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Biomedical subjects

I M Verma

Publications and source records attributed to I M Verma.

17 recordsLinked to original sources
PubMed1979

Genome organization of retroviruses. III. Restriction endonuclease cleavage maps of mouse sarcoma virus double-stranded DNA synthesized in vitro.

Genome length complementary DNA (cDNA) transcripts were synthesized in vitro by using purified virions of a cloned isolate of mouse sarcoma virus (MSV Clone 124). The cDNA transcripts were converted to double-stranded form by utilizing DNase-digested calf thymus DNA primers and E. coli DNA polymerase I. Restriction endonucleases Sal I, Hind III, Hpa I, Bgl II and Xba I were found to cleave the MSV double-stranded DNA once to generate two fragments, whereas restriction endonucleases Bgl I and Hae II cleaved twice to generate three fragments. Restriction endonucleases E. coli RI and Bam HI did not cleave MSV double-stranded DNA. The order of the restriction fragments was determined in relation to the 5' and 3' ends of the genomic RNA.

DNA Restriction Enzymes↗
PubMed1979

Heteroduplex analysis of the sequence relationships between the genomes of Kirsten and Harvey sarcoma viruses, their respective parental murine leukemia viruses, and the rat endogenous 30S RNA.

The sequence relations between Kirsten murine sarcoma virus (Ki-SV), Harvey murine sarcoma virus (Ha-SV), and a rat endogenous 30S RNA were studied by electron microscope heteroduplex analysis. The sequence relationships between the sarcoma viruses and their respective parental murine leukemia viruses (Kirsten and Moloney murine leukemia viruses), as well as between the two murine leukemia viruses, were also studied. The only observed nonhomology feature of the Kirsten murine leukemia virus/Moloney murine leukemia virus heteroduplexes was a substitution loop with two arms of equal length extending from 1.80 +/- 0.18 kilobases (kb) to 2.65 +/- 0.27 kb from the 3' end of the RNA. It is believed that this feature lies in the env gene region of the viral genomes. The Ha-SV and Moloney murine leukemia virus genomes (respective lengths, 6.0 and 9.0 kb) were homologous in a 1.0 +/- 0.05-kb region at the 3' end and possibly over a 200-nucleotide region at the 5' ends; otherwise, they were nonhomologous. Ha-SV and Ki-SV (length, 7.5 kb) were homologous in the first 4.36 +/- 0.37-kb region from the 3' end and in a 0.70 +/- 0.15-kb region at the 5' end. In between, there was a nonhomology region, possibly containing a short (0.23-kb) region of partial or total homology. The heteroduplex analysis between rat endogenous 30S RNA and Ki-SV shows that there are mixed regions of sequence homology and nonhomology at both the 5' and 3' ends. However, there is a large (4-kb) region of homology between Ki-SV and the rat 30S RNA in the center of the genomes, with only a small nonhomology hairpin feature. These studies help to define the regions of homology between the Ha-SV and Ki-SV genomes with each other and with the rat endogenous 30S RNA. These regions may be related to the sarcoma genicity of the viruses. In particular, the 0.7-kb region of homology of Ha-SV with Ki-SV at the 5' ends may be related to the formation of a 21,000-dalton phosphoprotein in cells transformed by either virus.

Animals↗
PubMed1979

Heteroduplex analysis of the RNA of clone 3 Moloney murine sarcoma virus.

Heteroduplex analysis of the RNA isolated from purified virions of clone 3 Moloney murine sarcoma virus (M-MSV) hybridized to cDNA's from Moloney murine leukemia virus (M-MLV) and clone 124 M-MSV shows that the main physical component of clone 3 RNA is missing all or most of the 1.5-kilobase (kb) clone 124 M-MSV specific sequence denoted beta s (S. Hu et al. Cell 10:469--477, 1977). This sequence is either deleted in clone 3 RNA or substituted by a very short (0.3-kilobase) sequence. In other respects, clone 3 and clone 124 RNAs show the same heteroduplex structure relative to M-MLV. Since beta s is believed to contain the src gene(s) of clone 124 RNA, this result leaves as an unresolved question the nature of the src gene(s) of the clone 3 M-MSV RNA complex.

Base Sequence↗
PubMed1979

Genome organization of retroviruses. VI. Heteroduplex analysis of ecotropic and xenotropic sequences of moloney mink cell focus-inducing viral RNA obtained from either a cloned isolate or a thymoma cell line.

The genome of a recombinant murine leukemia virus capable of inducing focal areas of morphological alteration in mink lung fibroblasts was studied by heteroduplex analysis. The dual-tropic recombinant virus was isolated from a thymoma cell line (Th16.3) and is referred to as BALB/Moloney mink cell focus-inducing virus (BALB/Mo-MCF virus). The nucleic acid sequences of RNA from virions obtained from either a thymoma cell line (Th16.3) or a clonal isolate (BALB/Mo-MCF81) were compared with the genomes of ecotropic and xenotropic viruses. The following inferences were drawn (i) A single nonhomologous region (substitution loop alpha) of about 0.7 kilobase was observed in a heteroduplex formed between Moloney murine leukemia virus complementary DNA (cDNA) and BALB/MoMCF81 RNA. This nonhomology region was mapped between 1.71 and 2.40 kilobases from the 3' end of the genome. (ii) The predominant class of heteroduplexes formed between virion RNA obtained from the thymoma cell line (Th16.3) and Moloney murine leukemia virus cDNA showed a substitution loop similar to that observed with the RNA obtained from a cloned isolate, BALB/Mo-MCF81. However, there were other molecules with additional regions of nonhomology. (iii) Heteroduplexes formed between NZB xenotropic RNA and ecotropic Moloney murine leukemia virus cDNA exhibited four major nonhomology regions extending 0.75 to 1.46, 2.0 to 2.8, 3.6 to 4.3, and 7.4 to 7.9 kilobases from the 3' end of the genome. (iv) The MCF-specific substitution loop alpha (1.71 to 2.40 kilobases) appeared as a duplex region when NZB xenotropic RNA was hybridized to cDNA transcripts synthesized by virions obtained from thymoma cell line Th16.3. The position of the other substitution loops observed in a heteroduplex formed between NZB xenotropic RNA and Moloney murine leukemia virus cDNA was not affected. (v) Heteroduplexes formed between xenotropic BALB virus 2 cDNA and NZB xenotropic RNA demonstrated a large degree of nucleic acid sequence homology. Of the 29 heteroduplexes examined, 24 appeared to be homoduplexes, and in the remaining 5 heteroduplexes only one region of nonhomology located between 3.2 and 3.8 kilobases from the 3' end of the genome could be identified. Hybridization of BALB virus 2 xenotropic RNA to NZB xenotropic cDNA followed by digestion with single-strand-specific nuclease S1 showed an 80% sequence homology.

Animals↗
PubMed1978

Reverse transcriptase of RNA tumor viruses. V. In vitro proteolysis of reverse transcriptase from avian myeloblastosis virus and isolation of a polypeptide manifesting only RNase H activity.

Purified avian myeloblastosis virus reverse transcriptase contains two subunits that are structurally related. The large subunit, beta (molecular weight, 95,000), was converted in vitro by chymotrypsin into a polypeptide of molecular weight 63,000. This polypeptide was indistinguishable from the small subunit, alpha (molecular weight, 65,000), in its chromatographic behavior on the phosphocellulose column and its tryptic peptide composition. During this proteolytic conversion, a polypeptide of molecular weight 32,000 (fragment B) was obtained. It was composed of tryptic peptides unique to beta and appeared to be derived from the portion of the beta subunit that was cleaved off during the conversion of beta into alpha. Upon continued proteolysis, a smaller polypeptide of molecular weight 24,000 (fragment A) was generated. This polypeptide manifested only RNase H activity and shared common amino acid sequences with beta and alpha subunits. Fragment A did not share any amino acid sequence homology with fragment B.

Avian Leukosis Virus↗
PubMed1978

Size analysis and relationship of murine leukemia virus-specific mRNA's: evidence for transposition of sequences during synthesis and processing of subgenomic mRNA.

Virus-specific mRNA from purified polyribosomes of mouse cells infected with Moloney murine leukemia virus (M-MuLV) was analyzed by electrophoresis in agarose gels, followed by hybridization of gel slices with M-MuLV-specific complementary DNA (cDNA). The size resolution of the gels was better than that of sucrose gradients used in previous analyses, and two virus-specific mRNA's of 38S and 24S were detected. The 24S virus-specific mRNA is predominantly derived from the 3' half of the M-MuLV genome, since cDNAgag(pol) (complementary to the 5' half of the M-MuLV genome) could not efficiently anneal with this mRNA. However, sequences complementary to cDNA synthesized from the extreme 5' end of M-MuLV 38S RNA (cDNA 5') are present in the 24S virus-specific mRNA, since cDNA 5' (130 nucleotides) efficiently annealed with this mRNA. The annealing of cDNA 5' was not due to repetition of 5' terminal nucleotide sequences at the 3' end of M-MuLV 38S RNA, since smaller cDNA 5' molecules (60 to 70 nucleotides), which likely lack the terminal repetition, also efficiently annealed with the 24S mRNA. The sequences in 24S virus-specific mRNA recognized by cDNA 5' are not present in 3' fragments of virion RNA that are the same length. Therefore, it appears that RNA sequences from the extreme 5' end of the M-MuLV genome may be transposed to sequences from the 3' half of the M-MuLV 38S RNA during synthesis and processing of the 24S virus-specific mRNA. These results may indicate a phenomenon similar to the RNA splicing processes that occur during synthesis of adenovirus and papovavirus mRNA's.

Base Sequence↗
PubMed1978

Genome organization of RNA tumor viruses. I. In vitro synthesis of full-genome-length single-stranded and double-stranded viral DNA transcripts.

Genome-length complementary DNA (cDNA) transcripts were synthesized in vitro by using purified virions of avian myeloblastosis virus. Moloney murine leukemia virus, and clone 124 mouse sarcoma virus. The size of the genomelenth cDNA transcripts was measured on either alkaline sucrose gradients or alkaline agarose gels. The longest cDNA transcripts synthesized by using avian myeloblastosis virus, Moloney murine leukemia virus, and clone 124 mouse sarcoma virus were 7, 9 and 6 kilobases (kb), respectively. The in vitro system used was capable of synthesizing double-stranded DNA, but the plus strands (same polarity as the viral RNA) were only 0.5 to 1.5 kb long. Lone Moloney murine leukemia virus cDNA transcripts were used as templates to synthesize the second plus strand. Essentially two strategies were employed as follows. (i) The 3' ends of the cDNA transcripts were extended by addition of 50 to 100 dAMP residues by terminal deoxynucleotidyl transferase. The (dA)n-tailed cDNA transcripts were used as templates along with an oligomer of dT as primer and Escherichia coli DNA polymerase to synthesize the plus strands. (ii) DNase-digested calf thymus DNA was used to prime the synthesis of plus strands on long cDNA with E. coli DNA polymerase I. In both cases, the synthesis of the plus strands was monitored by increased resistance of the cDNA templates to single-strand-specific S1 nuclease. The double-stranded DNA was fractionated on neutral sucrose gradients. Analysis of the double-stranded DNA synthesized by using oligo(dT) primer showed the plus strands to be about 5 to 6 kb long, whereas the plus strands synthesized by using DNase-digested calf thymus DNA primers were only 0.3 to 0.5 kb long. Double-stranded DNA synthesized by either method has an average size of 6 x 10(6) daltons. Double-stranded DNA was also synthesized by using cDNA transcripts as templates without the addition of any primers. In this case, the plus strands were covalently linked to the template strand and were not representative of the whole parent strand.

Avian Leukosis Virus↗
PubMed1978

Genome organization of RNA tumor viruses II. Physical maps of in vitro-synthesized Moloney murine leukemia virus double-stranded DNA by restriction endonucleases.

Physical maps of the genome of Moloney murine leukemia virus (M-MLV) DNA were constructed by using bacterial restriction endonucleases. The in vitro-synthesized M-MLV double-stranded DNA was used as the source of the viral DNA. Restriction endonucleases Sal I and Hind III cleave viral DNA at only one site and, thus, generate two DNA fragments. The two DNA fragments generated by Sal I are Sal IA (molecular weight, 3.5 x 10(6)) and Sal IB (molecular weight, 2.4 x 10(6)) and by Hind III are Hind IIIA (molecular weight, 3.6 x 10(6) and Hind IIIB (molecular weight, 2.3 x 10(6)). Restriction endonuclease Bam I generates four fragments of molecular weights of 2.1 x 10(6) (Bam IA), 2 X 10(6) (Bam IB), 1.25 X 10(6) (Bam IC), and 0.24 x 10(6) (Bam ID), whereas restriction endonuclease Hpa I cleaves the M-MLV double-stranded DNA twice to give three fragments of molecular weights of 4.4 x 10(6) (Hpa IA), 0.84 X 10(6) (Hpa IB), and 0.74 x 10(6) (Hpa IC). Digestion of M-MLV double-stranded DNA with restriction endonuclease Sma I produces four fragments of molecular weights of 3.9 x 10(6) (Sma IA), 1.3 X 10(6) (Sma IB), 0.28 X 10(6) (Sma IC), and 0.21 x 10(6) (Sma ID). A mixture of restriction endonucleases Bgl I and Bgl II (Bgl I + II) cleaves the viral DNA at four sites generating five fragments of approximate molecular weights of 2 x 10(6) (Bgl + IIA), 1.75 X 10(6) (Bgl I + IIB), 1.25 X 10(6) (Bgl I + IIC), 0.40 X 10(6) (Bgl I + IID), and 0.31 x 10(6) (Bgl I + IIE). The order of the fragments in relation to the 5' end and 3' end of the genome was determined either by using fractional-length M-MLV double-stranded DNA for digestion by restriction endonucleases or by redigestion of Sal IA, Sal IB, Hind IIIA, and Hind IIIB fragments with other restriction endonucleases. In addition, a number of other restriction endonucleases that cleave in vitro-synthesized M-MLV double-stranded DNA have also been listed.

Cell-Free System↗
PubMed1978

Heteroduplex analysis of the sequence relations between the RNAs of mink cell focus-inducing and murine leukemia viruses.

The sequence relationships betwen AKR ecotropic virus and an AKR-derived "mink cell focus-inducing" (MCF) isolate (AKR MCF 247), between Moloney murine leukemia virus (M-MLV) and an M-MLV MCF isolate (M-MLV83), and between AKR and M-MLV were studied by electron microscopic heteroduplex analysis. The MCF-specific sequences were found to map from 1.95 kilobases (kb) to 2.75 kb (+/- 0.15 kb) from the 3' end of the RNAs for both MCF isolates. The major sequence nonhomology regions between AKR and M-MLV lie between 0.9 and 3.5 kb from the 3' end. However, the AKR and M-MLV sequences immediately adjacent to the 1.95- and 2.75-kb junctions with MCF-specific sequences are relatively similar in AKR and M-MLV. Our results suggest that the env gene of MLVs maps from 1 kb to 3 kb from the 3' end of the genomic RNA and that the carboxyl end of the glycoprotein of each MCF strain is similar (or identical) to that of its ecotropic parent.

AKR murine leukemia virus↗
PubMed1975

Studies on reverse transcriptase of RNA tumor viruses. I. Localization of thermolabile DNA polymerase and RNase H activities on one polypeptide.

Purified reverse transcriptase from avian myeloblastosis virus or Rous sarcoma virus consists of two subunits of average mol wt of 100,000 and 60,000. The lower-molecular-weight subunit, alpha, has been isolated from avian myeloblastosis virus, Rous sarcoma virus and a temperature-sensitive mutant of Rous sarcoma virus, LA337. Subunit alpha manifests both the DNA polymerase and RNase H activities associated with purified reverse transcriptase of avian RNA tumor viruses. The thermal inactivation of these enzymatic activities of alpha subunit from the wild-type virus. The results show that both DNA polymerase and RNase H activities associated with the alpha subunit of LA337 are five to seven times more thermolabile then the corresponding alpha subunit from the wild-type virus. It is concluded that (i) both the polymerase and nuclease activities reside on the same polypeptide chain, and (ii) at least the lower-molecular-weight subunit alpha is coded for by the viral RNA.

Avian Leukosis Virus↗
PubMed1975

Studies on reverse transcriptase of RNA tumor viruses III. Properties of purified Moloney murine leukemia virus DNA polymerase and associated RNase H.

DNA polymerase was purified from a cloned isolate of Moloney murine leukemia virus (M-MuLV). Purified M-MuLV DNA polymerase, upon analysis by polyacrylamide gel electrophoresis, showed one major polypeptide of mol wt 80,000. Estimation of molecular weight from the sedimentation rate of the purifed enzyme in a glycerol gradient was consistent with a structure containing one polypeptide. M-MuLV DNA polymerase could transcribe ribopolymers, deoxyribopolymers, and heteropolymers as efficiently as did purified DNA polymerase from avian myeloblastosis virus (AMV). M-MuLV DNA polymerase, however, transcribed native 70S viral RNA less efficiently than did AMV DNA polymerase. Addition of oligo(dT) enhanced five to tenfold the transcription of 70S viral RNA by M-MuLV DNA polymerase. Purified enzyme also exhibited nuclease activity (RNase H) that selectively degraded the RNA moiety of the RNA-DNA hybrid. It did not degrade single-stranded RNA, single-stranded DNA, double-stranded RNA, and double-stranded DNA. M-MuLV DNA polymerase-associated RNase H acted as a random exonuclease. When [3-H]poly(A)-poly(dT) was used as a substrate, the size of the M-MuLV DNA polymerase-associated RHase H digested product was larger than the size of the digestion products by AMV DNA polymerase. The oligonucleotide digestion products could be further digested to 5'-AMP by snake venom phosphodiesterase, indicating that the products were terminated by 3'-OH groups. Alkaline hydrolysis of the oligonucleotide digestion products generated pAp, suggesting that M-MuLV DNA polymerase-associated RNase H cleaves at the 3' side of the 3',5'-phosphodiester bond. The ratios of the rates of DNA polymerase activity and RNase H activity were not significantly different in the murine and avian enzymes.

Centrifugation, Zonal↗
PubMed1975

Thermolabile reverse transcriptase of a mammalian leukemia virus mutant temperature sensitive in its replication and sarcoma virus helper functions.

Three temperature-sensitive mutants of the Rauscher strain of murine leukemia virus are defective in early post-penetration functions required both for leukemia virus infection and for initiation of transformation of cells by their pseudotypes of murine sarcoma virus. In the present study, the reverse transcriptase of one of these mutants (ts 29) is shown to be thermolabile compared with the enzymes of the wild-type virus and several other temperature-sensitive mutants. These findings provide evidence that the reverse transcriptase is required both for leukemia virus infection and for initition of transformation by the replication-defective murine sarcoma virus genome.

Absorption↗