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Prothymosin α Is Not Found in Yeast
PROTEIN EXPRESSION AND PURIFICATION ARTICLE NO.
13, 383–388 (1998)
PT980909
Prothymosin a Is Not Found in Yeast Mark W. Trumbore, Richard E. Manrow,1 and Shelby L. Berger Section on Genes and Gene Products, Division of Basic Sciences, National Cancer Institute, Bethesda, Maryland 20892
Received February 26, 1998, and in revised form April 29, 1998
According to published accounts, prothymosin a exhibits high evolutionary conservation from yeast to man (Makarova, T., Grebenshikov, N., Egorov, C., Vartapetian, A., and Bogdanov, A. FEBS Lett. 257, 247– 250, 1989). We report here our failure to find evidence for prothymosin a in yeast using three biochemical approaches: hybridization of yeast mRNA and genomic DNA with human prothymosin a coding region probes, performance of the polymerase chain reaction with yeast genomic template DNA and three sets of primers recognizing human prothymosin a coding region sequences, and isolation of yeast proteins essentially as described in the publication above. A survey of the Saccharomyces cerevisiae complete genome database using the program BLASTp verified our findings: there is no prothymosin a-homologue in yeast. Furthermore, DNA representing organisms from bacteria to amphibians also failed to hybridize with the same probes. Therefore, the presence of a prothymosin a gene in animals other than mammals is highly unlikely. © 1998 Academic Press
nucleic acid probes based on clones of the human prothymosin a gene and cDNA (9), we were well positioned to identify an evolutionarily related prothymosin a in lower eukaryotes. We were excited when we encountered the work of Bogdanov and colleagues in which a 13-kDa yeast prothymosin a was not only described, but also characterized and found to possess a tryptic peptide map almost indistinguishable from that of mouse prothymosin a (10 –12). Armed with evidence for the existence of prothymosin a in a simple eukaryote, we set out to clone the yeast gene. Because an exhaustive search for prothymosin a in yeast at the level of the protein, the mRNA, and the gene was unsuccessful, we undertook an extensive computer analysis of the Saccharomyces database. Now, it can be definitively stated based on practical and theoretical grounds that Saccharomyces cerevisiae does not contain a homologue of mammalian prothymosin a. MATERIALS AND METHODS
Nucleic Acids Prothymosin a participates in a vital nuclear process that is required for cell survival and growth (1–5). The precise function is unknown, but the extensive sequence homology among species, including human, goat, pig, cow, rat, and mouse (6 and Refs. therein), together with an abundance approaching that of histone cores (7) suggested a role performed throughout the animal kingdom. Prothymosin a’s properties are tantalizing; of the 109 amino acids in the mature human protein, 50% are acidic, none contain sulfur, and none are aromatic (1). No one has succeeded in raising specific antibodies with high titer. In contrast, a powerful tool for isolating the protein was developed by utilizing its capacity to partition to the aqueous phase of a phenol extraction in virtually 100% yield (8). With a simple means for protein purification and a host of 1 Present address: Journal of the National Cancer Institute, 7550 Wisconsin Ave., Rm. 114, Bethesda, MD 20814.
1046-5928/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
The preparation of yeast RNA (13,14) and DNA (15,16) made use of standard techniques. Polyadenylated RNA was selected by one pass through oligo(dT) cellulose columns without intermediate washes in order to prevent loss of mRNAs with short poly(A) tails (17). Wild-type yeast strains were proffered by Drs. Enrico Cabib and Sanford Silverman of NIDDK, NIH and NKY274SZ MAT a Ura3 lys2 ho::LYS2 was generously donated by Dr. Michael Lichten of NCI, NIH. Nucleic acids were analyzed electrophoretically, blotted, and hybridized with human coding region [32P]DNA generated either by introducing radioactivity into purified cloned fragments of human cDNA with a Multiprime DNA Labeling kit (Amersham) or by preparing 32P-labeled PCR DNA as noted below (18,19). Blots were washed at low stringency, namely, prolonged treatment under hybridization conditions, and at moderate or high stringency— 65°C for 1 h in 383
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0.1% SDS2 and 23 SSC or 0.23 SSC, respectively. (19). An EVO blot containing cleaved DNA from representative phyla was supplied by Bios. Yeast libraries in l were purchased from Stratagene and Clonetech and a yeast pWE15 cosmid library was obtained from Stratagene. Yeast libraries were also generated in pGEM3Zf(2) with HindIII-cut yeast DNA, and in ScaI-cut pBR322 with PvuII-cut yeast strain DC6 DNA. SURE cells (Stratagene) and DH10B cells (BRL) served as vehicles. The number of colonies or plaques evaluated exceeded the number required for 99.99% probability of finding a clone by several fold in all cases. Libraries were screened with PCR-generated 32 P-labeled human prothymosin a coding region probes prepared with human prothymosin a cDNA as the template, Sh 37 and Sh 39 as the primers (see below), and [32P]dCTP. Our procedures made use of either the recommendations of the manufacturer or published protocols (19). The polymerase chain reaction was carried out essentially as detailed by Berger et al. (20) with various purified yeast DNA preparations using primer pairs as follows: Sh 37 and Sh 39, Sh 37 and Rem 4, Rem 3 and Sh 39. The targets of the primers on the 1200-bp human prothymosin a full-length cDNA clone are: Sh 37 (forward), positions 181–208; Sh 39 (reverse), 482–510; Rem 3 (forward), 268 –286; and Rem 4 (reverse), 315– 331. The coding region of prothymosin a extends from the ATG codon at position 178 to the stop codon at position 511. A yeast library in lgt-11 (Clonetech) was also amplified using the primers noted above together with primers recognizing the flanking sequences in the l vector. Proteins Yeast proteins were obtained from DC6 cells which had been labeled for 6 h with 50 mCi/ml of [3H]glutamic acid in minimal medium (2) and disrupted by vigorous mixing with glass beads in lysis buffer (30 mM Tris HCl at pH 7.5, 100 mM NaCl, 1 mM MgCl2, and 1 mM CaCl2) to which protease inhibitors (pepstatin, leupeptin, and phenylmethylsulfonyl fluoride) had been added (2); after removing the beads centrifugally, a small subset of polypeptides was isolated from the aqueous phase after extracting several times with phenol in the presence of 0.5% SDS (8). Protein size was assessed electrophoretically in 15% polyacrylamide SDS gels with control proteins—prothymosin a from human myeloma cells and a synthetic prothymosin a processed pseudogene, PTMAP 4, both isolated by means of a phenol extraction (7,8). The protein product 2 Abbreviations used: PCR, polymerase chain reaction; PTMA, prothymosin a; PTMAP4, prothymosin a pseudogene 4; SDS, sodium dodecyl sulfate; SSC, a solution containing sodium chloride and citrate defined in Ref. (19).
of PTMAP 4 was prepared in vitro by translating a synthetic, capped mRNA in the wheat germ system in the presence of [3H]glutamic acid as described (3,7). The processed pseudogene is not expressed in human tissues and was not found in human cDNA libraries generated from normal skin fibroblats or transformed human myeloma cells (21). Computer Analyses For computer analysis, the S. cerevisiae genome database at the National Center for Biotechnology Information was searched in gapped mode with the BLASTp program in the BLAST 2.0 release. In gapped mode, allowances are made for interruptions in the regions of homology increasing the likelihood of detecting matching sequences. The filter, which masks sequences of low compositional complexity, was turned off in order to screen acid-rich regions. Open reading frames in common with the amino acid sequence of prothymosin a were aligned with the Bestfit and GAP routines in the Wisconsin GCG package, both of which gave identical results. RESULTS
Search for Prothymosin a Protein in Yeast Prothymosin a can be purified from mammalian cell lysates using techniques designed for the isolation of RNA (8,10). Accordingly, [3H]glutamate-labeled DC6 yeast cells were disrupted with glass beads and subjected to a phenol extraction. Figure 1 shows the results; the control proteins, human prothymosin a and a slightly longer synthetic prothymosin a pseudogene, are overexposed in order to search for the yeast homologue. Although a small amount of labeled yeast protein was obtained, no protein the size of the ;13-kDa putative yeast prothymosin a was observed. Search for Prothymosin a mRNA in Yeast Without a highly specific antibody, we turned instead to mRNA and searched Northern blots. Yeast was grown either in complete medium or in minimal medium, total RNA or polyadenylated RNA was isolated, and Northern blots were probed with 32P-labeled human prothymosin a coding region DNA. An example of these experiments with a blot washed at low stringency is shown in Fig. 2. Here, prothymosin a mRNA was readily observed in 5 mg of total RNA from human myeloma cells, but was not found in 5.6 or 9.8 mg of polyadenylated RNA from yeast cells grown in complete or minimal medium, respectively. Because the samples contain substantial amounts of rRNA visible on the autoradiograms, it is clear that the failure to visualize prothymosin a mRNA was not caused by degradation of the RNA. The absence of yeast prothymosin a
PROTHYMOSIN a IS NOT FOUND IN YEAST
FIG. 1. [3H]Glutamate-labeled yeast and human proteins. Materials partitioning into the aqueous phase of a phenol extraction were analyzed electophoretically in a 15% polyacrylamide SDS gel, fixed with Enlightning, and exposed to film for 1 month: 1, a mixture of human prothymosin a and the synthetic human PTMAP 4 protein (a human prothymosin a processed pseudogene protein); 2, yeast proteins from 75 ml of culture. The arrow marks the position of human prothymosin a.
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FIG. 3. Autoradiogram of an EVO blot. The purchased blot was hybridized as suggested by the manufacturer at 65°C overnight in 63 SSC, 53 Denhardt’s solution, 10% dextran sulfate, 1% SDS, and 100 mg/ml of salmon sperm DNA. The blot was washed at moderate stringency and exposed for 1 week. The positions of l HindIII markers in ascending order of size, namely, 2.0, 2.3, 4.4, 6.6, 9.4, and 23 kb are indicated. DNAs are as follows: 1, human; 2, mouse; 3, lobster; 4, clam; 5, sea urchin; 6, frog; 7, fly; 8, nematode; 9, yeast; and 10, E. coli.
whereas weak bands representing rRNAs and, to a much lesser extent, tRNA were the only signals visible in the yeast samples (data not shown). Search for Prothymosin a Genomic DNA in Yeast
mRNA was confirmed by performing experiments with 60 mg of yeast total RNA. On these blots, 12.5 mg of total RNA from human myeloma cells gave a clear signal,
FIG. 2. Autoradiogram of RNA. RNA was analyzed in a 1.2% agarose formaldehyde gel, blotted to Nytran, and hybridized with a [32P]human cDNA coding region probe at 30°C in 83 SSC, other components as described, and an equal volume of formamide. The blot was washed at low stringency: 1, 5 mg of total RNA from human myeloma cells; 2, 5.6 mg of polyadenylated RNA from yeast grown in complete medium; 3, 9.8 mg of polyadenylated RNA from yeast grown in minimal medium. The positions of rRNAs are noted. The arrow marks the position of human prothymosin a mRNA at 1.2 kb.
In mammals, the amount of prothymosin a and its mRNA correlates with the growth rate of the cell or tissue from which the materials are obtained (3). In yeast, there may be no such requirement. As a consequence, the genome was searched using three methods: Southern blots were probed with human prothymosin a cDNA coding region probes, yeast genomic DNA was used as a template for PCR carried out with specific prothymosin a cDNA primers, and several locally generated and commercial libraries were screened with prothymosin a-specific probes. Southern blots of yeast DNA were prepared from four independent preparations cleaved with BamHI, PvuII, EcoRI, HindIII, BstUI, HaeIII, HhaI, HpaII, MboI, RsaI, and Sau3AI. A 330-bp DNA obtained with the aid of PCR using human cDNA as the template and Sh 37 and Sh 39 as primers served as a positive control. After hybridizing and washing both blots at low stringency in the same bag, the control band was readily visible whereas only very faint, apparently nonspecific, bands were observed in both yeast and marker DNA (Fig. 3 and data not shown). To prove that the yeast Southern blots contained adequate amounts of DNA, the blots were stripped and probed with a 32P-labeled yeast Arg 4 39-end probe obtained from Dr. M. Lichten; there was at least one very bright Arg 4 DNA band in
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FIG. 4. Alignment of prothymosin a (top) with the ;88-kDa open reading frame, YPL190c (bottom), prothymosin a’s closest relative in the Saccharomyces cerevisiae genome database.
each sample. Since the assays contained sufficient amounts of quality DNA, the presence of sequences homologous with the human prothymosin a gene appeared to be highly problematic. PCR was carried out with yeast genomic DNA and the three sets of primers described under Materials and Methods. The results with all sets of primers demonstrated that no yeast PCR DNA with resemblance to human prothymosin a cDNA was encountered when the product DNA was sequenced or probed as stated at low, moderate, or high stringency. Similarly, PCR was also unsuccessful in generating prothymosin a-like sequences using a yeast library in lgt-11 regardless of whether the primers recognized prothymosin a or the flanking regions of the vector. Yeast libraries were also screened for prothymosin a-like sequences. We used two plasmid libraries generated in our laboratory; in one case, pBR322 was selected to decrease plasmid copy number in the event of toxicity of the very prothymosin a sequences we sought. There were no positive yeast DNA-containing clones regardless of the stringency with which the hybridized, colony lifts were washed (data not shown). Commercial libraries in l or in Escherichia coli and a cosmid library screened similarly corroborated these results. Our choice of a variety of restriction enzymes and cloning systems and the analysis of several yeast strains made it unlikely that the desired clone had escaped detection either because it was nonviable or because the yeast prothymosin a DNA had been cleaved into fragments too small to distinguish. In pursuit of prothymosin a genes in species other than yeast, a Southern blot containing EcoRI-cut DNA from representative phyla was probed with the 32Plabeled 330-bp human prothymosin a cDNA coding region. The autoradiogram is displayed in Fig. 3. Although the blot, washed at moderate stringency, was exposed to film for 1 week, it is evident that the human and mouse possess prothymosin a genes (and pseudogenes), that lobster and sea urchin DNAs might con-
tain them, and that clams, frogs, flies, nematodes, yeast, and E. coli do not have a prothymosin a gene. Comparison of the Prothymosin a Amino Acid Sequence with the Sacchaomyces cerevisiae Genome Database The BLASTp program employed in this study compares an amino acid query sequence, prothymosin a, against a protein sequence data base, the entire S. cerevisiae genome. Figure 4 illustrates the output for the best fit protein, YPL190c. It is evident that the overall match of prothymosin a with YPL190c is poor with 31.2% sequence identity and 54.1% similarity. Furthermore, the regions in common are overwhelmingly acidic elements known to appear in otherwise nonhomologous proteins. To expand the search, we viewed the second best fit, YGL150c, and found 27.5% identity and 48.6% similarity, again a molecule with acidic regions. When the data base was searched using the low-complexity filter, a mode that eliminates the acidic stretches from consideration, no matches to prothymosin a were found regardless of how the values that define random homology were adjusted. With the filter off, a search restricted to proteins approximately the same size as mammalian prothymosin a’s resulted in a poor match with the best fit, L22015, having 22% identity and 31.2% homology. Moreover, the predicted tryptic peptide maps of YPL190c, YGL150c, and L22015 are substantially different from that of prothymosin a, judging by the number of peptides produced and their sizes (Table 1). We conclude that yeast does not have a prothymosin a homologue. DISCUSSION
When two studies come to diametrically opposite conclusions, it is necessary to evaluate the quality of the data in each case. We searched for prothymosin a protein in yeast, but unlike Makarova et al. (11) we
PROTHYMOSIN a IS NOT FOUND IN YEAST
TABLE 1 Comparison of the Tryptic Peptides of Prothymosin a with Those of Three Best Fit Yeast Open Reading Frames Number of peptides Residues
PTMA
L22015
YPL190c
YGL150c
.80 71–80 61–70 51–60 41–50 36–40 31–35 26–30 21–25 16–20 11–15 6–10 2–5
0 0 0 1 0 0 0 0 0 0 2 1 4
0 0 0 0 0 1 0 1 1 2 2 5 6
1 1 1 0 2 1 0 0 5 4 6 8 26
1 0 0 1 0 4 2 1 4 8 21 38 84
Note. The data are expressed as the number of peptides in each size class. The size classes range from the smallest peptides with 2–5 residues to the largest with more than 80 residues. PTMA is prothymosin a, with 109 amino acids. The other designations refer to open reading frames in yeast.
failed to find evidence for it. In both laboratories, the purification makes use of the ability of prothymosin a to partition into the aqueous phase of a phenol extraction (8,10), a property which the aforementioned group attributed incorrectly (7) to the presence of a covalently bound RNA. We have used this technique to isolate prothymosin a from human cells and bovine thymus, and to obtain recombinant prothymosin a expressed in E. coli, whereas Pavlov et al. (12) demonstrated its efficacy by purifying recombinant human prothymosin a overexpressed in yeast. However, with the same approach, our attempts to find native yeast prothymosin a using large quantities of cells were uniformly unsuccessful. A search for prothymosin a sequences in nucleic acids failed. Messenger RNA from yeast, either with or without a poly(A) tail, was not capable of hybridizing to probes composed of the human prothymosin a coding region sequences, even at low stringency. At the level of the genome, no recognizable prothymosin a gene was found in Southern blots of yeast DNAs, or in libraries whether prepared in our laboratory or purchased from commercial sources. Although PCR provided improved sensitivity, there was no prothymosin a homologue produced with yeast genomic DNA templates using several different sets of primers. Since all hybridization methods require similarity of nucleic acid sequences, we reasoned that if yeast possessed a prothymosin a gene that did not hybridize to our probes, species closer to mammals on the evolutionary tree would also have the gene and its sequence
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would be more highly conserved. A blot with genomic DNAs representing many phyla revealed the presence of the gene in mouse and man, but failed to find prothymosin a unequivocably in any other group. When we were unable to identify a homologous prothymosin a gene in frogs, we concluded that the presence of such a gene, whether closely related or highly divergent, was highly improbable in yeast. In contrast, the Bogdanov group found the protein by extracting yeast with phenol and labeling the acquired polypeptides on lysine residues with Bolton Hunter reagent (10,11). Murine, bovine, and yeast prothymosin a were electrophoretically identical in size. Moreover, a two-dimensional tryptic peptide map indicated that yeast and mouse prothymosin a were more closely related, spot for spot, than the mouse and human proteins (11) despite the fact that the latter differ in only five respects: the addition of Q in mouse prothymosin a between positions 38 and 39 of the human protein; and mouse3human, SA3 AP, A3 V, and D3 E at positions 83– 84, 89, and 106, respectively. We believe that the vanishingly small amounts of prothymosin a reported in yeast are the key to understanding these data; the yeast map had clearly been exposed to film for a prolonged period of time and the amount of protein used to generate it was not disclosed. In a subsequent publication (12), an inability to isolate native prothymosin a from yeast was attributed to inadequate amounts of material. The availability of the entire yeast genome has made it possible to resolve the issue unambiguously. We have shown that the yeast genome does not have open reading frames homolgous to mammalian prothymosin a. Apparently, there is no such gene and no such protein in yeast. REFERENCES 1. Eschenfeldt, W. H., and Berger, S. L. (1986) The human prothymosin a gene is polymorphic and induced upon growth stimulation: Evidence using a cloned cDNA. Proc. Natl. Acad. Sci. USA 83, 9403–9407. 2. Sburlati, A. R., Manrow, R. E., and Berger, S. L. (1991) Prothymosin a antisense oligomers inhibit myeloma cell division. Proc. Natl. Acad. Sci. USA 88, 253–257. 3. Manrow, R. E., Sburlati, A. R., Hanover, J. A., and Berger, S. L. (1991) Nuclear targeting of prothymosin a. J. Biol. Chem. 266, 3916 –3924. 4. Clinton, M., Graeve, L., El-Dorry, H., Rodriguez-Boulan, E., and Horecker, B. L. (1991) Evidence for nuclear targeting of prothymosin and parathymosin synthesized in situ. Proc. Natl. Acad. Sci. USA 88, 6608 – 6612. 5. Wang, R-H., Tao, L., Trumbore, M. W., and Berger, S. L. (1997) Turnover of the acyl phosphates of human and murine prothymosin a in vivo. J. Biol. Chem. 272, 26405–26412. 6. Frillingos, S., Frangou-Lazaridis, M., Seferiadis, K., Hulmes, J. D., Pan, Y.-C. E., and Tsolas, O. (1991) Isolation and partial sequence of goat spleen prothymosin a. Mol. Cell. Biochem. 108, 85–94.
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7. Sburlati, A. R., De La Rosa, A., Batey, D. W., Kurys, G. L., Manrow, R. E., Pannell, L. K., Martin, B. M., Sheeley, D. M., and Berger, S. L. (1993) Phosphorylation of human and bovine prothymosin a in vivo. Biochemistry 32, 4587– 4596. 8. Sburlati, A. R., Manrow, R. E., and Berger, S. L. (1990) Human prothymosin a: Purification of a highly acidic nuclear protein by means of a phenol extraction. Protein Expression Purif. 1, 184 – 190. 9. Eschenfeldt, W. H., Manrow, R. E., Krug, M. S., and Berger, S. L. (1989) Isolation and partial sequencing of the human prothymosin a gene family. J. Biol. Chem. 264, 7546 –7555. 10. Vartapetian, A. B., Makarova, T. N., Koonin, E. V., Agol, V. I., and Bogdanov, A. A. (1988) Small cytoplasmic RNA from mouse cells covalently linked to a protein. FEBS Lett. 232, 35–38. 11. Makarova, T., Grebenshikov, N., Egorov, C., Vartapetian, A., and Bogdanov, A. (1989) Prothymosin a is an evolutionary conserved protein covalently linked to a small RNA. FEBS Lett. 257, 247–250. 12. Pavlov, N., Evstafieva, A., Rubtsov, Y., and Vartapetian, A. (1995) Human prothymosin a inhibits division of yeast Saccharomyces cerevisiae cells, while its mutant lacking nuclear localization signal does not. FEBS Lett. 366, 43– 45. 13. Rose, M. D., Winston, F., and Hieter, P. (1990) ‘‘Methods in Yeast Genetics, A Laboratory Course Manual,’’ pp. 140 –142, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
14. Feinberg, B., and McLaughlin, C. S. (1988) Isolation of yeast mRNA and in vitro translation in a yeast cell-free system, in ‘‘Yeast: A Practical Approach’’ (Campbell, I., and Duffus, J. H., Eds.), pp. 147–161, IRL Press, Oxford. 15. Johnston, J. R. (1988) Yeast genetics, molecular aspects, in ‘‘Yeast: A Practical Approach’’ (Campbell, I., and Duffus, J. H., Eds.), pp. 107–123, IRL Press, Oxford. 16. Phillippsen, P., Stotz, A., and Scherf, C. (1991) DNA of Saccharomyces cerevisiae. Methods Enzymol. 194, 169 –182. 17. Jacobsen, A. (1987) Purification and fractionation of poly(A)1RNA. Methods Enzymol. 152, 254 –261. 18. Ogden, R. C., and Adams, D. A. (1987) Electrophoresis in agarose and acrylamide gels. Methods Enzymol. 152, 61– 87. 19. Wahl, G. M., and Berger, S. L. (1987) Screening colonies or plaques with radioactive nucleic acid probes. Methods Enzymol. 152, 415– 423. 20. Berger, S. L., Manrow, R. E., and Lee, H. Y. (1993) Phoenix mutagenesis: One-step reassembly of multiply cleaved plasmids with mixtures of mutant and wild-type fragments. Anal. Biochem. 214, 571–579. 21. Manrow, R. E., Leone, A., Krug, M. S., Eschenfeldt, W. H., and Berger, S. L. (1992) The human prothymosin a gene family contains several processed pseudogenes lacking deleterious lesions. Genomics 13, 319 –331.
13, 383–388 (1998)
PT980909
Prothymosin a Is Not Found in Yeast Mark W. Trumbore, Richard E. Manrow,1 and Shelby L. Berger Section on Genes and Gene Products, Division of Basic Sciences, National Cancer Institute, Bethesda, Maryland 20892
Received February 26, 1998, and in revised form April 29, 1998
According to published accounts, prothymosin a exhibits high evolutionary conservation from yeast to man (Makarova, T., Grebenshikov, N., Egorov, C., Vartapetian, A., and Bogdanov, A. FEBS Lett. 257, 247– 250, 1989). We report here our failure to find evidence for prothymosin a in yeast using three biochemical approaches: hybridization of yeast mRNA and genomic DNA with human prothymosin a coding region probes, performance of the polymerase chain reaction with yeast genomic template DNA and three sets of primers recognizing human prothymosin a coding region sequences, and isolation of yeast proteins essentially as described in the publication above. A survey of the Saccharomyces cerevisiae complete genome database using the program BLASTp verified our findings: there is no prothymosin a-homologue in yeast. Furthermore, DNA representing organisms from bacteria to amphibians also failed to hybridize with the same probes. Therefore, the presence of a prothymosin a gene in animals other than mammals is highly unlikely. © 1998 Academic Press
nucleic acid probes based on clones of the human prothymosin a gene and cDNA (9), we were well positioned to identify an evolutionarily related prothymosin a in lower eukaryotes. We were excited when we encountered the work of Bogdanov and colleagues in which a 13-kDa yeast prothymosin a was not only described, but also characterized and found to possess a tryptic peptide map almost indistinguishable from that of mouse prothymosin a (10 –12). Armed with evidence for the existence of prothymosin a in a simple eukaryote, we set out to clone the yeast gene. Because an exhaustive search for prothymosin a in yeast at the level of the protein, the mRNA, and the gene was unsuccessful, we undertook an extensive computer analysis of the Saccharomyces database. Now, it can be definitively stated based on practical and theoretical grounds that Saccharomyces cerevisiae does not contain a homologue of mammalian prothymosin a. MATERIALS AND METHODS
Nucleic Acids Prothymosin a participates in a vital nuclear process that is required for cell survival and growth (1–5). The precise function is unknown, but the extensive sequence homology among species, including human, goat, pig, cow, rat, and mouse (6 and Refs. therein), together with an abundance approaching that of histone cores (7) suggested a role performed throughout the animal kingdom. Prothymosin a’s properties are tantalizing; of the 109 amino acids in the mature human protein, 50% are acidic, none contain sulfur, and none are aromatic (1). No one has succeeded in raising specific antibodies with high titer. In contrast, a powerful tool for isolating the protein was developed by utilizing its capacity to partition to the aqueous phase of a phenol extraction in virtually 100% yield (8). With a simple means for protein purification and a host of 1 Present address: Journal of the National Cancer Institute, 7550 Wisconsin Ave., Rm. 114, Bethesda, MD 20814.
1046-5928/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
The preparation of yeast RNA (13,14) and DNA (15,16) made use of standard techniques. Polyadenylated RNA was selected by one pass through oligo(dT) cellulose columns without intermediate washes in order to prevent loss of mRNAs with short poly(A) tails (17). Wild-type yeast strains were proffered by Drs. Enrico Cabib and Sanford Silverman of NIDDK, NIH and NKY274SZ MAT a Ura3 lys2 ho::LYS2 was generously donated by Dr. Michael Lichten of NCI, NIH. Nucleic acids were analyzed electrophoretically, blotted, and hybridized with human coding region [32P]DNA generated either by introducing radioactivity into purified cloned fragments of human cDNA with a Multiprime DNA Labeling kit (Amersham) or by preparing 32P-labeled PCR DNA as noted below (18,19). Blots were washed at low stringency, namely, prolonged treatment under hybridization conditions, and at moderate or high stringency— 65°C for 1 h in 383
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0.1% SDS2 and 23 SSC or 0.23 SSC, respectively. (19). An EVO blot containing cleaved DNA from representative phyla was supplied by Bios. Yeast libraries in l were purchased from Stratagene and Clonetech and a yeast pWE15 cosmid library was obtained from Stratagene. Yeast libraries were also generated in pGEM3Zf(2) with HindIII-cut yeast DNA, and in ScaI-cut pBR322 with PvuII-cut yeast strain DC6 DNA. SURE cells (Stratagene) and DH10B cells (BRL) served as vehicles. The number of colonies or plaques evaluated exceeded the number required for 99.99% probability of finding a clone by several fold in all cases. Libraries were screened with PCR-generated 32 P-labeled human prothymosin a coding region probes prepared with human prothymosin a cDNA as the template, Sh 37 and Sh 39 as the primers (see below), and [32P]dCTP. Our procedures made use of either the recommendations of the manufacturer or published protocols (19). The polymerase chain reaction was carried out essentially as detailed by Berger et al. (20) with various purified yeast DNA preparations using primer pairs as follows: Sh 37 and Sh 39, Sh 37 and Rem 4, Rem 3 and Sh 39. The targets of the primers on the 1200-bp human prothymosin a full-length cDNA clone are: Sh 37 (forward), positions 181–208; Sh 39 (reverse), 482–510; Rem 3 (forward), 268 –286; and Rem 4 (reverse), 315– 331. The coding region of prothymosin a extends from the ATG codon at position 178 to the stop codon at position 511. A yeast library in lgt-11 (Clonetech) was also amplified using the primers noted above together with primers recognizing the flanking sequences in the l vector. Proteins Yeast proteins were obtained from DC6 cells which had been labeled for 6 h with 50 mCi/ml of [3H]glutamic acid in minimal medium (2) and disrupted by vigorous mixing with glass beads in lysis buffer (30 mM Tris HCl at pH 7.5, 100 mM NaCl, 1 mM MgCl2, and 1 mM CaCl2) to which protease inhibitors (pepstatin, leupeptin, and phenylmethylsulfonyl fluoride) had been added (2); after removing the beads centrifugally, a small subset of polypeptides was isolated from the aqueous phase after extracting several times with phenol in the presence of 0.5% SDS (8). Protein size was assessed electrophoretically in 15% polyacrylamide SDS gels with control proteins—prothymosin a from human myeloma cells and a synthetic prothymosin a processed pseudogene, PTMAP 4, both isolated by means of a phenol extraction (7,8). The protein product 2 Abbreviations used: PCR, polymerase chain reaction; PTMA, prothymosin a; PTMAP4, prothymosin a pseudogene 4; SDS, sodium dodecyl sulfate; SSC, a solution containing sodium chloride and citrate defined in Ref. (19).
of PTMAP 4 was prepared in vitro by translating a synthetic, capped mRNA in the wheat germ system in the presence of [3H]glutamic acid as described (3,7). The processed pseudogene is not expressed in human tissues and was not found in human cDNA libraries generated from normal skin fibroblats or transformed human myeloma cells (21). Computer Analyses For computer analysis, the S. cerevisiae genome database at the National Center for Biotechnology Information was searched in gapped mode with the BLASTp program in the BLAST 2.0 release. In gapped mode, allowances are made for interruptions in the regions of homology increasing the likelihood of detecting matching sequences. The filter, which masks sequences of low compositional complexity, was turned off in order to screen acid-rich regions. Open reading frames in common with the amino acid sequence of prothymosin a were aligned with the Bestfit and GAP routines in the Wisconsin GCG package, both of which gave identical results. RESULTS
Search for Prothymosin a Protein in Yeast Prothymosin a can be purified from mammalian cell lysates using techniques designed for the isolation of RNA (8,10). Accordingly, [3H]glutamate-labeled DC6 yeast cells were disrupted with glass beads and subjected to a phenol extraction. Figure 1 shows the results; the control proteins, human prothymosin a and a slightly longer synthetic prothymosin a pseudogene, are overexposed in order to search for the yeast homologue. Although a small amount of labeled yeast protein was obtained, no protein the size of the ;13-kDa putative yeast prothymosin a was observed. Search for Prothymosin a mRNA in Yeast Without a highly specific antibody, we turned instead to mRNA and searched Northern blots. Yeast was grown either in complete medium or in minimal medium, total RNA or polyadenylated RNA was isolated, and Northern blots were probed with 32P-labeled human prothymosin a coding region DNA. An example of these experiments with a blot washed at low stringency is shown in Fig. 2. Here, prothymosin a mRNA was readily observed in 5 mg of total RNA from human myeloma cells, but was not found in 5.6 or 9.8 mg of polyadenylated RNA from yeast cells grown in complete or minimal medium, respectively. Because the samples contain substantial amounts of rRNA visible on the autoradiograms, it is clear that the failure to visualize prothymosin a mRNA was not caused by degradation of the RNA. The absence of yeast prothymosin a
PROTHYMOSIN a IS NOT FOUND IN YEAST
FIG. 1. [3H]Glutamate-labeled yeast and human proteins. Materials partitioning into the aqueous phase of a phenol extraction were analyzed electophoretically in a 15% polyacrylamide SDS gel, fixed with Enlightning, and exposed to film for 1 month: 1, a mixture of human prothymosin a and the synthetic human PTMAP 4 protein (a human prothymosin a processed pseudogene protein); 2, yeast proteins from 75 ml of culture. The arrow marks the position of human prothymosin a.
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FIG. 3. Autoradiogram of an EVO blot. The purchased blot was hybridized as suggested by the manufacturer at 65°C overnight in 63 SSC, 53 Denhardt’s solution, 10% dextran sulfate, 1% SDS, and 100 mg/ml of salmon sperm DNA. The blot was washed at moderate stringency and exposed for 1 week. The positions of l HindIII markers in ascending order of size, namely, 2.0, 2.3, 4.4, 6.6, 9.4, and 23 kb are indicated. DNAs are as follows: 1, human; 2, mouse; 3, lobster; 4, clam; 5, sea urchin; 6, frog; 7, fly; 8, nematode; 9, yeast; and 10, E. coli.
whereas weak bands representing rRNAs and, to a much lesser extent, tRNA were the only signals visible in the yeast samples (data not shown). Search for Prothymosin a Genomic DNA in Yeast
mRNA was confirmed by performing experiments with 60 mg of yeast total RNA. On these blots, 12.5 mg of total RNA from human myeloma cells gave a clear signal,
FIG. 2. Autoradiogram of RNA. RNA was analyzed in a 1.2% agarose formaldehyde gel, blotted to Nytran, and hybridized with a [32P]human cDNA coding region probe at 30°C in 83 SSC, other components as described, and an equal volume of formamide. The blot was washed at low stringency: 1, 5 mg of total RNA from human myeloma cells; 2, 5.6 mg of polyadenylated RNA from yeast grown in complete medium; 3, 9.8 mg of polyadenylated RNA from yeast grown in minimal medium. The positions of rRNAs are noted. The arrow marks the position of human prothymosin a mRNA at 1.2 kb.
In mammals, the amount of prothymosin a and its mRNA correlates with the growth rate of the cell or tissue from which the materials are obtained (3). In yeast, there may be no such requirement. As a consequence, the genome was searched using three methods: Southern blots were probed with human prothymosin a cDNA coding region probes, yeast genomic DNA was used as a template for PCR carried out with specific prothymosin a cDNA primers, and several locally generated and commercial libraries were screened with prothymosin a-specific probes. Southern blots of yeast DNA were prepared from four independent preparations cleaved with BamHI, PvuII, EcoRI, HindIII, BstUI, HaeIII, HhaI, HpaII, MboI, RsaI, and Sau3AI. A 330-bp DNA obtained with the aid of PCR using human cDNA as the template and Sh 37 and Sh 39 as primers served as a positive control. After hybridizing and washing both blots at low stringency in the same bag, the control band was readily visible whereas only very faint, apparently nonspecific, bands were observed in both yeast and marker DNA (Fig. 3 and data not shown). To prove that the yeast Southern blots contained adequate amounts of DNA, the blots were stripped and probed with a 32P-labeled yeast Arg 4 39-end probe obtained from Dr. M. Lichten; there was at least one very bright Arg 4 DNA band in
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FIG. 4. Alignment of prothymosin a (top) with the ;88-kDa open reading frame, YPL190c (bottom), prothymosin a’s closest relative in the Saccharomyces cerevisiae genome database.
each sample. Since the assays contained sufficient amounts of quality DNA, the presence of sequences homologous with the human prothymosin a gene appeared to be highly problematic. PCR was carried out with yeast genomic DNA and the three sets of primers described under Materials and Methods. The results with all sets of primers demonstrated that no yeast PCR DNA with resemblance to human prothymosin a cDNA was encountered when the product DNA was sequenced or probed as stated at low, moderate, or high stringency. Similarly, PCR was also unsuccessful in generating prothymosin a-like sequences using a yeast library in lgt-11 regardless of whether the primers recognized prothymosin a or the flanking regions of the vector. Yeast libraries were also screened for prothymosin a-like sequences. We used two plasmid libraries generated in our laboratory; in one case, pBR322 was selected to decrease plasmid copy number in the event of toxicity of the very prothymosin a sequences we sought. There were no positive yeast DNA-containing clones regardless of the stringency with which the hybridized, colony lifts were washed (data not shown). Commercial libraries in l or in Escherichia coli and a cosmid library screened similarly corroborated these results. Our choice of a variety of restriction enzymes and cloning systems and the analysis of several yeast strains made it unlikely that the desired clone had escaped detection either because it was nonviable or because the yeast prothymosin a DNA had been cleaved into fragments too small to distinguish. In pursuit of prothymosin a genes in species other than yeast, a Southern blot containing EcoRI-cut DNA from representative phyla was probed with the 32Plabeled 330-bp human prothymosin a cDNA coding region. The autoradiogram is displayed in Fig. 3. Although the blot, washed at moderate stringency, was exposed to film for 1 week, it is evident that the human and mouse possess prothymosin a genes (and pseudogenes), that lobster and sea urchin DNAs might con-
tain them, and that clams, frogs, flies, nematodes, yeast, and E. coli do not have a prothymosin a gene. Comparison of the Prothymosin a Amino Acid Sequence with the Sacchaomyces cerevisiae Genome Database The BLASTp program employed in this study compares an amino acid query sequence, prothymosin a, against a protein sequence data base, the entire S. cerevisiae genome. Figure 4 illustrates the output for the best fit protein, YPL190c. It is evident that the overall match of prothymosin a with YPL190c is poor with 31.2% sequence identity and 54.1% similarity. Furthermore, the regions in common are overwhelmingly acidic elements known to appear in otherwise nonhomologous proteins. To expand the search, we viewed the second best fit, YGL150c, and found 27.5% identity and 48.6% similarity, again a molecule with acidic regions. When the data base was searched using the low-complexity filter, a mode that eliminates the acidic stretches from consideration, no matches to prothymosin a were found regardless of how the values that define random homology were adjusted. With the filter off, a search restricted to proteins approximately the same size as mammalian prothymosin a’s resulted in a poor match with the best fit, L22015, having 22% identity and 31.2% homology. Moreover, the predicted tryptic peptide maps of YPL190c, YGL150c, and L22015 are substantially different from that of prothymosin a, judging by the number of peptides produced and their sizes (Table 1). We conclude that yeast does not have a prothymosin a homologue. DISCUSSION
When two studies come to diametrically opposite conclusions, it is necessary to evaluate the quality of the data in each case. We searched for prothymosin a protein in yeast, but unlike Makarova et al. (11) we
PROTHYMOSIN a IS NOT FOUND IN YEAST
TABLE 1 Comparison of the Tryptic Peptides of Prothymosin a with Those of Three Best Fit Yeast Open Reading Frames Number of peptides Residues
PTMA
L22015
YPL190c
YGL150c
.80 71–80 61–70 51–60 41–50 36–40 31–35 26–30 21–25 16–20 11–15 6–10 2–5
0 0 0 1 0 0 0 0 0 0 2 1 4
0 0 0 0 0 1 0 1 1 2 2 5 6
1 1 1 0 2 1 0 0 5 4 6 8 26
1 0 0 1 0 4 2 1 4 8 21 38 84
Note. The data are expressed as the number of peptides in each size class. The size classes range from the smallest peptides with 2–5 residues to the largest with more than 80 residues. PTMA is prothymosin a, with 109 amino acids. The other designations refer to open reading frames in yeast.
failed to find evidence for it. In both laboratories, the purification makes use of the ability of prothymosin a to partition into the aqueous phase of a phenol extraction (8,10), a property which the aforementioned group attributed incorrectly (7) to the presence of a covalently bound RNA. We have used this technique to isolate prothymosin a from human cells and bovine thymus, and to obtain recombinant prothymosin a expressed in E. coli, whereas Pavlov et al. (12) demonstrated its efficacy by purifying recombinant human prothymosin a overexpressed in yeast. However, with the same approach, our attempts to find native yeast prothymosin a using large quantities of cells were uniformly unsuccessful. A search for prothymosin a sequences in nucleic acids failed. Messenger RNA from yeast, either with or without a poly(A) tail, was not capable of hybridizing to probes composed of the human prothymosin a coding region sequences, even at low stringency. At the level of the genome, no recognizable prothymosin a gene was found in Southern blots of yeast DNAs, or in libraries whether prepared in our laboratory or purchased from commercial sources. Although PCR provided improved sensitivity, there was no prothymosin a homologue produced with yeast genomic DNA templates using several different sets of primers. Since all hybridization methods require similarity of nucleic acid sequences, we reasoned that if yeast possessed a prothymosin a gene that did not hybridize to our probes, species closer to mammals on the evolutionary tree would also have the gene and its sequence
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would be more highly conserved. A blot with genomic DNAs representing many phyla revealed the presence of the gene in mouse and man, but failed to find prothymosin a unequivocably in any other group. When we were unable to identify a homologous prothymosin a gene in frogs, we concluded that the presence of such a gene, whether closely related or highly divergent, was highly improbable in yeast. In contrast, the Bogdanov group found the protein by extracting yeast with phenol and labeling the acquired polypeptides on lysine residues with Bolton Hunter reagent (10,11). Murine, bovine, and yeast prothymosin a were electrophoretically identical in size. Moreover, a two-dimensional tryptic peptide map indicated that yeast and mouse prothymosin a were more closely related, spot for spot, than the mouse and human proteins (11) despite the fact that the latter differ in only five respects: the addition of Q in mouse prothymosin a between positions 38 and 39 of the human protein; and mouse3human, SA3 AP, A3 V, and D3 E at positions 83– 84, 89, and 106, respectively. We believe that the vanishingly small amounts of prothymosin a reported in yeast are the key to understanding these data; the yeast map had clearly been exposed to film for a prolonged period of time and the amount of protein used to generate it was not disclosed. In a subsequent publication (12), an inability to isolate native prothymosin a from yeast was attributed to inadequate amounts of material. The availability of the entire yeast genome has made it possible to resolve the issue unambiguously. We have shown that the yeast genome does not have open reading frames homolgous to mammalian prothymosin a. Apparently, there is no such gene and no such protein in yeast. REFERENCES 1. Eschenfeldt, W. H., and Berger, S. L. (1986) The human prothymosin a gene is polymorphic and induced upon growth stimulation: Evidence using a cloned cDNA. Proc. Natl. Acad. Sci. USA 83, 9403–9407. 2. Sburlati, A. R., Manrow, R. E., and Berger, S. L. (1991) Prothymosin a antisense oligomers inhibit myeloma cell division. Proc. Natl. Acad. Sci. USA 88, 253–257. 3. Manrow, R. E., Sburlati, A. R., Hanover, J. A., and Berger, S. L. (1991) Nuclear targeting of prothymosin a. J. Biol. Chem. 266, 3916 –3924. 4. Clinton, M., Graeve, L., El-Dorry, H., Rodriguez-Boulan, E., and Horecker, B. L. (1991) Evidence for nuclear targeting of prothymosin and parathymosin synthesized in situ. Proc. Natl. Acad. Sci. USA 88, 6608 – 6612. 5. Wang, R-H., Tao, L., Trumbore, M. W., and Berger, S. L. (1997) Turnover of the acyl phosphates of human and murine prothymosin a in vivo. J. Biol. Chem. 272, 26405–26412. 6. Frillingos, S., Frangou-Lazaridis, M., Seferiadis, K., Hulmes, J. D., Pan, Y.-C. E., and Tsolas, O. (1991) Isolation and partial sequence of goat spleen prothymosin a. Mol. Cell. Biochem. 108, 85–94.
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7. Sburlati, A. R., De La Rosa, A., Batey, D. W., Kurys, G. L., Manrow, R. E., Pannell, L. K., Martin, B. M., Sheeley, D. M., and Berger, S. L. (1993) Phosphorylation of human and bovine prothymosin a in vivo. Biochemistry 32, 4587– 4596. 8. Sburlati, A. R., Manrow, R. E., and Berger, S. L. (1990) Human prothymosin a: Purification of a highly acidic nuclear protein by means of a phenol extraction. Protein Expression Purif. 1, 184 – 190. 9. Eschenfeldt, W. H., Manrow, R. E., Krug, M. S., and Berger, S. L. (1989) Isolation and partial sequencing of the human prothymosin a gene family. J. Biol. Chem. 264, 7546 –7555. 10. Vartapetian, A. B., Makarova, T. N., Koonin, E. V., Agol, V. I., and Bogdanov, A. A. (1988) Small cytoplasmic RNA from mouse cells covalently linked to a protein. FEBS Lett. 232, 35–38. 11. Makarova, T., Grebenshikov, N., Egorov, C., Vartapetian, A., and Bogdanov, A. (1989) Prothymosin a is an evolutionary conserved protein covalently linked to a small RNA. FEBS Lett. 257, 247–250. 12. Pavlov, N., Evstafieva, A., Rubtsov, Y., and Vartapetian, A. (1995) Human prothymosin a inhibits division of yeast Saccharomyces cerevisiae cells, while its mutant lacking nuclear localization signal does not. FEBS Lett. 366, 43– 45. 13. Rose, M. D., Winston, F., and Hieter, P. (1990) ‘‘Methods in Yeast Genetics, A Laboratory Course Manual,’’ pp. 140 –142, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
14. Feinberg, B., and McLaughlin, C. S. (1988) Isolation of yeast mRNA and in vitro translation in a yeast cell-free system, in ‘‘Yeast: A Practical Approach’’ (Campbell, I., and Duffus, J. H., Eds.), pp. 147–161, IRL Press, Oxford. 15. Johnston, J. R. (1988) Yeast genetics, molecular aspects, in ‘‘Yeast: A Practical Approach’’ (Campbell, I., and Duffus, J. H., Eds.), pp. 107–123, IRL Press, Oxford. 16. Phillippsen, P., Stotz, A., and Scherf, C. (1991) DNA of Saccharomyces cerevisiae. Methods Enzymol. 194, 169 –182. 17. Jacobsen, A. (1987) Purification and fractionation of poly(A)1RNA. Methods Enzymol. 152, 254 –261. 18. Ogden, R. C., and Adams, D. A. (1987) Electrophoresis in agarose and acrylamide gels. Methods Enzymol. 152, 61– 87. 19. Wahl, G. M., and Berger, S. L. (1987) Screening colonies or plaques with radioactive nucleic acid probes. Methods Enzymol. 152, 415– 423. 20. Berger, S. L., Manrow, R. E., and Lee, H. Y. (1993) Phoenix mutagenesis: One-step reassembly of multiply cleaved plasmids with mixtures of mutant and wild-type fragments. Anal. Biochem. 214, 571–579. 21. Manrow, R. E., Leone, A., Krug, M. S., Eschenfeldt, W. H., and Berger, S. L. (1992) The human prothymosin a gene family contains several processed pseudogenes lacking deleterious lesions. Genomics 13, 319 –331.