Abstract:
The present invention relates to a novel human poly(A)polymerase enzyme (PAP), its nucleic and amino acid sequence and use thereof as well as an antibody against the novel enzyme and use thereof. The novel enzyme is named PAP gamma and is not related to the previously known PAPs.

Description:
FIELD OF THE INVENTION  
         [0001]    The present invention relates to a novel human poly(A) polymerase enzyme (PAP), its nucleic and amino acid sequence and use thereof as well as an antibody against the novel enzyme and use thereof.  
         BACKGROUND OF THE INVENTION  
         [0002]    The majority of mammalian messenger RNAs end with a 200-250 adenosine residues tail at their 3 40  ends. The function of the poly(A) tail is not fully understood but detailed studies have highlighted its role in regulating gene expression via translational regulation and mRNA stability control (reviewed in (Mitchell and Tollervey, 2000; Sachs and Varani, 2000).  
           [0003]    The mRNA&#39;s poly(A) tail is added post-transcriptionally to the pre-mRNA and the biochemistry of mammalian nuclear polyadenylation has been extensively studied (reviewed in (Wahle and Ruegsegger, 1999; Zhao et al., 1999)). The reaction is a multi-step and multi-component reaction which proceeds through two independent steps; RNA cleavage and adenosine addition. Both reactions are dependent on a highly conserved sequence-element, the hexanucleotide AAUAAA. At least six trans-acting protein factors are required for the reaction in vivo.  
           [0004]    Poly(A) polymerase (PAP), one of these factors, is the enzyme responsible for poly(A) tail addition. Mammalian PAP has been identified in and cloned from several species among them human, mouse and bovine (Raabe et al., 1991; Thuresson et al., 1994; Wahle et al., 1991; Zhao and Manley, 1996). Experimental data show that the degree of phosphorylation of PAP varies during Xenopus oocyte maturation (Ballantyne et al., 1995) and early development; in these cases the key regulatory mechanism in gene expression is the selective translational activation of maternal mRNAs, which get a long poly(A) tail and recruit to the polyribosomes. PAP phosphorylation varies as well as during the cell cycle (Colgan and Manley, 1997; Colgan et al., 1996). PAP is hyperphosphorylated in HeLa cells arrested in mitosis and the enzyme&#39;s activity is inhibited. It is also known that cells entering mitosis show a general repression of RNA and protein synthesis, and inhibition of PAP activity could contribute to this in several ways.  
           [0005]    Multiple forms of poly(A) polymerase (PAP) have been identified in mammalian cell lines and tissues (e.g. (Thuresson et al., 1994) reviewed in (Wahle and Ruegsegger, 1999; Zhao et al., 1999)). HeLa cell nuclear extracts contain several isoforms of PAP, having apparent molecular weights of 90, 100, 106 kDa, as identified by immunoblotting. It has previously been shown that the 106 kDa form is post-translationally modified by phosphorylation (Thuresson et al., 1994). Several forms of PAP are generated by alternative splicing since a series of alternatively spliced mRNAs have been identified (Raabe et al., 1991; Thuresson et al., 1994; Wahle et al., 1991; Zhao and Manley, 1996). The combination alternative splicing and post-translational modifications arises the possibility of a very complex pattern of PAP isoforms. The identification of multiple PAP isoforms and their functional significance still remains an open question. However, since PAP participates in a whole set of different reactions (e.g. RNA cleavage, AAUAAA dependent and independent adenosine addition), at different subcellular locations (nucleus, cytoplasm), it seems reasonable to hypothesize that different PAPs are responsible for different functions in vivo. It is known that poly(A) polymerase is associated with several diseases, for example breast cancer (Scorilas et al., 1998, Scorilas et al 2000).  
         SUMMARY OF THE INVENTION  
         [0006]    The present inventors have managed to biochemically separate and purify PAPs representing each of the native isoforms from HeLa cells. A functional comparison using the specific (CPSF and hexanucleotide—AAUAAA dependent) and non-specific (CPSF independent) in vitro polyadenylation assays revealed differences between the isoforms. These differences correlate to the stability of the CPSF/PAP/RNA complex. By RT-PCR five alternative spliced forms of PAP mRNAs were identified. These forms are alternative spliced variants of the previously cloned human and bovine PAP. These forms differ in their carboxy-terminal ends due to alternative splicing and result in carboxy-terminal PAP isoforms.  
           [0007]    Surprisingly, one of the presumed isoforms (90 kDa) turned out to be a novel human PAP enzyme encoded by a distinct gene, not linked to the previously identified human PAP gene (Kyriakopoulou et al. 2001).  
           [0008]    The novel PAP has been named PAPγ by the Human Gene Nomenclature Committee to distinguish it from the previously known PAP which is encoded by a gene now renamed to PAPOLA. PAPγ is encoded by a unique gene named PAPOLG and is not related to the previously identified mammalian PAPs. The nucleotide sequence and the deduced amino acid sequence are provided in the Sequence Listings. PAPγ shares some nucleotide sequence homology with the previously identified human PAP in its N-terminal part (the first 500 aminoacids), approximately 75%. The C-terminal part, starting from this position is unique for the novel PAPγ with the exception of the last 18 amino acids which are shared between the two forms of human PAP encoded by the genes PAPOLA and PAPOLG.  
           [0009]    Thus, the invention relates to an amino acid sequence for poly(A) polymerase γ (PAPγ) according to Sequence Listing Id No 2 and or Id No 4 and/or functional parts thereof having poly(A) polymerase (PAP) activity as well as essentially homologous, such as 90% homologous, variants of said sequence.  
           [0010]    The invention also relates to a nucleic acid sequence according to Sequence Listing Id No 1 and/or Id No 3 encoding the amino acid sequence according to claim  1  and variants of said nucleic acid sequence due to the degeneracy of the genetic code.  
           [0011]    Moreover, the invention relates to a vector comprising the above nucleic acid sequence as well as a host cell comprising the vector and other necessary elements for expression of the nucleic acid sequence encoding PAPγ.  
           [0012]    Furthermore, the invention relates to a method for production of recombinant PAPγ, comprising cultivating the above host cell in a suitable cultivation media; and recovering PAPγ from said media.  
           [0013]    The invention also relates to use of the above nucleic acid sequence or portions thereof for detection of PAPγ related diseases or disorders. In practice, the nucleic acid sequence or portions thereof will, for example, be used as a hybridisation probe for detection of corresponding sequences in patient samples. Preferably, a PAPγ specific part of the sequence is used.  
           [0014]    The invention also relates to an antibody against PAPγ which is selective for PAPγ and does not react with other PAPs. This means that the antibody is directed against an epitope present in the C-terminal part of PAPγ. Polyclonal or monoclonal or fragments of antibodies produced by conventional methods are contemplated. An exemplifying antibody is described in the detailed section below. The antibody according to invention may be used for detection of PAP related diseases, such as different forms of cancer, for example breast cancer.  
           [0015]    Finally, the invention relates to a reagent comprising PAPγ according to the invention. The reagent is intended for synthesizing and modifying RNA or as a target for the development of novel pharmaceutical drugs that either perturb or do not affect PAPγ activity. Preferably the drug target is derived from the unique C-terminal part of PAPγ. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0016]    [0016]FIG. 1 is a demonstration of poly(A) polymerase activity by specific incorporation of rATP. FIG. 1 shows polymerization activity of recombinant full length PAPγ in the presence of different nucleotide analogues.  
         [0017]    [0017]FIG. 2 shows specific polyadenylation activity of PAPγ according to the invention.  
         [0018]    [0018]FIG. 3 shows recognition of the native PAP isoforms in HeLa cell nuclear extracts, by different antibodies run on a 6% SDS polyacrylamide gel. 
     
    
     BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS  
       [0019]    Sequence ID NO 1: DNA sequence of the novel PAPγ according to the invention.  
         [0020]    Sequence ID NO 2: Amino acid sequence encoded by the DNA sequence according to SEQ ID NO 1.  
         [0021]    Sequence ID NO 3: DNA sequence of a homologous variant of the novel PAPγ according to the invention.  
         [0022]    Sequence ID NO 4: Amino acid sequence encoded by the DNA sequence according to SEQ ID NO 3.  
       DETAILED DESCRIPTION OF THE INVENTION  
       [0023]    The novel PAPγ was cloned and recombinant proteins were expressed in a prokaryotic expression system. Procedure for cloning and purification are found below. Initial kinetic analysis revealed that the new human PAPγ is equally good or better compared to the human PAPs encoded by the PAPOLA gene. All nucleotide and amino acid numbers below refer to SEQ Id No 3 and 4, respectively, as these are the preferred ones.  
         [0024]    In FIG. 1 it is shown that the recombinant PAPγ has high specificity for synthesing poly(A) tails, using the non-specific assay in the presence of Mn(II). The reaction mixture contains in final concentrations: 100 mM Tris/HCl buffer pH 8.6 (RT), 40 mM KCl, 40 μM EDTA, 10% glycerol, 1.0 mM DTT, 0.9 units Rnasin (ribonuclease inhibitor), 0.1% NP-40, 0.5 mM MnCl 2 , 0.5 mg/ml BSA and a mixture of cold and radiolabeled RNA substrate OligoA(15) (330 fmoles); PAP is added to the reaction at 3 different amounts of 17.5, 35, and 70 ng, respectively. The reactions proceed at 30 min, 37° C. The reaction is stopped by Proteinase K buffer, the RNA is extracted by phenol:chloroform and it is run in a 16% acrylamide: bis-acrylamide (19:1)-7 M urea gel. The gel is exposed overnight to a phosphoimager screen and further analyzed. The lanes represent the following: lane 1: negative control (no PAP), lanes 2-4: 0.5 mM rATP, lanes 5-7: 0.5 mM rUTP, lanes 8-10: 0.5 mM rGTP, lanes 11-13: 0.5 mM CTP and lanes 14-16: 0.5 mM dATP.  
         [0025]    In FIG. 2 it is shown that PAPγ also functions in the AAUAAA and CPSF dependent assay. This data provides conclusive proof that poly(A) polymerase activity is associated with PAPγ. The reaction mixture contains in final concentrations: 100 mM Tris/HCl buffer pH 8.3 (RT), 40 mM KCl, 40 μM EDTA, 9.6% glycerol, 0.24 mM DTT, 0.9 units Rnasin (ribonuclease inhibitor), 0.01% NP-40, 0.72 mM MgCl2, 0.2 mg/ml BSA, 1 mM ATP, 2.5% PVA, 20 mM creatinine phosphate and a mixture radiolabeled RNA substrate L3(53) (70 fmoles in total). CPSF (partially purified from calf thymus) 3 μl, and PAP 50 ng are added to the reaction. The reactions proceed at 15-30 min., 30° C. The reaction is stopped by proteinase K buffer, the RNA is extracted by phenol:chloroformand and it is run in a 10% acrylamide:bis-acrylamide(19:1)-7 M Urea gel. The gel is exposed overnight to a phosphoimager screen and further analyzed. The lanes represent the following: lane 1: negative control (no PAP), lane 2: PAP+CPSF.  
         [0026]    The development of specific polyclonal sera against the unique C-terminal part of the new enabled detection of PAPγ in HeLa nuclear extracts (FIG. 3). The proteins from the SDS gel are transferred to ImmobilonP membranes and immunostaining and visualization is done by ECLplus reagents. The lanes represent the following: lane 1: 20:14 monoclonal antibody (1:10 dilution) against the known human PAP gene: three isoforms with apparent MWs 90, 100, 106 kDA. This monoclonal antibody is directed against an epitope with the region of PAPγ that is shared between PAPs originating from the two different PAP genes. Lane 2, 3 polyclonal sera (dilution 1:2000, 1:4000 respectively) raised against a polypeptide comprising the C-terminal part located in the unique region starting at approximately amino acid 505 of PAPγ for specific recognition of one isoform, 90 kDa. Examples of such unique polypeptide is a polypeptide comprising amino acids located from amino acid 521 to the C-terminal end of PAPγ. This novel antibody is specific for PAPγ. This novel antibody was developed against the unique C-terminal part-of PAPγ. Below a detailed description of its production can be found. Lane 4: pre-immune serum (1:2000 dilution)—no detected signal.  
         [0027]    Cloning of PAPγ in different cloning vectors for expression in prokaryotic systems  
         [0028]    1.Cloning in pET-32a(+) vector  
         [0029]    The coding sequence of PAPγ was amplified by PCR using cDNA library derived from HeLa total RNA by reverse transcription. The primers used to amplify the first 1479 nt from 5′part were the following: primer (a) (5′-CACCATGGAAGAGATGTCTGCAAACACC-3′) introducing a NcoI site(in italics) upstream of the initiation codon and primer (d) (5′-GAGAGCTCTTAGGTACCGTGAAGTTGTTTTTTCTTTACATGAGTTGC introducing a SacI site downstream of the stop codon; (for cloning reasons to the pCAL-c vector that will be described further on, we had to introduce a KpnI restriction site simultaneously, which introduces 2 extra aa at the C-terminus in all the pET-32a clones expressing PAPγ. The NcoI cloning site introduces a point mutation at the second aa in the sequence by changing Lys to glutamate)). The PCR product was cloned into pGEM-T vector and then by digestion NcoI/SacI and ligation was inserted to the pET-32a (NcoI/SacI digested); the clone is named pET-32(H1-493)—(where H denotes that the tag is N-terminally located and the numbers 1-493 refer to a polypeptide segment starting at amino acid 1 and ending at amino acid 493 of the human PAP sequence). Another pair of primers were used to amplify by PCR a fragment reaching up to 2208 nt 3′ of PAPγ; internal primer (c) 5′-GCCTGTCTGGGATCCTCGGGT-3′ and primer (g) (5′-GAGAGCTCTAAGGTACCTTTTCTTTTTCTTTCTTCAGCAGTGCG-3′). PCR product was cloned in pGEM-T, digested by EcoRI/SacI and inserted between the EcoRI and SacI restriction enzyme sites of plasmid pPAPγ(H1-493); the resulting clone is named pPAPγ(H1-683). For determination of the full length of PAPγ sequence 3′RACE methodology was performed according to “Clontech Smart Race cDNA amplification kit” using two upstream specific primers: primer (h) (CAACACCTCACAACCCTGCCCA) and primer (i) (GAGATCCCATTCCCCATCCATAG). The PCR product after seminested was cloned into pGEM-T vector and sequenced for confirmation of the identification of stoping codon. The new full-length 3′end of PAPγ was cloned by a new round of PCR amplification using the pGEM-T vector insert and the primer pairs (c) and primer (j) (5′-GAGAGGTACCAAGCCGATTAAGGGTCAGTCG). The new 3′end was cloned into the same starting clone pPAPγ(H1-493) by digestion EcoRI/SacI. The pET-32(a) vector introduces N-terminally of the gene of PAPγ a thyrodoxine tag which increases the expression of the soluble fraction of the recombinant protein, a histidine tag and an S-tag for enabling easy purification via affinity chromatography. The tag is about 20 kDa.  
         [0030]    2. Cloning in pCAL-c vector.  
         [0031]    The same cloning strategy was used but in the restriction cut the pair EcoRi/KpnI was used. The pCAL-c vector introduces a 4 kD calmodulin peptide tag which can be used for affinity purification with calmodulin-affinity resin and results in clones named pPAPγ(1-736C) or similarly where the numbers indicate encoded PAPγ amino acids while the C after the numbers denote a C-terminally located tag.  
         [0032]    Expression and purification of recombinant form of PABγ cloned in PET32a vector, in  E.coli.    
         [0033]    PAPγ containing plasmids were used to transform BL21(DE3)pLysS  E.coli  strains. 1 colony is inoculated in 100 ml TB medium (containing phosphate) in the presence of 50 ug/ml carbenicillin and 34 μg/ml chloramphenicol and let grow by standing at 37° C. The 100 ml culture is inoculated in a final 1 lt culture in TB medium containing the required antibiotics. Bacteria are growing by vigorous shaking at 37° C. and are induced at OD 600  around 0.5-1.0 with 0.42 mM IPTG plus 0.524 mM MgCl 2 . Cells where harvested by centrifugation 3 hours post inducion and pellets frozen at −70° C.  
         [0034]    Extracts were made by thawing the cells on ice and lysing in 50 ml lysis buffer (20 mM Hepes/KOH pH7.5, 0.5M KCl, 1.0% NP-40, 1.0% Tween-20, 10% glycerol, 5 mM imidazole, 20 mM β-mercaptoethanol plus 1 tablet of EDTA-free protease inhibitors.); next follows sonication (3×10 sec), centrifugation 20 min at 39000 g and 0.45 μm filtration. The cell extracts are mixed batchwise to 1 ml Talon resin (Co++ affinity agarose) equilibrated in lysis buffer and proteins bound by 1 hr rotation at 4° C. The resin is packed in a manual chromatographic column and washed with 20 column volumes of lysis buffer; subsequently it is washed with 20 volumes wash buffer (lysis buffer without detergents and β-mercaptoethanol). The proteins are eluted by 5 volumes of elution buffer (20 mM Hepes/KOH pH7.5, 0.5M KCl, 10% glycerol, 200 mM imidazole, 0.5M KCl). The eluate is loaded in 1 ml HiTrap chelating column equilibrated in 20 mM Hepes/KOH pH7.5, 0.5M KCl, 1.0% NP-40, 1.0% Tween-20,10% glycerol, 50 mM imidazole). The column is washed with 10 column volumes equilibration buffer and 10 volumes wash buffer (equilibration buffer without detergents and containing 0.05 M KCl ). The proteins are eluted with 5 volumes elution buffer (20 mM Hepes/KOH pH 7.5, 0.05M KCl,10% glycerol, 0.5 mM DTT, 1.5 mM MgCl2, 200 mM imidazole). The eluate is loaded to a Heparin Hi-Trap column equilibrated in 20 mM Hepes/KOH pH 8.6, 0.05M KCl, 10% glycerol, 0.5 mM DTT, 1.5 mM MgCl2 buffer. It is washed with 5 volumes of the equilibration buffer and recombinant proteins eluted in fractions of 0.5 ml with 5 volumes of the same buffer containing 0.5 M KCl. In all buffer solutions they are added freshly 0.5 mM PMSF, 1.0 μg/ml leupeptin, 1.0 mg/ml pepstatin, 1.0 μg/ml aprotinin.  
         [0035]    Expression and purification of recombinant form of PABγ cloned in pCAL-c vector, in  E.coli.    
         [0036]    PCAL-c PAPγ containing plasmids were used to transform BL21 (DE3)pLysS  E.coli  strains. 1 colony is inoculated in 50 ml TB medium (containing phosphate buffer) plus 50 μg/ml carbenicillin and let grow by standing at 37° C. The 50 ml culture is inoculated in a final 500 ml culture in TB medium containing antibiotics. Bacteria are growing by vigorous shaking at 37° C. and induced at OD 600  around 0.6-1.0 with 0.42 mM IPTG plus 0.524 mM MgCl 2 . Cells where harvested by centrifugation 3 hours post induction and pellets frozen at −70° C. Extracts where made by unthawing the cells on ice and lysing in 30 ml lysis buffer—(Ca-binding buffer) (50 mM Tris/HCl pH7.5, 0.15 M KCl, 0.1 % Triton X-100, 10% glycerol, 1 mM Mg(CH 3 COO), 2 mM CaCl 2 , 1 mM imidazole, 10 mM β-mercaptoethanol plus 1 tablet of EDTA-free protease inhibitors.); next follows sonication (4×30 sec), centrifugation 20 min at 39000 g and 0.45 μm filtration. The cell extracts are mixed batchwise to 0.75 ml calmodulin resin (affinity agarose) equilibrated in binding buffer and proteins bound by rotation at 4° C. overnight. The resin is packed in a manual chromatographic column and washed with 20 column volumes of wash buffer I (50 mM Tris/HCl pH7.5, 0.2 M KCl, 0.1 % Triton X-100, 10% glycerol, 1 mM Mg(CHECOO), 2 mM CaCl 2 , 1 mM imidazole, 10 mM β-mercaptoethanol;subsequently it is washed with 20 volumes washII buffer (50 mM Tris/HCl pH7.5, 0.25 M KCl,10% glycerol, 1 mM. Mg(CH 3 COO), 2 mM CaCl 2 , 1 mM imidazole). The recombinant protein is eluted in fractions of 0.5 ml with 7 volumes of buffer containing 50 mM Tris/HCl pH 7.5, 1 M KCl, 2 mM EGTA, 10% glycerol, 0.5 mM DTT and 1.5 mM MgCl 2 . In all buffer solutions they are added freshly 0.5 mM PMSF, 1.0 μg/ml leupeptin, 1.0 mg/ml pepstatin, 1.0 μg/ml aprotinin.  
         [0037]    Production of polyclonal sera recognizing specifically PAPγ 
         [0038]    A unique part of the PAPγ sequence, as represented by a polypeptide starting at amino acid 521 and ending at amino acid 683, was cloned in pET-32(a) vector and the recombinant polypeptide was purified and used for immunization of rabbits. The 491 nt long fragment, comprising the above mentioned amino acids, was amplified using as template the plasmid pET-32(668) and a pair of primers: primer (p) (5′-CACCATGGAATCCAAAAGATTGTCTCTGGATAGC- 3 ′) and primer (g) (5′-GAGAGCTCTTAGGTACCTTATTTTCTTTTTCTTTCTTCAGCAGTGCG- 3 ′). The PCR product is cloned to pGEM-T vector and inserted to pET32(a) vector after restriction digestion with NcoI/BamHI. The recombinant polypeptide is expressed and purified, as described essentially, at the recombinant proteins&#39; purification schedule. The antigen was more than 95% pure, was diluted to 0.9 mg/ml protein concentration. 500 μl of antigen were used for injection of 2 independent rabbits in repetitive injections.  
       REFERENCES  
       [0039]    Ballantyne, S., Bilger A., {dot over (A)}ström, J., Virtanen, A. and Wickens, M. (1995) Poly(A) polymerases in the nucleus and cytoplasm of frog oocytes: Dynamic changes during oocyte maturation and early development.  RNA,  1, 64-78.  
         [0040]    Colgan, D. F. and Manley, J. L. (1997) Mechanism and regulation of mRNA polyadenylation.  Genes Dev.,  11, 2755-2766.  
         [0041]    Colgan, D. F., Murthy, K. G. K., Prives, C. and Manley, J. L. (1996) Cell-cycle related regulation of poly(A) polymerase by phosphorylation.  Nature,  384, 282-285.;  
         [0042]    Kyriakopoulou, C., Nordvarg, H., Virtanen, A. (2001). A novel nuclear human poly(A) polymerase, PAPγ.  J.Biol.Chem.  276, 33504-33511.  
         [0043]    Mitchell, P. and Tollervey, D. (2000) mRNA stability in eukaryotes.  Curr Opin Genet Dev,  10, 193-8.  
         [0044]    Raabe, T., Bollum, F. J. and Manley, J. L. (1991) Primary structure and expression of bovine poly(A) polymerase.  Nature,  353, 229-234.  
         [0045]    Sachs, A. B. and Varani, G. (2000) Eukaryotic translation initiation: there are (at least) two sides to every story.  Nat Struct Biol,  7, 356-61.  
         [0046]    Scorilas A., Courtis N., Yotis J., Talieri M., Michailakis M., Trangas T. (1998) Poly(A) polymerase activity levels in breast tumour cytosols.  J Exp Clin Cancer Res  1998 Dec;17(4):511-8.  
         [0047]    Scorilas A., Talieri M., Ardavanis A., Courtis N., Dimitriadis E., Yotis J., Tsiapalis C. M., and Trangas T. (2000) Polyadenylate polymerase enzymatic activity in mammary tumor cytosols: A new independent prognostic marker in primary breast cancer.  Cancer Research  60, 5427-5433.  
         [0048]    Thuresson, A. -C., {dot over (A)}ström, J., {dot over (A)}ström, A., Grönvik, K. -O. and Virtanen, A. (1994) Multiple forms of poly(A) polymerase in human cells.  Proc. Natl. Acad Sci. USA,  91, 979-983.  
         [0049]    Wahle, E., Martin, O., Schiltz, E. and Keller, W. (1991) Isolation and expression of cDNA clones encoding mammalian poly(A) polymerase.  Embo J.  10, 4251-7.  
         [0050]    Wahle, E. and Ruegsegger, U. (1999) 3′-End processing of pre-mRNA in eukaryotes.  FEMS Microbiol Rev,  23, 277-95.  
         [0051]    Zhao, J., Hyman, L. and Moore, C. (1999) Formation of mRNA 3′ ends in eukaryotes: mechanism, regulation, and interrelationships with other steps in mRNA synthesis.  Microbiol Mol Biol Rev,  63, 405-45.  
         [0052]    Zhao, W. and Manley, J. L. (1996) Complex Alternative RNA processing generates an unexpected diversity of poly(A) polymerase isoforms.  Mol. Cell. Biol.,  16, 2378-2386w.  
     
       
       
         1 
         
           
             4  
           
           
             1  
             2166  
             DNA  
             Homo sapiens  
           
            1 

atgaaagaga tgtctgcaaa caccgtgctg gacagccagc gtcaacaaaa gcattatgga     60 

attacctccc caattagttt ggcatctcct aaagaaattg atcatattta cacacagaaa    120 

ttaattgacg ccatgaaacc atttggagtg tttgaagatg aggaagaatt gaaccacagg    180 

ctggtggttc ttggtaaatt gaacaattta gtaaaagaat ggatttctga tgtcagcgag    240 

agtaagaacc tcccaccttc tgttgtggct actgttggtg gtaaaatttt cacatttgga    300 

tcctataggc ttggagtaca caccaaagga gctgacattg atgcactttg tgtagctcca    360 

agacatgtgg aaagatctga tttttttcag tctttttttg aaaaattgaa acatcaagat    420 

ggcattagaa acttaagagc tgtagaagat gcctttgtac ctgttataaa atttgaattt    480 

gatggtattg aaattgatct agtctttgca agactggcaa tacaaaccat atcagataat    540 

ttagatctaa gagacgactc tcgcctgaga agccttgata taaggtgtat tcgcagctta    600 

aatggttgta gagttactga tgaaattttg catttagtgc caaataaaga aacttttaga    660 

ctcaccctaa gagctgtcaa attatgggca aaacgacgtg gtatttattc caacatgcta    720 

ggattccttg gtggtgtctc ctgggcaatg ctagttgcaa gaacttgcca attgtatcca    780 

aatgcagcag catctacttt agttcataag ttctttttag ttttttccaa gtgggaatgg    840 

ccaaatcctg tgctgctgaa gcaaccagaa gaaagcaatt tgaatttgcc tgtctgggat    900 

cctcgggtaa atccatcaga taggtatcat ctcatgccca taatcacccc tgcctaccca    960 

caacagaatt ctacgtataa tgtgtccaca tcaactcgaa cagtaatggt agaagaattt   1020 

aaacaaggtc ttgcagtcac agatgaaatt cttcaaggaa agtcagattg gtccaaacta   1080 

cttgagccac cgaatttctt tcaaaagtat aggtacgttg gattagtaga atctaaaatc   1140 

cgtgtacttg ttggaaactt ggaacggaat gaatttatta ctcttgccca tgtgaatccc   1200 

cagtcattcc cagggaataa ggaacatcat aaagacaaca attacgtatc aatgtggttc   1260 

cttgggataa tttttcggag agtagaaaat gcagaaagtg tcaacataga cttgacatat   1320 

gatatacagt catttactga tacagtgtac agacaggcaa acaatataaa tatgctaaag   1380 

gagggaatga aaattgaagc aactcatgta aagaaaaaac aacttcacca ctaccttcct   1440 

gcagaaattc ttcaaaagaa gaaaaaggtg agtcaaagtc tctctgatgt caatcgaagc   1500 

tcgggcggac ttcaatccaa aagattgtct ctggatagca gttgtctgga tagctccaga   1560 

gacactgata atggaacacc ttttaattct ccagcgtcca agtctgatag cccttctgta   1620 

ggagaaacag aaaggaatag tgctgagcct gctgctgtaa ttgtggagaa gccactgagt   1680 

gtaccaccag cccaaggact ttccattcca gtgattggcg caaaagttga ctctacagta   1740 

aaaactgtat caccccccac tgtgtgtacc attcctaccg tagtaggacg aaatgtcatt   1800 

cctagaatca caacacctca caaccctgcc cagggacaac cgcatctgaa tggaatgtca   1860 

aatataacta agactgttac acctaagaga tcccattccc catccataga tgggactcct   1920 

aagaggttga aagacgtaga aaagtttatt cgacttgaat caacatttaa ggacccccgc   1980 

actgctgaag aaagaaaaag aaaatcagtg gatgccattg gaggagaatc tatgcctatt   2040 

ccaactattg atacatcacg caaaaagaga ctacccagta aagaactacc agattcatca   2100 

tctccagttc cagcaaacaa catccgtgtc atcaaaaatt ccattcgact gacccttaat   2160 

cggtaa                                                              2166 

 
           
             2  
             722  
             PRT  
             Homo sapiens  
           
            2 

Met Lys Glu Met Ser Ala Asn Thr Val Leu Asp Ser Gln Arg Gln Gln 
  1               5                  10                  15 

Lys His Tyr Gly Ile Thr Ser Pro Ile Ser Leu Ala Ser Pro Lys Glu 
             20                  25                  30 

Ile Asp His Ile Tyr Thr Gln Lys Leu Ile Asp Ala Met Lys Pro Phe 
         35                  40                  45 

Gly Val Phe Glu Asp Glu Glu Glu Leu Asn His Arg Leu Val Val Leu 
     50                  55                  60 

Gly Lys Leu Asn Asn Leu Val Lys Glu Trp Ile Ser Asp Val Ser Glu 
 65                  70                  75                  80 

Ser Lys Asn Leu Pro Pro Ser Val Val Ala Thr Val Gly Gly Lys Ile 
                 85                  90                  95 

Phe Thr Phe Gly Ser Tyr Arg Leu Gly Val His Thr Lys Gly Ala Asp 
            100                 105                 110 

Ile Asp Ala Leu Cys Val Ala Pro Arg His Val Glu Arg Ser Asp Phe 
        115                 120                 125 

Phe Gln Ser Phe Phe Glu Lys Leu Lys His Gln Asp Gly Ile Arg Asn 
    130                 135                 140 

Leu Arg Ala Val Glu Asp Ala Phe Val Pro Val Ile Lys Phe Glu Phe 
145                 150                 155                 160 

Asp Gly Ile Glu Ile Asp Leu Val Phe Ala Arg Leu Ala Ile Gln Thr 
                165                 170                 175 

Ile Ser Asp Asn Leu Asp Leu Arg Asp Asp Ser Arg Leu Arg Ser Leu 
            180                 185                 190 

Asp Ile Arg Cys Ile Arg Ser Leu Asn Gly Cys Arg Val Thr Asp Glu 
        195                 200                 205 

Ile Leu His Leu Val Pro Asn Lys Glu Thr Phe Arg Leu Thr Leu Arg 
    210                 215                 220 

Ala Val Lys Leu Trp Ala Lys Arg Arg Gly Ile Tyr Ser Asn Met Leu 
225                 230                 235                 240 

Gly Phe Leu Gly Gly Val Ser Trp Ala Met Leu Val Ala Arg Thr Cys 
                245                 250                 255 

Gln Leu Tyr Pro Asn Ala Ala Ala Ser Thr Leu Val His Lys Phe Phe 
            260                 265                 270 

Leu Val Phe Ser Lys Trp Glu Trp Pro Asn Pro Val Leu Leu Lys Gln 
        275                 280                 285 

Pro Glu Glu Ser Asn Leu Asn Leu Pro Val Trp Asp Pro Arg Val Asn 
    290                 295                 300 

Pro Ser Asp Arg Tyr His Leu Met Pro Ile Ile Thr Pro Ala Tyr Pro 
305                 310                 315                 320 

Gln Gln Asn Ser Thr Tyr Asn Val Ser Thr Ser Thr Arg Thr Val Met 
                325                 330                 335 

Val Glu Glu Phe Lys Gln Gly Leu Ala Val Thr Asp Glu Ile Leu Gln 
            340                 345                 350 

Gly Lys Ser Asp Trp Ser Lys Leu Leu Glu Pro Pro Asn Phe Phe Gln 
        355                 360                 365 

Lys Tyr Arg Tyr Val Gly Leu Val Glu Ser Lys Ile Arg Val Leu Val 
    370                 375                 380 

Gly Asn Leu Glu Arg Asn Glu Phe Ile Thr Leu Ala His Val Asn Pro 
385                 390                 395                 400 

Gln Ser Phe Pro Gly Asn Lys Glu His His Lys Asp Asn Asn Tyr Val 
                405                 410                 415 

Ser Met Trp Phe Leu Gly Ile Ile Phe Arg Arg Val Glu Asn Ala Glu 
            420                 425                 430 

Ser Val Asn Ile Asp Leu Thr Tyr Asp Ile Gln Ser Phe Thr Asp Thr 
        435                 440                 445 

Val Tyr Arg Gln Ala Asn Asn Ile Asn Met Leu Lys Glu Gly Met Lys 
    450                 455                 460 

Ile Glu Ala Thr His Val Lys Lys Lys Gln Leu His His Tyr Leu Pro 
465                 470                 475                 480 

Ala Glu Ile Leu Gln Lys Lys Lys Lys Val Ser Gln Ser Leu Ser Asp 
                485                 490                 495 

Val Asn Arg Ser Ser Gly Gly Leu Gln Ser Lys Arg Leu Ser Leu Asp 
            500                 505                 510 

Ser Ser Cys Leu Asp Ser Ser Arg Asp Thr Asp Asn Gly Thr Pro Phe 
        515                 520                 525 

Asn Ser Pro Ala Ser Lys Ser Asp Ser Pro Ser Val Gly Glu Thr Glu 
    530                 535                 540 

Arg Asn Ser Ala Glu Pro Ala Ala Val Ile Val Glu Lys Pro Leu Ser 
545                 550                 555                 560 

Val Pro Pro Ala Gln Gly Leu Ser Ile Pro Val Ile Gly Ala Lys Val 
                565                 570                 575 

Asp Ser Thr Val Lys Thr Val Ser Pro Pro Thr Val Cys Thr Ile Pro 
            580                 585                 590 

Thr Val Val Gly Arg Asn Val Ile Pro Arg Ile Thr Thr Pro His Asn 
        595                 600                 605 

Pro Ala Gln Gly Gln Pro His Leu Asn Gly Met Ser Asn Ile Thr Lys 
    610                 615                 620 

Thr Val Thr Pro Lys Arg Ser His Ser Pro Ser Ile Asp Gly Thr Pro 
625                 630                 635                 640 

Lys Arg Leu Lys Asp Val Glu Lys Phe Ile Arg Leu Glu Ser Thr Phe 
                645                 650                 655 

Lys Asp Pro Arg Thr Ala Glu Glu Arg Lys Arg Lys Ser Val Asp Ala 
            660                 665                 670 

Ile Gly Gly Glu Ser Met Pro Ile Pro Thr Ile Asp Thr Ser Arg Lys 
        675                 680                 685 

Lys Arg Leu Pro Ser Lys Glu Leu Pro Asp Ser Ser Ser Pro Val Pro 
    690                 695                 700 

Ala Asn Asn Ile Arg Val Ile Lys Asn Ser Ile Arg Leu Thr Leu Asn 
705                 710                 715                 720 

Arg Glx 

 
           
             3  
             2211  
             DNA  
             Homo sapiens  
           
            3 

atgaaagaga tgtctgcaaa caccgtgctg gacagccagc gtcaacaaaa gcattatgga     60 

attacctccc caattagttt ggcatctcct aaagaaattg atcatattta cacacagaaa    120 

ttaattgacg ccatgaaacc atttggagtg tttgaagatg aggaagaatt gaaccacagg    180 

ctggtggttc ttggtaaatt gaacaattta gtaaaagaat ggatttctga tgtcagcgag    240 

agtaagaacc tcccaccttc tgttgtggct actgttggtg gtaaaatttt cacatttgga    300 

tcctataggc ttggagtaca caccaaagga gctgacattg atgcactttg tgtagctcca    360 

agacatgtgg aaagatctga tttttttcag tctttttttg aaaaattgaa acatcaagat    420 

ggcattagaa acttaagagc tgtagaagat gcctttgtac ctgttataaa atttgaattt    480 

gatggtattg aaattgatct agtctttgca agactggcaa tacaaaccat atcagataat    540 

ttagatctaa gagacgactc tcgcctgaga agccttgata taaggtgtat tcgcagctta    600 

aatggttgta gagttactga tgaaattttg catttagtgc caaataaaga aacttttaga    660 

ctcaccctaa gagctgtcaa attatgggca aaacgacgtg gtatttattc caacatgcta    720 

ggattccttg gtggtgtctc ctgggcaatg ctagttgcaa gaacttgcca attgtatcca    780 

aatgcagcag catctacttt agttcataag ttctttttag ttttttccaa gtgggaatgg    840 

ccaaatcctg tgctgctgaa gcaaccagaa gaaagcaatt tgaatttgcc tgtctgggat    900 

cctcgggtaa atccatcaga taggtatcat ctcatgccca taatcacccc tgcctaccca    960 

caacagaatt ctacgtataa tgtgtccaca tcaactcgaa cagtaatggt agaagaattt   1020 

aaacaaggtc ttgcagtcac agatgaaatt cttcaaggaa agtcagattg gtccaaacta   1080 

cttgagccac cgaatttctt tcaaaagtat agacattata tagtattgac tgccagcgca   1140 

tcaacagaag aaaaccatct agagtgggtt ggattagtag aatctaaaat ccgtgtactt   1200 

gttggaaact tggaacggaa tgaatttatt actcttgccc atgtgaatcc ccagtcattc   1260 

ccagggaata aggaacatca taaagacaac aattacgtat caatgtggtt ccttgggata   1320 

atttttcgga gagtagaaaa tgcagaaagt gtcaacatag acttgacata tgatatacag   1380 

tcatttactg atacagtgta cagacaggca aacaatataa atatgctaaa ggagggaatg   1440 

aaaattgaag caactcatgt aaagaaaaaa caacttcacc actaccttcc tgcagaaatt   1500 

cttcaaaaga agaaaaagca aagtctctct gatgtcaatc gaagctcggg cggacttcaa   1560 

tccaaaagat tgtctctgga tagcagttgt ctggatagct ccagagacac tgataatgga   1620 

acacctttta attctccagc gtccaagtct gatagccctt ctgtaggaga aacagaaagg   1680 

aatagtgctg agcctgctgc tgtaattgtg gagaagccac tgagtgtacc accagcccaa   1740 

ggactttcca ttccagtgat tggcgcaaaa gttgactcta cagtaaaaac tgtatcaccc   1800 

cccactgtgt gtaccattcc taccgtagta ggacgaaatg tcattcctag aatcacaaca   1860 

cctcacaacc ctgcccaggg acaaccgcat ctgaatggaa tgtcaaatat aactaagact   1920 

gttacaccta agagatccca ttccccatcc atagatggga ctcctaagag gttgaaagac   1980 

gtagaaaagt ttattcgact tgaatcaaca tttaaggacc cccgcactgc tgaagaaaga   2040 

aaaagaaaat cagtggatgc cattggagga gaatctatgc ctattccaac tattgataca   2100 

tcacgcaaaa agagactacc cagtaaagaa ctaccagatt catcatctcc agttccagca   2160 

aacaacatcc gtgtcatcaa aaattccatt cgactgaccc ttaatcggta a            2211 

 
           
             4  
             736  
             PRT  
             Homo sapiens  
           
            4 

Met Lys Glu Met Ser Ala Asn Thr Val Leu Asp Ser Gln Arg Gln Gln 
  1               5                  10                  15 

Lys His Tyr Gly Ile Thr Ser Pro Ile Ser Leu Ala Ser Pro Lys Glu 
             20                  25                  30 

Ile Asp His Ile Tyr Thr Gln Lys Leu Ile Asp Ala Met Lys Pro Phe 
         35                  40                  45 

Gly Val Phe Glu Asp Glu Glu Glu Leu Asn His Arg Leu Val Val Leu 
     50                  55                  60 

Gly Lys Leu Asn Asn Leu Val Lys Glu Trp Ile Ser Asp Val Ser Glu 
 65                  70                  75                  80 

Ser Lys Asn Leu Pro Pro Ser Val Val Ala Thr Val Gly Gly Lys Ile 
                 85                  90                  95 

Phe Thr Phe Gly Ser Tyr Arg Leu Gly Val His Thr Lys Gly Ala Asp 
            100                 105                 110 

Ile Asp Ala Leu Cys Val Ala Pro Arg His Val Glu Arg Ser Asp Phe 
        115                 120                 125 

Phe Gln Ser Phe Phe Glu Lys Leu Lys His Gln Asp Gly Ile Arg Asn 
    130                 135                 140 

Leu Arg Ala Val Glu Asp Ala Phe Val Pro Val Ile Lys Phe Glu Phe 
145                 150                 155                 160 

Asp Gly Ile Glu Ile Asp Leu Val Phe Ala Arg Leu Ala Ile Gln Thr 
                165                 170                 175 

Ile Ser Asp Asn Leu Asp Leu Arg Asp Asp Ser Arg Leu Arg Ser Leu 
            180                 185                 190 

Asp Ile Arg Cys Ile Arg Ser Leu Asn Gly Cys Arg Val Thr Asp Glu 
        195                 200                 205 

Ile Leu His Leu Val Pro Asn Lys Glu Thr Phe Arg Leu Thr Leu Arg 
    210                 215                 220 

Ala Val Lys Leu Trp Ala Lys Arg Arg Gly Ile Tyr Ser Asn Met Leu 
225                 230                 235                 240 

Gly Phe Leu Gly Gly Val Ser Trp Ala Met Leu Val Ala Arg Thr Cys 
                245                 250                 255 

Gln Leu Tyr Pro Asn Ala Ala Ala Ser Thr Leu Val His Lys Phe Phe 
            260                 265                 270 

Leu Val Phe Ser Lys Trp Glu Trp Pro Asn Pro Val Leu Leu Lys Gln 
        275                 280                 285 

Pro Glu Glu Ser Asn Leu Asn Leu Pro Val Trp Asp Pro Arg Val Asn 
    290                 295                 300 

Pro Ser Asp Arg Tyr His Leu Met Pro Ile Ile Thr Pro Ala Tyr Pro 
305                 310                 315                 320 

Gln Gln Asn Ser Thr Tyr Asn Val Ser Thr Ser Thr Arg Thr Val Met 
                325                 330                 335 

Val Glu Glu Phe Lys Gln Gly Leu Ala Val Thr Asp Glu Ile Leu Gln 
            340                 345                 350 

Gly Lys Ser Asp Trp Ser Lys Leu Leu Glu Pro Pro Asn Phe Phe Gln 
        355                 360                 365 

Lys Tyr Arg His Tyr Ile Val Leu Thr Ala Ser Ala Ser Thr Glu Glu 
    370                 375                 380 

Asn His Leu Glu Trp Val Gly Leu Val Glu Ser Lys Ile Arg Val Leu 
385                 390                 395                 400 

Val Gly Asn Leu Glu Arg Asn Glu Phe Ile Thr Leu Ala His Val Asn 
                405                 410                 415 

Pro Gln Ser Phe Pro Gly Asn Lys Glu His His Lys Asp Asn Asn Tyr 
            420                 425                 430 

Val Ser Met Trp Phe Leu Gly Ile Ile Phe Arg Arg Val Glu Asn Ala 
        435                 440                 445 

Glu Ser Val Asn Ile Asp Leu Thr Tyr Asp Ile Gln Ser Phe Thr Asp 
    450                 455                 460 

Thr Val Tyr Arg Gln Ala Asn Asn Ile Asn Met Leu Lys Glu Gly Met 
465                 470                 475                 480 

Lys Ile Glu Ala Thr His Val Lys Lys Lys Gln Leu His His Tyr Leu 
                485                 490                 495 

Pro Ala Glu Ile Leu Gln Lys Lys Lys Lys Gln Ser Leu Ser Asp Val 
            500                 505                 510 

Asn Arg Ser Ser Gly Gly Leu Gln Ser Lys Arg Leu Ser Leu Asp Ser 
        515                 520                 525 

Ser Cys Leu Asp Ser Ser Arg Asp Thr Asp Asn Gly Thr Pro Phe Asn 
    530                 535                 540 

Ser Pro Ala Ser Lys Ser Asp Ser Pro Ser Val Gly Glu Thr Glu Arg 
545                 550                 555                 560 

Asn Ser Ala Glu Pro Ala Ala Val Ile Val Glu Lys Pro Leu Ser Val 
                565                 570                 575 

Pro Pro Ala Gln Gly Leu Ser Ile Pro Val Ile Gly Ala Lys Val Asp 
            580                 585                 590 

Ser Thr Val Lys Thr Val Ser Pro Pro Thr Val Cys Thr Ile Pro Thr 
        595                 600                 605 

Val Val Gly Arg Asn Val Ile Pro Arg Ile Thr Thr Pro His Asn Pro 
    610                 615                 620 

Ala Gln Gly Gln Pro His Leu Asn Gly Met Ser Asn Ile Thr Lys Thr 
625                 630                 635                 640 

Val Thr Pro Lys Arg Ser His Ser Pro Ser Ile Asp Gly Thr Pro Lys 
                645                 650                 655 

Arg Leu Lys Asp Val Glu Lys Phe Ile Arg Leu Glu Ser Thr Phe Lys 
            660                 665                 670 

Asp Pro Arg Thr Ala Glu Glu Arg Lys Arg Lys Ser Val Asp Ala Ile 
        675                 680                 685 

Gly Gly Glu Ser Met Pro Ile Pro Thr Ile Asp Thr Ser Arg Lys Lys 
    690                 695                 700 

Arg Leu Pro Ser Lys Glu Leu Pro Asp Ser Ser Ser Pro Val Pro Ala 
705                 710                 715                 720 

Asn Asn Ile Arg Val Ile Lys Asn Ser Ile Arg Leu Thr Leu Asn Arg 
                725                 730                 735