Biomedical subjects

V M Vogt

Publications and source records attributed to V M Vogt.

At least 55 records · Page 3Linked to original sources

PubMed1992

Characterization of the self-splicing products of a mobile intron from the nuclear rDNA of Physarum polycephalum.

We have characterized the splicing products formed in vitro from RNA derived from the mobile group I intron in the nuclear rDNA of Physarum polycephalum, Pp LSU 3. This intron is a close relative of the well known Tetrahymena intron Tt LSU 1, being inserted at exactly the same position in the rDNA and sharing about 90% sequence identity with Tt LSU 1 in the conserved elements characteristic of the catalytic core of all group I introns. However, Pp LSU 3 differs from Tt LSU 1 in that it encodes a site-specific endonuclease, which mediates the homing of the intron to unoccupied target sites. The endonuclease, I-Ppo, would appear to be a unique example of a protein encoded by an RNA polymerase I transcript. To gain clues to the splicing products formed in vivo, and to the nature of the messenger RNA for I-Ppo, we subjected Pp LSU 3 RNA to standard self-splicing conditions in vitro, and then analyzed the products by size, by northern blotting, and by primer extension. The results show two novel features. First, in addition to the expected 5' splice site, there is an alternative 5' splice site in the upstream exon, just preceding the first codon of the I-Ppo open reading frame. Second, at the position corresponding to the major circularization site in Tt LSU 1 there is an internal processing site, leading to the efficient separation of two halves of the excised intron, the 5' half encoding I-Ppo and 3' half containing the ribozyme. Surprisingly, this cleavage appears not to be due to circularization followed by hydrolytic opening of the circle, but rather to G addition. The formation of these products in vitro suggests how the messenger RNA for the I-Ppo endonuclease may be generated in vivo.

Animals↗

PubMed1992

Proteolysis in the maturation of avian retroviruses does not require calcium.

After budding from the plasma membrane, retrovirus particles undergo a process of maturation, which includes changes in morphology caused by several proteolytic cleavages of the precursor of the internal structural proteins, products of the gag gene. Cleavage is mediated by the viral protease, PR. The fact that in most systems cleavage appears to occur only after assembly is complete, suggests that PR may become enzymatically active as a consequence of release of the virion from the cell. Using avian leukosis virus as a model system, we tested the hypothesis that leakage of calcium ions into newly budded virions plays a role in their maturation. We found that in both quail Qt35 cells and monkey COS-1 cells, maturation occurred normally in calcium-free medium and in the presence of EGTA. A calcium ionophore also did not affect maturation. We conclude that calcium influx does not act as a trigger for PR-mediated maturation.

Alpharetrovirus↗

PubMed1992

Processing of avian retroviral gag polyprotein precursors is blocked by a mutation at the NC-PR cleavage site.

The avian sarcoma and leukosis viruses (ASLV) encode a protease (PR) at the C terminus of gag which in vivo catalyzes the processing of both gag and gag-pol precursors. The studies reported here were undertaken to determine whether PR is able to cleave these polyproteins while it is still part of the gag precursor or whether the release of its N terminus to form free PR is necessary for full proteolytic activity. To address this question, we created a mutation that disrupts the PR cleavage site between the NC and PR coding regions of the gag gene. This mutation was introduced into a eukaryotic vector that expresses only the gag precursor and into an otherwise infectious clone of ASLV that carries the neo gene as a selectable marker. These constructs were expressed in monkey COS cells or in quail QT35 cells, respectively. Processing was impaired in both systems. Mutant particles were formed, but they contained no mature processed gag proteins. We observed only the uncleaved gag precursor polypeptide Pr76 in one case or Pr76 and a cleaved product of about 60 kDa in the other. Processing of the mutant gag precursor could be complemented in trans by from a wild-type construct, suggesting that the mutation did not induce gross structural alterations in its precursor. Our results suggest that the PR first must be released from its precursor before it can attack other sites in the gag and gag-pol polyproteins and that cleavage at the NC-PR boundary is a prerequisite for the initiation of the PR-directed processing.

Amino Acid Sequence↗

PubMed1991

trans-acting viral protease is necessary and sufficient for activation of avian leukosis virus reverse transcriptase.

The structural and enzymatic components of retroviral cores are formed by proteolytic cleavage of precursor polypeptides, mediated by the viral protease (PR). We described previously the construction of PR-defective avian leukosis viruses. These mutant viruses are noninfectious, and their major internal components are the uncleaved gag and gag-pol polyproteins (Pr76gag and Pr180gag-pol). The reverse transcriptase (RT) activity associated with the PR-defective virions is approximately 500-fold reduced relative to that of wild-type virions, suggesting that specific cleavages activate RT activity. To gain a better understanding of the role that PR plays in the processing and activation of RT, we performed complementation experiments wherein wild-type or PR mutant gag precursors were separately coexpressed with frame-corrected wild-type or PR mutant gag-pol precursors. The results demonstrate that, as in other retrovirus systems, gag-pol precursors can be assembled into virions only when they are rescued by a gag precursor. If the gag precursor is wild type, then the rescued Pr180gag-pol is completely and properly matured, irrespective of whether its embedded PR domain is wild type or mutant. In both cases, the virions produced are fully and equally infectious. This indicates that an active-site mutation in the PR domain of the gag-pol precursor has no effect on avian leukosis virus infectivity when particles are assembled from wild-type gag precursors. In contrast, if the gag precursor has an active-site mutation in PR or is deleted for PR, then the virions are noninfectious and the gag and gag-pol precursors remain unprocessed, even if the embedded PR domain of Pr180gag-pol is wild type. Thus, in this system, virion-associated Pr180gag-pol displays no detectable cis- or trans-acting PR activity. As assayed with an exogenous template, virions with processed gag-pol polyprotein display high levels of RT activity while those with unprocessed Pr180gag-pol display greatly reduced RT activity. These results demonstrate that during virion assembly, the PR supplied by a gag precursor is both necessary and sufficient for trans-activation of RT through proteolytic maturation of copackaged gag-pol polyprotein.

Amino Acid Sequence↗

PubMed1991

Characterization of a telomere-binding protein from Physarum polycephalum.

We have partially purified a nuclear protein (PPT) from Physarum polycephalum that binds to the extrachromosomal ribosomal DNA telomeres of this acellular slime mold. Binding is specific for the (T2AG3)n telomere repeats, as evidenced by nitrocellulose filter binding assays, by gel mobility shift assays with both DNA fragments and double-stranded oligonucleotides, and by DNase I footprinting. PPT is remarkably heat stable, showing undiminished binding activity after incubation at 90 degrees C. It sediments at 1.2S, corresponding to a molecular weight of about 10,000 (for a globular protein), and its binding activity is undiminished by incubation with RNase, suggesting that it is not a ribonucleoprotein. We hypothesize that PPT plays a structural role in telomeres, perhaps preventing nucleolytic degradation or promoting telomere extension by a telomere-specific terminal transferase.

Base Sequence↗

PubMed1990

Ubiquitin in avian leukosis virus particles.

We have identified unconjugated ubiquitin as a component of avian leukosis virus (ALV). Quantitation both by immunoblotting and by protein staining showed that ubiquitin makes up about 0.5% of total viral protein, corresponding to 100 molecules per virion. This level is about fivefold higher than the level of unconjugated ubiquitin in the cytosol, when expressed as a fraction of total protein. Other abundant low molecular weight proteins in the cytosol were not detected in virions, indicating that packaging occurs in a specific manner. A naturally occurring ALV mutant that lacks the env gene was found to package normal levels of ubiquitin, ruling out involvement of the viral glycoproteins as carriers of ubiquitin. We examined disrupted virus particles as well as lysates of infected cells for the presence of gag protein-ubiquitin conjugates. No conjugates could be detected, suggesting that ubiquitin does not enter virions linked to gag protein.

Animals↗

PubMed1990

Properties of avian retrovirus particles defective in viral protease.

The structural and enzymatic components of retroviral cores are formed by proteolytic cleavage of precursor polypeptides, mediated by the viral protease (PR). We constructed an active-site mutation, D37I, in the PR of avian leukosis virus. The D37I mutation was introduced into an infectious DNA clone, and quail cell lines expressing the mutant virus were established. These cell lines produce normal amounts of virus particles, the major internal protein components of which are the uncleaved gag and gag-pol precursors. As in other retroviral systems, the protease-defective virions are noninfectious and retain the "immature" type A morphology as determined by thin-section transmission electron microscopy. The virion cores are stable at nonionic detergent concentrations that completely disrupt wild-type cores. Digestion of mutant virions with exogenous PR in the presence of detergent leads to complete and correct cleavage of the gag precursor but incomplete cleavage of the gag-pol precursor. The protease-defective virions encapsidate normal amounts of genomic RNA and tRNA(Trp) that is properly annealed to the primer-binding site, but some of the genomic RNA remains monomeric. Results from UV cross-linking experiments show that the gag polyprotein of mutant virions interacts with viral RNA and that this interaction occurs through the nucleocapsid (NC) domain. However, within mutant virions the interaction of the NC domain with RNA differs from that of mature NC with RNA in wild-type virions. Reverse transcriptase (RT) activity associated with mutant virions is diminished but still detectable. Digestion of the virions with PR leads to a fivefold increase in activity, but this PR-mediated activation of RT is incomplete. Since in vitro cleavage of the gag-pol precursor is also incomplete, we hypothesize that amino acid sequences N terminal to the reverse transcriptase domain inhibit RT activity.

Animals↗

PubMed1990

RNA-binding properties of the matrix protein (p19gag) of avian sarcoma and leukemia viruses.

We have reinvestigated the ability of the matrix protein (MA) (p19gag) of avian sarcoma and leukemia viruses to interact with RNA. Previous reports claimed on the one hand that MA can bind tightly and with a high degree of specificity to avian sarcoma and leukemia virus RNA in vitro and on the other that it cannot bind to RNA at all. We found that MA purified by any of several methods does bind to RNA, as measured by its ability to cause retention of radioactive RNA on nitrocellulose membranes in a filtration assay. However, this interaction is weak and lacks specificity. The interaction of MA with RNA was barely detectable by classical sedimentation analysis, and from this observation we estimate that the intrinsic MA-RNA association constant is ca. 10(3) M-1, at least 3 orders of magnitude smaller than the constant describing the interaction of the viral nucleocapsid protein (NC) (p12gag) with RNA, ca. 10(6) M-1. Separately purified phosphorylated and nonphosphorylated MA species bound RNA equally. We also found that MA can bind to DNA with an affinity similar to that for RNA. The large quantitative discrepancy between our results and earlier published reports can be traced in part to methods of data analysis.

Avian Leukosis Virus↗

PubMed1990

Characterization of I-Ppo, an intron-encoded endonuclease that mediates homing of a group I intron in the ribosomal DNA of Physarum polycephalum.

A novel and only recently recognized class of enzymes is composed of the site-specific endonucleases encoded by some group I introns. We have characterized several aspects of I-Ppo, the endonuclease that mediates the mobility of intron 3 in the ribosomal DNA of Physarum polycephalum. This intron is unique among mobile group I introns in that it is located in nuclear DNA. We found that I-Ppo is encoded by an open reading frame in the 5' half of intron 3, upstream of the sequences required for self-splicing of group I introns. Either of two AUG initiation codons could start this reading frame, one near the beginning of the intron and the other in the upstream exon, leading to predicted polypeptides of 138 and 160 amino acid residues. The longer polypeptide was the major form translated in vitro in a reticulocyte extract. From nuclease assays of proteins synthesized in vitro with partially deleted DNAs, we conclude that both polypeptides possess endonuclease activity. We also have expressed I-Ppo in Escherichia coli, using a bacteriophage T7 RNA polymerase expression system. The longer polypeptide also was the predominant form made in this system. It showed enzymatic activity in bacteria in vivo, as demonstrated by the cleavage of a plasmid carrying the target site. Like several other intron-encoded endonucleases, I-Ppo makes a four-base staggered cut in its ribosomal DNA target sequence, very near the site where intron 3 becomes integrated in crosses of intron 3-containing and intron 3-lacking Physarum strains.

Amino Acid Sequence↗

PubMed1989

Mobile introns: definition of terms and recommended nomenclature.

A number of introns in mitochondrial, chloroplast, nuclear or prokaryotic genes have recently been shown to encode double-strand sequence-specific endonucleases. Such introns are mobile genetic elements that insert themselves at or near the cleaved sites. A uniform nomenclature to designate the molecular elements involved in the phenomenon of intron mobility is proposed.

Base Sequence↗

PubMed1989

A mobile group I intron in the nuclear rDNA of Physarum polycephalum.

We have shown that a strain-specific group I intron (intron 3) in the nuclear extrachromosomal rDNA or Physarum polycephalum is a mobile element. Shortly after mating of amoebae from intron-lacking and intron-containing strains, intron 3 transposes in a site-specific manner into all available recipient molecules. The transposition appears to occur by gene conversion, as evidence by the co-conversion of adjacent sequences and by double strand breakage observed in some of the recipient rDNA molecules. We infer that the double strand break is induced by an endonuclease encoded by intron 3, since in vitro transcription and translation of the cloned intron leads to the synthesis of an enzymatically active, site-specific nuclease. This is the first demonstration of the transposition of a nuclear intron in an experimental setting, and provides a rare example of a protein encoded by an RNA polymerase I transcript.

Base Sequence↗

PubMed1987

A simple method for immunoaffinity purification of nondenatured avian sarcoma and leukemia virus gag-containing proteins.

We have developed a one-step purification procedure for proteins containing the N-terminal portion of the gag protein of avian sarcoma and leukemia viruses. In this procedure, a resin with a covalently attached monoclonal antibody to the gag protein p19 is used to bind gag-containing proteins from crude extracts. After washing of the resin, the bound proteins are eluted with 2 M MgCl2. For the transforming protein kinase encoded by Fujinami sarcoma virus p130gag-fps, this procedure gave an enrichment of several thousand-fold, a yield of over 10%, a final purity of over 20%, and no significant loss of protein kinase activity. Similar purifications were obtained with three other gag-containing proteins. The immunoaffinity purification described may be of general utility as a first step in purification of the several other avian retroviral transforming proteins that are synthesized from fusions of an oncogene with the viral gag gene.

Avian Leukosis Virus↗

PubMed1987

Characterization of ribosomal RNA synthesis in a gene dosage mutant: the relationship of topoisomerase I and chromatin structure to transcriptional activity.

The genes encoding 18S, 5.8S, and 28S ribosomal RNA (rRNA) are tandemly repeated at the nucleolus organizer region (NOR). The NORs in the chicken map to one pair of microchromosomes. A line of chickens that contains individuals that are either disomic, trisomic, or tetrasomic for this chromosome, and have two, three, or four nucleoli and NORs, per cell, respectively, has been described previously. Aneuploid animals display a proportional increase in the rRNA gene copy number per cell. But, despite an increase in rDNA dosage, the levels of mature rRNA are regulated to normal levels in cells from aneuploid chickens (Muscarella, D.E., V.M. Vogt, and S.E. Bloom, 1985, J. Cell Biol., 101:1749-1756). This paper addresses the question of how regulation of mature rRNA synthesis occurs in cells with elevated levels of rDNA. An analysis of rRNA transcription in chicken embryo fibroblasts (CEFs) revealed that the relative rates of rRNA synthesis and processing and the amounts of precursor rRNA per cell are similar for all three genotypes. A comparison of chromatin structure, as determined by sensitivity of rDNA in nuclei from CEFs to digestion by DNase I, revealed that some of the rRNA genes from aneuploid cells are more resistant to digestion than corresponding sequences in the disomic cells. A determination of the distribution of topoisomerase I on rDNA has also been performed using the compound camptothecin, which introduces single- and double-strand breaks in topoisomerase-DNA complexes. Quantitation of camptothecin-induced cleavages revealed that a larger proportion of the rRNA genes in aneuploid cells was resistant to cleavage than in disomic cells, and therefore have no detectable amounts of topoisomerase I. These results suggest that the regulation of rRNA synthesis in CEFs with elevated levels of rDNA is achieved by the use of a subset of the rRNA genes.

Aneuploidy↗

PubMed1987

Epitope mapping of monoclonal antibodies to gag protein p19 of avian sarcoma and leukaemia viruses.

We have characterized a set of 15 monoclonal antibodies to p19gag, one of the internal proteins of avian sarcoma and leukaemia viruses. All the antibodies work in immune precipitations as well as in immunoblotting, though with different efficiencies. We have developed a simple epitope mapping technique, which uses partial chemical cleavages at methionine or tryptophan residues followed by immunoblotting from SDS-polyacrylamide gels, to localize the epitopes of nine of these antibodies. The epitopes fall into at least four classes. The mapping procedure should also be useful for other antigens of known primary structure.

Antibodies, Monoclonal↗

PubMed1986

Structure and processing of the p2 region of avian sarcoma and leukemia virus gag precursor polyproteins.

We have purified two low-molecular-weight polypeptides from the Prague C strain of Rous sarcoma virus and have identified these as products of the gag precursor Pr76 by protein sequencing and by amino acid analysis. Both polypeptides are derived from a stretch of 22 amino acids within Pr76 that separates p19 and p10. We refer to this region as p2. Together the two cleavage products form the entire p2 region. The junctions of p19 with the amino-terminal fragment of p2 and of p10 with the carboxy-terminal fragment of p2 define two new processing sites within the gag precursor, Tyr-155-His-156 and Gly-177-Ser-178. Both polypeptides are major cleavage products of Pr76 that occur in Prague C Rous sarcoma virus at an estimated 1,000 copies per virion. They also are prominent components of avian myeloblastosis virus. The combination of gel filtration and reverse-phase high-pressure liquid chromatography, which was used for the isolation of the two fragments of p2, resolved over a dozen other low-molecular-weight polypeptides from avian sarcoma and leukemia viruses that previously were undetected. This technique thus should serve as a useful procedure for further characterization of viral components.

Amino Acid Sequence↗

PubMed1985

The ribosomal RNA gene cluster in aneuploid chickens: evidence for increased gene dosage and regulation of gene expression.

In the chicken, the nucleolus organizer regions, or sites of the genes encoding 18S, 5.8S, and 28S ribosomal RNA (rRNA), map to one pair of microchromosomes that can be identified by silver nitrate cytochemistry. This nucleolar organizer chromosome also contains the major histocompatibility complex. Chickens aneuploid for this chromosome have been identified and reproduced for over seven generations. Crossing two trisomic parents results in the production of viable disomic, trisomic, and tetrasomic progeny, showing two, three, and four nucleoli and nucleolar organizers per cell, respectively. A molecular analysis of rRNA genes was undertaken to establish the gene copy numbers in the aneuploid genotypes, and to determine if elevated numbers of rRNA genes are stably maintained and inherited over multiple generations. Gene copy numbers were determined using hybridization analysis of erythrocyte DNA obtained from individuals comprising a family which segregated disomic, trisomic, and tetrasomic genotypes. The values obtained were 290, 420, and 570 rDNA repeats per cell for disomic, trisomic, and tetrasomic animals, respectively. These results provide molecular confirmation of the two aneuploid states and show that elevated gene copy numbers have been maintained over multiple generations. Fibroblasts derived from disomic and tetrasomic embryos were found to grow at similar rates in culture, and mature rRNA levels in chicken embryo fibroblasts from disomic, trisomic and tetrasomic embryos were also found to have similar levels of mature rRNA. Therefore, despite the increase in rDNA content, the level of rRNA is regulated to diploid amounts in aneuploid fibroblasts.

Aneuploidy↗

PubMed1985

Primary structure of p19 species of avian sarcoma and leukemia viruses.

The internal structural proteins of avian sarcoma and leukemia viruses are derived from a precursor polypeptide that is the product of the viral gag gene. The N-terminal domain of the precursor gives rise to p19, a protein that interacts with the lipid envelope of the virus and that may also interact with viral RNA. The C terminus of p19 from the Prague C strain of Rous sarcoma virus was previously assigned to a tyrosine residue 175 amino acids from the N terminus. We have used metabolic labeling and carboxypeptidase digestion to show that the C terminus of p19 is actually tyrosine 155. This implies the existence of a sixth gag protein 22 amino acids in length and located between p19 and p10 on the gag precursor. The p19 species of some recombinant avian sarcoma viruses and of the defective endogenous virus derived from the ev-1 locus migrate on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as if they were about 4,000 daltons smaller than p19. We have elucidated the structure of these forms, called p19 beta, by analysis of the proteins and determination of the DNA sequence of the p19 region of the gag gene from ev-1 and ev-2. Esterification of carboxyl groups completely suppressed the differences in migration of p19 and p19 beta. Peptide mapping showed the altered mobility to be determined by sequences in the C-terminal cyanogen bromide fragment of the proteins. We conclude from the DNA sequence that a single glutamate-lysine alteration is responsible for the altered electrophoretic mobility.

Amino Acid Sequence↗

PubMed1984

Fine-structure analyses of lipid-protein and protein-protein interactions of gag protein p19 of the avian sarcoma and leukemia viruses by cyanogen bromide mapping.

In avian sarcoma and leukemia viruses, the gag protein p19 functions structurally as a matrix protein, connecting internal components with the viral envelope. We have used a combination of in situ cross-linking and peptide mapping to localize within p19 the regions responsible for two major interactions in this complex, p19 with lipid and p19 with p19. Lipid-protein cross-links were localized near the amino terminus within the first 35 amino acids of the polypeptide. Homotypic protein-protein disulfide bridges were found to originate from near the carboxy terminus of p19, from cysteine residues at amino acids 111 and 153. These results suggest that p19 is divided into domains with distinct functions. The peptide maps constructed for p19, and for the related proteins p23 in avian sarcoma and leukemia viruses and p19 beta in recombinant avian sarcoma viruses, should serve as useful tools for other types of studies involving these proteins.

Amino Acids↗