A human cDNA corresponding to a gene overexpressed during cell proliferation encodes a product sharing homology with amoebic and bacterial proteins.

A clone, designated pag, was isolated by differential screening of cDNA libraries made from the untransformed and ras-transformed human mammary epithelial cell line HBL100. This cDNA corresponds to a gene constitutively expressed in most human cells which is induced to higher levels upon serum stimulation in untransformed and ras-transformed HBL100 cells. However, the abundance of the pag transcript is approximately 3-fold higher in transformed as compared to untransformed cells after 7-15 h of serum stimulation. In the promyelocytic leukemia cell line HL60 induced to differentiate the level of pag mRNA starts to decrease between 48 and 72 h following induction. During this period, which represents the commitment phase of differentiation, HL60 cells cease to proliferate. Therefore, in HBL100 and HL60 cells, higher levels of pag gene expression are correlated with cell proliferation. The pag cDNA codes for a 22-kDa protein, devoid of known consensus motifs, and shares 66% homology with a murine gene product (MER5) that is preferentially expressed in erythroleukemia cells during the early period of cell differentiation. In addition, the pag gene product shares approximately 50% identity with a 29-kDa surface antigen of Entamoeba histolytica and a 26-kDa antigen of Helicobacter pylori. Distant relationship was also found with other prokaryotic proteins. The pag cDNA hybridizes to multiple sequences within human and other mammalian genomes and to fewer sequences in chicken and Saccharomyces cerevisiae. Although a true relationship between eukaryotic and prokaryotic genes is difficult to establish, the conservation of pag gene sequences throughout Eukaryotae rather suggests that the pag locus belongs to a new class of genes encoding highly conserved proteins.

A clone, designated pug, was isolated by differential screening of cDNA libraries made from the untransformed and ras-transformed human mammary epithelial cell line HBL100. This cDNA corresponds to a gene constitutively expressed in most human cells which is induced to higher levels upon serum stimulation in untransformed and ras-transformed HBLlOO cells. However, the abundance of the pag transcript is approximately %fold higher in transformed as compared to untransformed cells after 7-15 h of serum stimulation. In the promyelocytic leukemia cell line HL60 induced to differentiate the level of pag mRNA starts to decrease between 48 and 72 h following induction. During this period, which represents the commitment phase of differentiation, HL60 cells cease to proliferate. Therefore, in HBLlOO and HL60 cells, higher levels of pug gene expression are correlated with cell proliferation. The pug cDNA codes for a 22-kDa protein, devoid of known consensus motifs, and shares 66% homology with a murine gene product (MERB) that is preferentially expressed in erythroleukemia cells during the early period of cell differentiation. In addition, the pug gene product shares approximately 50% identity with a 29-kDa surface antigen of Entamoeba histolytica and a 26-kDa antigen of Helicobacter pylori. Distant relationship was also found with other prokaryotic proteins. The pug cDNA hybridizes to multiple sequences within human and other mammalian genomes and to fewer sequences in chicken and Saccharomyces cerevisiae. Although a true relationship between eukaryotic and prokaryotic genes is difficult to establish, the conservation of pag gene sequences throughout Eukaryotae rather suggests that the pug locus belongs to a new class of genes encoding highly conserved proteins.
The three ras oncogenes, Ha, Ki, N-ras, encode highly related 21-kDa proteins (p21) which are ubiquitously expressed and involved in the control of cellular proliferation and differentiation (1). It has been found that 10-20% of human tumors have a mutation in one of these genes leading to the production of p21" oncoproteins, which are thought The nucleotide sequence(s) reported in this paper has been submitted X67951.
The ras oncogenes produce many changes in cellular gene expression by turning on the expression of proteins involved in the regulation of transcription (3,4). This leads to the aberrant expression of a set of genes, which in turn induces metabolic, ultrastructural, and growth disorders of normal cell life. One approach to identifying genes whose deregulated expression could play a role in transformation is to use the differential cloning technique of cDNA libraries made from transformed and untransformed cells. This technique has been successfully used to identify deregulated genes in avian and rodent transformed cells (5)(6)(7), as well as in human tumors (8)(9)(10). However, this approach has been only rarely used to identify genes whose expression is altered following ras transformation (1 1, 12).
In an attempt to isolate abnormally expressed genes in rastransformed human cells, we have introduced a mutant version of the human Ha-ras gene into the nontumorigenic mammary epithelial cell line HBL100. We succeeded in achieving both morphological transformation and tumorigenic conversion (13). The untransformed and ras-transformed cell lines were used to isolate cDNAs corresponding to genes manifesting an increased expression in ras-transformed cells. We previously used this technique to demonstrate that the a member of the 89-kDa heat-shock protein family is constitutively overexpressed in HBLlOO ras-transformed cells (13).
Here, we report the characterization of a novel gene, pag (proliferation Gssociated gene), identified by differential hybridization. Expression of pug and hsp89a genes differ. Although they are both expressed ubiquitously and reach higher levels following serum stimulation in untransformed cells, the pug gene is not constitutively overexpressed in ras-transformed cells. We observed a transient overexpression in rmtransformed HBLlOO cells compared to untransformed cells only when cells were induced to proliferate upon serum stimulation. On the other hand, decreased expression was found in HL60 cells in the course of differentiation, suggesting that higher levels of expression are associated with cell proliferation. The pag polypeptide does not contain any known consensus motif and shares approximately 50% identity with parasite and bacterial proteins of unknown function. The possibility that the pug gene could belong to a new family of genes, highly conserved between Eukaryotae and Prokaryotae, is discussed.

MATERIALS AND METHODS
Cell Culture Conditions-For differential cloning experiments, approximately 3 X lo6 untransformed and ras-transformed cells were plated in 100-mm Petri dish and grown for 3 days at 37 "C in minimum essential medium, supplemented by nonessential amino acids and 10% newborn calf serum. Fresh medium was added overnight before RNA extraction. Serum-starved cells were obtained by seeding 4 x lo6 cells in 100-mm Petri dish containing 10 ml of minimum essential medium with 0.5% calf serum for at least 60 h. Serum stimulation was performed by feeding cells with 10 ml of minimum essential medium supplemented with 10% calf serum. HL60 cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum and induced to differentiate with 1.3% Me2S01 at a starting cell concentration of 2.5 X 105/ml.
Construction of cDNA Libraries and Sequencing-cDNA libraries were made from poly(A) RNA prepared from the HBLlOO/ras-I cell line (13) using the X g t l O cloning system (Amersham Corp.), as prescribed by the supplier, with oligo(dT) or random primers. Differential screening of the oligo(dT)-primed library was performed as previously described (13). Phage inserts were liberated by digestion with EcoRI, then subcloned into the M13mp18/mp19 bacteriophages and sequenced by the dideoxy sequencing method (14). Sequencing was performed by walking along the sequence using synthetic oligonucleotides as primers. Usually, the positive strand was sequenced using T7 polymerase and the negative strand using Taq polymerase.
Generation of Antibodies-The peptide Pro-Asp-Val-Gln-Lys-Ser-Lys-Glu-Tyr-Phe-Ser-Lys, corresponding to residues 186-197 of the pug protein, was synthesized (Neosystems, Strasbourg, France). This peptide was coupled via the tyrosine residue to ovalbumin or keyhole limpet hemocyanin with bis-diazotized benzidine (15). Female New Zealand rabbits were immunized subcutaneously with coupled material containing 250 gg of peptide in Freund's complete adjuvant and given booster injections with the same antigen in incomplete adjuvant at 1-month intervals. Rabbits were bled up to 20 ml by cardiac puncture, 7 and 20 days after injection. Expression of MBP-pug Polypeptide-A blunt end restriction fragment (DraI-Eco47III) from position 138-662, of pug cDNA, encoding amino acids , was inserted in the PMAL-CZ vector (New England Biolabs). Cloning procedure was performed according to the manufacturer. This expression vector uses the strong tac promoter and the malE translation initiation signal leading to high expression of a fusion protein containing the maltose-binding protein (MBP) and cloned pug sequences. Escherichia coli (strain TB1) was transformed with the construction and transformants were selected according to their resistance to ampicillin. Colonies containing recombinants were identified using differential staining by isopropyl-lthio-~-~-galactopyranoside/5-hromo-4-chloro-3-indoyl P-D-gahCtOside then plasmid DNA was analyzed with the appropriate restriction enzymes. Mass preparation of transformants for expression of MBPpug fusion polypeptide was carried out on 500 ml of induced cultures (1 mM isopropyl-1-thio-8-D-galactopyranoside, 2 h). After centrifugation, the cells were resuspended in 25 ml of lysis buffer (10 mM phosphate buffer, pH 7.0, containing 30 mM NaCI, 0.25% Tween 20, 10 mM EDTA, 10 mM EGTA, and 1 mg/ml lysosyme). 30 min later, NaCl was added to the mixture at a final concentration of 0.5 M, then sonicated and centrifuged for 30 min at 9,000 X g. The supernatant was mixed with amylose resin (New England Biolabs) and poured into a column. The column was washed three times with lysis buffer without Tween and the MBP-pug polypeptide was eluted with washing buffer supplemented with 10 mM maltose.
Western Blot Analysis-For immunoblotting, 10 pg of HBLlOO cell crude extract in radioimmune precipitation buffer or purified MBPpag protein were separated by SDS-PAGE. Blotting and immunodetection were carried out by standard procedures (16). The PAGAl antiserum and the alkaline phosphatase-conjugated second antibody (Sigma) were diluted 400-and 6000-fold, respectively.
RNA Preparation and Northern Blot Analysis-Cytoplasmic RNA preparations and Northern blots were performed using nick-translated probes as previously described (13). The integrity and the amount of RNA loaded onto the gel was checked by ethidium bromide staining. Efficiency of the transfer was monitored by hybridization using a 28 S oligonucleotide probe (17). Autoradiograms were scanned using an LKB Ultroscan (model XL).

Molecular Cloning and Nucleotide
Sequence of pug cDNA-A cDNA library, primed with oligo(dT), was prepared from polyadenylated mRNA extracted from ras-transformed HBLlOO cells. Fifty thousand primary recombinants were screened in duplicate by differential hybridization using 32Plabeled first strand cDNA probes from untransformed and ras-transformed HBLlOO cells. In order to detect overexpressed clones resulting only from transformation and not from cell culture conditions, ras-transformed, and untransformed G418-resistant HBLlOO cells were grown in parallel. Culture medium was replaced 16 h before RNA extraction.
Clones displaying a stronger hybridization signal with probes prepared from transformed cells were subjected to two further rounds of plaque purification. One of these carrying a new sequence, hybridized with a single mRNA species of 1.2 kb in human cells and showed a differential signal when used to probe Northern blots containing mRNA from untransformed and rus-transformed HBLlOO actively growing cells (data not shown). The size of the cDNA insert was shown to be around 900 bp. DNA sequence analysis confirmed the insert size (920 bp) and revealed a stretch of at least 80 adenine, indicating that this clone was probably complete at its 3' end. Since the 920-bp cDNA could be incomplete at its 5' end, due to rearrangement or recombination during the construction of the library, the 920-bp fragment was used to screen a randomly primed cDNA library prepared from ras-transformed HBLlOO cells. Three clones extending 17 nucleotides further at t h e 5' end were isolated. A potential full-length cDNA clone was sequenced. No mismatches were found when compared with the previously determined sequence. Thus, t h e 937-bp sequence presented in Fig. 1 is likely to represent the entire pug cDNA sequence. Molecular cloning and sequence analysis of t h e pug gene exons and gene promoter has confirmed this assumption.' The sequence of the pug cDNA carries a single open reading frame. Postulating the first ATG codon as an initiating codon, t h e pug sequence consists of a 5"untranslated region of 60 nucleotides, followed by 597 nucleotides encoding a presumed 199-amino acid polypeptide (Fig. I). The 3'-untranslated region of 280 nucleotides is composed of 55% A T base pairs and has an atypical polyadenylation signal, ATTAAA located 11 nucleotides upstream from the polyadenylation site. This hexamer has been shown to serve as an alternative signal for the addition of poly(A) tail. (18).
Pug cDNA Encodes u 22-kDa Protein Expressed in HBLIOO Cells-The deduced amino acid sequence of pug cDNA is shown in Fig.   1. The calculated molecular weight of the polypeptide is about 22,000. The estimated PI is 8.4. Since the pug cDNA was isolated solely by its mRNA overexpression, known peptide motifs that could suggest the cellular location or the function of t h e pug protein were searched in the PROSITE data bank (EMBL, release 8.1). No recognizable motif has been found.
To ensure that the deduced sequence indeed encodes a protein of 22 kDa, we raised a polyclonal antiserum, designated PAGA1, against a peptide located in the C-terminal region of pug (position 186-197). To establish the specificity of t h e PAGAl antibody for the pug gene product, a DNA fragment containing amino acids

TACAGGGGGTGGAGAGACCAGCCT~TCTTCC~TAGGAATGGCCTGAGTTGG
853 CGTTGTGGGCAGGCTACTGGTITGTATGATGTATTAGTAGAGCAACCCATTAATC 9 0 8 T"TGTAGTTTGT=CTTGAACTGAG recovered after affinity chromatography of crude bacterial extracts ( Fig. 2 A , lane 1 ). In contrast, the hybrid protein was weakly detectable when immunodetection was performed in presence of competing immunogenic peptide (Fig. 2 A , lane 2). Thus, the PAGAl antiserum indeed recognized the epitope of the pag protein chosen for immunization. Western blot analysis of HBLlOO cells, using crude rabbit pag antiserum, revealed multiple protein bands (Fig. 2B, lane 1 ). Immunodetection performed in presence of competing immunogenic peptide failed to detect two proteins of 22 and 105 kDa (Fig.  2B, lane 2 ) . Because the 1.2-kb messenger RNA is too short to encode a 105-kDa protein, the 22-kDa protein was likely to represent the protein product of the pag gene. Expression of pug in Various Human Tissues-Since the pag cDNA was cloned from a human mammary cell line, we wished to determine whether its expression was restricted to this cell type. Poly(A) RNA isolated from seven human organs, including heart, brain, placenta, lung, liver, skeletal muscle, and kidney, was analyzed by Northern blot hybridization (Fig. 3). A single 1.2-kb transcript was detected in all organs. The filter was washed and reprobed with @-actin to confirm RNA integrity (Fig. 3). The @-actin expression varies between different organs, thus hampering a direct quantitative comparison with respective pag transcripts. However, if we assume that equal amounts of RNA were indeed loaded onto the gel, pag mRNA levels seemed to be higher in organs having a higher level of proliferation such as kidney, placenta and lung as compared to organs having a low level of proliferation (brain, heart, skeletal muscle, and liver).

Induction of pag Expression by Serum in HBLlOO Cells-
The association between proliferation and enhanced pug expression, suggested by Northern blot analysis of human organs, was first examined in serum-stimulated untransformed and ras-transformed HBLlOO cells. For this purpose, untransformed and ras-transformed cells were serum-starved for 3 days then fed with culture medium containing 10% calf serum. In these culture conditions, growth-arrested HBLlOO cells were induced to proliferate. The level ofpag gene-specific transcripts was estimated by Northern blot analysis of total mRNA. In serum-starved HBLlOO or ras-transformed HBLlOO cells, pag mRNA could be easily detected by overnight autoradiography, and its level of expression did not show any significant variation during at least 60 h (Fig. 4). This level was likely to represent the constitutive level of pag mRNA expression in serum-starved HBLlOO cells. As already observed for some constitutively expressed genes, such as hsp genes (13,19), the level of expression increased after serum stimulation. Enhanced expression was seen after 3 h and was maximal within 7-15 h following serum addition (Fig. 4). Expression fell off thereafter to reach background levels by 60 h. Although the pug gene was induced by serum in both nontransformed HBLlOO or ras-transformed HBLlOO cells, its accumulation was approximately %fold higher in rastransformed cells (Fig. 4). This property was not peculiar to the HBLlOO cell line used in this assay. Transient overexpression, following serum stimulation, was also observed in other untransformed and ras-transformed HBLlOO cell lines (data not shown). Therefore, pug gene expression increased after induction of proliferation by serum stimulation of growth arrested HBLlOO cells.
Pag Expression Is Negatively Regulated during Differentiation of HLAO Cells-The association between enhanced pug expression and cell proliferation was studied in another cell system to ascertain whether higher levels of pug mRNA expression were indeed correlated with proliferation or could result from culture conditions. The promyelocytic leukemia cell line HL60 exposed to Me2S0 undergo growth arrest and ultimately reach a terminally differentiated state resulting in the expression of granulocytic markers (20). RNA was extracted a t various times after addition of Me2S0 and the steady-state of pug mRNA analyzed by Northern blot. HL60 cells grown in the same culture conditions but without Me2S0 were used as control. A slight decline of pag mRNA levels was observed after 48 h and was clearly seen after 72 h to reach very low levels after 80 h (Fig. 5, top left panel). Stepwise decrease of pag mRNA was not observed during the same period in HL60 cells grown in the absence of MezSO when corrected for variability in RNA loading (Fig. 5 , top right  panel). Therefore, decreasing levels of pug mRNA are associated with HL60 cell differentiation and do not result from culture conditions such as nutrient depletion. T o ensure that the differentiation process was indeed triggered upon addition of Me,SO, RNAs were subsequently hybridized with a human c-myc probe. As previously described (21-24), c-myc RNA was highly expressed in HL60 cells actively growing in the absence of Me2S0 (Fig 5, middle right panel), whereas it was dramatically reduced within few hours of exposure to MezSO and remains extremely low thereafter (Fig. 5 , middle left panel). Interestingly, the period from 48 to 72 h constitutes what has been termed the commitment phase of differentiation which also corresponds to the period HL60 cells cease to proliferate (20,(24)(25)(26)(27). Thus, the levels of pug mRNA and proliferation are decreasing in parallel. These data together with those demonstrating enhanced pag mRNA expression following serum stimulation of growth arrested HBLlOO cells indicate that higher levels of pag gene expression are associated with cell proliferation.
Proteins Related to the pug Gene Product-The deduced amino acid sequence of pug was compared to known protein sequences in the National Biomedical Research Foundation (release 33) and SWISSPROT (release 23) data banks using the FASTA program of Pearson and Lipman (28). Significant homology was found with the MER5 gene product. The cDNA of MER5 codes for a protein of 257 amino acids of unknown function. It was cloned from RNA preferentially synthesized in murine erythroleukemia (MEL) cells during the early period of cell differentiation induced by M e 8 0 (29). The homology lies from position 12 in pag and position 70 in MER5 to the C-terminal region. In this region the identity is 66% and rises to 83% when chemically related amino acids are considered (Table I). Only two gaps, one between position 79 and 80 in the MER5 protein and one at position 197 in the pug protein, were introduced during the alignment. Thus, if we exclude the 69 first amino acids of the N-terminal domain of MER5, the pug protein is closely related to MER5.
In addition to the similarity observed with the MER5 gene product, the pag protein revealed significant similarities with amoebic and bacterial proteins. Most closely related to pug is a 29-kDa antigen of pathogenic Entamoeba histolytica, located on the surface of trophozoites (30). The identity was 57% and rose to 73% when chemically related amino acids were considered ( Table I). The 26-kDa antigen of Helicobucter pylori (31), a pathogenic bacteria of the stomach, exhibits 48% identity with pag protein and rose to 64%, when conserved amino acid substitutions were considered (Table I). An unpublished sequence of Clostridium pasteurianum, present in the protein data bases shares approximately 50% identity with pug. Distant relationship was also found with an uncharacterized open reading frame (NADH dehydrogenase) located upstream of the coding region of the NADH dehydrogenase of Bacillus subtilis (32), and with a subunit (C22) of the alkyl hydroperoxide reductase of Salmonella typhimurium (33) ( Table I). found to correspond to this conserved sequence, or to other less conserved sequences among the consensus amino acid sequences of the PROSITE data bank (EMBL, release 8.1).
Evolutionary Conservation of the pag Gene-The evolutionary conservation of the pug gene was examined in cellular DNA from different mammals, chickens, and yeasts. Under moderate stringent hybridization (30% formamide, 5 x SSC, 42 "C) and washing (0.1 x SSC, 50 "C) conditions, the pug cDNA hybridized to multiple sequences within human and mammalian genomes and to fewer sequences in chicken genome (Fig. 7). It also hybridized with a 2.2-kbp fragment of yeast Saccharomyces cerevkiae DNA. Additional fragments of 10 and 3.3 kbp were also seen on overexposed autoradiograms (data not shown). Therefore, the pug cDNA represents a highly conserved gene sequence.  12-197 12-194 12-194 2.5-1R9

DISCUSSION
Activation of a ras gene has a pleiotropic effect on cell growth leading ultimately to a transformed phenotype. We have chosen to compare a rm-transformed human cell with its untransformed counterpart in order to identify genes whose deregulation may participate in transformation. A novel cDNA clone, pag was thus isolated.
The pug cDNA encodes a 199-amino acid polypeptide with a predicted molecular mass of 22 kDa, assuming that initiation of translation starts at the first ATC codon. Using an antibody raised against a peptide located within the C-terminal part of pug, Western blot analysis revealed a 22-kDa protein expressed in HBLlOO cells. This suggests that the predicted structure of the pug polypeptide is correct. However, we cannot rule out that an another codon is used for initiation of translation. For example posttranslational modifications of a smaller polypeptide could lead to a product with an apparent molecular mass of 22 kDa. Direct sequencing of the purified pug protein will identify the initiation codon. In the absence of such data, we can, however, conclude that the polypeptide encoded by the open reading frame is expressed Computer-generated alignment of homologous regions of pag and MER5 with related proteins. Alignment was performed using the program CLUSTAL of PCGENE software package (release 6.5). Boxed residues, conservation with both pug and MER5; residues that are identical in all proteins are boxed and shaded. Numbers on the left refer to the first amino acid of the line. Spaces represent gaps inserted to maximize matches. ORH is previous name for pug.
in HBLlOO cells. The size predicted from the cDNA sequence is similar to that estimated by Western blot analysis.
The pug gene was found to be transcribed in most human tissues suggesting that a constitutive level of expression is necessary for cell life. Following serum stimulation of growtharrested HBLlOO cells, pug expression dramatically increased above the constitutive level. On the other hand, pug mRNA expression decreased during differentiation of HL60 cells, in particular when they ceased to divide (48-72 h), whereas it remained high in actively growing HL60 cells. Therefore, in this cell system, variations of the pug mRNA expression did not appear to be due to culture conditions such as nutrient depletion or acidification of the medium. Although we ignore its precise function, this gene was designated pug (proliferation gssociated gene) since higher levels of pug mRNA expression are associated with cell proliferation.
A moderate but significant elevation of pag expression was found in ras-transformed HBLlOO cells as compared to untransformed HBLlOO cells following serum stimulation. This difference in the level of expression may not be directly related to transformation but instead result from alteration of the transcriptional machinery induced by the p21" oncoprotein. Alternatively, this overexpression could reflect a direct role in transformation. The clone of ras-transformed cells (HBLlOO/rasl) used in this study exhibited 4-&fold higher levels of Ha-ras mRNA compared with nontransformed cells (13). This higher level of expression is in the same range as the %fold overexpression of pug mRNA and raises the possibility of coordinate expression of pug and ras genes in actively growing cells. Whether pug interacts directly with the p21" or is involved in the same pathway of transmission of a mitogenic signal remains to be determined.
Comparison of the pug amino acid sequence with MER5 revealed a high degree of homology (Table I). The MER5 gene was first identified as a cDNA, cloned from RNA preferentially synthesized in murine erythroleukemia during the early period of cell differentiation (29). Antisense RNA of the MER5 gene inhibits the differentiation of murine erythroleukemia, suggesting that the MER5 gene product is necessary for completing differentiation (34). The homology spans most of the pug protein, and corresponds to region 70-257 of the MER5 protein. The difference in size between the pug and MER5 gene products as well as comparison of their gene promoter (29)' indicate that pug is not the human counterpart of the murine MER5 gene. Moreover, pug is expressed in most human tissues whereas MER5 is preferentially expressed in immature erythroblast cells (29). pug mRNA expression decreases during the course of differentiation of HL60 cells, whereas MER5 mRNA expression increases during the early period of differentiation of MEL cells (24-48 h). Although pug and MER5 display a different pattern of expression during differentiation, it is tempting to speculate that pug and MER5 share a common domain involved in the cellular response to either proliferation or differentiation signals. This domain may encompass the whole region of homology or be limited to common motifs. In either case, the absence of homology with known proteins, indicates that pug and MER5 represent a new class of proteins which could be involved in cellular proliferation or differentiation.
A striking relationship was also found with proteins of distantly related organisms such as E. histolyticu and H. pylori. E. histolyticu is a human parasite which causes extensive mortality and morbidity worldwide through diarrheal disease and organ abscess formation. We found a high degree of Zoo blot analysis of the pug gene. Southern blot containing 8 pg of genomic DNA per lane of the indicated species was hybridized in 5 X SSC, 30% formamide, a t 42 "C, then washed in 0.1 X SSC a t 40 "C. The final wash was performed in 0.1 X SSC a t 50 "C for 15 min. The filter was exposed overnight to Kodak X-OMAT AR film a t -70 "C in the presence of intensifier screen. X-Hind111 DNA fragments served as molecular mass markers.
identity (57%) with a 29-kDa surface antigen. Homology reached 73% when conserved amino acid substitutions were considered. The 26-kDa antigen of H. pylori was also found closely related to pug. H. pylori is a pathogenic strain of Gramnegative bacteria associated with diseases of the gastrointestinal tract including carcinoma (35,36). Mild treatment of H. pylori removes the 26-kDa antigen from the cells, suggesting that the protein might be located on the cell surface (31). Work is in progress to obtain antibodies against the native pag protein, to determine whether pag is associated with the plasma membrane despite the absence of consensus sequences for membrane localization.
We have shown that pug is a serum-inducible gene highly related to eukaryotic and prokaryotic proteins. Two hypotheses can be drawn from these observations. Close analogy of protein sequences between prokaryotes and eukaryotes can reflect the conservation of gene products necessary for cellular survival. Alternatively, this homology may represent a case of convergent evolution of proteins. Since E. histolytica and H. pylori are both pathogenic organisms, one can speculate that antigens sharing structural relationship with a host protein have been selected for their role in the infectivity of human cells. Although a true evolutionary relationship between eukaryotic and prokaryotic proteins may be difficult to establish, the high homology shared between these proteins, together with the high degree of conservation throughout Eukaryotae, rather favors the hypothesis that the pug gene product belongs to a novel class of highly conserved proteins.