Biomedical subjects

K Beemon

Publications and source records attributed to K Beemon.

At least 19 recordsLinked to original sources

PubMed1994

Rous sarcoma virus RNA stability requires an open reading frame in the gag gene and sequences downstream of the gag-pol junction.

The intracellular accumulation of the unspliced RNA of Rous sarcoma virus was decreased when translation was prematurely terminated by the introduction of nonsense codons within its 5' proximal gene, the gag gene. Subcellular fractionation of transfected cells suggested that nonsense codon-mediated instability occurred in the cytoplasm. Analysis of constructs containing an in-frame deletion in the nucleocapsid domain of gag, which prevents interaction between the Gag protein and viral RNA, showed that an open reading frame extending to approximately 30 nucleotides from the natural gag termination codon was needed for RNA stability. Sequences at the gag-pol junction necessary for ribosomal frameshifting were not required for RNA stability; however, sequences located 100 to 200 nucleotides downstream of the natural gag termination codon were found to be necessary for stable RNA. The stability of RNAs lacking this downstream sequence was not markedly affected by premature termination codons. We propose that this downstream RNA sequence may interact with ribosomes translating gag to stabilize the RNA.

Animals↗

PubMed1993

Mutation of an RSV intronic element abolishes both U11/U12 snRNP binding and negative regulation of splicing.

A cis-acting negative regulator of splicing (NRS) within the gag gene of RSV is involved in control of the relative levels of spliced and unspliced viral mRNAs. Insertion of the NRS into the intron of an adenovirus pre-mRNA resulted in inhibition of splicing in vitro before the first cleavage step. Analyses of spliceosome assembly with this substrate showed that it formed large RNP complexes that did not migrate like mature spliceosomes on native gels. Affinity selection of the RNP complexes formed on NRS-containing pre-mRNAs showed an association with U11 and U12 snRNPs, as well as with the spliceosomal snRNPs. Immunoprecipitation with antisera specific for U1 and U2 snRNPS showed binding of both snRNPs to NRS RNA. A 7-nucleotide missense mutation in the NRS that prevented binding of U11 and U12 snRNPs impaired NRS activity in vivo, suggesting a functional role for U11 and U12 snRNPs in the inhibition of splicing mediated by the RSV NRS RNA.

Adenoviridae↗

PubMed1993

Mutation of the C/EBP binding sites in the Rous sarcoma virus long terminal repeat and gag enhancers.

Several C/EBP binding sites within the Rous sarcoma virus (RSV) long terminal repeat (LTR) and gag enhancers were mutated, and the effect of these mutations on viral gene expression was assessed. Minimal site-specific mutations in each of three adjacent C/EBP binding sites in the LTR reduced steady-state viral RNA levels. Double mutation of the two 5' proximal LTR binding sites resulted in production of 30% of wild-type levels of virus. DNase I footprinting analysis of mutant DNAs indicated that the mutations blocked C/EBP binding at the affected sites. Additional C/EBP binding sites were identified upstream of the 3' LTR and within the 5' end of the LTRs. Point mutations in the RSV gag intragenic enhancer region, which blocked binding of C/EBP at two of three adjacent C/EBP sites, also reduced virus production significantly. Nuclear extracts prepared from both chicken embryo fibroblasts (CEFs) and chicken muscle contained proteins binding to the same RSV DNA sites as did C/EBP, and mutations that prevented C/EBP binding also blocked binding of these chicken proteins. It appears that CEFs and chicken muscle contain distinct proteins binding to these RSV DNA sites; the CEF binding protein was heat stable, as is C/EBP, while the chicken muscle protein was heat sensitive.

Animals↗

PubMed1992

Intronic sequences and 3' splice sites control Rous sarcoma virus RNA splicing.

cis-acting sequences of Rous sarcoma virus (RSV) RNA involved in control of the incomplete splicing that is part of the retroviral life cycle have been studied. The 5' and two alternative 3' splice sites, as well as negative regulator of splicing element in the intron, have been introduced into chimeric constructs, and their responsive roles in splicing inhibition have been evaluated by transient transfection experiments. Although the RSV 5' splice site was used efficiently in these assays, substrates containing either the RSV env or the RSV src 3' splice site were not spliced completely, resulting in 40 to 50% unspliced RNA. Addition of the negative regulator of splicing element to substrates containing RSV 3' splice sites resulted in greater inhibition of splicing (70 to 80% unspliced RNA), suggesting that the two elements function independently and additively. Deletion of sequences more than 70 nucleotides upstream of the src 3' splice site resulted in efficient splicing at this site, suggesting that inefficient usage is not inherent in this splice site but is instead due to to sequences upstream of it. Insertion of these upstream sequences into the intron of a heterologous pre-mRNA resulted in partial inhibition of its splicing. In addition, secondary structure interactions were predicted to occur between the src 3' splice site and the inhibitory sequences upstream of it. Thus, RSV splicing control involves both intronic sequences and 3' splice sites, with different mechanisms involved in the underutilization of the env and src splice acceptor sites.

Avian Sarcoma Viruses↗

PubMed1991

Characterization of Rous sarcoma virus intronic sequences that negatively regulate splicing.

Retroviruses splice only a fraction of their primary RNA transcripts to subgenomic mRNA. The unspliced RNA is transported to the cytoplasm, where it serves as genomic RNA as well as mRNA for the gag and pol genes. Deletion of sequences from the Rous sarcoma virus gag gene, which is part of the intron of the subgenomic mRNAs, was previously observed to result in an increase in the ratio of spliced to unspliced RNA. These sequences, which we termed a negative regulator of splicing (NRS), can be moved to the intron of a heterologous gene resulting in an accumulation of unspliced RNA in the nucleus. We have used such constructs, assayed by transient expression in chicken embryo fibroblasts, to define the minimal sequences necessary to inhibit splicing. Maximal NRS activity was observed with a 300-nt fragment containing RSV nts 707-1006; two noncontiguous domains within this fragment, one of which contains a polypyrimidine tract, were both found to be essential. The NRS element was active exclusively in the sense orientation in two heterologous introns tested and in both avian and mammalian cells. Position dependence was also observed, with highest activity when the NRS was inserted in the intron near the 5' splice site. The NRS element was also active at an exon position 136 nts upstream of the 5' splice site but not at sites further upstream. In addition, it did not affect the splicing of a downstream intron.

Animals↗

PubMed1991

Nonsense codons within the Rous sarcoma virus gag gene decrease the stability of unspliced viral RNA.

The intracellular accumulation of the unspliced RNA of Rous sarcoma virus was decreased when translation was prematurely terminated by the introduction of nonsense codons within its 5' proximal gene, the gag gene. In contrast, the levels of spliced viral RNAs were not affected in our transient expression assays in chicken cells. Experiments using the transcription inhibitor dactinomycin showed that mutant unspliced RNAs were degraded more rapidly than wild-type RNA. Furthermore, mutant RNAs could be partially stabilized by coexpression of wild-type gag proteins in trans; however, intact gag proteins were not required to maintain the stability of RNAs which did not contain premature termination codons. Thus, termination codons seemed to destabilize the RNA not because of their effect on gag protein function but instead because they disrupted the process of translating the gag region of the RNA. Analysis of double-mutant constructs containing both deletions and termination codons within the gag gene also suggested that the stability of the unspliced RNA was affected by a cis-acting interaction between the RNA and ribosomes.

Animals↗

PubMed1990

Sequence specificity of mRNA N6-adenosine methyltransferase.

The sequence specificity of chicken mRNA N6-adenosine methyltransferase has been investigated in vivo. Localization of six new N6-methyladenosine sites on Rous sarcoma virus (RSV) virion RNA has confirmed our extended consensus sequence for methylation: RGACU, where R is usually a G (7/12). We have also observed A (2/12) and U (3/12) at the -2 position (relative to m6A at +1) but never a C. At the +3 position, the U was observed 10/12 times; an A and a C were observed once each in weakly methylated sequences. The extent of methylation varied between the different sites up to a maximum of about 90%. To test the significance of this consensus sequence, it was altered by site-specific mutagenesis, and methylation was assayed after transfection of mutated RSV DNA into chicken embryo fibroblasts. We found that changing the G at -1 or the U at +3 to any other residue inhibited methylation. However, inhibition of methylation at all four of the major sites in the RSV src gene did not detectably alter the steady-state levels of the three viral RNA species or viral infectivity. Additional mutants that inactivated the src protein kinase activity produced less virus and exhibited relatively less src mRNA in infected cells.

Animals↗

PubMed1989

Avian retroviral long terminal repeats bind CCAAT/enhancer-binding protein.

DNA-protein interactions involving enhancer and promoter sequences within the U3 regions of several avian retroviral long terminal repeats (LTRs) were studied by DNase I footprinting. The rat CCAAT/enhancer-binding protein, C/EBP, bound to all four viral LTRs examined. The Rous sarcoma virus binding site corresponded closely to the 5' limit of the LTR enhancer; nucleotides -225 to -188 were protected as a pair of adjacent binding domains. The Fujinami sarcoma virus LTR bound C/EBP at a single site at nucleotides -213 to -195. C/EBP also bound to the promoter region of the enhancerless Rous-associated virus-0 LTR at nucleotides -77 to -57. The avian myeloblastosis virus LTR bound C/EBP at three sites: nucleotides -262 to -246, -154 to -134, and -55 to -39. We have previously observed binding of C/EBP to an enhancer in the gag gene of avian retroviruses. A heat-treated nuclear extract from chicken liver bound to all of the same retroviral sequences as did C/EBP. Alignment of the avian retroviral binding sequences with the published binding sites for C/EBP in two CCAAT boxes and in the simian virus 40, polyoma, and murine sarcoma virus enhancers suggested TTGNNGCTAATG as a consensus sequence for binding of C/EBP. When two bases of this consensus sequence were altered by site-specific mutagenesis of the Rous sarcoma virus LTR, binding of the heat-stable chicken protein was eliminated.

Animals↗

PubMed1988

Proposed gag-encoded transcriptional activator is not necessary for Rous sarcoma virus replication or transformation.

It has been reported that gene expression directed by the long terminal repeat of Rous sarcoma virus (RSV) is trans activated by a protein encoded in an alternate reading frame within the RSV gag gene (S. Broome and W. Gilbert, Cell 40:537-546, 1985). We have made specific mutations to test the role of the putative transcriptional activator in RSV replication. Termination codons were created within the alternate reading frame coding for the trans activator, and the mutations were introduced into an infectious RSV plasmid. We were unable to demonstrate specific trans activation of the RSV long terminal repeat by either wild-type or mutant RSV plasmids in transient cotransfection assays. Experiments using mutant or wild-type RSV-infected chick embryo fibroblasts indicated that the proposed RSV transcriptional activator was not required for viral replication or transformation and did not increase steady-state levels of viral RNA.

Animals↗

PubMed1988

Localization and footprinting of an enhancer within the avian sarcoma virus gag gene.

A cis-acting regulatory element within the gag gene of avian retroviruses has been localized by deletion analysis, and sites of protein interaction have been studied by DNase I footprinting. Unidirectional deletions were made from both the 5' and 3' ends of a 656-base-pair fragment of the gag gene of Fujinami sarcoma virus. These deletion mutants were tested for enhancer activity in a chloramphenicol acetyltransferase transient expression assay. A sharp 5' boundary for enhancer activity was observed between 776 and 786 nucleotides downstream from the transcription initiation site. In contrast, deletion from the 3' side resulted in a gradual loss of enhancer activity, reaching a near basal level of activity by nucleotide 868. Internal deletion of 76 nucleotides just downstream of the 5' boundary abolished enhancement. Mutagenesis of a consensus enhancer core sequence (GTGGTTTG) showed that this sequence was not necessary for enhancer activity in our transient assays. DNase I footprinting with both a highly purified enhancer-binding protein from rat liver (EBP20) and a partially purified chicken liver nuclear extract showed specific protection of nucleotides 813 to 872 within the localized enhancer region. Footprinting of unidirectional deletion mutants that had lost activity indicated that this binding was not sufficient to confer enhancement.

Animals↗

PubMed1988

Regulation of Rous sarcoma virus RNA splicing and stability.

Only a fraction of retroviral primary transcripts are spliced to subgenomic mRNAs; the unspliced transcripts are transported to the cytoplasm for packaging into virions and for translation of the gag and pol genes. We identified cis-acting sequences within the gag gene of Rous sarcoma virus (RSV) which negatively regulate splicing in vivo. Mutations were generated downstream of the splice donor (base 397) in the intron of a proviral clone of RSV. Deletion of bases 708 to 800 or 874 to 987 resulted in a large increase in the level of spliced RSV RNA relative to unspliced RSV RNA. This negative regulator of splicing (nrs) also inhibited splicing of a heterologous splice donor and acceptor pair when inserted into the intron. The nrs element did not affect the level of spliced RNA by increasing the rate of transport of the unspliced RNA to the cytoplasm but interfered more directly with splicing. To investigate the possible role of gag proteins in splicing, we studied constructs carrying frameshift mutations in the gag gene. While these mutations, which caused premature termination of gag translation, did not affect the level of spliced RSV RNA, they resulted in a large decrease in the accumulation of unspliced RNA in the cytoplasm.

Avian Sarcoma Viruses↗

PubMed1987

Inhibition of methylation at two internal N6-methyladenosine sites caused by GAC to GAU mutations.

We previously have mapped N6-methyladenosine (m6A) sites within the genomic RNA of Rous sarcoma virus (RSV). The results of that study and of experiments using inhibitors of methylation suggest that m6A might be involved in mRNA processing events. We describe an approach for directly analyzing the function of m6A in RNA and for studying the sequence specificity of the m6A methylase. Two sites of methylation in RSV (nucleotides 7414 and 7424) were altered by oligonucleotide-directed mutagenesis. The highly conserved GAC consensus sequence at those sites was changed to GAU. The new sequences were no longer methylated in the RSV genomic RNA; the GAC sequence was required for efficient base modification at those two adenosines. The altered m6A pattern did not affect viral RNA processing or the viral life cycle within infected cells.

Adenosine↗

PubMed1987

pp60c-src has less affinity for the detergent-insoluble cellular matrix than do pp60v-src and other viral protein-tyrosine kinases.

A difference in affinity for a Nonidet P-40-insoluble cellular matrix was observed between the products of the viral and cellular src genes. It has previously been demonstrated that pp60v-src is associated with a detergent-insoluble matrix containing the cellular cytoskeleton (J. G. Burr, G. Dreyfuss, S. Penman, and J. M. Buchanan, Proc. Natl. Acad. Sci. USA 77:3484-3488, 1980). We observed a similar association of the transforming proteins of Fujinami sarcoma virus (P130gag-fps) and Yamaguchi 73 avian sarcoma virus (P90gag-yes), both of which are tyrosine-specific protein kinases. However, we found that the endogenous c-src product, pp60c-src, was not tightly bound to the detergent-insoluble matrix. This does not appear to have been due to differences in the cytoskeleton between transformed and nontransformed cells since pp60c-src was also solubilized by nonionic detergent in cells transformed by Rous sarcoma virus. This difference in the affinities of the v-src and c-src products for cytoskeletal proteins may contribute to the inability of pp60c-src to transform cells.

Avian Sarcoma Viruses↗

PubMed1987

cis-acting regulatory elements within gag genes of avian retroviruses.

A cis-acting enhancer element has been detected within the gag gene of several avian retroviruses, including Rous sarcoma virus, Fujinami sarcoma virus, and the endogenous Rous-associated virus-0. A consensus enhancer core sequence, GTGGTTTG, is present in all of these viral genomes, approximately 900 bases downstream from the site of initiation of transcription. When an internal fragment derived from the gag gene of any of these viruses (spanning nucleotides 533 to approximately 1149) was inserted into a plasmid containing the chloramphenicol acetyltransferase (cat) gene under control of the simian virus 40 promoter, 9- or 21-fold enhancement of CAT expression was observed after transfection into mouse L cells and chicken embryo fibroblasts, respectively. This enhancement was not dependent on the position of insertion of the gag fragment into the plasmid. However, there was a strong dependence on orientation, with higher levels of CAT expression in constructs in which the 5' end of the gag fragment was nearest to the promoter, suggesting a possible negative regulatory element at the 3' end of this fragment. Deletion of the 3' end of the insert resulted in a gag fragment, containing nucleotides 533 to 1017, which enhanced expression equally in either orientation. When the gag fragment was inserted into a plasmid containing the cat gene under the control of an intact Rous sarcoma virus long terminal repeat, it induced a two- to threefold increase in CAT activity and CAT mRNA levels. Translation of the gag fragment did not appear to be necessary for the observed enhancement, since two insertional mutations resulting in frameshifts in the gag insert did not affect CAT expression. However, deletion of a 330-base internal fragment from the gag insert restored a basal level of CAT activity. These results suggest that retroviruses have regulatory elements within their genes distinct from those in the long terminal repeats that flank the genes.

Animals↗

PubMed1986

Avian sarcoma virus gag-fps and gag-yes transforming proteins are not myristylated or palmitylated.

The transforming proteins of several avian sarcoma viruses were examined for evidence of covalently attached fatty acids. While the product of the viral src gene could be readily labeled biosynthetically with [3H]myristic acid, the gag-onc transforming proteins of Fujinami sarcoma virus, PRCII, PRCIIp, and Y73 avian sarcoma viruses were not readily labeled with either [3H]myristate or [3H]palmitate. Thus, avian gag-onc proteins appear to lack modifications shared by mammalian gag and gag-onc proteins, and the products of the oncogenes src, tck, and ras.

Animals↗

PubMed1985

Precise localization of m6A in Rous sarcoma virus RNA reveals clustering of methylation sites: implications for RNA processing.

N6-methyladenosine (m6A) residues are present as internal base modifications in most higher eucaryotic mRNAs; however, the biological function of this modification is not known. We describe a method for localizing and quantitating m6A within a large RNA molecule, the genomic RNA of Rous sarcoma virus. Specific fragments of 32P-labeled Rous sarcoma virus RNA were isolated by hybridization with complementary DNA restriction fragments spanning nucleotides 6185 to 8050. RNA was digested with RNase and finger-printed, and individual oligonucleotides were analyzed for the presence of m6A by paper electrophoresis and thin-layer chromatography. With this technique, seven sites of methylation in this region of the Rous sarcoma virus genome were localized at nucleotides 6394, 6447, 6507, 6718, 7414, 7424, and 8014. Further, m6A was observed at two additional sites whose nucleotide assignments remain ambiguous. A clustering of two or more m6A residues was seen at three positions within the RNA analyzed. Modification at certain sites was found to be heterogeneous, in that different molecules of RNA appeared to be methylated differently. Previous studies have determined that methylation occurs only in the sequences Gm6AC and Am6AC. We observed a high frequency of methylation at PuGm6ACU sequences. The possible involvement of m6A in RNA splicing events is discussed.

Adenosine↗

PubMed1984

The avian sarcoma virus PRCII lacks 1020 nucleotides of the fps transforming gene.

Fujinami sarcoma virus (FSV) and PRCII avian sarcoma virus both encode gag-fps transforming proteins associated with tyrosine-specific protein kinase activity; however, PRCII has a lower oncogenic potential than does FSV. In this study, the genomes of PRCII and FSV have been compared. By hybridization of PRCII [32P]RNA to FSV DNA on Southern blots, a large internal deletion in the 5' half of the fps gene in PRCII has been mapped. To determine the exact size and location of the deletion in PRCII, dideoxy sequencing of PRCII RNA with FSV DNA fragments as primers was used. The FSV sequence corresponding to the deletion in PRCII was flanked by 6-base direct repeats ( AGCTGG ) at 1614-1619 and 2634-2639 nucleotides. One copy of the direct repeat was retained in the PRCII genome. The length of the deleted region was 1020 nucleotides. The deletion in fps did not alter the kinase domain or ATP-binding site of the P105 transforming protein of PRCII. It was shown that the specific kinase activity of P105 was as high as that of FSV P130 . The sequence deleted from PRCII was found to encode part of a large hydrophilic domain. In the accompanying paper [J. Woolford and K. Beemon (1984) Virology 135, 168-180], evidence that the PRCII and FSV proteins have different subcellular locations and solubility properties, possibly due to the loss of this domain, is presented. These alterations in the structure and location of the PRCII protein may prevent it from phosphorylating certain substrates involved in oncogenic transformation.

Animals↗

PubMed1984

Transforming proteins of fujinami and PRCII avian sarcoma viruses have different subcellular locations.

The subcellular locations of transforming proteins encoded by the related avian sarcoma viruses, PRCII and Fujinami sarcoma virus (FSV), were compared by cell fractionation and by indirect immunofluorescence. Whereas both viruses encode gag-fps proteins associated with tyrosine-specific kinase activity, FSV is more highly tumorigenic than PRCII in vivo. Cell fractionation studies showed that the PRCII transforming protein, P105, became associated with the high-speed particulate fraction shortly after synthesis. However, PRCII P105 did not fractionate with the plasma membrane marker, but rather with high-density membranes. It is unique in this subcellular localization among viral tyrosine kinases. This membrane association was found to be relatively insensitive to salt concentration and did not require divalent cations. Immunofluorescent studies, using anti-fps serum, showed that the PRCII protein was present in discrete, large, cytoplasmic patches, as well as in a juxtanuclear location. In contrast, FSV-encoded P130 was found to fractionate with the plasma membrane marker when cells were analyzed in low salt in the presence of magnesium. However, at higher salt concentrations and in the absence of magnesium, the bulk of P130 was found to be soluble. Immunofluorescent staining of FSV P130 revealed a diffuse, cytoplasmic pattern that was distinct from that of the PRCII product. The observed difference in the subcellular localization of these transforming proteins may be the cause of the difference in tumorigenicity between the two viruses.

Animals↗