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Biomedical subjects

C Weissmann

Publications and source records attributed to C Weissmann.

At least 163 records · Page 9Linked to original sources
PubMed1977

Revised amino acid sequence of Qbeta coat protein between positions 1 and 60.

A reinvestigation of the primary structure of Qbeta coat protein between positions 1 and 60 was undertaken to resolve two discrepancies between the published amino acid seuqence (Maita, T., and Konigsberg, W. (1971) J. Biol. Chem. 246, 5003-5024) and the cognate nucleotide sequence recently determined in our laboratory (C. Escarmis and M. A. Billeter, unpublished results). The 22nd amino acid was asparagine rather than aspartic acid, and an additional amino acid, serine, was present between proline in position 55 and arginine in position 56. The revised structure agrees with the nucleotide sequence determined so far.

Amino Acid Sequence↗
PubMed1977

Avian myeloblastosis virus RNA is terminally redundant: implications for the mechanism of retrovirus replication.

We have determined the terminal heteropolymeric sequences of AMV RNA by the following procedures: first, RNA sequence determination on the 5' terminal and the poly(A)-linked 3' terminal T1 oligonucleotides, and second, analysis by the Maxam and Gilbert (1977) method of AMV strong stop DNA and of DNA complementary to the poly(A)-linked T1 oligonucleotide, synthesized with reverse transcriptase and (pdT)13 as primer. The structure deduced for the 5' terminal region is (5')7mGpppGmCCAUUCUACCUCUCACCACAUUGGUGUGCACCUGGGUUGAUGGCCGGACCGUCGAUUCCCUGACGACUACGAGCACCUGCAUGAAGCAGAAGGCUUCAU... Two distinct 3' terminal sequences were deduced: GCCAUUCUACCUCUCAAA...AOH and GCCAUUCUACCUCUCACCAAA...AOH. The two termini, differing by a C-C-A sequence, may reflect genetic heterogeneity of the AMV stock or, more probably, may be generated at or after RNA transcription. These results demonstrate a terminal redundancy of the hetero polymeric sequence of 16 and 19 nucleotides, respectively. The terminal redundancy allows for mechanisms which involve transfer of the DNA segment synthesized on the 5' terminal redundant sequence to the 3' terminal redundant sequence.

Alpharetrovirus↗
PubMed1977

Presence of a putative 15S precursor to beta-globin mRNA but not to alpha-globin mRNA in Friend cells.

Dimethyl sulfoxide-induced Friend cells were labeled for periods of 5-60 min. The denatured RNA was fractionated by sucrose gradient centrifugation and the distribution of alpha- and beta-globin-specific [(3)H]RNA was determined by hybridization to hybrid plasmids containing mouse alpha- and beta-globin DNA, respectively. After 5 min of labeling, a 15S peak of beta-globin-specific (but not alpha-globin-specific) [(3)H]RNA was detected, next to an equal amount of 10S beta-globin [(3)H]RNA. With increasing periods of labeling, the amount of 15S beta-globin [(3)H]RNA remained constant but the amount 10S beta-globin [(3)H]RNA increased steadily. alpha-Globin-specific [(3)H]RNA sedimented at 11 S after 5 min of labeling and at 9.5 S after longer labeling periods. Analysis of 15S globin-specific [(3)H]RNA purified by the poly(dC)-cDNA method [Curtis, P. J. & Weissmann, C. (1976) J. Mol. Biol. 106, 1061-1075] showed oligonucleotides characteristic of beta-globin mRNA but not of alpha-globin mRNA, as well as about 20 new oligonucleotides. Our results suggest that 10S beta-globin mRNA arises via a 15S precursor that has a half-life of 5 min or less; 9.5S alpha-globin mRNA may be derived from an 11S precursor.

Animals↗
PubMed1976

Specific excision of the inserted DNA segment from hybrid plasmids constructed by the poly(dA). poly (dT) method.

The hybrid plasmid PBETAG, consisting of plasmid PMB9 DNA with an insert of rabbit globin DNA (about 600 base pairs) flanked by poly(dA) poly(dT) regions (Maniatis, T., Kee, S.G., Efstratiadis, A. and Kafatos, F.C. (1976) Cell 8, 163-182), was cleaved into two fragments by endonuclease S1 under conditions of partial denaturation. Only the smaller fragment (575 base pairs) contained globin-specific sequences, showing that excision had occurred in the A-T-rich regions. This method of cleavage provides a useful procedure for assessing the length of inserts in hybrid plasmids prepared by the poly(dA)-POLY(DT) tail method, and allows the preparative recovery of the insert.

Animals↗
PubMed1976

Initiation sites of Rous sarcoma virus RNA-directed DNA synthesis in vitro.

Rous sarcoma virus DNA synthesis in disrupted virions is initiated mainly at a site about 200 nucleotides or less from the 5' terminus, but other initiation sites throughtout the RNA seem to be used as well. No AUG triplet occurs within a 5' terminal segment of about 25 nucleotides.

Avian Sarcoma Viruses↗
PubMed1976

In vitro site-directed mutagenesis: generation and properties of an infectious extracistronic mutant of bacteriophage Qbeta.

An infectious extracistronic mutant of phage Qbeta has been prepared by site-directed mutagenesis. Qbeta RNA minus strands containing the mutagenic base analog N4-hydroxy-CMP instead of UMP at position 39 from the 5' end were synthesized in vitro and used as template for Qbeta replicase to synthesize one generation of plus strands. E. coli spheroplasts were infected with the newly synthesized plus strands and phage recovered from single plaques. RNA sequence analysis revealed that four out of the eighteen phage clones analyzed contained RNA with an A leads to G transition at position 40 from the 3' end (which corresponds to position 39 of the minus strand). Thus, the viability of phage Qbeta does not depend on a unique nucleotide sequence in the 3'-extracistronic RNA segment. Upon in vivo propagation of mutant 40, spontaneous true revertants arose with high frequency and overgrew the parental clone within about 10 passages, indicating a selective disadvantage of the extracistronic mutant. Replication of mixtures of wild type and mutant RNA in vitro resulted in a decrease of the proportion of mutated RNA in the progeny plus strands. The fact that Qbeta RNA containing an A leads to G transition in nucleotide--40 of Qbeta RNA is less efficiently replicated in vitro may explain the selective disadvantage of the mutant phage in vivo. The preparation of an infectious mutated RNA by site-directed mutagenesis shows that the method is suitable to produce specific nucleotide exchanges without impairing the biological competence of the RNA.

Bacteriophages↗
PubMed1975

Evidence for the participation of a host enzyme in the activation of poly (A)-Qbeta RNA as an infectious agent.

It has been reported earlier that phage Qbeta RNA (Gilvarg, C., Bollum, F.J. and Weissmann, C. (1975) Proc. Natl. Acad. Sci. U.S. 72, 428-432) elongated at its 3' terminus with up to 100 or more AMP residues retained its full infectivity for Escherichia coli spheroplasts, and that the resulting progeny did not inherit the poly (A) appendage. We now show that while poly (A)-Qbeta RNA appears to function normally as messenger for the synthesis of virus-specific proteins it has lost its capacity to serve as template for Qbeta replicase. Template function could be restored by phosphorolysis with polynucleotide phosphorylase. Taken in conjunction, these results imply that after poly (A)-Qbeta RNA enters the spheroplast a host enzyme (perhaps polynucleotide phosphorylase) removes part or all of the adenylate residues prior to replication of the RNA.

Coliphages↗
PubMed1975

Site-directed mutagenesis: effect of an extracistronic mutation on the in vitro propagation of bacteriophage Qbeta RNA.

It has been proposed that the nucleotide sequences of the 3' terminal extracistronic regions of phage RNA plus and minus strands have been strictly conserved during evolution because they are stringently required for recognition by the viral replicase. We have devised a method to generate point mutations at selected sites in the genome of phage Qbeta. The in vitro synthesis of Qbeta RAN with G leads to A transition in the 16th position from the 3' end, i.e., in the terminal extracistronic region of the genome, is outlined. When a mixture of about 60% wild-type and 40% mutant RNA was repeatedly replicated, the mutant RNA was enriched to 80%, showing that at least this point mutation in the terminal sequence of Qbeta RNA does not impair its in vitro replication, but in fact slightly accelerates it.

Base Sequence↗
PubMed1975

Mapping of biological functions on RNA of avian tumor viruses: location of regions required for transformation and determination of host range.

A map of the large T1 oligonucleotides of the RNA of Prague Rous sarcoma virus, strain B (Pr RSVb) has recently been established (Coffin and Billeter, submitted for publication). Since the RNA of Rous associated virus, type 1 (RAV-1) lacks many of the large 1 oligonucleotides of Pr RSV-B and contains others not present in the latter, the RNA of recombinants between RAV-1 and Pr RSV-B could be analyzed with regard to the origin of its sequences. Recombinants were selected for transforming capacity (characteristic for Pr RSV-B) and ability to grow on C/B chicken fibroblasts (characteristic for RAV-1). Four out of five recombinants examined had undergone at least two crossovers. The set of Pr RSV-B-specific oligonucleotides present in all recombinants defined an RNA region near the poly(A) segment; this must contain genetic information required for transformation required for transformation (the onc function). All recombinants lost a set of contiguous Pr RSV-B-specific oligonucleotides and concomitantly acquired a set of RAV-1-specific oligonucleotides. These define a region in the middle section of the oligonucleotide map, all or some of which must be required for determining growth capacity on C/B cells (the env function).

Avian Sarcoma Viruses↗