Biomedical subjects
Publications and source records attributed to R Dhar.
The oncogene of the human EJ bladder carcinoma cell lines arose via alteration of a cellular proto-oncogene. Experiments are presented that localize the genetic lesion that led to activation of the oncogene. The lesion has no affect on levels of expression of the oncogene. Instead, it affects the structure of the oncogene-encoded protein.
The transforming protein coded for by the onc gene (v-rasHa) of Harvey murine sarcoma virus (Ha-MuSV) is the 21,000-dalton protein (p21) which is the immediate agent responsible for the virus-induced malignant transformation of normal cells. The p21 proteins of Ha-MuSV and the closely related Kirsten murine sarcoma virus are heavily phosphorylated in vivo. In the partially purified Ha-MuSV p21, the protein shows a guanine nucleotide-binding activity and, in addition, a very unique autophosphorylating activity at a threonine residue using as phosphoryl donor GTP but not ATP. In the present study, we compared the tryptic peptide maps of the Ha-MuSV p21 phosphorylated in vivo and in vitro. The results show that the major phosphorylation site is identical. Since the GTP-specific phosphorylation is very unique and distinct from all other known protein kinases, the present observation suggests that the in vitro enzymatic activity is responsible for the p21 phosphorylation in vivo. We have analyzed the amino acid sequence surrounding the major phosphorylation site of the Ha-MuSV p21 by automated Edman degradations of the tryptic phosphopeptides. Threonine residue 59 from the initiator methionine residue 1 of the p21 protein is the phosphorylated amino acid residue, and the surrounding amino acid sequence is NH2...-Thr-Cys-Leu-Leu-Asp-Ile-Leu-Asp-Thr-Thr(P)-Gly-Gln-Glu-Glu-Tyr-...COOH. The p21 proteins of both the Ha-MuSV and the closely related Kirsten murine sarcoma virus share the same phosphopeptide. The amino acid sequence of the phosphorylation site is distinct from all other known protein kinases.
Harvey murine sarcoma virus is a retrovirus which transforms cells by means of a single virally encoded protein called p21 has. We have determined the nucleotide sequence of 1.0 kilobase in the 5' half of the viral genome which encompasses the has coding sequences and its associated regulatory signals. The nucleotide sequence has identified the amino acid sequence of two additional overlapping polypeptides which share their reading frames and the carboxyl termini with p21 but which contain additional NH2-terminal amino acids.
The p21 transforming protein coded for by the v-ras gene of Harvey murine sarcoma virus (Ha-MuSV) migrates as a doublet band between 21,000 and 23,000 daltons during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lower band of the doublet is designated p21, and the upper band is designated pp21 since it comigrates with the phosphorylated form of p21. By pulse-labeling with [35S] methionine, we detected a p21 precursor, pro-p21, which migrated as if it was approximately 1,000 daltons larger than p21. The precursor-product relationship was established by pulse-chase experiments with [25S] methionine in the presence of 100 micrograms of cycloheximide per ml, which inhibited all de novo protein biosynthesis. Within 4 h, pro-p21 was completely chased into p21, and during the next 24 h pp21 accumulated. Thus, formation of pp21 from p21 did not require de novo protein synthesis. By subcellular fractionation into cytosol amd membrane fractions, we found that pro-p21 was synthesized in a non-membrane-bound state and that shortly after its complete synthesis, the p21 product was associated with the membrane fraction. By selective cleavage of p21 at a unique aspartic acid-proline residue with 70% formic acid or with Staphylococcus aureus V8 protease, we found that the intramolecular site of pro-p21 processing was located in the C-terminal portion of the pro-p21 molecule. The possibilities that the precursor was involved in the assembly of p21 into the plasma membrane and, alternatively, that the processing was a step in the activation of p21 biochemical activities are discussed.
On the late side of the simian virus 40 (SV40) DNA replication origin are several sets of tandem repeated sequences, the largest of which is 72 base pairs long. The role of these sequences was examined through construction of deletion mutants of SV40. A mutant from which one of the 72-base-pair repeated units was removed is viable upon transfection of monkey kidney cells with viral DNA. Extension of this deletion into the second repeated unit, however, leads to nonviability, as recognized by the absence of early transcription and of tumor antigen production. These observations indicate that the 72-base-pair repeated sequences form an essential element in the early viral transcriptional promoter and explain the inability of such a deleted genome to complement an early temperature-sensitive mutant of SV40, tsA, as well as the failure to replicate its DNA. In a parallel experiment it was found that the extended deletion mutant was also unable to complement a late temperature-sensitive mutant of SV40, tsB. This suggests that the extended mutant is also defective in DNA replication or late transcription (or both).
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In the present study we tried to determine whether the presence of DNA sequences homologous to the Escherichia coli tuf gene (encodes peptide chain elongation factor Tu) in many taxonomically-unrelated prokaryotes is due to selective pressure for these sequences or due to the transfer of chromosomal material subsequent to the divergence of the genera from their progenitors. We found that the degree of sequence homology to the DNA immediately adjacent to the E. coli tuf A gene is either nonexistent or much less than that found for the tuf gene. Furthermore, the tuf-homologous sequences present in one prokaryote were found to be in large part the same as or a subset of those present in others. That is, various prokaryotes share a common subset of tuf-homologous sequences. These findings suggest that strong selective pressure and not recent intergeneric chromosomal transfer is responsible for the ubiquitous presence of certain tuf-homologous sequences. Because the genetic code is degenerate, DNA sequence need not be conserved to conserve protein sequence. Therefore, if the only function of these sequences is to encode protein, their persistence must mean that in some instances codon sequence is selected for.
We have adapted a rapid sequencing technique from the enzymatic nick-translation method of Maat and Smith. The Forward-Backward procedure employs both synthetic and 3' to 5' exonucleolytic activities of E. coli DNA polymerase I to achieve greater reliability, especially in reading stretches of the same nucleotide. The technique has been employed to determine sequence alterations in four early SV40 temperature-sensitive (tsA) point mutants and five early SV40 viable deletion mutants. The nucleotide sequence of these mutants provides an insight into their biological properties.
We obtained influenza viral DNA clones containing sequences derived from cytoplasmic viral mRNA and genomic viral RNA. Sequence analysis of terminal nucleotides of five independentlyisolated viral DNA segments showed that additional oligonucleotides were covalently linked to the 5' terminus of viral mRNA transcripts. The sequences of these additional nucleotides varied among DNA clones of the same gene and of different genes as well. These inserts also varied in length, ranging from 6 to 14 nucleotides. The heterogeneity of these sequences suggests that they were derived originally from cellular RNA molecules. These findings provide evidence that cellular RNA sequences are used to prime influenza viral mRNA transcription in infected cells. In addition, the sequences at both termini of vRNA were fully represented in clone pFV 88, indicating that the cloned DNA contained the complete viral gene coding for the hemagglutinin.
Analysis of total cytoplasmic and polyadenylylated cytoplasmic RNA from cells lytically infected with simian virus 40 (SV40) has demonstrated the presence of a small RNA, approximately 65 nucleotides long, that is induced late in lytic infection. This small RNA is apparently specific in size and sequence and is not selected on columns of oligo(dT)-cellulose. It is homologous to a region of the early SV40 mRNAs (and to the late DNA strand), starting approximately 250 nucleotides from the 3' end of the early mRNAs (SV40 map position 0.21). The function of this RNA in the viral cycle and its source are unknown at this time; however, its temporal expression, unique sequence, and interesting region of homology within the SV40 genome suggest a possible role in the control of SV40 gene expression.
Integrated Moloney murine sarcoma provirus (MSV) has direct terminal repeat sequences (TRS). We determined the nucleotide sequence of both 588-base-pair TRS elements and the adjacent host and viral junctions of an integrated MSV cloned in bacteriophage lambda. Sequences were identified corresponding to the tRNAPro primer binding site in genomic RNA and the reverse-transcribed minus strong stop DNA. Each 588-base-pair repeat contains putative sites for promoting RNA synthesis and RNA polyadenylylation. The first and last 11 nucleotides of the TRS are inverted with respect to each other, and the same four-nucleotide host sequence is found bracketing integrated MSV. Some similarities of TRS and prokaryotic insertion sequence elements are discussed.
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We have determined the DNA sequences which correspond to the splicing regions in the transcripts of human papovavirus BKV, an evolutionary variant of SV40. To precisely localize the excision points in the BKV sequence, we have conducted a preliminary analysis of numerous viral and eukaryotic splice site sequences. This analysis suggests that the preferred sequence for the donor site belongs to at least one of four groups: Pu↓GTAxG, Pu↓GTAxxT, Pu↓GTxxGT, Pu↓GTxAG (↓ = the cleavage site). These four groups derive from the two basic sequences, Pu↓GTxxG and Pu↓GTA. An optimal donor site might be: AG↓GTAAGT. The preferred sequence for the acceptor site is of the form PyPyxPyAG↓; no dinucleotide AG occurs within 13 nucleotides prior to the terminal AG of the 3' end of the intervening sequences. As we have found in this study of BKV splice sites, the sequences for the preferred donor and acceptor sites provide predictive value in localizing RNA splice points when the DNA sequence is known.
The complete DNA sequence of human papovavirus BKV(Dun), consisting of 5153 nucleotide pairs, is presented. We describe the segments of the genome which correspond to the replication origin, the tandem repeated sequences, the 5' and 3' ends of the mRNAs, the splice sites, the early and late viral proteins and the putative viral polypeptides. These BKV DNA sequences are compared with analogous regions in the SV40 and Py virus genomes in an attempt to localize viral functions for lytic growth and transformation.
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The nucleotide sequence from 0.64 to 0.53 map units in the BK virus genome coding for the small t protein has been determined. There is only one open reading frame that can code for a polypeptide of 172 amino acids, the putative small t protein. Beyond this segment, multiple termination codons are present in all three reading frames. There is considerable nucleotide and amino acid sequence homology between this region of BK virus and the analogous region of simian virus 40, especially in the proximal portion from 0.64 to 0.60 map units which is most likely common to the small t and large T BK virus proteins. A comparison of the conserved sequences within the early papovavirus genes both confirms the evolutionary relationship between these viruses and suggests the amino acid composition of the regions required for T antigen functions.
Simian virus 40 deletion mutants were constructed lacking specifically the intervening sequences for a late viral mRNA. The construction method involved the replacement of portions of the late simian virus 40 genes with the DNA segment from reverse transcription of the viral mRNAs. Restriction endonuclease cleavage and sequence analysis confirmed the precise structure of the mutant DNAs and demonstrated that they contained the genetic information for VP1, including all potential 5' ends for the late viral RNAs. Thus, the primary late transcription product(s) of this mutant should have the structure of functional 16S mRNAs. Complementation analysis as well as immunoprecipitation showed, however, that deletion of the intervening sequences from this mutant prevented the expression of VP1. The nature of this failure appears to be a defect in the posttranscriptional processing of the viral RNA. These results indicate that splicing is an essential function in the biogenesis of certain mRNAs.