Microbial communities of Auka hydrothermal sediments shed light on vent biogeography and the evolutionary history of thermophily.

Hydrothermal vents have been key to our understanding of the limits of life, and the metabolic and phylogenetic diversity of thermophilic organisms. Here we used environmental metagenomics combined with analysis of physicochemical data and 16S rRNA gene amplicons to characterize the sediment-hosted microorganisms at the recently discovered Auka vents in the Gulf of California. We recovered 325 metagenome assembled genomes (MAGs) representing 54 phyla, over 30% of those currently known, showing the microbial community in Auka hydrothermal sediments is highly diverse. 16S rRNA gene amplicon screening of 224 sediment samples across the vent field indicates that the MAGs retrieved from a single site are representative of the microbial community in the vent field sediments. Metabolic reconstruction of a vent-specific, deeply branching clade within the Desulfobacterota suggests these organisms metabolize sulfur using novel octaheme cytochrome-c proteins related to hydroxylamine oxidoreductase. Community-wide comparison between Auka MAGs and MAGs from Guaymas Basin revealed a remarkable 20% species-level overlap, suggestive of long-distance species transfer over 400 km and subsequent sediment colonization. Optimal growth temperature prediction on the Auka MAGs, and thousands of reference genomes, shows that thermophily is a trait that has evolved frequently. Taken together, our Auka vent field results offer new perspectives on our understanding of hydrothermal vent microbiology.

MEF2-mediated recruitment of class II HDAC at the EBV immediate early gene BZLF1 links latency and chromatin remodeling.

In B lymphocytes induced to proliferate in vitro by the Epstein-Barr virus (EBV), extra-chromosomal viral episomes packaged in chromatin persist in the nucleus, and there is no productive cycle. A switch from this latency to the productive cycle is observed after induced expression of the EBV BZLF1 gene product, the transcription factor EB1. We present evidence that, during latency, proteins of the myocyte enhancer binding factor 2 (MEF2) family are bound to the BZLF1 promoter and recruit class II histone deacetylases. Furthermore, we propose that latency is determined primarily by a specific and local recruitment of class II histone deacetylase (HDAC) by MEF2D to the BZLF1 gene promoter. The switch from latency to the productive cycle could be due in part to post-translational modification of MEF2 proteins and changes in the local acetylation state of the chromatin.

Kaposi's sarcoma-associated herpesvirus and Kaposi's sarcoma.

Kaposi's sarcoma-associated herpesvirus (KSHV) is present in all epidemiologic forms of Kaposi's sarcoma (KS). The KSHV genome contains several open reading frames which are potentially implicated in the development of KS. Some are unique to KSHV; others are homologous to cellular genes. The putative role of these genes in the genesis of KS is discussed.

Kaposi's sarcoma-associated herpesvirus (human herpesvirus-8) encodes a homologue of the Epstein-Barr virus bZip protein EB1.

Analysis of the recently completed genomic sequence of Kaposi's sarcoma-associated herpesvirus (human herpesvirus-8) revealed that ORF 50 encodes a protein with homology to the Epstein-Barr virus (EBV) transcription factor R. In this report, we show that ORF K8, contiguous to ORF 50, is interrupted by two introns and that the spliced RNA is translated into a bZip protein that has homology to the EBV transcription factor EB1. The newly characterized K8 protein forms homodimers but does not heterodimerize with other members of the bZip protein family.

Transcriptional repression by the Epstein-Barr virus EBNA3A protein tethered to DNA does not require RBP-Jkappa.

The Epstein-Barr virus (EBV) proteins EBNA1, EBNA2, EBNA3A, EBNA3C, LMP1 and EBNA-LP are essential for the in vitro immortalization of primary B lymphocytes by EBV. EBNA2 is a transcriptional activator of viral and cellular genes. Both EBNA3A and EBNA3C have been shown to specifically inhibit EBNA2-activated transcription by direct interaction with RBP-Jkappa, a cellular DNA-binding factor known to recruit EBNA2 to EBNA2-responsive genes. This interaction interferes with the binding of RBP-Jkappa to DNA in vitro, and this is probably the mechanism by which EBNA3A and EBNA3C repress EBNA2-activated transcription in vivo. EBNA3A and EBNA3C also directly repress transcription when tethered to a promoter via the DNA-binding domain of the yeast Gal4 protein. As RBP-Jkappa has been previously shown to be a repressor in mammalian cells, this repression could be due to the recruitment of RBP-Jkappa by Gal4-EBNA3A and 3C. In this study, we have precisely mapped the domain of EBNA3A involved in the interaction with RBP-Jkappa and we have shown that interaction with RBP-Jkappa is not required for the Gal4-EBNA3A-mediated repression. Furthermore, we have characterized in EBNA3A a domain of 143 amino acids which is necessary and sufficient for EBNA3A-dependent repression.

Epstein-Barr virus EBNA3A and EBNA3C proteins both repress RBP-J kappa-EBNA2-activated transcription by inhibiting the binding of RBP-J kappa to DNA.

Following infection by Epstein-Barr virus (EBV), the production of viral nuclear proteins EBNA1, EBNA2, EBNA3A, and EBNA3C and the viral membrane protein LMP1 is essential for the permanent proliferation of primary B lymphocytes to occur. Among these, the transcription factor EBNA2 is central to the immortalizing process, since it activates not only the transcription of all the EBNA proteins and LMP1, TP1, and TP2 but also certain cellular genes. EBNA2 is targeted to its DNA-responsive elements through direct interaction with the DNA-binding cellular repressor RBP-J kappa. In a transient-expression assay, the EBNA2-activated transcription was found to be downregulated by EBNA3A, EBNA3B, and EBNA3C. However, since it has been reported that EBNA3C, but not EBNA3A, directly contacts RBP-J kappa in vitro, these proteins appear to repress through different mechanisms. Here, we report for the first time that EBNA3A and EBNA3C both stably interact with RBP-J kappa and most probably repress EBNA2-activated transcription by destabilizing the binding of RBP-J kappa to DNA.

RBP-J kappa repression activity is mediated by a co-repressor and antagonized by the Epstein-Barr virus transcription factor EBNA2.

The Epstein-Barr virus (EBV) protein EBNA2 is a transcriptional activator that can be targeted to its DNA responsive elements by direct interaction with the cellular protein RBP-J kappa. RBP-J kappa is a ubiquitous factor, highly conserved between man, mouse and Drosophila, whose function in mammalian cells is largely unknown. Here we provide evidence that RBP-J kappa is a transcriptional repressor and, more importantly, that RBP-J kappa repression is mediated by a co-repressor. The function of the co-repressor could be counterbalanced by making a fusion protein (RBP-VP16) between RBP-J kappa and the VP16 activation domain. This RBP-VP16-mediated activation could be strongly increased by an EBNA2 protein deprived of its activation domain, but not by an EBNA2 protein incapable of making physical contact with RBP-J kappa. Our results suggest that EBNA2 activates transcription by both interfering with the function of a co-repressor recruited by RBP-J kappa and providing an activation domain.

The human J kappa recombination signal sequence binding protein (RBP-J kappa) targets the Epstein-Barr virus EBNA2 protein to its DNA responsive elements.

The Epstein-Barr virus (EBV) protein EBNA2, which is essential for the immortalization of human primary B cells by EBV, acts as a transcriptional activator of cellular and viral genes. Specific responsive elements have been characterized in several of the promoters activated by EBNA2. They all share the core sequence GTGGGAA. EBNA2 does not, however, bind to these sequences directly, but appears to be targeted to them by a cellular protein. A similar core sequence has recently been identified as a high-affinity binding site for the human recombination signal sequence binding protein RBP-J kappa. Here we provide evidence that RBP-J kappa binds to specific sequences in EBNA2-responsive elements. Our results also demonstrate that RBP-J kappa makes direct physical contact with EBNA2 in solution and recruits EBNA2 to its cognate DNA sequences, suggesting that RBP-J kappa may mediate EBNA2 transactivation of both cellular and viral genes.

The bZIP motif of the Epstein-Barr virus (EBV) transcription factor EB1 mediates a direct interaction with TBP.

The EBV transcription factor EB1, is a key determinant of the switch from the latent infection to the lytic cycle. EB1 belongs to the Jun, Fos, ATF, CREB, C/EBP and GCN4 family of proteins, carrying a sequence-specific DNA-binding domain called "basic-Zipper" (bZIP). The N-terminal region of EB1 is required for transcriptional activation, whereas the C-terminal region contains the DNA-binding domain. The mechanism by which site-specific transcription factors increase specific initiation at polymerase II dependent promoters is thought to occur via recruitment and stabilization of components that form the initiation complex, i.e., TFIID, TFIIA, TFIIB, TFIIE, TFIIG, TFIIH, TFIIJ and pol II. TFIID is not a single protein but consists of the TATA-binding protein TBP plus several distinct and tightly associated proteins called TAFs. More specifically, in vitro studies have revealed that the TAFs are not required for basal transcription, but are essential for mediating regulated transcription by different upstream activators. TFIID binding at the promoter sites is one of the limiting steps in the assembly of the initiation complex. Direct interactions with TBP or with one or several TAFs, mediated by the activation domain of site specific activators, could influence the binding rate of TFIID, and thus provide one of the mechanisms by which transcription is regulated. We show here that EB1 interacts directly with TBP in vitro, and that it is the bZIP domain, likely the region contacting the DNA rather than the activation domain, which is required for physical contact between EB1 and TBP.

Use of monoclonal antibodies to distinguish pathogenic Naegleria fowleri (cysts, trophozoites, or flagellate forms) from other Naegleria species.

Monoclonal antibodies (MAbs) reactive to the pathogenic amoeba Naegleria fowleri were analyzed by enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay, Western blotting (immunoblotting), and radioimmunoprecipitation assay (RIPA). Two MAbs (3A4 and 5D12) showed reactivity by ELISA with all N. fowleri strains tested and no reactivity with the five other Naegleria species, N. lovaniensis, N. gruberi, N. australiensis, N. jadini, and N. andersoni. These MAbs reacted with the three morphological forms of N. fowleri (trophozoites, cysts, and flagellates). The reactivity on Western blots was suppressed by treatment with metaperiodate, suggesting a carbohydrate epitope. Differences in reactivity patterns between trophozoites and cysts observed with radioimmunoprecipitation assay might reflect differences in biological properties. The formalin stability of the epitope may be useful in detecting N. fowleri in fixed biopsies and in investigating the pathological process.

The acidic activation domain of the Epstein-Barr virus transcription factor R interacts in vitro with both TBP and TFIIB and is cell-specifically potentiated by a proline-rich region.

In cells latently infected with Epstein-Barr virus (EBV), the expression of two viral transactivators, EB1 and R, is responsible for the switch from latency to a productive cycle. R contains a DNA-binding/dimerization domain localized at the N-terminus. The domain required for transcriptional activation is localized at the C-terminus and contains two regions of very different amino acid composition. The first is very rich in prolines, whereas the second is rich in acidic residues and contains two potential alpha-helices. We investigated the activation potential of these subregions when linked to the heterologous Gal4 DNA-binding domain. We found that the acidic region--more precisely, the second putative alpha-helix--is an activating domain. In contrast, the proline-rich region is insufficient by itself for activation but collaborates with the acidic region in a cell-specific manner to make transactivation more efficient. We demonstrated that R interacts in vitro with the basal transcription factors TBP and TFIIB, and that the acidic domain of R mediates these interactions.

Domains of the Epstein-Barr virus (EBV) transcription factor R required for dimerization, DNA binding and activation.

In cells latently infected with EBV, the switch from latency to a productive infection is linked to the expression of two transcriptional activators, the upstream element factor EB1 and the enhancer factor R. R activates by interacting directly with specific DNA sequences called RREs (R Responsive Elements). Each binding site covers about 18 bp, where R simultaneously contacts two core sequences separated by 5 to 7 bp (1). Here we show that R binds in vitro as a homodimer to an RRE, and that stable homodimers can also form in solution in the absence of DNA. By functional analysis of deletion and insertion mutants of R, we have localized the DNA binding region within the 280 N-terminal amino acids and the dimerization region within the 232 N-terminal amino acids. As no obvious homologies were detected with other known DNA binding or dimerization motifs, R could contain novel protein structures mediating these functions. The transcriptional activation domain has been located in the C-terminal half of the protein. This domain contains two regions with structures already identified in other transcription factors: one region is rich in proline, the other rich in acidic residues.

Transcriptional interference between the EBV transcription factors EB1 and R: both DNA-binding and activation domains of EB1 are required.

The switch from latency to a productive infection in EBV-infected B cells is linked to the expression of two viral sequence-specific DNA-binding transcription factors called EB1 and R. EB1 shares sequence homologies with the bZIP family of proteins in the basic region required for specific DNA interaction. Here, we provide evidence that EB1 and R can synergistically activate specific transcription, and that overexpressed, unbound EB1, represses the R-induced transcription ('squelching'). In order to identify the EB1 domains involved in transcriptional activation, transcriptional synergy and transcriptional repression, we performed extensive mutagenesis of the EB1 protein. Results show that five segments (region 1 to region 5), localized at the N-terminus of EB1 exhibit characteristics of activating domains, since they are required for full transcriptional activity, without obvious role in DNA-binding, or the nuclear localization. Two domains rich in basic amino-acids are required for the nuclear localization of EB1. One domain is within the basic region B, also necessary for specific and stable interaction between EB1 and its cognate DNA sequences. It is also shown that the 'activation' domain, and more surprisingly the DNA-binding domain of EB1, may interact with a factor(s), essential for R-induced activation, and probably required for synergy between EB1 and R.

The enhancer factor R of Epstein-Barr virus (EBV) is a sequence-specific DNA binding protein.

In cells latently infected with EBV, the switch from latency to productive infection is linked to the expression of two EBV transcription factors called EB1 (or Z) and R. EB1 is an upstream element factor which has partial homology to the AP1/ATF family, whereas R is an enhancer factor. In the R-responsive enhancer of the replication origin only active during the EBV lytic cycle (ORIIyt), R-responsive elements are located in a region of about 70 bp (RRE-DR). Here we show that R, produced either by in vitro translation, or present in nuclear extracts from HeLa cells constitutively producing R, binds directly to and protects against DNAase I digestion, two regions in RRE-DR. Using mobility shift assay and DMS interference, we have characterized the contact-points between R and the DNA. Two binding sites, RRE-DR1 and RRE-DR2, were characterized and are contiguous in RRE-DR. R binds to these two sites probably by simultaneously contacting two sequences within the sites, which are separated by 7 bp in RRE-DR1, cctGTGCCttgtcccGTGGACaatgtccc, and by 6bp in RRE-DR2, caatGTCCCtccagcGTGGTGgctg. Direct interaction of R with its cognate sequences is conferred by its N-terminal 355 amino-acids. Directed mutagenesis in RRE-DR, of either R-binding site, impaired binding of R in vitro and, as assayed by transient expression in HeLa cells, impaired R-activation by a factor of two. This suggests that RRE-DR1 and RRE-DR2 do not respond cooperatively to R.

The Epstein-Barr virus (EBV) early protein EB2 is a posttranscriptional activator expressed under the control of EBV transcription factors EB1 and R.

From the cloning and characterization of cDNAs, we found that the Epstein-Barr virus (EBV) open reading frame (ORF) BMLF1-BSLF2 coding for the early protein EB2 is present in several mRNAs generated by alternative splicing and expressed in the leftward direction from two promoters PM and PM1. The PM promoter controls the expression of two abundant mRNA species of 1.9 and 2 kilobases (kb), whereas the PM1 promoter controls the expression of at least three mRNAs 3.6, 4.0, and 4.4 kb long. The PM promoter probably overlaps with the PS promoter which controls the transcription of a 3.6-kb mRNA expressed in the rightward direction and containing the ORF BSRF1. Although it increases the amount of chloramphenicol acetyltransferase enzyme expressed from the chimeric pMCAT gene, EB2 is not a promiscuous trans-activator of gene expression and does not positively regulate its own expression from promoter PM. The EB2 activation is not promoter dependent but could possibly act by stabilizing mRNAs and increasing their translation. The PM promoter is, however, activated by the two EBV transcription trans-acting factors, EB1 and R, encoded by the EBV ORFs BZLF1 and BRLF1, respectively. EB1 activates the PM promoter from a consensus AP-1 binding site, and R activates the PM promoter from an enhancer.

Epstein-Barr virus bicistronic mRNAs generated by facultative splicing code for two transcriptional trans-activators.

The Epstein-Barr virus (EBV) genome codes for several transcriptional trans-activators. One of them, the BZLF1 open reading frame (ORF)-encoded product EB1, is able to induce the productive cycle in infected B cells. From the cloning and characterization of full-length cDNAs, we found that EB1 could be made from three overlapping messenger RNAs expressed under the control of two different promoters that we call P1 and P2. The first mRNA, 1 kb long, is made from the P1 promoter and codes for EB1 alone. The two other mRNAs, respectively 3 and 4 kb long and made by facultative splicing, are bicistronic mRNAs. They code not only for the trans-activator EB1 but also for a second EBV transcriptional trans-activator R, encoded by the BRLF1 ORF. In effect, authentic EB1 and R proteins are expressed from the 3 and 4 kb long cDNAs as demonstrated by identification of the proteins with specific antisera. In addition, EB1 and R expressed from the 3 and 4 kb cDNAs activate transcription from their specific targets in the EBV early promoter DR.

The Epstein-Barr virus (EBV) DR enhancer contains two functionally different domains: domain A is constitutive and cell specific, domain B is transactivated by the EBV early protein R.

The Epstein-Barr virus (EBV) DR promoter is located upstream of the PstI repeats, and besides the TATA box, it contains two cis-acting regulatory elements. One of them has enhancer properties. To define more precisely the functional region(s) in the DR enhancer, we generated 5' and 3' deletion mutants. These deletion mutants, which were transfected into various recipient cells of different origins, allowed us to identify two functionally distinct domains, A and B. Domain A was constitutively active in all cell lines tested, except in lymphoid B cells. Domain B was active in lymphoid B cells, and its activity required both EB1 (the BZLF1-encoded EBV trans-acting factor) and the presence of the EBV genome. This suggested that an EBV-encoded, EB1-inducible factor was activating the enhancer B domain. In effect, the B domain was trans-activated by R, an EBV early product encoded by the open reading frame BRLF1, and the activation by R occurred in epithelial, fibroblastic, and lymphoid cells. The R-responsive element has been reduced to 28 base pairs containing the double palindromic sequence TTGTCCCGTGGACAATGTCC. Both domains A and B act by increasing the initiation of specific RNAs.

Structure and sequence of the Drosophila zeste gene.

The zeste gene of Drosophila affects the expression of other genes in a manner that depends on the homologous pairing of the chromosomes bearing the target gene. Zeste mediates transvection effects, the ability of one gene to control the expression of its homologous copy on another chromosome. We have determined the structure of the zeste gene and several mutants bearing partial deletions and the sequence of the z+, z1, zop6 and z11G3 alleles. The predicted zeste protein has an unusual structure including runs of Gln, Ala and alternating Gln Ala. Contrary to expectations the z1, zop6 and z11G3 mutations can each be attributed to single amino acid changes. The analysis of the mutants suggests that the zeste gene product is required for normal expression of at least some genes and we argue that za mutants may have residual function.