Reducing agent can be omitted in the incubation medium of the batch in vitro fermentation model of the pig intestines.

Over the past decade, in vitro methods have been developed to study intestinal fermentation in pigs and its influence on the digestive physiology and health. In these methods, ingredients are fermented by a bacterial inoculum diluted in a mineral buffer solution. Generally, a reducing agent such as Na2S or cysteine-HCl generates the required anaerobic environment by releasing metabolites similar to those produced when protein is fermented, possibly inducing a dysbiosis. An experiment was conducted to study the impact of two reducing agents on results yielded by such in vitro fermentation models. Protein (soybean proteins, casein) and carbohydrate (potato starch, cellulose) ingredients were fermented in vitro by bacteria isolated from fresh feces obtained from three sows in three carbonate-based incubation media differing in reducing agent: (i) Na2S, (ii) cysteine-HCl and (iii) control with a mere saturation with CO2 and devoid of reducing agent. The gas production during fermentation was recorded over 72 h. Short-chain fatty acids (SCFA) production after 24 and 72 h and microbial composition of the fermentation broth after 24 h were compared between ingredients and between reducing agents. The fermentation residues after 24 h were also evaluated in terms of cytotoxicity using Caco-2 cell monolayers. Results showed that the effect of the ingredient induced higher differences than the reducing agent. Among the latter, cysteine-HCl induced the strongest differences compared with the control, whereas Na2S was similar to the control for most parameters. For all ingredients, final gas produced per g of substrate was similar (P>0.10) for the three reducing agents whereas the maximum rate of gas production (R max) was reduced (P0.10) after 24 h of fermentation with Na2S and in the control without reducing agent. Molar ratios of branched chain-fatty acids were higher (P<0.05) for protein (36.5% and 9.7% for casein and soybean proteins, respectively) than for carbohydrate (<4%) ingredients. Only fermentation residues of casein showed a possible cytotoxic effect regardless of the reducing agent (P<0.05). Concerning the microbial composition of the fermentation broth, most significant differences in phyla and in genera ascribable to the reducing agent were found with potato starch and casein. In conclusion, saturating the incubation media with CO2 seems sufficient to generate a suitable anaerobic environment for intestinal microbes and the use of a reducing agent can be omitted.

Use of medium without reducing agent for in vitro fermentation studies by bacteria isolated from pig intestine.

Over the past decade, several in vitro methods have been developed to study intestinal fermentation in pigs and its influence on health. In these methods, samples are fermented by a bacterial inoculum diluted in a mineral buffer solution. Generally, a reducing agent such as Na(2)S or cysteine HCl generates the required anaerobic environment by release of H(2)S inducing an imbalance among bacterial species by the production of toxic metabolites. Therefore, an experiment was conducted to study the impact of reducing agent on fermentation patterns. Protein (soybean protein and/or casein) and carbohydrate (potato starch and/or cellulose) ingredients were fermented in vitro by pig intestinal bacteria from fresh feces obtained from 3 sows fed an antibiotic-free commercial diet in 3 incubation media differing in reducing agent: (i) Na(2)S, (ii) cysteine HCl, or (iii) without reducing agent. Gas fermentation kinetics were monitored over 72 h (pressure was measured every 2 min). Short-chain fatty acid (SCFA) production after 24 and 72 h were compared among ingredient and reducing agents (n = 2). Gas production was higher (P < 0.05) when fermenting carbohydrate than protein ingredients. Except for soybean protein, total SCFA production after 24 and 72 h was similar (P > 0.05) for each ingredient regardless the incubation medium. The SCFA molar ratios did not differ (P > 0.05) between Na(2)S and without reducing agent. In conclusion, saturation of incubation media with CO(2) seems sufficient to generate an anaerobic environment. So incubation media could be simplified by omitting the reducing agent without influencing the fermentation kinetics and SCFA production.

Adaptation of a sandwich enzyme-linked immunosorbent assay to determine the concentration of bovine leukemia virus p24 and optimal conditions for p24 expression in short-term cultures of peripheral blood mononuclear cells.

Bovine leukemia virus (BLV) is a common retroviral infection of cattle. Infection is accompanied by integration of BLV into the host cell genome and is persistent for the life of the individual as is the presence of anti-BLV antibodies. Lymphosarcoma occurs in a small fraction of infected adult individuals but otherwise there is little or no associated disease. Viremia is undetectable, however, BLV is expressed readily once infected cells are cultured in vitro. A sandwich enzyme-linked immunosorbent assay (sELISA) was optimized, using murine monoclonal antibodies, to quantify the major internal structural protein (p24) produced in short-term cultures of peripheral blood mononuclear cells (PBMCs). Optimal production of BLV p24 was achieved utilizing RPMI supplemented with 10% fetal bovine serum (FBS), pH 7, and 5 x 10(6) cells per ml. Cultures were terminated at 24 h. The sELISA was linear between 30 and 900 ng/ml and the limit of detection was 1.2 ng/ml. At three concentrations of p24, intra- and inter-assay coefficients of variation (CV) varied between 9.2 and 13.3 and 5.1 and 12.9%, respectively.

Inhibition of histone deacetylases induces bovine leukemia virus expression in vitro and in vivo.

Packaging into nucleosomes results in a global transcriptional repression as a consequence of exclusion of sequence-specific factors. This inhibition can be relieved by using inhibitors of histone deacetylases, acetylation being a major characteristic of transcriptionally active chromatin. Paradoxically, the expression of only approximately 2% of the total cellular genes is modulated by histone hyperacetylation. To unravel the potential role of this transcriptional control on BLV expression, we tested the effect of two highly specific inhibitors of deacetylases, trichostatin A (TSA) and trapoxin (TPX). Our results demonstrate that treatment with TSA efficiently enhanced long terminal repeat-directed gene expression of integrated reporter constructs in heterologous D17 stable cell lines. To further examine the biological relevance of these observations made in vitro, we analyzed ex vivo-isolated peripheral blood mononuclear cells (PBMCs) from bovine leukemia virus (BLV)-infected sheep. TSA deacetylase inhibitor induced a drastic increase in viral expression at levels comparable to those induced by treatment with phorbol-12-myristate 13-acetate and ionomycin, the most efficient activators of BLV expression known to date. TSA acted directly on BLV-infected B lymphocytes to increase viral expression and does not seem to require T-cell cooperation. Inhibition of deacetylation after treatment with TSA or TPX also significantly increased viral expression in PBMCs from cattle, the natural host for BLV. Together, our results show that BLV gene expression is, like that of a very small fraction of cellular genes, also regulated by deacetylation.

Genetic determinants of bovine leukemia virus pathogenesis.

The understanding of HTLV-induced disease is hampered by the lack of a suitable animal model allowing the study of both viral replication and leukemogenesis in vivo. Although valuable information has been obtained in different species, such as rabbits, mice, rats, and monkeys, none of these systems was able to conciliate topics as different as viral infectivity, propagation within the host, and generation of leukemic cells. An alternate strategy is based on the understanding of diseases induced by viruses closely related to HTLV-1, like bovine leukemia virus (BLV). Both viruses indeed belong to the same subfamily of retroviruses, harbor a similar genomic organization, and infect and transform cells of the hematopoietic system. The main advantage of the BLV system is that it allows direct experimentation in two different species, cattle and sheep.

Discordance between bovine leukemia virus tax immortalization in vitro and oncogenicity in vivo.

Bovine leukemia virus (BLV) Tax protein, a transcriptional activator of viral expression, is essential for viral replication in vivo. Tax is believed to be involved in leukemogenesis because of its second function, immortalization of primary cells in vitro. These activities of Tax can be dissociated on the basis of point mutations within specific regions of the protein. For example, mutation of the phosphorylation sites at serines 106 and 293 abrogates immortalization potential in vitro but maintains transcriptional activity. This type of mutant is thus particularly useful for unraveling the role of Tax immortalization activity during leukemogenesis independently of viral replication. In this report, we describe the biological properties of BLV recombinant proviruses mutated in the Tax phosphorylation sites (BLVTax106+293). Titration of the proviral loads by semiquantitative PCR revealed that the BLV mutants propagated at wild-type levels in vivo. Furthermore, two animals (sheep 480 and 296) infected with BLVTax106+293 developed leukemia or lymphosarcoma after 16 and 36 months, respectively. These periods of time are within the normal range of latencies preceding the onset of pathogenesis induced by wild-type viruses. The phenotype of the mutant-infected cells was characteristic of a B lymphocyte (immunoglobulin M positive) expressing CD11b and CD5 (except at the final stage for the latter marker), a pattern that is typical of wild-type virus-infected target cells. Interestingly, the transformed B lymphocytes from sheep 480 also coexpressed the CD8 marker, a phenotype rarely observed in tumor biopsies from chronic lymphocytic leukemia patients. Finally, direct sequencing of the tax gene demonstrated that the leukemic cells did not harbor revertant proviruses. We conclude that viruses expressing a Tax mutant unable to transform primary cells in culture are still pathogenic in the sheep animal model. Our data thus provide a clear example of the discordant conclusions that can be drawn from in vitro immortalization assays and in vivo experiments. These observations could be of interest for other systems, such as the related human T-cell leukemia virus type 1, which currently lack animal models allowing the study of the leukemogenic process.

Long-term protection against bovine leukaemia virus replication in cattle and sheep.

In this report, we have evaluated the ability of two different types of live attenuated bovine leukaemia virus (BLV) variants (BLV DX and BLV 6073) to protect cattle and sheep against a heterologous wild-type BLV challenge. Four months after challenge, the protection of the vaccinated animals was effective in contrast to unvaccinated controls. However, long-term protection (18 months after challenge) was observed only in six out of seven animals, one of the vaccinated cattle being infected 12 months after challenge. A second prospective approach investigated the injection of naked plasmid DNA. Two sheep were injected with plasmid DNA encoding the BLV envelope proteins; the challenge virus infection was delayed but could not be completely abrogated. Our results demonstrate that vaccines based on live attenuated viruses and naked DNA injections are able to delay BLV infection, although complete protection cannot be achieved. In addition, our data cast light onto the need to perform long-term vaccination trials because challenge superinfection can occur even after apparent protection for 12 months.

Identification of cytosolic Mg2+-dependent soluble inorganic pyrophosphatases in potato and phylogenetic analysis.

Using polyclonal antibodies raised against a previously cloned potato Mg2+-dependent soluble inorganic pyrophosphatase (ppa1 gene) [8], a second gene, called ppa2, could be isolated. A single locus homologous to ppa2 was mapped on potato chromosomes, unlinked to the two loci identified for ppa1. From a phylogenetic and structural point of view, the PPA1 and PPA2 polypeptides are more closely related to prokaryotic than to eukaryotic Mg2+-dependent soluble inorganic pyrophosphatases (soluble PPases). Subcellular localization by immunogold electron microscopy, using sections from leaf parenchyma cells, showed that PPA and PPA2 are localized to the cytosol. Based on these observations, the likely phylogenetic origin and the physiological significance of the cytosolic soluble pyrophosphatases are discussed.

Phosphorylation of bovine leukemia virus Tax protein is required for in vitro transformation but not for transactivation.

The Tax proteins of the oncovirinae viruses are phosphorylated transcriptional activators that exhibit oncogenic potential. The role of phosphorylation in their functional activities remains unknown. As a model for the Human T-cell leukemia virus type I (HTLV-I), Bovine Leukemia Virus (BLV) permits the characterization of viral replication and leukemogenesis in vivo. Here, we show that the BLV Tax protein is phosphorylated on serine residues 106 and 293 both in insect and in mammalian cells. These sites can also be efficiently phosphorylated by the cdc2 and MAP kinases in vitro. Mutation of these residues does not affect the capacity of the Tax protein to function as a transactivator. Indeed, the Tax proteins mutated at one or both serines increase LTR-directed viral transcription at levels similar to those obtained with wild-type Tax in cell culture. Moreover, inhibition of Tax phosphorylation by W7, a calmodulin antagonist, does not alter its transactivation activity. Thus, phosphorylation on serines 106 and 293 is not required for transactivation by Tax. However, simultaneous substitution of both serines into alanine residues destroys the capacity of Tax to cooperate with the Ha-ras oncogene to transform primary rat embryo fibroblasts and induce tumors in nude mice. When the serines were replaced with aspartic acid residues, the oncogenic potential of Tax was maintained indicating that the negative charge rather than the phosphate group itself was required for Tax oncogenicity. Finally, to assess the role of the serine residues in vivo, recombinant viruses which express the Tax mutants were constructed and injected into sheep. It appeared that the mutated proviruses replicate at levels similar to the wild-type virus in vivo. We conclude that Tax phosphorylation is dispensable for transactivation and viral replication in vivo but is required for its oncogenic potential in vitro.

Conservative mutations in the immunosuppressive region of the bovine leukemia virus transmembrane protein affect fusion but not infectivity in vivo.

Many retroviruses, including bovine leukemia virus (BLV), contain a highly conserved region located about 40 amino acids downstream from the fusion peptide within the sequence of the external domain of the transmembrane (TM) protein. This region is notably thought to be involved in the presentation of the NH2-terminal peptide to allow cell fusion. By using hydrophobic cluster analysis and by analogy with the influenza A hemagglutinin structures, the core of the TM structure including this particular region was predicted to consist, in the BLV and other retroviral envelope proteins, of an alpha-helix followed by a loop region, both docked against a subsequent alpha-helix that forms a triple-stranded coiled coil. The loop region could undergo, as in hemagglutinin, a major refolding into an alpha-helix integrating the coiled coil structure and putting the fusion peptide to one tip of the molecule. Based on this model, we have identified amino acids that may be essential to the BLV TM structure, and a series of mutations were introduced in the BLV env gene of an infectious molecular clone. A first series of mutations was designed to disturb the coiled coil structure (substitutions with proline residues), whereas others would maintain the general TM structure. When expressed by Semliki Forest virus recombinants, all the mutated envelope proteins were stable and efficiently synthesized in baby hamster kidney cells. Both proline-substituted and conservative mutants were strongly affected in their capacity to fuse to CC81 indicator cells. In addition, it appeared that the integrity of the TM coiled coil structure is essential for envelope protein multimerization, as analyzed by metrizamide gradient centrifugation. Finally, to gain insight into the role of this coiled coil in the infectious potential of BLV in vivo, the mutated TM genes were introduced in an infectious and pathogenic molecular clone and injected into sheep. It appeared that only the conservative mutations (A60V and A64S) allowed maintenance of viral infectivity in vivo. Since these mutations destroyed the ability to induce syncytia, we conclude that efficient fusion capacity of the recombinant envelopes is not a prerequisite for the infectious potential of BLV in vivo. Viral propagation of these mutants was strongly affected in some of the infected sheep. However, the proviral loads within half of the infected animals (2 out of 2 for A60V and 1 out of 4 for A64S) were close to the wild-type levels. In these sheep, it thus appears that the A60V and A64S mutants propagate efficiently despite being unable to induce syncytia in cell culture.

Polyclonal bovine sera but not virus-neutralizing monoclonal antibodies block bovine leukemia virus (BLV) gp51 binding to recombinant BLV receptor BLVRcp1.

Bovine leukemia virus (BLV), a transactivating lymphotropic retrovirus, is the etiologic agent of enzootic lymphosarcoma or leukemia in cattle. Sera from BLV-infected animals possess high BLV-neutralizing antibody titres. The availability of the recombinant BLV receptor candidate, BLVRcp1, allowed us to determine a mechanism of virus neutralization by polyclonal sera and monoclonal antibodies (MAbs). Bovine sera from animals naturally infected with BLV blocked gp51 binding to recombinant BLVRcp1. In contrast, virus-neutralizing MAbs specific for gp51 F, G, and H epitopes did not prevent gp51-receptor attachment. Furthermore, gp51 neutralization epitopes F, G, and H were accessible to antibodies following gp51 attachment to BLVRcp1. This finding implies that virus neutralization by MAbs to defined BLV gp51 epitopes can occur subsequent to virus engagement of the receptor while polyclonal sera can specifically block virus attachment to the receptor. In conclusion, these data suggest that cell infection by BLV is a multistep process requiring receptor binding (inhibited by polyclonal sera) followed by a second, postbinding event(s) at the cell membrane (inhibited by anti-gp51 MAbs).

The major homology region of bovine leukaemia virus p24gag is required for virus infectivity in vivo.

In order to gain insight into the role of the major homology region (MHR) in the infectious potential of bovine leukaemia virus (BLV), mutations were introduced into the capsid gene of an infectious molecular clone. A provirus that was designed to contain only a slightly modified version of the MHR (substitution of phenylalanine 147 with a tyrosine) was still infectious in vivo. Furthermore, the provirus loads were not significantly different from those obtained with a wild-type virus. A second mutant was designed to analyse a mild modification of the MHR at the level of arginine 150. The substitution of this residue with a lysine completely destroyed the infectious potential of the recombinant virus. Finally, a third mutant that was deleted in the MHR region was unable to infect the host. Thus it appears that the integrity of the MHR domain is essential for BLV infectivity in vivo.

Both wild-type and strongly attenuated bovine leukemia viruses protect peripheral blood mononuclear cells from apoptosis.

Bovine leukemia virus (BLV) and the human T-cell leukemia viruses belong to the same subfamily of oncoviruses. Although much attention has focused on the mechanisms of cell proliferation and transformation by these viruses, experiments on the apoptotic process have yielded conflicting data in in vitro cell culture. Experimental infection of sheep with BLV proviruses offers the opportunity to analyze apoptosis in vivo. Here, we show that BLV-infected peripheral mononuclear cells, cultivated ex vivo, are protected from spontaneous programmed cell death. Moreover, the virus is able to specifically interfere with the apoptotic program of infected B lymphocytes. Strongly attenuated mutant proviruses that harbor deletions in the G4 and/or R3 genes also decrease the global susceptibility to apoptosis at levels similar to those obtained with the wild-type virus. In addition, cell culture supernatants from wild-type and mutant viruses can prevent uninfected cells from undergoing programmed cell death. These observations demonstrate that the R3 and G4 genes are not required to maintain both direct and indirect protection against apoptosis. They also imply that the level of programmed cell death observed ex vivo is independent of the amounts of proviruses in the animals. The failure of these cells to undergo apoptosis might be related to the pathogenesis induced by BLV.

Fusion of bovine leukemia virus with target cells monitored by R18 fluorescence and PCR assays.

PCR and R18 fluorescence dequenching assays have been combined to monitor the kinetics of fusion of bovine leukemia virus with target cells (CC81, OVK, or Raji). Antibodies raised against gp51 allow us to demonstrate that not only the hydrophobic N-terminal domain of the transmembrane glycoprotein gp30 but also specific domains of gp51 (amino acids 39 to 103) are involved in bovine leukemia virus-cell fusion.

Cellular pathways involved in the ex vivo expression of bovine leukemia virus.

Bovine leukemia virus (BLV) is the etiologic agent of enzootic bovine leukosis. The virus adopts a strategy based on the lack of viral expression in vivo; only very rare BLV-infected B lymphocytes express viral information. When the cells are isolated from animals in persistent lymphocytosis and cultivated ex vivo, a tremendous increase in viral expression occurs. To gain insight into this mechanism, we employed a general approach using chemicals that interfere specifically with cellular pathways involved in signal transduction from the cell membrane to the nucleus. Our data demonstrate that BLV expression is not correlated with the activity of protein kinase A (PKA) and is even inhibited by cyclic AMP (cAMP). The cAMP/PKA pathway is thus apparently not involved in ex vivo viral expression. In contrast, PKC appears to play a key role in this process. Phorbol myristate acetate can directly activate viral expression in B cells (in the absence of T cells). Furthermore, calphostin C, a highly specific inhibitor of PKC, partly decreases ex vivo BLV expression. Our data further demonstrate that calmodulin and calcineurin, a calmodulin-dependent phosphatase, play a key role in the induction of viral expression. The involvement of this calmodulin-dependent pathway could explain the induction of expression that cannot be assigned to PKC. Furthermore, it appears that the activation of viral expression requires a calmodulin but not a PKA-dependent pathway. These data highlight major differences between transient transfection and ex vivo experiments. Finally, despite their homologies, BLV and human T-cell leukemia virus appear to use different signal transduction pathways to induce viral expression.

Dissociation of increases in plasma insulin-like growth factor I and testosterone during the onset of puberty in bulls.

The present study was conducted to examine the relationship between plasma concentrations of testosterone, insulin-like growth factor I (IGF-I) and IGF-binding proteins (IGFBPs) during puberty, in male calves treated with GnRH or testosterone propionate. Twelve male Holstein calves (10 weeks old) were assigned to the control group (n = 6), the GnRH-treated group (n = 3) or the testosterone-treated group (n = 3). For 8 weeks, the GnRH-treated group received a single i.v. injection of GnRH (0.5 microgram kg-1 body mass) each day while the testosterone-treated group received an i.m. injection of testosterone propionate (0.5 mg kg-1 body mass) twice a day. The calves were studied until they were 200 days old. Hormone treatments were stopped one month after puberty was reached in the control group. Blood samples were collected every 30 min for 8 h every third day. Hormone concentrations were determined by radioimmunoassay. Western ligand blotting and immunoblotting, using monoclonal antibodies against IGFBP-2 and IGFBP-3, were used to characterize the IGF-binding proteins. In the control group, puberty occurred at about 120 days of age and was associated with an increase in concentrations of testosterone, IGF-I and IGFBP-3 and a decrease in concentration of IGFBP-2. In the GnRH-treated group, plasma testosterone remained low until 8 weeks after establishment of puberty in the control group (4 weeks after the end of treatment). In the testosterone-treated group, testosterone was high during the treatment period and then decreased to prepubertal values when treatment was stopped. Testosterone values increased again to reach postpubertal values 5 weeks after the end of hormone treatment. Nevertheless, independent of testosterone status, the profile of IGF-I and the IGFBPs in the GnRH- and testosterone-treated groups were parallel to that reported for the control group with the transition from prepubertal to adult values at about 120 days of age. In conclusion, concentrations of testosterone, IGF-I and IGFBP-3 increase together, but probably independently, during the onset of puberty in male calves.

The sequence of an 11.1 kb DNA fragment between ADH4 and ADE5 on the left arm of chromosome VII reveals the presence of eight open reading frames.

The complete sequence of a 11, 132 bp segment located on the left arm of chromosome VII of Saccharomyces cerevisiae has been determined and analysed. Eight open reading frames (ORFs) of at least 100 amino acids were identified. Five show similarities to known amino acid sequences. Another ORF encoding the chromosome segregation protein CSE1 is not entirely located in our sequenced fragment and is incomplete at its C-terminus. The two remaining ORFs do not display similarities to known sequences.

The YXXL signalling motifs of the bovine leukemia virus transmembrane protein are required for in vivo infection and maintenance of high viral loads.

The bovine leukemia virus (BLV) transmembrane protein (gp30) contains three YXXL motifs at its carboxyterminal end. Two of these motifs have been implicated in vitro in signal transduction pathways from the external to the intracellular compartment. In order to analyze the biological relevance of these motifs in vivo, recombinant BLV proviruses were constructed. A mutation of the tyrosine residue of the second YXXL motif completely destroyed the infectious potential of the virus in sheep. In contrast, the tyrosine of the first motif appeared to be dispensable for infectivity. However, the propagation of the recombinant virus within the animal was greatly impaired (as demonstrated by PCR and enzyme-linked immunosorbent assay). These recombinant BLVs thus exhibit an attenuated phenotype. Altogether, our data demonstrate the importance of the YXXL motifs of the BLV transmembrane protein for in vivo infection and viral propagation.

Attenuation of bovine leukemia virus by deletion of R3 and G4 open reading frames.

Complex oncoviruses contain, in addition to the classical retroviral genes (gag, pol, and env), a region (X) located between the envelope sequences and the 3' long terminal repeat. The X region contains two genes, tax and rex, whose protein products are involved in transcriptional and posttranscriptional regulation of viral expression. In addition to these activators, the bovine leukemia virus (BLV) and the human T-cell leukemia virus (HTLV) contain alternative open reading frames (R3 and G4 for BLV; p30, p13, and p12 for HTLV). As a virus/animal model for HTLV-induced leukemogenesis, BLV provirus can be injected intradermally into sheep, where it induced B-lymphocyte transformation. Deletion of the R3 and G4 sequences from an infectious and tumorigenic BLV provirus greatly impaired the in vivo propagation of the viruses as demonstrated by DNA polymerase chain reaction, RNA blots, structural-protein ELISA, and immunofluorescence analysis. Our results show that the alternative open reading frames are required for maintaining high virus loads during the course of persistent infection in vivo. Thus, R3 and G4 are candidates for antiviral drug development. Furthermore, viruses with a deletion in these sequences should be tested as live attenuated vaccines.

Cloning and characterization of MN, a human tumor-associated protein with a domain homologous to carbonic anhydrase and a putative helix-loop-helix DNA binding segment.

MN is a transmembrane glycoprotein that has been detected in HeLa cells and in some human carcinomas. The expression of MN protein in HeLa cells is regulated by cell density. In HeLa x fibroblast cell hybrids its expression correlates with tumorigenicity. Using a specific monoclonal antibody we have identified a cDNA clone coding for MN. Analysis of the deduced amino acid sequence revealed strong structural homology between the central region of the MN protein and carbonic anhydrases (CA). MN sequence retains the conserved zinc-binding site as well as the enzyme's active center. In accord with these findings, MN protein from HeLa cells was found to bind zinc and to have carbonic anhydrase activity. The N-terminal region of MN shares some similarity with DNA binding proteins of the helix-loop-helix (HLH) family, and the protein was found to have affinity for DNA by DNA-cellulose chromatography. The region between the CA-like domain and the putative HLH domain is rich in imperfect repeats of serine, proline, glycine and acidic residues with few hydrophobic amino acids, resembling thus an activation region of transcription factors. The fact that MN protein is detectable in several types of human carcinomas, but not in corresponding non-cancerous tissues, suggests its possible role in neoplasia. In addition, the analysis of biological consequences of MN expression of NIH3T3 cells provides the evidence in favour of MN protein involvement in control of cell proliferation and transformation.