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PubMed1992

Ty3 GAG3 and POL3 genes encode the components of intracellular particles.

Ty3 is a Saccharomyces cerevisiae retrotransposon that integrates near the transcription initiation sites of polymerase III-transcribed genes. It is distinct from the copialike Ty1 and Ty2 retrotransposons of S. cerevisiae in both the sequences of encoded proteins and gene order. It is a member of the gypsylike family of retrotransposons which resemble animal retroviruses. This study was undertaken to investigate the nucleocapsid particle of a transpositionally active gypsylike retrotransposon. Characterization of extracts from cells in which Ty3 expression was induced showed the presence of Ty3 nucleoprotein complexes, or viruslike particles, that migrated on linear sucrose gradients with a size of 156S. These particles are composed of Ty3 RNA, full-length, linear DNA, and proteins. In this study, antibodies raised against peptides predicted from the Ty3 sequence were used to identify Ty3-encoded proteins. These include the capsid (26 kDa), nucleocapsid (9 kDa), and reverse transcriptase (55 kDa) proteins. Ty3 integrase proteins of 61 and 58 kDa were identified previously (L. J. Hansen and S. B. Sandmeyer, J. Virol. 64:2599-2607, 1990). Reverse transcriptase activity associated with the particles was measured by using exogenous and endogenous primer-templates. Immunofluorescence studies of cells overexpressing Ty3 revealed cytoplasmic clusters of immunoreactive proteins. Transmission electron microscopy showed that Ty3 viruslike particles are about 50 nm in diameter. Thus, despite the unusual position specificity of Ty3 upstream of tRNA-coding regions, aspects of the Ty3 life cycle are fundamentally similar to those of retroviruses.

Amino Acid Sequence↗
PubMed1992

Temperature-sensitive mutations in the vaccinia virus H4 gene encoding a component of the virion RNA polymerase.

Four previously isolated temperature-sensitive (ts) mutants of vaccinia virus WR (ts1, ts31, ts55, and ts58) comprising a single complementation group (R. C. Condit, A. Motyczka, and G. Spizz, Virology 128:429-443, 1983) have been mapped by marker rescue to the H4L open reading frame located within the genomic HindIII-H DNA fragment. The H4 gene is predicted to encode a 93.6-kDa polypeptide expressed at late times during infection. Nucleotide sequence alterations responsible for thermolabile growth lead to amino acid substitutions in the H4 gene product. All four ts alleles display "normal" patterns of early and late viral protein synthesis at the nonpermissive temperature (40 degrees C). Mature virion particles, microscopically indistinguishable from wild-type virions, are produced in the cytoplasm of cells infected with ts1 at 40 degrees C. Western immunoblot analysis localizes the H4 protein to the virion core. After solubilization from cores, the H4 protein is associated during purification with transcriptionally active vaccinia virus DNA-dependent RNA polymerase.

Amino Acid Sequence↗
PubMed1992

The S. cerevisiae SEC65 gene encodes a component of yeast signal recognition particle with homology to human SRP19.

Translocation of proteins across the endoplasmic reticulum (ER) membrane represents the first step in the eukaryotic secretory pathway. In mammalian cells, the targeting of secretory and membrane protein precursors to the ER is mediated by signal recognition particle (SRP), a cytosolic ribonucleoprotein complex comprising a molecule of 7SL RNA and six polypeptide subunits (relative molecular masses 9, 14, 19, 54, 68 and 72K). In Saccharomyces cerevisiae, a homologue of the 54K subunit (SRP54) co-purifies with a small cytoplasmic RNA, scR1 (refs 4, 5). Genetic data indicate that SRP54 and scR1 are involved in translocation in vivo, suggesting the existence of an SRP-like activity in yeast. Whether this activity requires additional components similar to those found in mammalian SRP is not known. We have recently reported a genetic selection that led to the isolation of a yeast mutant, sec65-1, which is conditionally defective in the insertion of integral membrane proteins into the ER. Here we report the cloning and sequencing of the SEC65 gene, which encodes a 31.2K protein with significant sequence similarity to the 19K subunit of human SRP (SRP19). We also report the cloning of a multicopy suppressor of sec65-1, and its identification as the previously defined SRP54 gene, providing genetic evidence for an interaction between these gene products in vivo.

Amino Acid Sequence↗
PubMed1992

Functional replacement of genes for individual polyketide synthase components in Streptomyces coelicolor A3(2) by heterologous genes from a different polyketide pathway.

Streptomyces coelicolor A3(2) and Streptomyces violaceoruber Tü22 produce the antibiotics actinorhodin and granaticin, respectively. Both the aglycone of granaticin and the half-molecule of actinorhodin are derived from one acetyl coenzyme A starter unit and seven malonyl coenzyme A extender units via the polyketide pathway to produce benzoisochromane quinone moieties with identical structures (except for the stereochemistry at two chiral centers). In S. coelicolor and S. violaceoruber, the type II polyketide synthase (PKS) is encoded by clusters of five and six genes, respectively. We complemented a series of S. coelicolor mutants (act) defective in different components of the PKS (actI for carbon chain assembly, actIII for ketoreduction, and actVII for cyclization-dehydration) by the corresponding genes (gra) from S. violaceoruber introduced in trans on low-copy-number plasmids. This procedure showed that four of the act PKS components could be replaced by a heterologous gra protein to give a functional PKS. The analysis also served to identify which of three candidate open reading frames (ORFs) in the actI region had been altered in each of a set of 13 actI mutants. It also proved that actI-ORF2 (whose putative protein product shows overall similarity to the beta-ketoacyl synthase encoded by actI-ORF1 but whose function is unclear) is essential for PKS function. Mutations in each of the four complemented act genes (actI-ORF1, actI-ORF2, actIII, and actVII) were cloned and sequenced, revealing a nonsense or frameshift mutation in each mutant.

Anthraquinones↗
PubMed1992

The six conserved helicase motifs of the UL5 gene product, a component of the herpes simplex virus type 1 helicase-primase, are essential for its function.

The UL5 protein of herpes simplex virus type 1, one component of the viral helicase-primase complex, contains six sequence motifs found in all members of a superfamily of DNA and RNA helicases. Although this superfamily contains more than 20 members ranging from bacteria to mammalian cells and their viruses, the importance of these motifs has not been addressed experimentally for any one of them. In this study, we have examined the functional significance of these six motifs for the UL5 protein through the introduction of site-specific mutations resulting in single amino acid substitutions of the most highly conserved residues within each motif. A transient replication complementation assay was used to test the effect of each mutation on the function of the UL5 protein in viral DNA replication. In this assay, a mutant UL5 protein expressed from an expression clone is used to complement a replication-deficient null mutant with a mutation in the UL5 gene for the amplification of herpes simplex virus origin-containing plasmids. Eight mutations in conserved regions and three similar mutations in nonconserved regions of the UL5 gene were analyzed, and the results indicate that all six conserved motifs are essential to the function of UL5 protein in viral DNA replication; on the other hand, mutations in nonconserved regions are tolerated. These data provide the first direct evidence for the importance of these conserved regions in any member of the superfamily of DNA and RNA helicases. In addition, three motif mutations were introduced into the viral genome, and the phenotypic analyses of these mutants are consistent with results from the transient replication complementation assay. The ability of these three mutant UL5 proteins to form specific interactions with other members of the helicase-primase complex, UL8 and UL52, indicates that the functional domains required for replication activity of UL5 are separable from domains responsible for protein-protein interactions. It is anticipated that this type of structure-function analysis will lead to the identification of protein domains that contribute not only to the enzymatic activities of helicase or primase but also to protein-protein interactions within members of the complex.

Amino Acid Sequence↗
PubMed1992

Two mRNAs exist for the Hs PROS-30 gene encoding a component of human prosomes.

Screening of a lambda gt11 cDNA expression library of the HeLa cell genome with a monoclonal antibody that specifically recognizes prosomal 30-33-kDa proteins, allowed isolation of a 1264-nucleotide (nt) recombinant cDNA containing a 327-nt untranslated 5'-end. The amino acid (aa) sequence deduced from this cDNA revealed a protein of 269 aa (M(r) of 30,227) that includes a consensus box characteristic for Tyr phosphorylation, also observed in other prosomal proteins. Comparison with another prosomal 27-kDa protein, cloned in our laboratory, indicated the presence of three prosome-specific homology boxes observed in these proteins from archaebacteria to man. Interestingly, except for the untranslated 5'-end, as well as the sequence coding for the N-terminal six aa, this cDNA is identical to two recently published cDNAs encoding subunit C2 of human liver proteasome [Tamura et al., Biochim. Biophys. Acta 1089 (1991) 95-102] and subunit NU of human erythrocyte macropain [DeMartino et al., Biochim. Biophys. Acta 1079 (1991) 29-38]. Primer extension and Northern blot analysis using two specific 18-mer oligodeoxyribonucleotides indicated the presence of two mRNAs that have divergent 5'-ends. These results, as confirmed by the polymerase chain reaction, establish the existence of two distinct Hs PROS-30 mRNAs, differing in their 5'-noncoding regions and in the N-terminal six aa of their protein products.

Amino Acid Sequence↗
PubMed1992

Site-directed mutations in the potyvirus HC-Pro gene affect helper component activity, virus accumulation, and symptom expression in infected tobacco plants.

Helper component (HC-Pro) is a virus-encoded nonstructural protein required for aphid transmission of potyviruses. In the tobacco vein mottling virus (TVMV) polyprotein, HC-Pro represents a 457 residue polypeptide from amino acid position 257 to 713. Previous sequence comparison studies have suggested that mutations of one or two specific amino acid residues in the HC-Pro protein might result in loss of aphid transmission activity. To test this hypothesis, the initial targets were the residues corresponding to these specific amino acids, a lys to glu change and an ile to val change at amino acid positions 307 and 482, respectively, of the TVMV polyprotein, as well as the combination of the two. Two additional mutations within the HC-Pro representing dipeptide changes thr-ser to ile-asp and thr-ala to leu-glu at amino acid positions (283/284) and (368/369), respectively, were also tested to further define the effects of mutations in this region on helper component activity. The mutations at positions 482 and (368/369) had no effect on aphid transmission activity, while mutation at position 307 completely abolished the activity. Except for the 482 mutation, all the mutations also affected symptomatology and virus accumulation in infected plants. Due to the very low concentrations of HC-Pro in plants infected with the (283/284) mutant, the effect of this dipeptide change on aphid transmission activity could not be assessed. The majority of the tested mutations fall within a putative zinc-finger motif postulated in the cysteine-rich N-terminus of HC-Pro. The possible role of this motif in the potyviruses is further discussed in the light of our present results with TVMV.

Animals↗
PubMed1975

Gene activities for ribosomal components in Escherichia coli B/r.

The relative transcriptional activities of genes coding for ribosomal RNA (rRNA) and ribosomal proteins (r-proteins) at a steady-state growth rates ranging from 0.65 to 2.1 doublings/h can be estimated from previous measurements of the synthesis rates of stable and unstable RNA (Pato & von Meyenburg, 1970; Nierlich, 1972a,b; Bremer et al., 1973; Dennis & Bremer, 1973b, 1974b) and ribosomal proteins (Schleif, 1967; Dennis & Bremer, 1974a). Comparison of these transcriptional activities suggests that the expression of the r-protein genes and rRNA genes is controlled seperately.

Amino Acids↗
PubMed1979

Molecular genetics of herpes simplex virus: the terminal a sequences of the L and S components are obligatorily identical and constitute a part of a structural gene mapping predominantly in the S component.

In herpes simplex virus 1 (HSV-1) DNA, a small sequence, designated the a sequence, flanks the reiterated sequences at the ends of both the L and S components. The a sequence is the only sequence shared by the termini of all isomeric arrangements of HSV-1 DNA that arise from inversions of the covalently linked L and S components. We report that the a sequence, although present in both components, is a part of a structural gene mapping predominantly in the reiterated sequences of the S component. This conclusion is based on the observations that the mutant HSV-1(13)tsC75 is rescued by transfection of cells with the mutant DNA and any one of the four terminal or four L-S junction fragments of wild-type DNA. Furthermore, in doubly infected cells, this mutant shows little or no recombination or complementation with other ts mutants previously mapped within the reiterated sequences of the S component. Because it is otherwise difficult to explain the isolation of a mutant with several independent, equivalent mutations, the data argue for a mechanism that maintains the identity of the multiple copies of the a sequence.The paradox arising from the two observations that all termini rescue the ts mutant but that in coinfection tests the ts lesion is closely linked to the reiterated sequences of the S component could be accounted for by postulating that either recombination occurs while the DNA is in a circular form-in which case all a sequences would be adjacent to the reiterated sequence of the S component-or recombination can occur while the DNA is in a linear form. In this case the only effective substitution of the a sequence that is perpetuated is the one occurring at the L-S junction or in the S component. In light of the observations that tsC75 and the other mutants tested in this study map in the reiterated sequences and fail to yield appreciable recombinational frequencies, it is unlikely that isomerization of the DNA occurs by intramolecular recombination between reiterated sequences.

Base Sequence↗
PubMed1976

Effect of gene dispersion on estimates of components of generation means and variances.

The components of generation means that measure gene action and interaction at homozygous loci have expectations that depend on the degree of association or dispersion of alleles of like effect at different loci. With anything less than complete association, estimates of these components do not necessarily reflect the relative directions, magnitudes or kinds of gene action or interaction present. To illustrate these expected consequences, the F1, F2 and backcross generation of two contrasting crosses between pairs of inbred lines of Nicotiana rustica have been raised in which the same alleles are segregating at the same loci but for loci contributing to variation in final height the alleles of like effect are predominantly associated in one cross and predominantly dispersed in the other. The simultaneous analysis of the data from the two crosses show all the expected effects of the differences in the degree of association or dispersion. In the dispersion cross the effects of gene action at homozygous loci are underestimated and the interaction between homozygous loci is not detected at all. The large directional dominance component that is common to both crosses is in fact no greater than the additive component once the deflating effect of dispersion is removed, thus ruling out the presence of overdominance. No effects of association or dispersion on the components of variation could be detected nor would any be expected unless there were differences in the predominant linkage phase between the two crosses. Large and predictable effects on estimates of the number of effective factors are, however, demonstrable.

Crosses, Genetic↗
PubMed1978

Regulation and characterization of protein products coded by the nif (nitrogen fixation) genes of Klebsiella pneumoniae.

Two hundred and thirty-five Nif- strains of Klebsiella pneumoniae were characterized by two-dimensional polyacrylamide gel electrophoresis. Forty-two of these strains were tested further by in vitro acetylene reduction assays. By these techniques, nine nif-coded polypeptides were identified, and eight of these were assigned to specific nif genes. Nitrogenase component I required nifK and nifD, which coded for the beta and alpha subunits, and nifB, -E, and -N were required for the iron-molybdenum cofactor, which is a part of the active site of nitrogenase. nifH coded for the structural protein of component II, and nifM and nifS products seemed to be necessary for the synthesis of an active component II. There were two genes, nifF and nifJ, that were required for N2 fixation in vivo but not for N2 fixation in vitro. There were at least two cases (nifE and nifN, nifK and nifD) of two proteins that seemed to require each other for stability in vivo. Regulation of N2 fixation is apparently complex, and this is reflected by the assignment of regulatory functions to the gene products of nifA, nifL, nifK, nifD, nifH, and NIFJ.

Bacterial Proteins↗
PubMed1979

Mapping of the structural gene for the second component of complement with respect to the human major histocompatibility complex.

Families have been HLA typed, and allotypes of the second component of complement and properdin factor B determined. The lod score for the C2 structural gene and HLA-B from the study of 11 families and 55 informative meioses was 14.39 at maximum likelihood estimate of the recombination fraction of .02. This is related to other estimates of the distance between these two genes. The relative kinetic activities of the C2 allotypes were studied and no differences were demonstrated. No crossovers between Bf and C2 were observed.

Adult↗
PubMed2026

Phage bioinformatics tools: a review of computational approaches for bacteriophage research.

Rising clinical interest in phage therapy and the exponential growth of metagenomic sequence catalogues have driven a rapid expansion of bacteriophage bioinformatics. More than 80 dedicated tools, mostly published since 2020, now span identification, assembly, annotation, taxonomy, lifestyle prediction, defence-system detection, and host prediction. Aimed at experienced practitioners and developers, this review synthesizes the field through the lens of three successive computational paradigms: sequence homology, bounded by database completeness; machine learning, constrained by labelled training data; and foundation models, which now achieve Matthews correlation coefficients above 0.95 in identification tasks and, through structure-informed prediction, raise functional annotation to over half of phage genes. Furthermore, we map the upstream components, namely, gene callers, homology engines, protein language models, and structural search tools, that underpin most downstream pipelines, exposing shared infrastructure and ecosystem-level fragility when dependencies change. To translate this into practice, we propose web-based and command-line reference workflows calibrated to user expertise and sample types. Finally, we set an agenda for the next wave of tool development. Roughly half of phage genes still resist functional annotation despite structural methods; no broadly generalizable strain-level host predictor exists for phage therapy; varying true-positive rates (0%-97%) underscore the absence of standardized community benchmarks analogous to Critical Assessment of Structure Prediction or Critical Assessment of Metagenome Interpretation. As generative genome models begin designing synthetic phages, progress will depend less on producing standalone tools than on rigorous evaluation, interoperable infrastructure, and clinically meaningful prediction targets.

Computational Biology↗
PubMed2026

Are components of the histone gene expression machinery functionally repurposed in terminally differentiated cells?

The expression of metazoan replication-dependent histone genes is controlled by the nuclear protein at the ataxia-telangiectasia locus (NPAT) and U7 small nuclear ribonucleoprotein particle (snRNP). NPAT activates transcription of histone genes during S-phase, whereas U7 snRNP is a multi-subunit endonuclease that cleaves the resultant transcripts at the 3' end, yielding mature histone mRNAs. In cycling cells, NPAT and U7 snRNP with its four unique components, U7 snRNA, Lsm10, Lsm11 and FLASH, are highly enriched in histone locus bodies (HLBs), the nuclear condensates formed near histone gene loci. Here, we show that in muscle and neural cells that have ceased to replicate their chromatin and permanently exited the cell cycle, HLBs are dismantled and NPAT, FLASH and Lsm11 are detected in the cytoplasm. This observation suggests that in postmitotic cells, NPAT and U7 snRNP become repurposed for functions unrelated to generating histone mRNAs. We identified a highly conserved region in Lsm11 that engages in various protein-protein interactions and likely acts as a universal platform that controls the assembly, localization and function of Lsm11 complexes, including U7 snRNP, during cell growth and differentiation. Since the assembly of U7 snRNP requires survival motor neuron, the protein mutated in spinal muscular atrophy, our results may provide a new perspective on the pathophysiology of this neuromuscular disorder.

Histones↗
PubMed1977

Multiple interactions of a DNA-binding protein in vivo. III. Phage T4 gene-32 mutations differentially affect insertion-type recombination and membrane properties.

We have investigated in in vivo roles of T4 gene-32 protein in recombination. We have studied the effects of gene-32 mutations under conditions that allow normal DNA replication and are permissive for progeny production. Under these conditions, certain gene-32 mutations specifically reduce insertion-type (short-interval) recombination but none affect crossover-type (long-interval) recombination (see Figure 5). Heterozygote frequencies in all gene-32 mutants are similar to or higher than in a gene-32+ background and are not correlated with recombination deficiencies. "Recombination-deficient" alleles are dominant or codominant over the "recombination-proficient" gene 32 mutation tsL171. This explains apparent discrepancies between a gene-32 map deduced from two-factor crosses and the map derived from three-factor crosses. We have also found that the "recombination proficient" mutation tsL171 and it homdoalleles suppress the characteristic plaque morphology of rII mutants. Under restrictive conditions, tsL171 is partially suppressed by rII mutations, which allow the use of host ligase in recombination. Our present and previous results are discussed in terms of current recombination models. We conclude that gene-32 protein functions in recombination by forming a complex with DNA, with recombination enzymes and with membrane components. Since gene-32 protein interacts with many components of this recombination complex, gene-32 mutations may differentially affect various recombination steps.

Alleles↗
PubMed1979

Organization of genes for transcription and translation in the rif region of the Escherichia coli chromosome.

The lambdarifd18 transducing phage is known to carry several genes for components of transcriptional and translational machineries; these genes are clustered in the rif region at 88 min on the Escherichia coli genetic map. They include a set of genes for rRNA's (rrnB), a gene for spacer tRNA, tRNA2Glu (tgtB), one of the two genes for EF-Tu (tufB), genes for four ribosomal proteins (rplK, A, J, and L), genes for the beta and beta' subunits of RNA polymerase (rpoB and rpoC), and genes for three tRNA's (tyrU, gluT, and thrT). An additional tRNA gene (subsequently identified as thrU by Landy and his co-workers) and a gene for a protein (protein U) with unknown functions were found to be carried by lambdarif d18. We analyzed the organization of these genes by using various deletion and hybrid phages derived from lambdarif d18 and lambdarif d12, a phage related to lambdarif d18. The expression of various genes was examined in UV-irradiated cells infected with these transducing phages. Two main conclusions were obtained. First, the four tRNA genes are not cotranscribed with the genes in rrnB, even though these tRNA genes are located close to the distal end of rrnB. Second, the four ribosomal protein genes are organized into two separate transcriptional units; rplK and A are in one unit and rplJ and L are in the second unit. The first group of genes was shown to have a promoter separate from that for tufB or protein U. The second group of genes shares the promoter with rpoB and C, as described in a separate paper (M. Yamamoto and M. Nomura, Proc. Natl. Acad. Sci. U.S.A., 75:3891--3895). These and other results described in this paper show that the genes are organized in the following order: promoter, genes in rrnB; promoter, thrU, tyrU, (promoter?) glyT, thrT; (promoter?) tufB; promoter, a gene for protein U; promoter, rplK, rplA; promoter, rplJ, rplL, rpoB, rpoC.

Bacterial Proteins↗
PubMed1992

Studies on processing, particle formation, and immunogenicity of the HIV-1 gag gene product: a possible component of a HIV vaccine.

Antigens in a particulate conformation were shown to be highly immunogenic in mammals. For this reason, the particle forming capacity of derivatives of the HIV-1 group specific core antigen p55 gag was assayed and compared dependent on various expression systems: recombinant bacteria, vaccinia- and baculoviruses were established encoding the entire core protein p55 either in its authentic sequence or lacking the myristylation consensus signal. Moreover, p55 gag was expressed in combination with the protease (p55-PR) or with the entire polymerase (p55-pol), respectively. Budding of 100-160 nm p55 core particles, resembling immature HIV-virions, was observed in the eucaryotic expression systems only. In comparison to the vaccinia virus driven expression of p55 in mammalian cells, considerably higher yields of particulate core antigen were obtained by infection of Spodoptera frugiperda (Sf9) insect cells with the recombinant Autographa californica nuclear polyhedrosis (AcMNPV) baculovirus. Mutation of the NH2-terminal myristylation signal sequence prevented budding of the immature core particles. Expression of the HIV p55-PR gene construct by recombinant baculovirus resulted in complete processing of the p55 gag precursor molecule in this system. The introduction of an artificial frameshift near the natural frameshift site resulted in constitutive expression of the viral protease and complete processing of p55, both in Escherichia coli and in vaccinia virus infected cells. Interestingly, significant processing of p55 resembling that of HIV infected H9 cells could also be achieved in the vaccinia system by fusing the entire pol gene to the gag gene. Moreover, processing was not found to be dependent on amino-terminal myristylation of the gag procursor molecule, which is in contrast to observations with type C and type D retrovirus. However, complete processing of p55 into p24, p17, p9 and p6 abolished particle formation. Purified immature HIV-virus like particles were highly immunogenic in rabbits, leading to a strong humoral immune response after immunization. Empty immature p55 gag particles represent a noninfectious and attractive candidate for a basic vaccine component.

AIDS Vaccines↗