Northern blot in accordance with the manufacturer's (Clontech) instruction. Specifically, the RNA originated from a melanoma (G361), a lung carcinoma (A549), an adenocarcinoma of the colon (SW480), from a Raji Burkitt's lymphoma, from a leukaemia cell line (MOLT-4), from a chronic leukaemia cell line (K-562), from a cervical tumour (HeLa) and from the leukaemia cell line HL60. The transcripts marked 4.4 kb, 6 kb and 9.5 kb are specific for hTC (compare Example 10). Fig. B: About 2 g of poly-A+ RNA from different human tissues were immobilized on the Northern blot in accordance with the manufacturer's (Clontech) instructions. Specifically, the RNA was isolated from heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. An RNA size standard is shown.

Fig. 16: Western blot analysis of the rabbit sera against peptides from the human telomerase amino acid sequence (Example 12). In each case, 20 pl of the bacterial lysates from Example 13 were analysed in a western blot (Ausubel et al., 1987) using the antisera from Example 12. Lysates from bacteria which harbour the pMALEST construct were loaded in lanes 1, 2, 6 and 7. Lysates from bacteria which harbour the pMALAl construct were loaded in lanes 3, 4, 8 and 9. Lysates from bacteria which were not induced with IPTG (isopropyl-betathiogalactopyranoside) were loaded in lanes 1, 3, 6 and 8. Lysates from IPTGinduced bacteria were loaded in lanes 2, 4, 7 and 9. A standard size marker kDa protein ladder from Life Technologies, Cat. No. 10064-012) was loaded -19in lane 5. The 50 kDa and 120 kDa bands are marked at the edges of the membranes. The PVDF membrane in Fig. A containing lanes 1 to 4 was incubated with preimmune sera against peptideB (compare Example 12). The PVDF membrane in Fig. B containing lanes 6 to 9 was incubated with preimmune sera against peptide C (compare Example 12). The PVDF membrane in Fig. B containing lanes 1 to 4 was incubated with immune sera against peptide B (compare Example 12). The PVDF membrane in Fig. B containing lanes 6 to 9 was incubated with immune sera against peptide C (compare Example 12).

Fig. 17: Autoradiogram of 35 S-labelled, in vitro-translated protein. The complete in vitrotranslated hTC (compare Example 15) was loaded in lane 1. A C-terminally truncated version of phTC was loaded in lane 2. Lane 3 shows a positive control for the in vitro translation which was supplied by the manufacturer (compare Example 15). A protein size standard for estimating protein sizes is marked on the right-hand side.

Fig. 18: Autoradiogram of 32 P-labelled products from the TRAP assay (compare Example 15). A TRAP assay mixture without any added enzyme or protein was loaded, as a negative control, in lanes 1 and 2. A TRAP assay mixture containing partially purified human telomerase from HeLa cells was loaded, as a positive control, in lanes 3 and 4. A TRAP assay mixture containing in vitro-translated phTC was loaded, undiluted, in lanes 5 and 6. A TRAP assay mixture containing in vitro-translated phTC, at a 1:4 dilution, was loaded in lanes 7 and 8. A TRAP assay mixture containing in vitro-translated phTC, at a dilution of 1:16, was loaded in lanes 9 and 10. A TRAP assay mixture containing in vitro-translated luciferase was loaded, as a negative control, in lanes 11 and 12.

Fig. 19: Autoradiogram of 32 P-labelled products from the direct telomerase assay (compare Example 15). A radioactively labelled 10 bp marker was loaded in lane 1. A telomer oligonucleotide ([TTAGGG] 3 which was radioactively labelled 5' was loaded in lane 2. Lane 3 is an empty lane. Partially purified human telomerase from HeLa cells was used in a direct assay and the synthesis product was loaded, as a positive control, in lane 4. The in vitro-translated phTC from Example 15 was used in a direct assay and the synthesis product was loaded in lane -21 Examples Example 1 It is nowadays accepted that less than 5% of the human genome is in fact transcribed and translated into protein. Even before the genome has been completely sequenced, it is possible to obtain important information about the 60,000-70,000 genes in a human cell by investigating these coding moieties of the genome in a specific manner. The automation of high-throughput DNA sequencing technology in the last 10 to 15 years has made it possible to collect many cDNAs from plasmid cDNA libraries of widely differing origin and sequence the 5' or 3' end in each case. These short DNA sequences, which are typically of from 300 to 400 bp in length, are termed expressed sequence tags or ESTs for short and are compiled in various specialized data bases. The EST approach was initially described by Okubo et al.

(1992) and transferred to a larger scale by Adams et al. (1992). At present, approximately 50,000 human cell genes are partially sequenced and documented as EST entries.

By comparing with the DNA and amino acid sequences of known genes, it is possible to identify related, but hitherto unkown, genes in these EST databases (Gerhold and Caskey, 1996). tBLASTn (Altschul et al., 1990) is a search algorithm which has proved particularly useful for this purpose. This algorithm translates every DNA clone in the EST data base in all six possible reading frames and compares these amino acid sequences with the known protein sequence.

The EST data base at the National Center for Biotechnology Information (NCBI) was searched with the recently published protein sequence for the Euplotes aediculatus catalytic telomerase subunit p1 2 3 (Lingner et al., 1997). This resulted in a human EST with the accession number AA281296 being identified which exhibits significant homology with p 1 2 3 in reading frame This amino acid sequence in reading frame +1 is termed Est+l in that which follows.

L

-22- The homology between p123 and the Est+ 1 is most conspicuous in two sequence regions which are separated by 30 amino acids. The longer sequence region, which in p123 extends from amino acid 438 to amino acid 484, is 38% identical to the corresponding region Est+,. If similar amino acids are also taken into consideration, the congruence is even 59%. The second block of homology extends, in the p123 protein, from amino acid 513 to amino acid 530 and exhibits 44% identity with the corresponding sequence segment in the identified Est+l. A congruence of 61% is obtained when amino acid residues having similar properties are taken into account.

The P (probability) value is an important parameter for assessing a BLAST search. P indicates the probability of also finding a specific segment pair in a BLAST search using a random sequence and varies numerically between 0 (highly significant result) and 1 (insignificant result). Thus, comparison of the p123 equivalent from yeast (est2p) with the NCBI EST data base, for example, gave a negative result: The EST which was found had a probability of P=1 (Tab. On the other hand, human telomerase-associated protein 1 (hTP1), which was found in an EST data base which is not available to the general public (Harrington et al., 1997), gives a probability of P=0.004.

{PRIVATE }known P identified gene origin of the cDNA gene (species) library est2p (Saccharomyces 0.999 Rat EST Kidney cerevisiae (Tetrahymena 0.004 hTPl (Harrington et al., Crypts of the intestinal termophilia) 1997) epithelium p123 (Euplotes 3.5x10- 6 AA281296 Germinal centres of the aediculatus) tonsils Tab. 1: Comparison of three tBlastn search runs using different known genes.

The human EST AA281296 which was identified by the comparison with p123 has a probability of P=3.5 o

O

-23 These data suggest that the identified EST in all probability encodes a fragment of the catalytic subunit of human telomerase. For this reason, the corresponding gene is abbreviated below to hTC (human Telomerase, catalytic) and the deduced protein is abbreviated to phTC.

Example 2 The EST which was identified by the comparison with p123 was fed into the EST data base on 2 April 1997 and has not been published in any journal. According to information obtained from the National Center for Biotechnology Information, the cDNA library which contains this EST clone was prepared as follows: After the mRNA had been prepared from the germinal centres of the tonsils, a cDNA synthesis was carried out and the double-stranded cDNA fragments were cloned in an orientated manner, using the Not I and Eco RI restriction enzyme cleavage sites, into the vector pT7T3D-Pac.

The 389 bp which had been fed into the EST database were sequenced using the -28m13 rev2 primer supplied by Amersham (DNA sequence, see Fig. 1 Position 1685 to 2073).

Lasergene program software (Dnastar Inc.) was used to translate the DNA sequence of EST AA281296 in accordance with the human genetic code. The resulting amino acid sequence (Est+l) corresponds to Position 542 to 670 in Fig. 2.

The deduced protein sequence of Est+t is composed of 129 amino acids, including 27 basic, 11 acidic, 51 hydrophobic and 28 polar amino acid residues.

The EST (AA281296) which was identified in Example 1 was obtained commercially from Research Genetics, Inc. (Huntsville) in the form of a plasmid transformed into E. coli and analyse experimentally: 7 -24- As shown in the ethidium bromide-stained agarose gel depicted in Fig. 3, a fragment from EST AA281296 of approximately 2.2 kb in size is liberated from the vector pT7T3D after subjecting the prepared plasmid DNA to restriction digestion. With the aid of a polymerase chain reaction (PCR), which was carried out in parallel and which made use of specific internal primers, EST AA281296 was inspected: the lengths of the expected PCR products are 325 and 380 bp and are in agreement with the lengths of the fragments which were found experimentally (cf. tracks 4 and 5 in Fig. This therefore demonstrated that the E.coli clone supplied by Research Genetics, Int. (Huntsville) therefore harbours the identified EST as a plasmid.

After the DNA had been prepared, the 2176 bp of the insert in total were identified by means of double-strand sequencing. A comparison of the DNA sequences of clone AA281296 and of the C5F fragment (compare Example 7) showed that there was a 182 bp deletion (Positions 2352 to 2533, Fig. 1) and that the open reading frame is consequently displaced in this region.

In summary, the DNA sequence of clone AA281296 is composed of the sequence information shown in Fig. 1 (Positions 1685 to 2351 and Positions 2534 to 4042).

Example 3 The tBLASTn comparison only identifies the regions in which there is the greatest agreement between p123 and Est+l (amino acids 438-530, in p123), whereas the intervening amino acids are not taken into account. A Lipman-Pearson protein comparison was carried out in order to be able to draw conclusions about the relatedness of the protein sequences over a larger region (amino acids 437-554, in p123) (see Fig. When this was done, 34% of the amino acids were found to be identical while 59% of the amino acids were found to be either identical or biochemically similar. This result demonstrates that the relatedness of these proteins also continues outside the regions of homology which were found using the tBLASTn program.

As has recently been reported (Lingner et al., 1997), Euplotes aediculatus p123 and Saccharomyces cerevisiae est2p are homologous to each other. In order to relate the degree of c affinity between p123 and est2p to the homology between p123 and Est+ which is described here, the Lipman-Pearson protein comparison was employed to compare the above-described region of p123 (amino acids 437-554) with est2p, too, using identical parameters. This showed that, in this chosen region, p123 and est2p are 21% identical and that 22% of their amino acid residues are either identical or biochemically similar (see Fig. Accordingly, the homology between Est+l and Euplotes p123 is significantly higher than between p123 and est2p.

Example 4 The homology of p123 with Est+l and est2p suggests that all 3 proteins belong to the same protein family. In order to confirm this assumption, est2p was compared with Est+l under the conditions described in Example 3 (see Fig. This showed that Est+l is 20% identical to est2p, that is exhibits a degree of homology which is comparable to that of p123 to est2p.

This comparatively low level of congruence also confirms the finding that no significant EST was identified in the tBLASTn search using est2p (see Example 1).

Example A computer comparison using p123, est2p and phTC was carried out in order to identify possibly functional domains which are important for the protein family consisting of catalytic telomerase subunits derived from different species (see Fig. In this analysis, two regions which are present in all three proteins are particularly conspicuous (see Fig. At present, no unambiguous function can be assigned to the region which, in p123, corresponds to amino acids 447 to 460 (Fig. 13, telomerase motif). A motif search using the Genetics Computer Group (GCG) Wisconsin Sequence Analysis Package and a search in a protein data base (Swissprot, version of 8.6.1997) did not provide any significant insights.

On the other hand, a second region which is homologous between p123, est2p and phTC, corresponding in p123 to amino acids 512-526, exhibits a consensus motif for a reverse transcriptase (RT) (Figs. 7 and 13). Lingner et al., (1997) showed that p123/est2p contain a -~LI -26total of 6 such RT motifs, which are essential for the catalytic function of p123/est2p. As depicted in Figs. 7 and 13, two such RT motifs are also conserved in the sequence of phTC which has been investigated. These motifs are the RT motifs which are located to the furthest extent N-terminally in p 123/est2p (Lingner et al., 1997).

The primary sequences of reverse transcriptases are strongly divergent; only a few amino acids are fully conserved within a separate motif (Poch et al., 1989 and Xiong and Eickbush, 1990). In addition, due to having different distances between the conserved RT motifs, reverse transcriptases which are encoded by retroviruses or long terminal repeat (LTR) retroposons differ from those reverse transcriptases which are encoded by non-LTR retroposons or group 1I introns (Xiong and Eickbush, 1990). Based on the structure of their RT motifs, p123, est2p and phTC are to be assigned to the latter RT group. Interestingly, in this context, the consensus sequences of the RT motifs in phTC correspond most closely to the postulated RT consensus motif: of eight amino acid residues within the two RT motifs, 6 are present in the case of phTC while only 5 are present in the case of p 123 and esp2p (Figs. 7 and 13). It is striking in this context how the hydrophobic amino acids, such as leucine and isoleucine, and the amino acids lysine and arginine, in particular, are in specific positions (Figs. 7 and 13).

In summary, it was hereby possible to demonstrate, at the descriptive level, that the AA281296 clone, identified due to its homology with p123, is a fragment of the catalytic subunit of human telomerase.

-27- Example 6 For cloning the 5' end of the hTC-cDNA, three consecutive RACE (rapid amplification of cDNA ends) reactions were carried out in addition to the homology screening described in Example 8. Marathon-Ready cDNA (Clontech) form the human leukaemia cell line K562 or from human testis tissue was employed as the cDNA source. The implementation of the individual RACE rounds, as well as the results obtained, are described below.

In addition to this, the sequence information obtained in the RACE rounds was used in order to amplify the individual fragments from a contiguous cDNA clone by means of PCR.

RACE round 1: In a final volume of 50 pl, 10 pmol of dNTP-mix were added to 5 tl of K562 Marathon- Ready cDNA (from Clontech, Catalogue Number 7441-1), and a PCR reaction was carried out in 1 x Klen Taq PCR reaction buffer and 1 x advantage Klen Taq polymerase mix (from Clontec). 10 pmol of the internal gene-specific primer GSP2 -GCAACTTGCTCCAGACACTTCTTCCGG-3') from the 5' region of the hTC-EST clone and 10 pmol of the Marathon Adaptor primer API (5'-CCATCCTAATACGACTCACTATAGGGC-3'; from Clontech) were added as primers.

The PCR was carried out in 4 steps. After a one-minute denaturation at 94 0 C, denaturation was then carried out for 5 cycles of 30 sec at 94 0 C and the primers were then subsequently annealed for 4 min at 72 0 C and the DNA chain was extended. There then followed 5 cycles in which the DNA was denatured for 30 sec at 94 0 C but the subsequent primer extension took place for 4 min at 70 0 C. Finally, 22 cycles were then carried out in which, after the 30 sec DNA denaturation, the primer annealing and chain extension took place for 4 min at 68 0

C.

Following this PCR, the PCR product was diluted 1:50. 5 gl of this dilution were used in a second "nested" PCR together with 10 pmol of dNTP-mix in 1 x 10 Klen Taq PCR reaction buffer and 1 x Advantage Klen Taq polymerase mix and also 10 pmol of primer GSP2 and 10 pmol of the "nested" Marathon Adaptor primer AP2 -28- (5'-ACTCACTATAGGGCTCGAGCGGC-3'; from Clontech). The PCR conditions corresponded to the parameters selected in the first PCR. As the only exception, only 16 cycles were chosen, instead of 22 cycles, in the last PCR step.

A DNA fragment of 1153 bp in length was obtained as the product of this nested RACE PCR. This fragment was cloned into the TA cloning vector pCR2.1 from Invitrogen and subjected to complete double-strand sequencing (Fig. 8 and SEQ ID No. 3).

Nucleotides 974 to 1153 represent the nucleotide region 1629 to 1808 of the hTC-cDNA which is depicted in Fig. 1. The nucleotide region extending from bp 1 to bp 973, which does not exhibit any homology with the hTC-cDNA sequence shown in Fig. 1, represents intron sequences of the hTC gene (data not shown). A 3' splice consensus sequence is located at the exon-intron transition. The presence of intron sequences could be due to using incompletely spliced mRNA as the starting substance for the cDNA synthesis. Genomic DNA contamination in the cDNA could also be an explanation for intron sequences being found.

RACE round 2: Based on the sequence data obtained in the first RACE round, a second RACE was carried out using the gene-specific primer GSP5 from the 5'region of RACE product 1 (5'-GGCAGTGACCAGGAGGCAACGAGAGG-3') and the API primer. Marathon-Ready cDNA from human testis (from Clontech; Catalogue Number 7414-1) was used as the cDNA source. The same PCR conditions were selected as in the 1st PCT in RACE round 1. The 1st PCR was also followed, in RACE round 2, by a 2nd "nested" PCR using diluted PCR product as the cDNA source. The gene-specific primer GSP6 from the 5' region of RACE product 1 (5'-GGCACACTCGGCAGGAAACGCACATGG-3') and the AP2 primer were used as the "nested" PCR primers. The conditions corresponded to parameters for the nested PCR from RACE round 1.

The PCR product of 412 bp in length from the nested PCR of RACE round 2 was cloned into the TA cloning vector pCRII-Topo from Invitrogen and sequence completely (Fig. 9 and SEQ 'i -29- ID No. The sequence segment from bp 267 to bp 412 is completely homologous with the region of the product from RACE 1. The region from bp 1 to bp 266 extends RACE product 1 at the 5' end. This RACE product 2 is probably, in its entirety, an intron region of the hTC gene (data not shown).

RACE round 3: A third RACE round led to the identification of hTC-cDNA regions which were located further on in the 5' direction. Using the sequence results from RACE round 2 as a base, a gene-specific primer GSP9 (5'-CCTCCTCTGTTCACTGCTCTGGCC-3') was selected from the 5' region of RACE product 2 and used in a new RACE together with the AP primer and Marathon-Ready cDNA from human testis (from Clontech). The RACE conditions were the same as those used in the 1st PCR in RACEs 1 and 2. In the "nested" RACE which followed, and which took place, in accordance with the "nested" RACEs in rounds 1 and 2, using the gene-specific primer GSP10 from the 5' region of RACE product 2 (5'-CGTAAGTTTATGCAAACTGGACAGG-3') and AP2, a fragment of 1012 bp in length (Fig. 10 and SEQ ID No. 5) was amplified and cloned into the TA cloning vector pCRII- -TOPO. Subsequent sequencing showed that the 3' region of this RACE fragment (bp 817 bp 1012) evidently still constitutes an intron sequence of the hTC gene. The region from bp 889 to bp 1012 is completely homologous with the 5' region of RACE product 2. On the other hand, the 5' region of this fragment, from bp 1 to bp 816, is identical to the bp 814 bp 1629 region of the hTC-cDNA which is shown in Fig. 1. A potential 5' splice consensus sequence is located at the exon-intron transition.

Example 7 A PCR was carried out in order to clone a contiguous fragment from the sequence information obtained from RACE 2 and clone AA281296. Marathon-Ready cDNA from human testis (from Clontech; Catalogue Number 7414-1) was used as the cDNA source. The PCR mixture was as described under RACE 1 (compare Example 6) but using the primers (5'-CGAGTGGACACGGTGATCTCTGCC-3') from the 5' region of RACE 2 and primer C3B (5'-GCACACCTTTGGTCACTCCAAATTCC-3') from a 3' region of clone AA281296. The PCR was carried out in 2 steps. After a one-minute denaturation at 94 0

C,

denaturation was then carried out for 36 cycles of 30 sec at 94 0 C and, after that, the primers were annealed, and the DNA chain was extended, for 4 min at 68 0

C.

A DNA fragment of 2486 bp in length, which is designated the C5F fragment below, was obtained as the product of this PCR. This fragment was cloned into the TA cloning vector pCRII-TOPO from Invitrogen and subjected to complete double-strand sequencing. A comparison of the DNA sequences of the C5F fragment and the AA281296 clone showed that there was an in-frame insertion of 182 bp between RT motif 3 and RT motif 4 (Positions 2352 to 2533, Fig. A further comparison of DNA of the C5F fragment with the sequences from the three RACE rounds made it clear that an intron which was already identified in RACE 2 was present at the 3' end of C5F. A 3' splice consensus sequence is located at the exon-intron transition. In summary, the DNA sequence of the C5F fragment is consequently composed of the sequence information shown in Fig. 9 (Position 64 to 278) and the sequence data shown in Fig. 1 (Positions 1636 to 3908).

Example 8 For cloning the 5' end of the hTC-cDNA, a homology screening (Ausubel et al., 1987) was carried out in addition to the RACE protocol described in Example 6. A human erythroleukaemia 5'-stretch plus cDNA library (from Clontech, cat. No. HL5016b) from the human leukaemia cell line K562 was used as the cDNA source. Approximately 3 x 106 Pfu of this random and oligo-dT-primed library were plated out and used for screening as described in Ausubel et al. (1987). A radioactively labelled hTC-DNA fragment of 719 bp in length (Positions 1685 to 2404, corresponding to Fig. 1) was used as the probe.

Following a rescreening with the same hTC probe, the k clone 12 was verified as being positive out of 20 putatively positive clones. Following plaque purification and X DNA preparation (Ausubel et al., 1987), the 4 kb insert was recloned into the pBluescript vector and sequenced (Fig. 11 and SEQ ID No. 6).

-31- A comparison of the X clone 12 sequence with the sequences of the RACE clones and the DNA sequence of clone AA281296 showed that this clone, which was identified in the homology screening, encodes a 5' part of the hTC-cDNA and possesses a putative ATG start codon in Position 63 in accordance with Fig. 1. There is no stop codon in the same reading frame 5' of this ATG. Subsequent sequence analyses make it clear that X clone 12 probably contains an intron from Positions 1656 to 2004. Very well conserved 5' and 3' splice sites provide support for this hypothesis. The hTC-cDNA-encoding sequence then continues from Position 2005 to Position 2382. The sequence from 2383 to the 3' end of clone 12 exhibits a conspicuous open reading frame in reading frame A bioinformatic analysis of the corresponding DNA sequence showed that, over about 400 bp, this reading frame is identical to a variety of ESTs which have no connection with the hTC cDNA. Consequently, X clone 12 is a chimeric clone which essentially consists of the 5' end of the hTC cDNA and another cDNA clone of unknown function.

A diagrammatic summary showing the relative orientations of the RACE products, and the homology screening, is depicted in Fig.12. The complete sequence of the hTC cDNA (Fig. 1) was assembled from X clone 12 (Positions 21 to 1655 in accordance with Fig. 11), the PCR product (Positions 1636 to 3908 in accordance with Fig. 1) and EST AA281296 (Positions 3909 to 4042, in accordance with Fig. 1).

Example 9 A total of seven motifs for reverse transcriptases (RT motifs) was identified by comparing the phTG protein sequence (Fig. 2 and SEQ ID NO. 2) with a reverse transcriptase consensus sequence (Poch et al., 1989, Xiong and Eickbush, 1990) (Fig. 13). Within these motifs, some amino acids are highly conserved not only between the RT consensus sequence and phTC but also in comparison with the Euplotes telomerase protein. Thus, two aspartic acids (Positions 868 and 869 in Fig. 2) are, for example, completely conserved in RT motif 5 (Fig. 13). RT motif 7, which was deduced from other reverse transcriptases (Poch et al., 1989, Xiong and 1 ~-z~i -32- Eickbush, 1990), was only demonstrated in the human catalytic telomerase subunit and not in the Euplotes protein (Fig. 13).

Structural features which can only be found in the telomerase proteins and not in other reverse transcriptases are also conspicuous. The telomerase motif (Positions 553 and 565 in Fig. 2) is a structure which is specific for this protein family since it does not occur in any previously known protein. A further feature which has only been identified in the catalytic telomerase proteins is the difference between RT motifs 3 and 4, which distance, at 107 amino acids, is markedly greater than in other RTs. These special features indicate that the catalytic subunits of the telomerases from different species probably constitute a separate subgroup of RNA-dependent DNA polymerases.

Example Expression of the telomerase RNA subunit (hTR) does not correlate with telomerase activity but, instead, is observed ubiquitously (Feng et al., 1995). Consequently, the question arises as to whether expression of the catalytic telomerase subunit is associated with telomerase activity.

Northern blot experiments (Ausubel et al., 1987) were carried out in order to analyze the level of hTC expression. The commercially available Northern blots were supplied with a number of RNA preparations from normal human tissue (from Clontech; catalogue No. 7760-1) or with RNA samples from human cancer cell lines (from Clontech; Catalogue Number 7757-1). A radioactively labelled hTC DNA fragment of 719 bp in length (Positions 1685 to 2404, in accordance with Fig. 1) was used as the probe. The membranes were incubated with the probe in accordance with the manufacturer's (Clontech) instructions.

Two main RNA transcripts, of about 9.5 kb and 4.4 kb in size, and an additional RNA transcript of about 6 kb, which transcripts cross-hybridize with the probe, were detected in the eight human cell lines (3 leukaemia cell lines, 3 carcinoma cell lines, one melanoma and one lymphoma) tested (Fig. 15, Fig. In the comparison, the hTC mRNA was expressed most -0 -33strongly in the leukaemia cell lines K-562 and HL-60 (Fig. 15, Fig. By contrast, it was not possible to detect the hTC transcript in the normal tissues (heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas) which were tested (Fig. 15, Fig. This observation is not surprising since it was not possible to detect any telomerase activity, either, in these tissues (Kim et al., 1994).

These data indicate that the induction of hTC expression plays an important role in activating the telomerase during tumour development.

Example 11 Several PCR products, whose sizes only differed from each other to a minimal extent, were always obtained when the hTC cDNA fragments from various cDNA libraries (Clontech Marathon Ready cDNA from the human leukaemia cell line K562 and from human testis and also cDNA from the human premyeloid leukaemia cell line HL60) were subjected to PCR amplification. In order to elucidate the differences between the different hTC-PCR products, a fragment of the hTC cDNA depicted in Fig. 1 extending from bp 1783 to bp 3901 was amplified using the primers C5A (5'-CCGGAAGAGTGTCTGGAGCAAGTTGC-3') and C3B (5'-GCACACCTTTGGTCACTCCAAATTCC-3'). Marathon-Ready cDNA from K562 leukaemia cells (from Clontech; Catalogue Number 7441-1) was used as the cDNA source (PCR1 and In a third PCR, a hTC fragment, from bp 1695 to bp 3463, of the hTC cDNA in Fig. 1 was amplified from HL60 cDNA using the primers GSP1 front (5'-GGCTGATGAGTGTGTACGTCGTCGAG-3') and HTRT3A (5'-GGGTGGCCATCAGTCCAGGATGG-3').

The conditions of the 3 PCR reactions are described below: In the first PCR, and in a final volume of 50 1p, 10 pmol of dNTP mix were added to 5 pl of K562 Marathon-Ready cDNA, and a PCR reaction was carried out in 1 x Klen Taq PCR reaction buffer and 1 x Advantage Klen Taq polymerase mix (from Clontech). 10 pmol of Seach of the primers C5A and C5B were added. The PCR was carried out in 3 steps. A -34one-minute denaturation at 94 0 C was followed by 35 PCR cycles in which the DNA was firstly denatured for 30 sec at 94 0 C and the primers were then annealed, and the DNA chain was extended, for 4 min at 68 0 C. In conclusion, there followed a chain extension for 10 min at 68 0 C. The resulting PCR products were cloned into the TA cloning vector pCRII-TOPO from Invitrogen.

In a second PCR, 10 pmol of each of the primers C5A and C3B, 10 pmol of dNTP mix and 2 U of Taq DNA polymerase (from Gibco-BRL) were added to 5 pl of K562 Marathon- Ready cDNA, and a PCR reaction was carried out in 1 x PCR buffer (from Perkin Elmer) in a final volume of 50 pl. The PCR reaction was carried out in 3 steps. The DNA was firstly denatured for 3 min at 94 0 C. There then followed 34 cycles in which, consecutively, the DNA was denatured for 45 sec at 94C, primer annealing then took place for 1 min at 68 0

C

and, after that, the DNA chain was extended for 3 min at 72 0 C. In the last PCR step, a concluding chain extension was carried out for 10 min at 72 0 C. The resulting PCR products were cloned into the TA cloning vector pCR2.1 from Invitrogen.

For the third PCR, the cDNA synthesis kit from Boehringer Mannheim was first of all used to carry out a cDNA synthesis from 2 pg of DNaseI-treated poly-A RNA from the human premyeloid cell line HL60 in accordance with the manufacturer's instructions. 1 pl of this HL60 cDNA was then mixed with 10 pmol of each of the primers GSP1 front and HTRT3A and also 10 pmol of dNTP mix, in a final volume of 50 pl, and, after 1.25 pl of DMSO in 1 x Klen Taq PCR reaction buffer and 1 x Advantage Klen Taq polymerase mix (from Clontech) had been added, a PCR reaction was carried out. The PCR reaction proceeded in 3 steps. After a denaturation for 3 min at 94C, the DNA was initially denatured for 1 min at 94 0 C and the primers were then annealed, and the DNA chain extended, for 4 min at 68 0

C,

over 37 cycles. The reaction was concluded by a further incubation for 10 min at 68 0 C. The PCR products were cloned into the TA cloning vector pCR2.1-TOPO.

Complete double-strand sequencing of the cloned hTC cDNA fragments from PCRs 1 and 2, and partial sequencing of the hTC cDNA fragments obtained from PCR 3, showed that, in addition to the hTC cDNA depicted in Fig. 1, 4 variants of this cDNA exist in human cells, i.e.: Variant 1 of human hTC cDNA is distinguished by a deletion of 182 bp in length extending from nucleotides 2345 to 2526. This deletion results in the ORF being displaced, with a truncated hTC protein, which lacks RT motifs 4 to 7, being read off.

Variant 2 of human hTC cDNA exhibits a deletion of 36 bp in length extending from nucleotides 2184 to 2219. RT motif 3 is lost as a result of this deletion. However, the reading frame is retained and a protein is produced which selectively lacks RT motif 3.

Variant 3 of human hTC cDNA is a combination of variants 1 and 2. It exhibits both a deletion from bp 2184 to 2219 and a deletion from bp 2345 to 2526.

Variant 4 of human hTC cDNA is distinguished by the loss of the nucleotide region from bp3219 to bp3842. This missing sequence is replaced by a sequence which is not homologous with hTC. From bp 3843 onwards, the sequence is once again completely identical to the hTC sequence depicted in Fig. 1. The sequence of variant 4 is shown in Fig. 14. In accordance with the 5' primer chosen, it begins with bp 1783 of the hTC cDNA shown in Fig. 1. The region which is not homologous is emphasized in bold and, from Position 3219 to Position 3451 (Fig. 14 and SEQ ID No. 7) is, to the extent of 98.7%, in agreement, at the DNA level, with an EST (Accession No. AA299878) from a human uterus tumour.

Exanple 12 In order to obtain antisera having specificity for the catalytic subunit of human telomerase, the available nucleotide sequence (Fig. 1) was translated into an amino acid sequence (Fig. Using a secondary structure prediction program (PROTEAN, from the DNAStar software package, DNASTAR Inc., Madison, WI, USA), two peptides were chosen which, -36with a certain degree of probability, evoke an immune response. These are the following peptides, which are depicted in the one-letter code for amino acids: B: C-K-R-V-Q-L-R-E-L-S-E-A-E-V-R-Q

CONH

2 /Pos. 594 608 C: C-Q-E-T-S-P-L-R-D-A-V-V-I-E-Q-S-S-S-L-N-E

CONH

2 /Pos. 781-800 The cysteines which are underlined are not derived from the telomerase sequence but were additionally added on as linkers for the coupling.

The peptides were coupled to keyhole limpet hemocyanin (KLH) using the thiol-reactive coupling reagent m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). Two rabbits were in each case immunized with these coupled peptides at intervals of from 2 to 4 weeks.

Prior to immunization, 5 ml of blood were withdrawn in order to obtain preimmune sera.

After 4 immunizations, 5 ml of blood were likewise withdrawn for obtaining immune sera.

These sera were tested for reactivity with fusion proteins (Example 13) in a Western blotting experiment (Ausubel et al., 1987).

Example 13 Bacterial expression experiments were carried out in order to be able to analyse the protein of the catalytic telomerase subunit.

The constructs of these experiments are described below: For the expression construct pMalEST, the insert in the AA281296 clone mentioned in Example 2 was excised with restriction enzymes Eco RI and Not I and the cleavage sites were filled in using the Klenow fragment (Ausubel et al., 1987); the insert was then cloned into the given reading frame of the maltose-binding protein of the bacterial expression vector pMAL-C2 (from New England Biolabs). Vector pMAL-C2 was digested with restriction enzyme Pst I and the protruding single-strand ends were removed with T4 DNA polymerase (Ausubel et al., 1987).

IR-

-37- The expression construct pMaIAl contains the nucleotide sequence of Fig. 1 from Position 1789 to Position 3908. This DNA fragment was amplified from a commercially available K562 Marathon-Ready cDNA library (from Clontech, Catalogue Number 7441-1) by means of PCR using the primers C5A (5'-ACCGGAAGAGTGTCTGGAGCAAGTTG-3') and C3B (5'-GCACACCTTTGGTCACTCCAAATTCC-3'), and cloned into the TA cloning vector pCRII-TOPO from Invitrogen. The PCR conditions were as described in Example 7.

For the expression construct pMalAl, the insert was excised using the restriction enzyme Eco RI and the cleavage sites were filled in using the Klenow fragment (Ausubel et al., 1987); the insert was then cloned into the bacterial expression vector pMAL-C2 (from New England Biolabs) which had been cleaved with the restriction enzyme Xmn I.

These constructs were then used for protein expression in the bacterial strain E. coli The expression conditions were those as described in the instructions provided by New England Biolabs (Catalogue Number 800). The bacterial lysates which were prepared were tested in a Western blotting experiment (Ausubel et al., 1987).

Example 14 The bacterial lysates from Example 13 were analysed in a Western blot (Ausubel et al., 1987) using the antisera from Example 12.

Since the proportion of the fusion represented by the maltose-binding protein is about 43 kDa in size, fusion proteins of about 74 kDa and 106 kDa are expected for the pMalEST and pMalAl constructs, respectively.

When comparing the preimmune sera with the sera following the first immunization, it becomes evident that specific antibodies were formed against the B and C epitopes (Fig. 16).

Furthermore, in addition to the expected 74 kDa and 106 kDa proteins, respectively, smaller protein fragments were also observed which react with the antisera. These smaller products probably originate from premature products.

~4~A ,7- -38- Only the epitope for serum B is present on the fusion protein from the expression using pMalEST. By contrast, the epitopes for sera B and C are present on the fusion protein from pMalAl. For this reason, antiserum C does not recognize the pMalEST expression product and only recognizes the larger protein fragments from the expression experiments using pMalAl. This observation underlines the high degree of specificity of the antisera which were generated.

Example In order to be able to analyse the protein of the catalytic telomerase subunit, the protein component should be reconstituted in vitro together with the RNA component.

The constructs for these experiments are described below: The RNA component of 504 nt in length (Feng et al., 1995) was amplified from a 293 cell cDNA library using the primers HTR9BAM ATCCTAATACGACTCACTATAGGGTTGCGGAGGGTGGGCCTG-3') and HTR2BAM (5'-CGCGGATCCCGGCGAGGGGTGACGGATGC-3). Primer HTR9BAM contains a T7 promoter from nucleotide 10 to 29. In the PCR, 10 pmol of dNTP mix were added, in a final volume of 100 pl, to 3 pl of cDNA from 293 cells, and a PCR reaction was carried out in 1 x PCR reaction buffer containing 0.5 tl of Taq polymerase (from Gibco). 10 pmol of each of the primers HTR9BAM and HTR2BAM were added. The PCR was carried out in 3 steps.

A ten-minute denaturation at 94C was followed by 35 PCR cycles in which the DNA was first of all denatured for one minute at 94 0 C and, after that, the primers were annealed, and the DNA chain was extended, for 2 min at 62 0 C. In conclusion, there followed a chain extension for 4 min at 72 0 C. The resulting PCR products were cloned, after a restriction digestion with Bam HI, into the Bam HI cleavage site of vector pUC19 in such a way that the RNA component is under the control of the T7 promoter. This construct is designated HTR504 in that which follows.

il t i 7 -39- The cDNA fragment of 3411 bp in length (Position 60 to Position 3470, Fig. 1) was cloned into the vector PCRII TOPO (from Invitrogen). Detailed information on the cloning is given in Examples 8 and 7, and also in Fig. 12. In this construct, which is designated HTC FL, the T7 promoter is located 5' before the hTC cDNA.

The catalytic telomerase protein component was synthesized in a commercially available transcription/translation system, after adding the hTC FL construct, in accordance with the manufacturer's (Promega; Catalogue Number L4610) instructions. Whether the in vitro translation of the expected 127 kDa product had been successful was checked in an SDS-PAGE (Ausubel et al., 1987) using 5S-labelled cysteine (Fig. 17).

The telomerase RNA component was synthesized using a transcription system in accordance with the manufacturer's (Ambion; Catalogue Number 1344) instructions or using the method described by Pokrovskaya and Gurevich (1994).

For the in vitro re-constitution, 0.5 pg of hTRNA was added to 50 pl of the above-described translation mixture containing the hTC FL construct and the whole was incubated at 37 0 C for min. The enzymatic activity of 2 pl of this mixture was investigated using the TRAP assay Kim et al., 1994). The measurement of the activity, by the same method, of telomerase which was purified from HeLa cells (Shay et al., 1994) was used as the positive control. As can be seen in Fig. 18, both the reconstituted enzyme and the native enzyme produce the same product pattern, i.e. the nucleotide ladder which is characteristic for telomerase. This result also verifies that a single protein component, together with the RNA, is sufficient for the enzymatic telomerase activity.

In addition to the described TRAP assay, 5 pl of the reconstitution mixture were tested for its activity in a direct telomerase assay (Shay et al., 1994). In this experiment, too, the characteristic nucleotide ladder verifies the successful reconstitution of recombinant hTC protein and telomerase RNA component.

Le A 32 486-Foreign countries 40 characteristic nucleotide ladder verifies the successful reconstitution of recombinant hTC protein and telomerase RNA component.

In summary, it was hereby possible to demonstrate, at the functional level, that the identified, and completely cloned, hTC-cDNA constitutes the catalytic subunit of human telomerase.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge in Australia.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers or steps but not the exclusion of any other integer or group of integers or steps.

It will be appreciated by those of skill in that ato which theinvention relates that an -:'isolated' or 'purified' nucleic acid as referred to in the claims herein after, is one which has been identified and separated from at least one contaminant nucleic acid molecule with which it is associated in its natural state. Accordingly, it will be understood that isolated or purified nucleic acids are in a form which differs from the form or setting in which they are found in nature. An isolated or purified nucleic acid may be obtained using a number of techniques known in the art, for example, recombinant DNA aetechnologies or chemical synthesis. It will further be appreciated that the terms 'isolated' a: or 'purified' do not reflect the extent to which the nucleic acid molecule has been purified.

Similarly, an 'isolated' or 'purified' protein or polypeptide will be understood to be- one which has been identified and separated from the environment in which it naturally resides. An isolated or purified protein may be obtained using a number of techniques known in the art, for example expression from recombinant cells, or chemical synthesis.

It will further be appreciated that the terms 'isolated' or "purified" do not reflect the extent to which the protein has been purified or separated from the environment in which it naturally resides.

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(1992). Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. EMBO J. 11, 1921-1929.

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Feng, Funk, Wang, Weinrich, Avilion, Chiu, Adams, Chang, E., Allsopp, Yu, Le, West, Harley, Andrews, Greider, C.W. and Villeponteau, B. (1995). The RNA component of human telomerase. Science 269, 1236-1241.

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Greider, C.W. and Blackburn, E.H. (1985). Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 43, 405-413.

Greider, C.W. and Blackburn, E.H. (1987). The telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity. Cell 51, 887-898.

Greider, C.W. and Blackburn, E.H. (1989). A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis. Nature 337, 331-337.

Harley, Futcher, A.B. and Greider, C.W. (1990). Telomeres shorten during ageing of human fibroblasts.

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and Robinson, M.O. (1997). A mammalian telomerase-associated protein. Science 275: 973-977.

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and Yamakido, M. (1995). Activation of telomerase in human lymphocytes and hematopoietic progenitor cells.

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L lL-- i~ .7L ~~I -43- Meyne, Ratliff, R.L. and Moyzis, R.K. (1989). Conservation of the human telomere sequence (TTAGGG), among vertebrates. Proc. Natl. Acad. Sci. 86, 7049-7053.

Okubo, K. Hori, Matoba, Niiyama, Fukushima, Kojima, Y. and Matsubra, K. (1992). Large scale cDNA sequencing for analysis of quantitative and qualitative aspects of gene expression. Nature Genetics 2: 173-179.

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SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: Bayer AG STREET: Bayerwerk CITY: Leverkusen COUNTRY: Germany POSTAL CODE: D-51368 TELEPHONE: 0214-303688 TELEFAX: 0214-303482 (ii) TITLE OF THE INVENTION: Human catalytic telomerase subunit and its diagnostic and therapeutic use (iii) NUMBER OF SEQUENCES: 7 (iv) COMPUTER-READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30B (EPA) INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 4042 Basenpaare TYPE: Nucleotide STRANDEDNESS: Einzelstrang TOPOLOGY: Linear (ii) ART DES MOLEKSLS: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (vi) ORIGINAL SOURCE: INDIVIDUAL/ISOLATE: Human (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GTTTCAGGCA GCGCTGCGTC CTGCTGCGCA CGTGGGAAGC CCTGGCCCCG CGATGCCGCG CGCTCCCCGC TGCCGAGCCG TGCGCTCCCT GCTGCGCAGC AGGTGCTGCC GCTGGCCACG TTCGTGCGGC GCCTGGGGCC CCAGGGCTGG AGCGCGGGGA CCCGGCGGCT TTCCGCGCGC TGGTGGCCCA GTGCCTGGTG GGGACGCACG GCCGCCCCCC GCCGCCCCCT CCTTCCGCCA GGTGTCCTGC TGGTGGCCCG AGTGCTGCAG AGGCTGTGCG AGCGCGGCGC GAAGAACGTG GCTTCGCGCT GCTGGACGGG GCCCGCGGGG GCCCCCCCGA GGCCTTCACC GCAGCTACCT GCCCAACACG GTGACCGACG CACTGCGGGG GAGCGGGGCG

GCCACCCCCG

CACTACCGCG

CGGCTGGTGC

TGCGTGCCCT

CTGAAGGAGC

CTGGCCTTCG

ACCAGCGTGC

TGGGGGCTGC

120 180 240 300 360 420 480 I

,,I

-46-

TGCTGCGCCG

TGCTGGTGGC

CTGCCACTCA

AACGGGCCTG

GTGCGAGGAG

GTGGCGCTGC

GCAGGACGCG

AAGAAGCCAC

GCCGCCAGCA

CTTGTCCCCC

AGCTGCGGCC

TCGTGGAGAC

CCCGCCTGCC

ACGCGCAGTG

CCCCAGCAGC

AGGAGGACAC

AGGTGTACGG

CCAGGCACAA

ATGCCAAGCT

TGCGCAGGAG

TCCTGGCCAA

TCTTTTATGT

TCTGGAGCAA

AGCTGTCGGA

GACTCCGCTT

TGGGAGCCAG

CACTGTTCAG

TGCTGGGCCT

AGGACCCGCC

TCCCCCAGGA

GCGTGCGTCG

X

C%

CGTGGGCGAC

TCCCAGCTGC

GGCCCGGCCC

GAACCATAGC

GCGCGGGGGC

CCCTGAGCCG

TGGACCGAGT

CTCTTTGGAG

CCACGCGGGC

GGTGTACGCC

CTCCTTCCTA

CAT CT TTCT C

CCAGCGCTAC

CCCCTACGGG

CGGTGTCTGT

AGACCCCCGT

CTTCGTGCGG

CGAACGCCGC

CTCGCTGCAG

CCCAGGGGTT

GTTCCTGCAC

CACGGAGACC

GTTGCAAAGC

AGCAGAGGTC

CATCCCCAAG

AACGTTCCGC

CGTGCTCAAC

GGACGATATC

GCCTGAGCTG

CAGGCTCACG

GTATGCCGTG

GACGTGCTGG

GCCTACCAGG

CCGCCACACG

GTCAGGGAGG

AGTGCCAGCC

GAGCGGACGC

GACCGTGGTT

GGTGCGCTCT

CCCCCATCCA

GAGACCAAGC

CTCAGCTCTC

GGTTCCAGGC

TGGCAAATGC

GTGCTCCTCA

GCCCGGGAGA

CGCCTGGTGC

GCCTGCCTGC

TTCCTCAGGA

GAGCTGACGT

GGCTGTGTTC

TGGCTGATGA

ACGTTTCAAA

ATTGGAATCA

AGGCAGCATC

CCTGACGGGC

AGAGAAAAGA

TACGAGCGGG

CACAGGGCCT

TACTTTGTCA

GAGGTCATCG

GTCCAGAAGG

TTCACCTGCT

TGTGCGGGCC

CTAGTGGACC

CCGG GGT CCC

GAAGTCTGCC

CCGTTGGGCA

TCTGTGTGGT

CTGGCACGCG

CATCGCGGCC

ACT TCCTCTA

TGAGGCCCAG

CCTGGATGCC

GGCCCCTGTT

AGACGCACTG

AGCCCCAGGG

AGCTGCTCCG

GCCGGCTGGT

ACACCAAGAA

GGAAGATGAG

CGGCCGCAGA

GTGTGTACGT

AGAACAGGCT

GACAGCACTT

GGGAAGCCAG

TGCGGCCGAT

GGGCCGAGCG

CGCGGCGCCC

GGCGCACCTT

AGGTGGATGT

CCAGCATCAT

CCGCCCATGG

GGCACGCTGC

GCCGCTGTAC

CCGAAGGCGT

CCTGGGCCTG

GTTGCCCAAG

GGGGTCCTGG

GTCACCTGCC

CCACTCCCAC

ACCACGTCCC

CTCCTCAGGC

CCTGACTGGC

AGGGACTCCC

TCTGGAGCTG

CCCGCTGCGA

CTCTGTGGCG

CCAGCACAGC

GCCCCCAGGC

GTTCATCTCC

CGTGCGGGAC

GCACCGTCTG

CGTCGAGCTG

CTTTTTCTAC

GAAGAGGGTG

GCCCGCCCTG

TGTGAACATG

TCTCACCTCG

CGGCCTCCTG

CGTGCTGCGT

GACGGGCGCG

CAAACCCCAG

GCACGTCCGC

GCGCTCTTTG

CAGCTCGGCG

CTGGGATGCG

CCAGCCCCGG

AGGCCCAGGC

GCCCACCCGG

AGACCCGCCG

CCATCCGTGG

TGGGACACGC

GACAAGGAGC

GCTCGGAGGC

CGCAGGTTGC

CTTGGGAACC

GCTGCGGTCA

GCCCCCGAGG

AGCCCCTGGC

CTCTGGGGCT

CTGGGGAAGC

TGCGCTTGGC

CGTGAGGAGA

CTCAGGTCT T

CGGAAGAGTG

CAGCTGCGGG

CTGACGTCCA

GACTACGTCG

AGGGTGAAGG

GGCGCCTCTG

GTGCGGGCCC

TAGGACACCA

AACACGTACT

AAGGCCTTCA

540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 -47-

AGAGCCACGT

TGCAGGAGAC

AGGCCAGCAG

TCAGGGGCAA

TGCTCTGCAG

ACGGGCTGCT

CGAAAACCTT

TGCGGAAGAC

TTCAGATGCC

TGGAGGTGCA

TCAACCGCGG

TGAAGTGTCA

ACATCTACAA

CATTTCATCA

CCTCCCTCTG

GCGCCGCCGG

TCAAGCTGAC

AGACGCAGCT

ACCCGGCACT

AGGCCGAGAG

GGCGGCCCAC

AGGCCTGCAT

AAGGGCTGAG

CACCCCAGGG

TCCATCCCCA

ACCATCCAGG

AGGTGTGCCC

TTGGGGGGAG

AAAAAAAA

CTCTACCTTG

CAGCCCGCTG

TGGCCTCTTC

GTCCTACGTC

CCTGTGCTAC

CCTGCGTTTG

CCTCAGGACC

AGTGGTGAAC

GGCCCACGGC

GAGCGACTAC

CTTCAAGGCT

CAGCCTGTTT

GATCCTCCTG

GCAAGTTTGG

CTACTCCATC

CCCTCTGCCC

TCGACACCGT

GAGTCGGAAG

GCCCTCAGAC

CAGACACCAG

ACCCAGGCCC

GTCCGGCTGA

TGTCCAGCAC

CCAGCTTTTC

GATTCGCCAT

TGGAGACCCT

TGTACACAGG

GTGCTGTGGG

A AAAA AAAA

ACAGACCTCC

AGGGATGCCG

GACGTCTTCC

CAGTGCCAGG

GGCGACATGG

GTGGATGATT

CTGGTCCGAG

TTCCCTGTAG

CTATTCCCCT

TCCAGCTATG

GGGAGGAACA

CTGGATTTGC

CTGCAGGCGT

AAGAACCCCA

CTGAAAGCCA

TCCGAGGCCG

GTCACCTACG

CTCCCGGGGA

TTCAAGACCA

CAGCCCTGTC

GCACCGCTGG

AGGCTGAGTG

ACCTGCCGTC

CTCACCAGGA

TGTTCACCCC

GAGAAGGACC

CGAGGACCCT

AGTAAAATAC

AA

AGCCGTACAT

TCGTCATCGA

TACGCTTCAT

GGATCCCGCA

AGAACAAGCT

TCTTGTTGGT

GTGTCCC!TGA

AAGACGAGGC

GGTGCGGCCT

CCCGGACCTC

TGCGTCGCAA

AGGTGAACAG

ACAGGTTTCA

CATTTTTCCT

AGAACGCAGG

TGCAGTGGCT

TGCCACTCCT

CGACGCTGAC

TCCTGGACTG

ACGCCGGGCT

GAGTCTGAGG

TCCGGCTGAG

TTCACTTCCC

GCCCGGCTTC

TCGCCCTGCC

CTGGGACCTC

GCACCTGGAT

TGAATATATG

GCGACAGTTC

GCAGAGCTCC

GTGCCACCAC

GGGCTCCATC

GTTTGCGGGG

GACACCTCAC

GTATGGCTGC

CCTGGGTGGC

GCTGCTGGAT

CATCAGAGCC

ACTCTTTGGG

CCTCCAGACG

CGCATGTGTG

GCGCGTCATC

GATGTCGCTG

GTGCCACCAA

GGGGTCACTC

TGCCCTGGAG

ATGGCCACCC

CTACGTCCCA

CCTGAGTGAG

GCCTGAGCGA

CACAGGCTGG

CACTCCCCAC

CTCCTTTGCC

TGGGAATTTG

GGGGGTCCCT

AGTTTTTCAG

GTGGCTCACC

TCCCTGAATG

GCCGTGCGCA

CTCTCCACGC

ATTCGGCGGG

CTCACCCACG

GTGGTGAACT

ACGGCTTTTG

ACCCGGACCC

AGTCTCACCT

GTCTTGCGGC

GTGTGCACCA

CTGCAGCTCC

TCTGACACGG

GGGGCCAAGG

GCATTCCTGC

AGGACAGCCC

GCCGCAGCCA

GCCCACAGCC

GGGAGGGAGG

TGTTTGGCCG

GTGTCCAGCC

CGCTCGGCTC

ATAGGAATAG

TTCCACCCCC

GAGTGACCAA

GTGGGTCAAA

TTTTGAAAAA

2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4042 INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: -48- LENGTH: 1132 amino acids TYPE: Amino acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) ART DES MOLEKSLS: Protein (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (vi) ORIGINAL SOURCE: INDIVIDUAL/ISOLATE: Human (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Pro Arg Ala Pro Arg Cys Arg Ala Val Arg Ser Leu Leu Arg Ser 1 5 10 His Pro Ala Pro Val Leu Glu Asp Gly 145 Leu Gin Pro Glu Gly 225 Tyr Gin Leu Pro Ala Ala Ala Ala 130 Asp Val Leu Arg Ala 210 Gly Arg Gly Val Ala Arg Phe Phe 115 Leu Asp Ala Gly Arg 195 Gly Ser Glu Trp Ala Ala Val Gly 100 Thr Arg Val Pro Ala 180 Arg Val Ala Leu Leu Cys Ser 70 Gin Ala Ser Ser Val 150 Cys Thr Gly Leu Arg 230 Leu Gin 40 Val Arg Leu Leu Arg 120 Ala Leu Tyr Ala Glu 200 Leu Leu Ala 25 Arg Cys Gin Cys Asp 105 Ser Trp Leu Gin Arg 185 Arg Pro Pro Phe Asp Pro Ser 75 Arg Ala Leu Leu Arg 155 Cys Pro Trp Pro Pro 235 Arg Ala Asp Leu Ala Gly Asn 125 Leu Ala Pro His His 205 Ala Arg Arg Ala Ala Lys Lys Gly 110 Thr Arg Leu Pro Ala 190 Ser Arg Pro Leu Phe Arg .Glu Asn Pro Val Arg Phe Leu 175 Ser Val Arg Arg Gly Arg Pro Leu Val Pro Thr Val Val 160 Tyr Gly Arg Arg Arg 240 Z- -49- Gly Ala Val Leu Ala 305 Cys Asp Ser Arq Arg 385 Ala Ala Gly Va1 Val 465 Arg Leu Ser Val Leu 545 Ala His Ser Ser 290 Gly Pro Lys Leu Pro 370 Tyr Gln Ala Ser Gln 450 Arg His Gly Val Pro 530 His Ala Pro Pro 275 Gly Pro Pro Glu Thr 355 Trp Trp Cys Val Va1 435 Leu Ala Asn Lys Arg 515 Ala Trp Pro Gly 260 Ala Thr Pro Va1 Gin 340 Gly Met Gin Pro Thr 420 Ala Leu Cys Glu His 500 Asp Ala Leu Glu 245 Arg Arg Arg Ser Tyr 325 Leu Ala Pro Met Tyr 405 Pro Ala Arg Leu Arg 485 Ala Cys Glu Met Glu Arg Ala Ser 295 Ser Glu Pro Arq Thr 375 Pro Val Ala Glu His 455 Arg Phe Leu Trp Arq 535 Val Arg Gly Glu 280 His Arg Thr Ser Leu 360 Pro Leu Leu Gly Glu 440 Ser Leu Leu Ser Leu 520 Leu Tyr Pro 250 Ser Ala Ser Pro His 330 Leu Glu Arq Leu Lys 410 Cys Asp Pro Pro Asn 490 Gin Arg Glu Val Lys 570 Gly Arq Ser Gly 300 Pro Leu Ser Ile Pro 380 Leu His Arg Asp Gin 460 Gly Lys Leu Pro Ile 540 Leu Gin Gly Leu 285 Arg Trp Tyr Ser Phe 365 Arg Leu Cys Glu Pro 445 Va1 Leu Lys Thr Gly 525 Leu Leu Gly Phe 270 Glu Gin Asp Ser Leu 350 Leu Leu Gly Pro Lys 430 Arq Tyr Trp Phe Trp 510 Va1 Ala Arg Trp Va1 Ala His Pro 320 Gly Pro Ser Gin His 400 Arg Gin Leu Phe Ser 480 Ser Met Cys Phe Phe 560 Phe Tyr Val Thr Glu Thr Thr Phe Gin 565 Asn Arg Leu Phe Phe Tyr 575 i- 7 7 Arg Leu His Pro 625 Gly Arg Pro Ala Glu 705 Pro Asn Gly Leu Pro 785 Ala Ala Gin Met Arg 865 Lys Val Lys Lys Arg 610 Lys Ala Val Gly Trp 690 Leu Gin Thr His Gin 770 Leu Ser Val Gly Glu 850 Leu Thr Val Ser Arg 595 Glu Pro Arg Lys Leu 675 Arg Tyr Asp Tyr Val 755 Pro Arg Ser Arg Ser 835 Asn Val Phe Asn Val 580 Val Ala Asp Thr Ala 660 Leu Thr Phe Arg Cys 740 Arg Tyr Asp Gly Ile 820 Ile Lys Asp Leu Leu Trp Gin Arg Gly Phe 645 Leu Gly Phe Val Leu 725 Val Lys Met Ala Leu 805 Arg Leu Leu Asp Arg 885 Arg Ser Leu Pro Leu 630 Arg Phe Ala Val Lys 710 Thr Arg Ala Arg Val 790 Phe Gly Ser Phe Phe 870 Thr Lys Lys Arg Ala 615 Arg Arg Ser Ser Leu 695 Val Glu Arg Phe Gin 775 Val Asp Lys Thr Ala 855 Leu Leu Thr Leu Glu 600 Leu Pro Glu Val Val 680 Arg Asp Val Tyr Lys 760 Phe Ile Val Ser Leu 840 Gly Leu Val Val Gin 585 Leu Leu Ile Lys Leu 665 Leu Val Val Ile Ala 745 Ser Val Glu Phe Tyr 825 Leu Ile Val Arg Val Ser Ser Thr Val Arg 650 Asn Gly Arg Thr Ala 730 Val His Ala Gin Leu 810 Val Cys Arg Thr Gly 890 Asn Ile Glu Ser Asn 635 Ala Tyr Leu Ala Gly 715 Ser Val Val His Ser 795 Arg Gin Ser Arg Pro 875 Val Phe Gly Ala Arg 620 Met Glu Glu Asp Gin 700 Ala Ile Gin Ser Leu 780 Ser Phe Cys Leu Asp 860 His Pro Pro Ile Glu 605 Leu Asp Arg Arg Asp 685 Asp Tyr Ile Lys Thr 765 Gin Ser Met Gin Cys 845 Gly Leu Glu Val Arg 590 Val Arg Tyr Leu Ala 670 Ile Pro Asp Lys Ala 750 Leu Glu Leu Cys Gly 830 Tyr Leu Thr Tyr Glu Gin Arg Phe Val Thr 655 Arg His Pro Thr Pro 735 Ala Thr Thr Asn His 815 Ile Gly Leu His Gly 895 Asp His Gin Ile Val 640 Ser Arg Arg Pro Ile 720 Gin His Asp Ser Glu 800 His Pro Asp Leu Ala 880 Cys Glu c i~s~- L~;l l r- -51 900 905 910 Ala Leu Gly Gly Thr Ala Phe Val Gin Met Pro Ala His Gly Leu Phe 915 920 925 Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu Glu Val Gin Ser 930 935 940 Asp Tyr Ser Ser Tyr Ala Arg Thr Ser Ile Arg Ala Ser Leu Thr Phe 945 950 955 960 Asn Arg Gly Phe Lys Ala Gly Arg Asn Met Arg Arg Lys Leu Phe Gly 965 970 975 Val Leu Arg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gin Val Asn 980 985 990 Ser Leu Gin Thr Val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu Gin 995 1000 1005 Ala Tyr Arg Phe His Ala Cys Val Leu Gin Leu Pro Phe His Gin Gin 1010 1015 1020 Val Trp Lys Asn Pro Thr Phe Phe Leu Arg Val Ile Ser Asp Thr Ala 1025 1030 1035 1040 Ser Leu Cys Tyr Ser Ile Leu Lys Ala Lys Asn Ala Gly Met Ser Leu 1045 1050 1055 Gly Ala Lys Gly Ala Ala Gly Pro Leu Pro Ser Glu Ala Val Gin Trp 1060 1065 1070 Leu Cys His Gin Ala Phe Leu Leu Lys Leu Thr Arg His Arg Val Thr 1075 1080 1085 Tyr Val Pro Leu Leu Gly Ser Leu Arg Thr Ala Gin Thr Gin Leu Ser 1090 1095 1100 Arg Lys Leu Pro Gly Thr Thr Leu Thr Ala Leu Glu Ala Ala Ala Asn 1105 1110 1115 1120 Pro Ala Leu Pro Ser Asp Phe Lys Thr Ile Leu Asp 1125 1130 INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 1153 base pairs TYPE: Nucleotide STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (vi) ORIGINAL SOURCE: INDIVIDUAL/ISOLATE: Human C1 N jj 52 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GTGCCTGCAG AGACCCGTCT GGTGCACTCT GATTCTCCAC TTGCCTGTTG CATGTCCTCG

TTCCCTTGTT

ATCCTCTCGT

CCCCCTGATC

ACCCAGGCTG

AAGCAGTTCT

CTGGCTAATT

CAAACTCCTG

TGCAAGCC-AC

CCTTGTCCTG

TGCTGTTTTC

CAT TATTAAT

CCTCCTTTGT

TCCTTTGCGT

CCTCCATGGC

GAGACTCACG

CGTTCCTTAG

CTGGCCAAGT

TTTTATGTCA

TGGAGCAAGT

TCTCACCACC

TGCCTCCTGG

TTTTTATTGT

GAGTGTAATG

CATTCCTCAA

TTTGTATTTT

ACCTCAAGTG

CGTGCCCGGC

AGCAATAAGA

CCTGCTGACT

ATTGTTTTCC

TCCCCGTCTG

GGTTCTTCTG

ATCTAGCGAC

AGGAGGGCGG

CCAGGGTTGG

TCCTGCACTG

CGGAGACCAC

TGC

TCTTGGGTTG

TCACTGGGCA

CGTTGTTTGC

GCACAATCTC

CCTCATGAGT

TAGTAGAGAT

ATCTGCCCGC

ATACCTTGAT

CCCTTAGTGT

TAGTTCTATC

GTGTTGAGTG

TCTTCTGTCT

TCTTGTTATT

GTCCGGGGAC

TCATCTTGGC

CTGTGTTCCG

GCTGATGAGT

GTTTCAAAAG

CCATGTGCGT

TTTGCTTTTA

TTTTGTTTAT

GGCTCACTGC

AGCTGGGATT

AGGCTTTCAC

CTTGGCCTCC

CTTTTAAAAT

ATTTTAGCTC

TCAGGCATCT

TTTCTTTAGC

CAGGCCCGCC

GCTGGTAAAC

CTCTGCTTAT

CCGTGAGTGT

GCCGCAGAGC

GTGTACGTCG

AACAGGCTCT

TTCCTGCCGA

TTTCTCTTTG

TGAGACAGTC

AACCTCTGCC

ACAGGCGCCC

CATGTTGGCC

CACAGTGCTG

GAAGTCTGAA

TGGCCACCCC

TGACACCCCC

TTTGCCCCCG

GTCTGGGGTC

CCCAGCTTTA

GATGCACAGA

CTGGAGCACC

ACCGTCTGCG

TCGAGCTGCT

TTTTCTACCG

GTGTGTGTTG

CTTAGTGTTA

TCACTCTGTC

TCCTCGGTTC

ACCACCACGC

AGGCTGGTCT

GGATTACAGG

ACATTGCTAC

CCAGCCTGTG

ACAAGCTAAG

CCCTGCTTTT

CCCTTCCTTG

CCTGTGCTGG

TGAAGATGTG

ACGTGGCCAG

TGAGGAGATC

CAGGTCTTTC

GAAGAGTGTC

120 180 240 300 360 420 480 540 600 660 720 78 0 840 900 960 1020 1080 1140 1153 INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 412 base pairs TYPE: Nucleotide STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (vi) ORIGINAL SOURCE: INDIVIDUAL/ISOLATE: Human (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: 53 CAGAGCCCTG GTCCTCCTGT CTCCATCGTC ACGTGGGCAC CGTCGAGTGG ACACGGTGAT CTCTGCCTCT GCTCTCCCTC TACGAGGTTC ACCTTCACGT TTTGATGGAC ACGCGGTTTC GTGAACAGAG GAGGCTGGGC GCGGCAGTGG AGCCGGGTTG CTGGAAGCAC AGACGCTCTG GCGAGGGTGC CTGCAGAGAC CTCCACTTGC CTGTTGCATG TCCTCGTTCC CTTGTTTCTC GTGCGTTTCC TGCCGAGTGT GTGTTGATCC TCTCGTTGCC INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 1012 base pairs TYPE: Nucleotide STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLCULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (vi) ORIGINAL SOURCE: INDIVIDUAL/ISOLATE: Human

ACGTGGCTTT

CTGTCCAGTT

CAGGCACCGA

CCGGCAATGG

CCGCCTGGTG

ACCACCTCTT

TCCTGGTCAC

TCGCTCAGGA

TGCATAAACT

GGCCAGAGCA

GGAGAAGTGT

CACTCTGATT

GGGTTGCCAT

TG

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: GGGGTCCTGG GCCCACCCGG GCAGGACGCG TGGACCGAGT

GTCACCTGCC

CCACTCCCAC

ACCACGTCCC

CTCCTCAGGC

CCTGACTGGC

AGGGACTCCC

TCTGGAGCTG

CCCGCTGCGA

CTCTGTGGCG

CCAGCACAGC

GCCCCCAGGC

GTTCATCTCC

AGACCCGCCG

CCATCCGTGG

TGGGACACGC

GACAAGGAGC

GCTCGGAGGC

CGCAGGTTGC

CTTGGGAACC

GCTGCGGTCA

GCCCCCGAGG

AGCCCCTGGC

CTCTGGGGCT

CTGGGGAAGC

TGCGCTTGGC

AAGAAGCCAC

GCCGCCAGCA

CTTGTCCCCC

AGCTGCGGCC

TCGTGGAGAC

CCCGCCTGCC

ACGCGCAGTG

CCCCAGCAGC

AGGAGGACAC

AGGTGTACGG

CCAGGCACAA

ATGCCAAGCT

TGCGCAGGAG

CTCTTTGGAG

CCACGCGGGC

GGTGTACGCC

CTCCTTCCTA

CATCTTTCTG

CCAGCGCTAC

CCCCTACGGG

CGGTGTCTGT

AGACCCCCGT

CTTCGTGCGG

CGAACGCCGC

CTCGCTGCAG

CCCAGGT GAG

GACCGTGGTT

GGTGCGCTCT

CCCCCATCCA

GAGACCAAGC

CTCAGCTCTC

GGTTCCAGGC

TGGCAAATGC

GTGCTCCTCA

GCCCGGGAGA

CGCCTGGTGC

GCCTGCCTGC

TTCCTCAGGA

GAGCTGACGT

GAGGTGGTGG

TCTGTGTGGT

CTGGCACGCG

CATCGCGGCC

ACTTCCTCTA

TGAGGCCCAG

CCTGGATGCC

GGCCCCTGTT

AGACGCACTG

AGCCCCAGGG

AGCTGCTCCG

GCCGGCTGGT

ACACCAAGAA

GGAAGATGAG

CCGTCGAGGG

54- CCCAGGCCCC AGAGCTGAAT GCAGTAGGGG CTCAGAAAAG GGGGCAGGCA GAGCCCTGGT CCTCCTGTCT CCATCGTCAC GTGGGCACAC GTGGCTTTTC GCTCAGGACG TCGAGTGGAC ACGGTGATCT CTGCCTCTGC TCTCCCTCCT GTCCAGTTTG CATAAACTTA CG INFORM4ATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 3972 base pairs TYPE: Nucleotide STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (vi) ORIGINAL SOURCE: INDIVIDUAL/ISOLATE: Human (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 900 960 1012 GAATTCGCGG CCGCGTCGAC GTTTCAGGCA GCGCTGCGTC

CCTGGCCCCG

GCTGCGCAGC

CCAGGGCTGG

GTGCCTGGTG

GGTGTCCTGC

GAAGAACGTG

GGCCTTCACC

GAGCGGGGCG

GGCACGCTGC

GCCGCTGTAC

CCGAAGGCGT

CCTGGGCCTG

GTTGCCCAAG

GGGGTCCTGG

GTCACCTGCC

CCACTCCCAC

GCCACCCCCG

CACTACCGCG

CGGCTGGTGC

TGCGTGCCCT

CTGAAGGAGC

CTGGCCTTCG

ACCAGCGTGC

TGGGGGCTGC

GCGCTCTTTG

CAGCTCGGCG

CTGGGATGCG

CCAGCCCCGG

AGGCCCAGGC

GCCCACCCGG

AGACCCGCCG

CCATCCGTGG

CGATGCCGCG

AGGTGCTGCC

AGCGCGGGGA

GGGACGCACG

TGGTGGCCCG

GCTTCGCGCT

GCAGCTACCT

TGCTGCGCCG

TGCTGGTGGC

CTGCCACTCA

AACGGGCCTG

GTGCGAGGAG

GTGGCGCTGC

GCAGGACGCG

AAGAAGCCAC

GCCGCCAGCA

CGCTCCCCGC

GCTGGCCACG

CCCGGCGGCT

GCCGCCCCCC

AGTGCTGCAG

GCTGGACGGG

GCCCAACACG

CGTGGGCGAC

TCCCAGCTGC

GGCCCGGCCC

GAACCATAGC

GCGCGGGGGC

CCCTGAGCCG

TGGACCGAGT

CTCTTTGGAG

CCACGCGGGC

CTGCTGCGCA

TGCCGAGCCG

TTCGTGCGGC

TTCCGCGCGC

GCCGCCCCCT

AGGCTGTGCG

GCCCGCGGGG

GTGACCGACG

GACGTGCTGG

GCCTACCAGG

CCGCCACACG

GTCAGGGAGG

AGTGCCAGCC

GAGCGGACGC

GACCGTGGTT

GGTGCGCTCT

CCCCCATCCA

CGTGGGAAGC

TGCGCTCCCT

GCCTGGGGCC

TGGTGGCCCA

CCTTCCGCCA

AGCGCGGCGC

GCCCCCCCGA

CACTGCGGGG

TTCACCTGCT

TGTGCGGGCC

CTAGTGGACC

CCGGGGTCCC

GAAGTCTGCC

CCGTTGGGCA

TCTGTGTGGT

CTGGCACGCG

CATCGCGGCC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 72 55

ACCACGTCCC

CTCCTCAGGC

CCTGACTGGC

AGGGACTCCC

TCTGGAGCTG

CCCGCTGCGA

CTCTGTGGCG

CCAGCACAGC

GCCCCCAGGC

GTTCATCTCC

CGTGCGGGAC

CCCAGGCCCC

CCTCCTGTCT

ACGGTGATCT

CTTCACGTTT

GC-CTGGGCGC

ACGCT CT GGC

GTGAGGAGAT

TCAGGTCTTT

GGAAGAGTGT

AGCTGCGGGA

TGACGTCCAG

ACTACGTCGT

ACTTCCTTTT

GCCCGGAGGA

CGCACGGTGA

GAGCTCCTGG

ATGCAGCACG

TCGTAATAGC

GCCAAATGGG

TCCGGTTCTT

TGGGACACGC

GACAAGGAGC

GCTCGGAGGC

CGCAGGTTGC

CTTGGGAACC

GCTGCGGTCA

GCCCCCGAGG

AGC CC C TGGC

CTCTGGGGCT

CTGGGGAAGC

TGCGCTTGGC

AGAGCTGAAT

CCATCGTCAC

CTGCCTCTGC

TGATGGACAC

GGCAGTGGAG

GAGGGTGCCT

CCTGGCCAAG

CTTTTATGTC

CTGGAGCAAG

GCTGTCGGAA

ACTCCGCTTC

GGGAGCCAGA

TAAACAGAAG

GGGGCCACGG

GGTGGCCGAG

GGCAGGGACA

GCCCGAGCGG

CGGCCCAGGC

GCCACACCTT

CCTGCTCAGT

CTTGTCCCCC

AGCTGCGGCC

TCGTGGAGAC

CCCGCCTGCC

ACGCGCAGTG

CCCCAGCAGC

AGGAGGACAC

AGGTGTACGG

CCAGGCACAA

ATGCCAAGCT

TGCGCAGGAG

GCAGTAGGGG

GTGGGCACAC

TCTCCCTCCT

GCGGTTTCCA

CCGGGTTGCC

GCAGGGGTTG

TTCCTGCACT

ACGGAGACCA

TTGCAAAGCA

GCAGAGGTCA

ATCCCCAAGC

ACGTTCCGCA

TGCGTTTGAG

GACACAGCCA

GTGCCGGTGC

GGCTCTGAGG

GTGGGGGCCC

GCTCTGAACC

GTCCTGGAAG

GGGGCTACGA

GGTGTACGCC

CTCCTTCCTA

CATCTTTCTG

CCAGCGCTAC

CCCCTACGGG

CGGTGTCTGT

AGACCCCCGT

CTTCGTGCGG

CGAACGCCGC

CTCGCTGCAG

CCCAGGTGAG

CTCAGAAAAG

GTGGCTTTTC

GTCCAGTTTG

GGCGCCGAGG

GGCAATGGGG

GCTGTGTTCC

GGCTGATGAG

CGTTTCAAAA

TTGGAATCAG

GGCAGCATCG

CTGACGGGCT

GAGAAAAGAG

CCCCACATTT

GGGCCATGGC

CTCCAGAAAA

ACCACAAGAA

ACCACGCCAT

TTCAGAGTCT

AAATCATGGT

CCACCTAGGT

GAGACCAAGC

CTCAGCTCTC

GGTTCCAGGC

TGGCAAATGC

GTGCTCCTCA

GCCCGGGAGA

CGCCTGGTGC

GCCTGCCTGC

TTCCTCAGGA

GAGCTGACGT

GAGGTGGTGG

GGGGCAGGCA

GCTCAGGACG

CATAAACTTA

CCAGAGCAGT

AGAAGTGTCT

GGCCGCAGAG

TGTGTACGTC

GAACAGGCTC

ACAGCACTTG

GGAAGCCAGG

GCGGCCGATT

GGTGGCTGTG

GGTATCAGCT

ACGGCGCCAA

GCAGCGTGGG

GCAGCTGGGC

TCTGGTCAAA

CAAAAGCTGG

CCACTTCCAG

AGTTGCTACC

ACTTCCTCTA

TGAGGCCCAG

CCTGGATGCC

GGCCCCTGTT

AGACGCACTG

AGCCCCAGGG

AGCTGCTCCG

GCCGGCTGGT

ACACCAAGAA

GGAAGATGAG

CCGTCGAGGG

GAGCCCTGGT

TCGAGTGGAC

CGAGGTTCAC

GAACAGAGGA

GGAAGCACAG

CACCGTCTGC

GTCGAGCTGC

TTTTTCTACC

AAGAGGGTGC

CCCGCCCTGC

GTGAACATGG

CTTTGGTTTA

TAGATGAAGG

CCCATTTGTG

GGTGTAGGGG

CAGGGCCTGG

GGTGTTGTAG

GACCCTCAGG

GTTCGCCGGG

TAATCCTTCC

1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 -56- CGGCGAAhAT

GGGCAGTGCC

AGGGTGCCAG

CCGGCTGCGT

TTCACATGGA

CGTTGAGATG

TGGACCTTTC

ATCCCATAAG

TGATCAGGAG

AGGCTCCACT

ACACATACAC

GCGGCCCCTC

TGATCACAAT

CTTCATGGAT

AAGAGACAAA

GCCGAAGCCC

ACGCCAAAGC

ACATCAGAGA

GGTGTCTGCA

ATACCTTTTC

TCCAACACCA

GGCGCATTCC

CCTTTTCAA.G

ACGTGTCACC

CTGGAGCAGA

CACTCCCTCT

AACTTTGGCT

TTCTGAAGCC

GACTACGGCC

GATGTTCTCA

CTGTGAATAC

CAGCTTCTTC

ATCCGAGTGC

CAGGATCGAC

G CC TCCT CT G

AACCCTCCGA

ACAAGCGGAG

CTCGGCTCCA

GCCAGTGCTG

ACAGGCTGGT

ACCACCGCTT

TCTCCCTGGC

CACCAGGGGT

GTGTTTATCC

CCCTCCTGCC

AGCAAGAGGT

AGGTACTCAT

GGCAATGAGA

GTGTGGTCCC

AGCCAATAGG

TCGGGAGGCA

TTGATCTTCT

CGCGTGCCTG

ATCATAGCCC

TCTCTAGGCC

CAGGTAGCTT

CGATTTGCGC

ACTCCAGGCT

TGTCATCTGT

AGAAAAGGAC

CAAACCAACC

AGGGAAACTT

TCTGCACTTT

AGCCCGAGGG

TGATGTTCCG

ACTGCTGACA

GTTCCACCAC

CCACAGACAA

GGTGGCTGGT

TCTTAATCTC

GCCTCCGGGT

CTCTGAGCCC

TGGTCCCTGG

GGTGCCCTGC

AGACCAGGCT

GCGGTCCATC

GGTCAACATG

TCCCAAGGAC

TTCGTCCTCC

GTTCCGAAGC

CACGTTGCTC

GTTGAACCGG

TAGAAAGCTG

AATCCCACCT

TAGCACTCGA

GCCAAGCACC

GTCTTGCAGT

AGACACCTTT

CAAGAGGCCC

GGGTCGACGC

TCCATGGCTG CAGCATCCTT 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 3972

AGGTCCAGGG

GCTGAAAAGA

ACTGAGAGCC

ATAGCACCTC

CCACCTCCCA

AGATCACAGG

CCATCCCAGT

CGGCCCATGC

GGCCGCGAAT

GAGGAGCATC

GGGAGAGCTG

CAGTGGAGGC

GTCTGCTCTC

CGATCACCAC

ACCTTCCAGG

CCAGAGAAAA

CGTGCGGCAG

TC

INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 2089 base pairs TYPE: Nucleotidie STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA FRAGMENT TYPE: Linear (vi) ORIGINAL SOURCE: INDIVIDUAL/ISOLATE: Human (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CCGGAAGAGT GTCTGGAGCA AGTTGCAAAG CATTGGAATC AGACAGCACT TGAAGAGGGT GCAGCTGCGG GAGCTGTCGG AAGCAGAGGT CAGGCAGCAT CGGGAAGCCA GGCCCGCCCT GCTGACGTCC AGACTCCGCT TCATCCCCAA GCCTGACGGG CTGCGGCCGA TTGTGAACAT 57

GGACTACGTC

GAGGGTGAAG

GGGCGCCTCT

TGTGCGGGCC

GTACGACACC

GAACACGTAC

CAAGGCCTTC

CGTGGCTCAC

CTCCCTGAAT

CGCCGTGCGC

CCTCTCCACG

GATTCGGCGG

CCTCACCCAC

CGTGGTGAAC

CACGGCTTTT

TACCCGGACC

CAGTCTCACC

GGTCTTGCGG

GGTGTGCACC

GCTGCAGCTC

CTCTGACACG

GTGCCTGGCC

TGAATCTGGG

ACTGTCAGGC

AGGAGCTGTC

GCCTGGTCTC

CCTGTGGTGG

GCCAAGGGCT

GCGTATCACC

CTGGACACTT

AACCTGCGGT

GTGGGAGCCA

GCACTGTTCA

GTGCTGGGCC

CAGGACCCGC

ATCCCCCAGG

TGCGTGCGTC

AAGAGCCACG

CTGCAGGAGA

GAGGCCAGCA

ATCAGGGGCA

CTGCTCTGCA

GACGGGCTGC

GCGAAAACCT

TTGCGGAAGA

GTTCAGATGC

CTGGAGGTGC

TTCAACCGCG

CTGAAGTGTC

AACATCTACA

CCATTTCATC

GCCTCCCTCT

TCAGTGGCAG

CTTAGGAAGT

TCGTCTGCCC

TGGGAGCTGC

TCCTGTTTGC

GATTGGGCTG

TAGGAGGAGG

ACGACAGAGC

TGTCCAGCAT

CCTGAGCTTA

GAACGTTCCG

GCGTGCTCAA

TGGACGATAT

CGCCTGAGCT

ACAGGCTCAC

GGTATGCCGT

TCTCTACCTT

CCAGCCCGCT

GTGGCCTCTT

AGTCCTACGT

GCCTGTGCTA

TCCTGCGTTT

TCCTCAGGAC

CAGTGGTGAA

CGGCCCACGG

AGAGCGACTA

GCTTCAAGGC

ACAGCCTGTT

AGATCCTCCT

AGCAAGTTTG

GCTACTCCAT

CAGTGCCTGC

TCTTACCCCT

GCCCTCTCGT

CATCCTTCCC

CCCATGGTGG

TCTCCCGTCC

CCAGGCCCAG

CCCGCGCCGT

CAGGGAGGTT

ACAGCTTCTA

CAGAGAAAAG

CTACGAGCGG

CCACAGGGCC

GTACTTTGTC

GGAGGTCATC

GGTCCAGAAG

GACAGACCTC

GAGGGGTGCC

CGACGTCTTC

CCAGTGCCAG

CGGCGACATG

GGTGGATGAT

CCTGGTCCGA

CTTCCCTGTA

CCTATTCCCC

CTCCAGCTAT

TGGGAGGAAC

TCTGGATTTG

GCTGCAGGCG

GAAGAACCCC

CCTGAAAGCC

CTGCTGGTGT

TTTCGCATCA

GGGGTGAGCA

ACCTTGCTCT

GATTTGGGGG

ATGGCACTTA

GCTACCCCAC

CCTCTGCTTC

TCTGATCCGT

CTTTCTGTTC

AGGGCCGAGC

GCGCGGCGCC

TGGCGCACCT

AAGGTGGATG

GCCAGCATCA

GCCGCCCATG

CAGCCGTACA

GTCGTCATCG

CTACGCTTCA

GGGATCCCGC

GAGAACAAGC

TTCTTGTTGG

GGTGTCCCTG

GAAGACGAGG

TGGTGCGGCC

GCCCGGACCT

ATGCGTCGCA

CAGGTGAACA

TACAGGTTTC

ACATTTTTCC

AAGAACGCAG

TAGTGTGTCA

GGAAGTGGTT

GAGCACCTGA

GCCTGGGGAA

GCCTGGCCTC

GGGCCCTTGT

CCCTCTCAGG

CCAGTCACCG

GTGAAATTCA

TTTCTGTGTT

GTCTCACCTC

CCGGCCTCCT

TCGTGCTGCG

TGACGGGCGC

TCAAACCCCA

GGCACGTCCG

TGCGACAGTT

AGCAGAGCTC

TGTGCCACCA

AGGGCTCCAT

TGTTTGCGGG

TGACACCTCA

AGTATGGCTG

CCCTGGGTGG

TGCTGCTGGA

CCATCAGAGC

AACTCTTTGG

GCCTCCAGAC

ACGCATGCGT

TGCGCGTCAT

GTATGTGCAG

GGAGACTGAG

TAACCCAACC

TGGAAGGGAC

GCGCTGGGGG

TCCTGTTTGC

GCAAACCCAG

AGCAGAGGCC

TCCTCTGCCC

AGCCATGTCG

GTGGAGACCC

240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 58 TGAGAAGGAC CCTGGGAGCT CTGGGAATTT GGAGTGACCA AAGGTGTGC 2089

Claims (9)

1. Functional equivalents, variants and catalytically active fragments of the catalytically active human telomerase subunit in isolated or purified form, comprising the amino acid sequence depicted in Fig. 2, characterized in that they comprise an amino acid sequence encoded by the DNA sequence depicted in Fig. 1 with a deletion of 182 bp in length extending from nucleotide 2345 to 2526, the DNA sequence depicted in Fig. I with a deletion of 36 bp in length extending from nucleotide 2184 to 2219, the DNA sequence depicted in Fig. 1 with a deletion of 36 bp in length ~.5.extending from nucleotide 2184 to 2219 and a deletion of 182 bp in length extending from nucleotide 2345 to 2526 or the DNA sequence depicted in Fig. 14. 2(0 2. Nucleic acid sequences in isolated or purified form, encoding functional equivalents, variants and catalytically active fragments of the catalytically active human telomerase subunit comprising the amino acid sequence depicted in Fig 2, characterized in that they comprise the DNA sequence depicted in Fig. 1 with a deletion of 182 bp in length extending from nucleotide 2345 to 2526, the DNA sequence depicted in Fig. 1 with a deletion of 36 bp in length extending from nucleotide 2184 to 2219, the DNA sequence depicted in Fig. 1 with a deletion of 36 bp in length extending from nucleotide 2184 to 2219 and a deletion of 182 bp in length extending from nucleotide 2345 to 2526 or the DNA sequence depicted in Fig. 14.

3. Antisense nucleic acids in isolated or purified form which bind to the nucleic acid sequence according to claim 2.

4. Antibodies against telomerase according to claim 1, where appropriate labelled with one or more labels.

5. Use of nucleic acid sequences according to claim 2 for preparing telomerase.

6. Use of antibodies according to claim 4 for diagnosis.

7. Use of antibodies according to claim 4 for preparing medicaments.

8. Vector comprising a nucleic acid sequence, in particular DNA, according to claim 2.

9. Microorganisms harbouring the vector according to claim 8. Screening assay for identifying modulators of human telomerase comprising the telomerase according to claim 1. S* S S S S S S.. .55555 5. 5 S. S a S 95 S 5* S S a *5 0 P:\WPDOCS\CRN\ShbleySpcc\7462436.spedoc-29/01/02 -61-

11. Process for preparing the telomerase according to claim 1, characterized in that the microorganism according to claim 9 is cultured and the telomerase is isolated. DATED this 29th day of January, 2002 BAYER AKTIENGESELLSCHAFIT By its Patent Attorneys DAVIES COLLISON CAVE 0eS@ 0S 0 S. 0* S S.. 00 065050 SO S 0 S S S 0 S I RA -0 ^Vrof 1: wl i