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Biomedical subjects

R Kamen

Publications and source records attributed to R Kamen.

15 recordsLinked to original sources
PubMed1979

Multiple 5' terminal cap structures in late polyoma virus RNA.

Nuclear and cytoplasmic polyoma virus-specific RNA extracted from 32P-labeled mouse embryo cells late during productive viral infection was analyzed for the presence of 5' terminal capped structures by complete digestion with RNAases T1, T2 and A, followed by two-dimensional electrophoretic fractionation. Seven major cap I structures (m7 GpppNm1pN2p) were observed in both cases. These termini were further characterized by digestion with penicillium nuclease P1, followed by product analysis in a variety of alternative separate systems. Each structure had an individual combination of N1 and N2 nucleotides, where N1 was always a purine nucleotide but N2 was any nucleotide subject to the single exception that m7GpppGmpCp is found only in low yield. Four different cap II derivatives (m7GpppNm1pNm2pN3p) of four of the cap I structures were also detected in cytoplasmic RNA. None of the termini described derived from contaminating host cell RNA. All of these cap structures mapped on the polyoma viral DNA genome between 66 and 71 map units, a region distant from the 5' end of the bodies of two of the three late polyoma mRNAs. All the polyoma virus-specific cap structures, however, were present in each of the purified 16S, 18S and 19s late mRNAs. These data suggested that families of capped leader sequences of varying sizes are attached to the main body of each late polyoma mRNA species by a splicing mechanism.

Animals↗
PubMed1979

Amplification in the leader sequence of late polyoma virus mRNAs.

Ribonuclease T1 fingerprints of the three "late" polyoma virus mRNAs show that oligonucleotides of the leader sequence are present in multiple copies in each mRNA. These oligonucleotides, however, appear unimolar in fingerprints of complete, continuous transcripts of the late strand of the viral DNA. Oligonucleotides which are represented only once in the DNA are thus reiterated in the mature mRNAs. Consequently, when mRNA was hybridized to the leader region of immobilized viral DNA, those copies present in excess of their genomic representation failed to hybridize and were released by RNAase treatment. Analysis of the RNAase-resistant hybrids revealed a series of leader species with complex sequence arrangements. We suggest that these complicated reiterated sequences are generated during the processing of a precursor RNA which extends several times around the genome. This RNA would be shortened by a series of splicing reactions which conserve sequences from the leader region and attach them to a suitable coding sequence.

Animals↗
PubMed1979

State and organization of polyoma virus DNA sequences in transformed rat cell lines.

Polyoma virus-transformed rat cell lines were isolated as colonies growing in agar after infection of F2408 cells with low multiplicities of wild-type virus. Viral DNA present in the transformed cells was analyzed by fractionating the cellular DNA on agarose gels before and after digestion with various restriction endonucleases, followed by detection of the DNA fragments containing viral sequences using the procedure described by Southern (E. Southern, J. Mol. Biol., 98:503--515, 1975). Five lines, independently derived, were studied in detail. All five lines, when examined after a minimum number of passages in culture, contained both free and apparently integrated viral DNA. The free polyoma DNA in three of the lines was indistinguishable, by restriction enzyme analysis, from wild-type viral DNA, whereas the two other lines also contained smaller free DNA molecules which lacked parts of the wild-type genome. The integrated DNA in the five lines studies existed as head-to-tail tandem repeats of unit-length polyoma DNA covalently attached to nonviral DNA. The same five polyoma-transformed rat lines were examined after further passage in culture. Free viral DNA was then either undetectable or greatly reduced in amounts, whereas the high-molecular-weight, integrated units persisted after passage of the cells. The subclones, derived from one of the five lines selected for detailed analysis, showed some variations in the quantity and size of the free viral DNA as well as minor alterations in the pattern of the apparently integrated sequences.

Animals↗
PubMed1978

Transcription of a defective polyoma virus genome.

The circular genome of the cloned defective polyoma virus D-50 consists of tandemly repeated copies of the DNA sequence between 67 and 84 units on the wild-type polyoma virus DNA map. Each repeated copy thus contains the origin of viral DNA replication, which is located at about 71 map units. Viral RNA was synthesized in vitro using viral transcription complexes extracted late (30 hr) after infection from mouse cells co-infected with D-50 and helper wild-type virus. Both wild-type and D-50 DNA molecules were active as templates for in vitro transcription. Approximately 84% of the RNA transcribed in vitro from wild-type DNA was complementary to the L DNA strand. This is normal for wild-type transcription late after infection. By contrast, at least 90% of the RNA transcribed from D-50 DNA molecules was complementary to the E DNA strand. After normalization of the data to account for the observed molar ratio of D-50 DNA repeated sequences to unit length wild-type DNA, we estimate that transcription of the E DNA strand of each D-50 repeated unit is about 1.4 times as efficient as transcription of the wild-type E DNA strand. Transcription of the D-50 L DNA strand, however, is only 0.03 times as efficient as transcription of the wild-type L DNA strand. The implications of these results concerning the nature and location of promoter sequences in polyoma DNA are discussed.

Binding Sites↗
PubMed1978

Polyoma virus-specific RNA synthesis in an inducible line of polyoma virus-transformed rat cells.

Viral RNA present in the inducible LPT clone 1A of polyoma virus-transformed rat cells was characterized before and after mitomycin C induction by hybridization with 32P-labeled separated E and L strands of polyoma viral DNA restriction endonuclease fragments. In clone 1A cells maintained under normal growth conditions, the cytoplasm contained a transcript of the E-strand DNA from the "early" region similar to that previously identified in lytically infected cells, as well as minor quantities of RNA complementary to less than one-half of the L- and the E-strand DNA from the "late" region. Nuclei of normally growing cells contained the same species found in the cytoplasm, as well as an additional abundant RNA complementary to one-half of the L-strand DNA of the late region. No significant changes occurred in the cytoplasmic viral RNA after mitomycin C treatment before the onset of viral DNA replication, but the concentration of the nuclear L-strand DNA transcript diminished. After the onset of viral DNA replication after mitomycin C treatment, transcripts of virtually the entire L-strand DNA were found in the nuclei, and a 10-fold increase was observed in the abundance of RNA transcribed from the E strand of the early region. In the cytoplasm, the abundance of the early RNA increased about 25-fold and late RNA complementary to the L-strand DNA of the late region was found in a similar quantity. The synthesis of both the early and the late RNA species was inhibited if viral DNA replication was blocked with 5-fluorodeoxyuridine. We conclude that the induction of viral DNA replication in LPT cells is not determined at the level of mRNA synthesis.

Animals↗
PubMed1978

Biofeedback and meditation: effects on muscle tension and locus of control.

A total of 48 subjects participated in a relaxation experiment to determine whether frontalis muscle EMG biofeedback, Transcendental Meditation, and meditation (Benson technique) produced decreased muscle tension and concomitant changes in locus of control. All three treatments resulted in significant decreases in frontalis muscle tension when compared to a control. Concomitant changes towards an internal locus of control occurred only in the subjects given biofeedback.

Biofeedback, Psychology↗
PubMed1977

Analysis of polyoma virus nuclear RNA by mini-blot hybridization.

The size and sequence composition of virus-specific RNA extracted from the nuclei of mouse cells late during polyoma virus productive infection were studied by blot-hybridization analysis of 32P-labeled RNA fractionated on CH3HgOH/agarose gels. Viral RNA molecules between approximately 0.4 and 4 times the length of a complete transcript of the 5.4-kilobase circular viral DNA were found. Less than 20% of such molecules were polyadenylylated. Although viral RNA of all sizes contained species that together hybridized to the entire polyoma genome, sequences complementary to the late region were more abundant than sequences complementary to the early region in transcripts less than 10-12 kilobases long.

Base Sequence↗
PubMed1976

Location of the sequences coding for capsid proteins VP1 and VP2 on polyoma virus DNA.

The 19S and 16S polyoma virus late mRNAs have been separated on sucrose-formamide density gradients and translated in vitro. The 16S RNA codes only for polyoma capsid protein VP1, while the 19S RNA codes in addition for capsid protein VP2. Since the 19S and 16S species have been previously mapped on the viral genome, these results allow us to deduce the location of the sequences coding for VP1 and VP2. Comparison of the chain lengths of the capsid proteins with the size of the viral mRNAs coding for them suggests that VP1 and VP2 are entirely virus-coded. Purified polyoma 19S RNA directs the synthesis of very little VP1 in vitro, although it contains all the sequences required to code for the protein. The initiation site for VP1 synthesis which is located at an internal position on the messenger is probably inactive either because it is inaccessible or because it lacks an adjacent "capped" 5' terminus. Similar inactive internal initiation sites have been reported for other eucarotic viral mRNAs (for example, Semliki forest virus, Brome mosaic virus, and tobacco mosaic virus), suggesting that while eucaryotic mRNAs may have more than one initiation site for protein synthesis, only those sites nearer the 5' terminus of the mRNA are active.

Base Sequence↗
PubMed1976

Topography of polyoma virus messenger RNA molecules.

The different species of polyoma virus-spedific RNA molecules present in the cytoplasm of 3T6 cells 30 hr after viral infection have been characterized by molecular hybridization between nonradioactive polyadenlated RNA, fractionated by sedimentation through sucrose-formamide density gradients, and the 32P-labeled separated strands of restriction endonuclease fragments of polyoma DNA. Two relatively abundant RNA molecules, sedimenting at 16S and at 19S, transcribed from the L strand of the viral DNA, as well as a minor 20S species transcribed from the E strand of the DNA, were detected. The most abundant viral transcript, the 16S RNA molecule, was estimated to be complementary to the 22% of the L-strand DNA extending from 47 to 25 map units. The less abundant 19S L DNA strand transcript included all the sequences present in the 16S RNA and mapped between 68 and 25 map units. The minor 20S RNA molecule was tentatively identified as a transcript of the E-strand DNA from the entire early region of the polyoma genome. These three viral RNA molecules together exhaust greater than 95% of the coding capacity of the viral DNA. A small region of the DNA (4-5%), including the origin of DNA replication, does not appear to determine sequences present among the major stable species of vital mRNA.

Cell Line↗
PubMed1975

Orientation of the complementary strands of polyoma virus DNA with respect to the DNA physical map.

The chemical polarities of the two strands of polyoma virus DNA with respect to the DNA physical map have been determined by hybridization of restriction endonuclease fragments specifically labeled with [125I]dCMP at their 3' termini to asymmetric polyoma complementary RNA (the product of in vitro transcription of viral DNA by Escherichia coli RNA polymerase). The orientations of the polyoma-specific stable RNA transcripts present in the cytoplasm of productively linfected mouse cells have been deduced from this result: the 5' ends of the early and late viral transcripts map very near the origin of viral DNA replication.

Cell-Free System↗