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The phylogenetic diversity of eukaryotic transcription.

Eukaryotic transcription is a highly regulated process involving interactions between large numbers of proteins. To analyse the phylogenetic distribution of the components of this process, six crown eukaryote group genomes were queried with a reference set of transcription-associated (TA) proteins. On average, one in 10 proteins encoded by these genomes were found to be homologous to sequences in the reference set. Analysis of families identified using an accurate sequence clustering algorithm and containing both TA proteins and eukaryotic sequences showed that in two-thirds of the families the homologues originate from a single kingdom. Furthermore, in only 15% of the fungal-specific clusters are the homologues present in both budding and fission yeast, as compared with the metazoan-specific clusters where 53% of the homologues originate from two or more species. Families whose members comprise general transcription factor or RNA polymerase subunits exhibit a low degree of taxon specificity, suggesting that the transcription initiation complex is highly conserved. This contrasts with transcriptional regulator families, that are primarily taxon-specific, indicating proteins controlling gene activation exhibit considerable sequence diversity across the eukaryotic domain.

Animals↗

Nucleotide sequence of complementary DNA and derived amino acid sequence of murine complement protein C3.

The nucleotide sequences coding for murine complement component C3 have been determined from a cloned genomic DNA fragment and several overlapping cloned complementary DNA fragments. The amino acid sequence of the protein was deduced. The mature beta and alpha subunits contain 642 and 993 amino acids respectively. Including a 24 amino acid signal peptide and four arginines in the beta-alpha transition region, which are probably not contained in the mature protein, the unglycosylated single chain precursor protein preproC3 would have a molecular mass of 186 484 Da and consist of 1663 amino acid residues. The C3 messenger RNA would be composed of a 56 +/- 2 nucleotide long 5' non-translated region, 4992 nucleotides of coding sequence, and a 3' non-translated region of 39 nucleotides, excluding the poly A tail. The beta chain contains only three cysteine residues, the alpha chain 24, ten of which are clustered in the carboxy terminal stretch of 175 amino acids. Two potential carbohydrate attachment sites are predicted for the alpha chain, none for the beta chain. From a comparison with human C3 cDNA sequence (of which over 80% has been determined) an extensive overall sequence homology was observed. Human and murine preproC3 would be of very similar length and share several noteworthy properties: the same order of the subunits in the precursor, the same basic residue multiplet in the beta-alpha transition region, and a glutamine residue in the thioester region. The equivalent position of the known factor I cleavage sites in human C3 alpha could be located in the murine C3 alpha chain and the size and sequence of the resulting peptide were deduced. A comparison of the amino acid sequences of murine C3 and human alpha 2-macroglobulin is given. Several areas of strong sequence homology are observed, and we conclude that the two genes must have evolved from a common ancestor.

Amino Acid Sequence↗

Identification of the products of a varicella-zoster virus glycoprotein gene.

Two of the genes identified from the previously published DNA sequence of the Us component of the varicella-zoster virus (VZV) genome were predicted to encode membrane proteins with polypeptide molecular weights of 39000 (39K) and 70K. A rabbit antiserum directed against a unique peptide containing the seven amino acid residues at the carboxy terminus of the 39K gene product specifically precipitated glycoproteins with apparent molecular weights of 55K and 45K from VZV-infected cells labelled with [3H]mannose. The complete inhibition of precipitation of gp55 by free peptide and the partial inhibition of precipitation of gp45 support the conclusion that the 39K gene encodes gp55 and perhaps gp45. The number of VZV genes currently thought to encode glycoproteins is discussed in view of this finding.

Cell Line↗

Anaerovirgula multivorans gen. nov., sp. nov., a novel spore-forming, alkaliphilic anaerobe isolated from Owens Lake, California, USA.

A novel, alkaliphilic, obligately anaerobic bacterium, strain SCAT, was isolated from mud sediments of a soda lake in California, USA. The rod-shaped cells were motile, Gram-positive, formed spores and were 0.4-0.5x2.5-5.0 microm in size. Growth occurred within the pH range 6.7-10.0 and was optimal at pH 8.5. The temperature range for growth was 10-45 degrees C, with optimal growth at 35 degrees C. NaCl was required for growth. Growth occurred at 0.5-9.0% (w/v) NaCl and was optimal at 1-2% (w/v). The novel isolate was a catalase-negative chemo-organoheterotroph that fermented sugars, proteolysis products, some organic and amino acids, glycerol, d-cellobiose and cellulose. It was also capable of growth by the Stickland reaction. Strain SCAT was sensitive to tetracycline, chloramphenicol, rifampicin and gentamicin, but it was resistant to ampicillin and kanamycin. The G+C content of the genomic DNA was 34.2 mol%. Major fatty acid components were C14:0, iso-C15:0, C16:1omega9c and C16:0. 16S rRNA gene sequence analysis of strain SCAT showed a similarity of approximately 97% with the type strains of Clostridium formicaceticum and Clostridium aceticum in clostridial cluster XI and a similarity of less than 94.2% to any other recognized Clostridium species and those of related genera in this cluster. Strain SCAT was clearly differentiated from C. formicaceticum and C. aceticum based on comparison of their phenotypic properties and fatty acid profiles, as well as low levels of DNA-DNA relatedness between strain SCAT and the type strains of these two species. Therefore, strain SCAT is considered to represent a novel species of a new genus, Anaerovirgula multivorans gen. nov., sp. nov., in clostridial cluster XI. The type strain is SCAT (=ATCC BAA-1084T=JCM 12857T=DSM 17722T=CIP 107910T).

Amino Acids↗

Molecular characterization of Penicillium chrysogenum virus: reconsideration of the taxonomy of the genus Chrysovirus.

Molecular cloning and complete nucleotide sequencing of Penicillium chrysogenum virus (PcV) dsRNAs indicated that PcV virions contained four dsRNA segments with sizes of 3562, 3200, 2976 and 2902 bp. Each dsRNA segment had unique sequences and contained a single large open reading frame (ORF). In vitro translation of transcripts derived from full-length cDNA clones of PcV dsRNAs yielded single products of sizes similar to those predicted from the deduced amino acid sequences of the individual ORFs. Sequence similarity searches revealed that dsRNA1 encodes a putative RNA-dependent RNA polymerase. In this study, it was determined that dsRNA2 encodes the major capsid protein and that p4, encoded by dsRNA4, is virion-associated as a minor component. All four dsRNAs of PcV, like the genomic segments of viruses with multipartite genomes, were found to have extended regions of highly conserved terminal sequences at both ends. In addition to the strictly conserved 5'-terminal 10 nt, a second region consisting of reiteration of the sequence CAA was found immediately upstream of the AUG initiator codon. These (CAA)(n) repeats are reminiscent of the translational enhancer elements of tobamoviruses. The 3'-terminal 14 nt were also strictly conserved. As PcV and related viruses with four dsRNA segments (genus Chrysovirus) have not been previously characterized at the molecular level, they were provisionally classified in the family Partitiviridae, comprising viruses with bipartite genomes. This study represents the first report on molecular characterization of a chrysovirus and the results suggest the creation of a new family of mycoviruses with multipartite dsRNA genomes to accommodate PcV and related viruses.

Amino Acid Sequence↗

Structure determination of the head-tail connector of bacteriophage phi29.

The head-tail connector of bacteriophage phi29 is composed of 12 36 kDa subunits with 12-fold symmetry. It is the central component of a rotary motor that packages the genomic dsDNA into preformed proheads. This motor consists of the head-tail connector, surrounded by a phi29-encoded, 174-base, RNA and a viral ATPase protein, both of which have fivefold symmetry in three-dimensional cryo-electron microscopy reconstructions. DNA is translocated into the prohead through a 36 A diameter pore in the center of the connector, where the DNA takes the role of a motor spindle. The helical nature of the DNA allows the rotational action of the connector to be transformed into a linear translation of the DNA. The crystal structure determination of connector crystals in space group C2 was initiated by molecular replacement, using an approximately 20 A resolution model derived from cryo-electron microscopy. The model phases were extended to 3.5 A resolution using 12-fold non-crystallographic symmetry averaging and solvent flattening. Although this electron density was not interpretable, the phases were adequate to locate the position of 24 mercury sites of a thimerosal heavy-atom derivative. The resultant 3.2 A single isomorphous replacement phases were improved using density modification, producing an interpretable electron-density map. The crystallographically refined structure was used as a molecular-replacement model to solve the structures of two other crystal forms of the connector molecule. One of these was in the same space group and almost isomorphous, whereas the other was in space group P2(1)2(1)2. The structural differences between the oligomeric connector molecules in the three crystal forms and between different monomers within each crystal show that the structure is relatively flexible, particularly in the protruding domain at the wide end of the connector. This domain probably acts as a bearing, allowing the connector to rotate within the pentagonal portal of the prohead during DNA packaging.

Bacillus Phages↗

An automated film reader for DNA sequencing based on homomorphic deconvolution.

An automated reader for electrophoresis based DNA sequencing methods is described that provides fast and accurate sequence determination. Digitized sequencing lanes are processed with homomorphic blind deconvolution in preparation for peak detection, interlane alignment, peak refinement and base calling. Initial reads from direct blot sequencing films have error rates of about 1% at the rate of 5 nucleotides/s. Typical read lengths are 500-600 nucleotides. The described reader is a significant improvement over existing readers and could be an essential component in the sequencing efforts of the Human Genome Project.

Base Sequence↗

High-quality homology models derived from NMR and X-ray structures of E. coli proteins YgdK and Suf E suggest that all members of the YgdK/Suf E protein family are enhancers of cysteine desulfurases.

The structural biology of proteins mediating iron-sulfur (Fe-S) cluster assembly is central for understanding several important biological processes. Here we present the NMR structure of the 16-kDa protein YgdK from Escherichia coli, which shares 35% sequence identity with the E. coli protein SufE. The SufE X-ray crystal structure was solved in parallel with the YdgK NMR structure in the Northeast Structural Genomics (NESG) consortium. Both proteins are (1) key components for Fe-S metabolism, (2) exhibit the same distinct fold, and (3) belong to a family of at least 70 prokaryotic and eukaryotic sequence homologs. Accurate homology models were calculated for the YgdK/SufE family based on YgdK NMR and SufE crystal structure. Both structural templates contributed equally, exemplifying synergy of NMR and X-ray crystallography. SufE acts as an enhancer of the cysteine desulfurase activity of SufS by SufE-SufS complex formation. A homology model of CsdA, a desulfurase encoded in the same operon as YgdK, was modeled using the X-ray structure of SufS as a template. Protein surface and electrostatic complementarities strongly suggest that YgdK and CsdA likewise form a functional two-component desulfurase complex. Moreover, structural features of YgdK and SufS, which can be linked to their interaction with desulfurases, are conserved in all homology models. It thus appears very likely that all members of the YgdK/SufE family act as enhancers of Suf-S-like desulfurases. The present study exemplifies that "refined" selection of two (or more) targets enables high-quality homology modeling of large protein families.

Animals↗

Generation and characterization of a PhoP homologue mutant of Neisseria meningitidis.

Two-component regulatory systems are important regulators of virulence genes in a number of bacteria. Genes encoding a two-component regulator system, with homology to the phoP/phoQ system in salmonella, were identified in the meningococcal genome. Allele replacement was used to generate a meningococcal knock-out mutant of the regulator component of this system, and its phenotype was examined. The mutant displayed many differences in protein profiles compared with wild type, consistent with it being a gene-regulatory mutation. Many of the growth characteristics of the mutant were similar to those of phoP mutants of salmonella: it was unable to grow at low concentrations of magnesium and was sensitive to defensins and other environmental stresses. Magnesium-regulated differences in protein expression were abrogated in the mutant, indicating that the meningococcal PhoP/PhoQ system may, as in salmonella, respond to changes in environmental magnesium levels. These results are consistent with the PhoP homologue playing a similar role in the meningococcus to PhoP in salmonella and suggest that it may similarly be involved in the regulation of virulence genes in response to environmental stimuli in the meningococcus. In support of this conclusion, we found the mutant grew was unable to grow in mouse serum and was attenuated in its ability to traverse through a layer of human epithelial cells. Identification of those genes regulated by the meningococcal PhoP may provide a route towards the identification of virulence genes in the meningococcus.

Alleles↗

Genetic loci affecting resistance to human malaria parasites in a West African mosquito vector population.

Successful propagation of the malaria parasite Plasmodium falciparum within a susceptible mosquito vector is a prerequisite for the transmission of malaria. A field-based genetic analysis of the major human malaria vector, Anopheles gambiae, has revealed natural factors that reduce the transmission of P. falciparum. Differences in P. falciparum oocyst numbers between mosquito isofemale families fed on the same infected blood indicated a large genetic component affecting resistance to the parasite, and genome-wide scanning in pedigrees of wild mosquitoes detected segregating resistance alleles. The apparently high natural frequency of resistance alleles suggests that malaria parasites (or a similar pathogen) exert a significant selective pressure on vector populations.

Alleles↗

Analysis of proline reduction in the nosocomial pathogen Clostridium difficile.

Clostridium difficile, a proteolytic strict anaerobe, has emerged as a clinically significant nosocomial pathogen in recent years. Pathogenesis is due to the production of lethal toxins, A and B, members of the large clostridial cytotoxin family. Although it has been established that alterations in the amino acid content of the growth medium affect toxin production, the molecular mechanism for this observed effect is not yet known. Since there is a paucity of information on the amino acid fermentation pathways used by this pathogen, we investigated whether Stickland reactions might be at the heart of its bioenergetic pathways. Growth of C. difficile on Stickland pairs yielded large increases in cell density in a limiting basal medium, demonstrating that these reactions are tied to ATP production. Selenium supplementation was required for this increase in cell yield. Analysis of genome sequence data reveals genes encoding the protein components of two key selenoenzyme reductases, glycine reductase and d-proline reductase (PR). These selenoenzymes were expressed upon the addition of the corresponding Stickland acceptor (glycine, proline, or hydroxyproline). Purification of the selenoenzyme d-proline reductase revealed a mixed complex of PrdA and PrdB (SeCys-containing) proteins. PR utilized only d-proline but not l-hydroxyproline, even in the presence of an expressed and purified proline racemase. PR was found to be independent of divalent cations, and zinc was a potent inhibitor of PR. These results show that Stickland reactions are key to the growth of C. difficile and that the mechanism of PR may differ significantly from that of previously studied PR from nonpathogenic species.

Amino Acid Isomerases↗

Detection of alternative nitrogenases in aerobic gram-negative nitrogen-fixing bacteria.

Strains of aerobic, microaerobic, nonsymbiotic, and symbiotic dinitrogen-fixing bacteria were screened for the presence of alternative nitrogenase (N2ase) genes by DNA hybridization between genomic DNA and DNA encoding structural genes for components 1 of three different enzymes. A nifDK gene probe was used as a control to test for the presence of the commonly occurring Mo-Fe N2ase, a vnfDGK gene probe was used to show the presence of V-Fe N2ase, and an anfDGK probe was used to detect Fe N2ase. Hitherto, all three enzymes have been identified in Azotobacter vinelandii OP, and all but the Fe N2ase are present in Azotobacter chroococcum ATCC 4412 (MCD1). Mo-Fe N2ase and V-Fe N2ase structural genes only were confirmed in this strain and in two other strains of A. chroococcum (ATCC 480 and ATCC 9043). A similar pattern was observed with Azotobacter beijerinckii ATCC 19360 and Azotobacter nigricans ATCC 35009. Genes for all three systems are apparently present in two strains of Azotobacter paspali (ATCC 23367 and ATCC 23833) and also in Azomonas agilis ATCC 7494. There was no good evidence for the existence of any genes other than Mo-Fe N2ase structural genes in several Rhizobium meliloti strains, cowpea Rhizobium strain 32H1, or Bradyrhizobium japonicum. Nitrogenase and nitrogenase genes in Azorhizobium caulinodans behaved in an intermediate fashion, showing (i) the formation of ethane from acetylene under Mo starvation, a characteristic of alternative nitrogenases, and (ii) a surprising degree of cross-hybridization to the vnfDGK, but not the anfDGK, probe. vnfDGK- and anfDGK-like sequences were not detected in two saccharolytic Pseudomonas species or Azospirillum brasilense Sp7. The occurrence of alternative N2ases seems restricted to members of the family Azotobacteraceae among the aerobic and microaerobic diazotrophs tested, suggesting that an ability to cope with O2 when fixing N2 may be an important factor influencing the distribution of alternative nitrogenases.

Azotobacter↗

A two-component signal transduction system essential for growth of Bacillus subtilis: implications for anti-infective therapy.

A two-component signal transduction system encoded by the yycF and yycG genes is part of an operon containing three genes, yycH, yycI, and yycJ, with no known function and a gene, yycK, coding for an HtrA-like protease. This operon was transcribed during growth, and its transcription shut down as the cells approached stationary phase. This decreased transcription was not Spo0A dependent. The HtrA protease gene was separately controlled during sporulation from a sigmaG promoter. Studies using insertional inactivation plasmids revealed that neither yycF nor yycG could be inactivated, whereas the other genes were inactivated without loss of viability. A temperature-sensitive YycF response regulator mutant was isolated and shown to have an H215P mutation in a putative DNA-binding domain which is closely related to the OmpR family of response regulators. At the nonpermissive temperature, cultures of the mutant strain stopped growth within 30 min, and this was followed by a decrease in optical density. Microscopically, many of the cells appeared to retain their structure while being empty of their contents. The essential processes regulated by this two-component system remain unknown. A search of the genome databases revealed YycF, YycG, and YycJ homologues encoded by three linked genes in Streptococcus pyogenes. The high level of identity of these proteins (71% for YycF) suggests that this system may play a similar role in gram-positive pathogens.

Amino Acid Sequence↗

The twin-arginine translocation pathway of Mycobacterium smegmatis is functional and required for the export of mycobacterial beta-lactamases.

The twin-arginine translocation (Tat) pathway exports folded proteins across the bacterial cytoplasmic membrane and is responsible for the proper extracytoplasmic localization of proteins involved in a variety of cellular functions, including pathogenesis. The Mycobacterium tuberculosis and Mycobacterium smegmatis genomes contain open reading frames with homology to components of the Tat export system (TatABC) as well as potential Tat-exported proteins possessing N-terminal signal sequences with the characteristic twin-arginine motif. Due to the importance of exported virulence factors in the pathogenesis of M. tuberculosis and the limited understanding of mycobacterial protein export systems, we sought to determine the functional nature of the Tat export pathway in mycobacteria. Here we describe phenotypic analyses of DeltatatA and DeltatatC deletion mutants of M. smegmatis, which demonstrated that tatA and tatC encode components of a functional Tat system capable of exporting characteristic Tat substrates. Both mutants displayed a growth defect on agar medium and hypersensitivity to sodium dodecyl sulfate. The mutants were also defective in the export of active beta-lactamases of M. smegmatis (BlaS) and M. tuberculosis (BlaC), both of which possess twin-arginine signal sequences. The Tat-dependent nature of BlaC was further revealed by mutation of the twin-arginine motif. Finally, we demonstrated that replacement of the native signal sequence of BlaC with the predicted Tat signal sequences of M. tuberculosis phospholipase C proteins (PlcA and PlcB) resulted in the Tat-dependent export of an enzymatically active 'BlaC. Thus, 'BlaC can be used as a genetic reporter for Tat-dependent export in mycobacteria.

Anti-Bacterial Agents↗

Identification of a transactivating function mapping to the putative immediate-early locus of human herpesvirus 6.

Sequencing studies have indicated that the unique component of the human herpesvirus 6 (HHV-6) genome and the unique long segment of the human cytomegalovirus genome are genetically colinear. Of particular interest is the identification of a region of local CpG dinucleotide suppression in the genome of HHV-6, a feature conserved in the genomes of human cytomegalovirus, murine cytomegalovirus, and simian cytomegalovirus, and a characteristic of the major immediate-early loci of these viruses. Adjacent to this region in HHV-6 are approximately 30 copies of a 103- to 108-bp sequence element, which contains consensus binding sites for the transcription factors AP2 and NF kappa B, in addition to a single KpnI recognition site. Together, these KpnI repeat units may compose an immediate-early enhancer, analogous to those found in the cytomegaloviruses. We present the sequence of this region of HHV-6 and demonstrate that a transactivating function is encoded by this region. We have used polymerase chain reaction to synthesize fragments containing open reading frames and 5' sequences with or without the upstream KpnI repeat units. Effector plasmids containing these HHV-6 coding and 5' sequences were able to effect activation of heterologous promoter-chloramphenicol acetyltransferase (CAT) constructs, including adenovirus E3-CAT and E4-CAT, human T-cell lymphotropic virus type I long terminal repeat (LTR)-CAT, and human immunodeficiency virus LTR-CAT, in cotransfection experiments in Vero cells and peripheral blood lymphocytes. Furthermore, we have identified the major open reading frame (RF4; 2.3 kb) as being essential for activation, and we have shown that the NF kappa B, SP1, and TATA box motifs in the human immunodeficiency virus LTR are all required for full induction of the promoter by the HHV-6-encoded transactivator.

Amino Acid Sequence↗

Myosin light chain genes in the turkey (Meleagris gallopavo).

Myosin light chains associate with the motor protein myosin and are believed to play a role in the regulation of its actin-based ATPase activity. Myosin light chain cDNA clones from the turkey (Meleagris gallopavo) were isolated and sequenced. One sequence corresponded to an alternative transcript, the skeletal muscle essential light chain (MYL1 isoform 1) and a second to the smooth muscle isoform of myosin light chain (MYL6). The DNA and predicted amino acid sequences of both light chain genes were compared to that of the chicken. Based on the cDNA sequence, oligonucleotide primers were designed to amplify genomic DNA from six of the seven introns of the MYL1 gene. Approximately 5 kb of DNA was sequenced (introns and 3' UTR) and evaluated for the presence of single nucleotide polymorphisms (SNPs). SNPs were verified by sequencing common intron regions from multiple individuals and three polymorphisms were used to genotype pedigreed families. MYL1 is assigned to a turkey linkage group that corresponds to a region of chicken chromosome 7 (GGA7). The results of this study provide genomic reagents for comparative studies of avian muscle components and muscle biology.

3' Untranslated Regions↗

Salt in the wound: a possible role of Na+ gradient in chlamydial infection.

Genome analysis has revealed the presence of key components of the Na(+) chemiosmotic cycle, including the primary Na(+) pump (Na(+)-translocating NADH:ubiquinone oxidoreductase), in the cytoplasmic membrane of two ubiquitous human pathogens, Chlamydia trachomatis and Chlamydiophyla pneumoniae. This observation seemed paradoxical in the case of obligatory intracellular parasites because the Na(+) cycle is thought to be primarily a mechanism that enhances the adaptive potential in free-living bacteria that are often facing drastic changes in the salinity and pH of the environment. We present a model suggesting that operation of the Na(+) cycle may play an important role in the course of chlamydial infection, when the Na(+) and H(+) homeostasis of the host cell become severely impaired. This introduces the intriguing possibility of the application of drugs targeting Na(+)-transporting enzymes to chlamydial infections, which are notoriously difficult to treat.

Animals↗

Human leukemic intermediate DNA components.

The DNA extracted from human leukemic leucocytes has been fractionated on a methylated albumin kieselguhr (MAK) column. The different fractions obtained have been reannealed to a Cot value of 20 mol times sec/1 to study the distribution of the intermediate DNA on the MAK column. Intermediate DNA contains two components, one (CsCl density after reannealing, 1.703 g/ml) obtained by reannealing high molecular weight DNA, the other (CsCl density after reannealing, 1.707 g/ml) obtained only by reannealing sonicated low molecular weight DNA. High molecular weight intermediate DNA (1.703 component) is eluted early from the MAK column in the fractions corresponding to the main DNA peak, while low molecular weight intermediate DNA (1.707 component) is more widespread on the MAK column, but appears to be enriched in the fractions eluted later. The possibility is discussed that the latter component is interspersed in that part of the genome which is apparently more homogeneous in density in an analytical CsCl gradient, and is absent on the skewed, more heterogeneous, heavy side of the main DNA in CsCl.

Centrifugation, Density Gradient↗

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