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S D Putney

Publications and source records attributed to S D Putney.

46 records · Page 3Linked to original sources
PubMed1986

HTLV-III/LAV-neutralizing antibodies to an E. coli-produced fragment of the virus envelope.

Immunization with either an Escherichia coli recombinant segment of the human T-cell lymphotropic virus (HTLV-III/LAV) envelope protein (gp 120) or with deglycosylated gp 120 envelope protein produced antibodies that neutralize HTLV-III/LAV infection in vitro. Virus neutralization titers of these antisera were equivalent to those obtained with purified native gp120 as immunogen. This localizes at least one class of neutralizing epitopes to the carboxyl-terminal half of the molecule. In addition, native gp120 prevented HTLV-III/LAV--mediated cell fusion, whereas the recombinant gp120 fragment did not. This shows that although glycosylation is not required for induction of neutralizing antibodies, it may be important for interaction with CD4, the virus receptor. A segment of the HTLV-III/LAV envelope produced in E. coli may be an important ingredient of a vaccine for acquired immune deficiency syndrome.

Antibodies, Viral↗
PubMed1984

The complete nucleotide sequence for rabbit muscle aldolase A messenger RNA.

The complete nucleotide sequence of rabbit muscle aldolase mRNA has been determined from recombinant cDNA clones and from a primer-extended cDNA synthesis on the mRNA template. The sequence is composed of 1375 nucleotides, exclusive of the poly(A) tails. The 5'-untranslated region contains 62 bases including a potential ribosome-binding site. The 3'-untranslated region is 221 bases long. The remaining 1092 nucleotides are in an open reading frame coding for 364 amino acids. There is a single methionine residue preceding the NH2-terminal proline. The deduced amino acid sequence corrects a number of discrepancies between published structures as well as assignments previously missed. The sequences of the cloned cDNAs suggests there may be microheterogeneity in the messenger RNA population.

Amino Acid Sequence↗
PubMed1983

A new troponin T and cDNA clones for 13 different muscle proteins, found by shotgun sequencing.

Complete amino acid sequences have been established for 19 muscle-related proteins and these proteins are each sufficiently abundant to suggest that their mRNA levels are about 0.4% or higher. Based on these considerations, a simple theoretical analysis shows that clones for most of these proteins can be identified within a complementary DNA library by sequencing cDNA inserts from 150-200 randomly selected clones. This procedure should not only rigorously identify specific clones, but it could also uncover amino acid sequence variants of major muscle proteins such as the troponins. We have determined sequences for about 20,000 nucleotides within 178 randomly selected clones of a rabbit muscle cDNA library, and report here that in addition to finding sequences encoding the two known skeletal muscle isotypes of troponin C, we have discovered sequences encoding two forms of troponin T. Over the region of nucleotide sequence overlap in the troponin T clones, the new isotype diverges significantly from its counterpart. Altogether, clones for 13 of the 19 known muscle-specific proteins were identified, in addition to the clone for the new troponin T isotype.

Amino Acid Sequence↗
PubMed1981

An aminoacyl tRNA synthetase binds to a specific DNA sequence and regulates its gene transcription.

Alanine tRNA synthetase represses transcription of its own gene by binding specifically to a palindromic sequence which flanks the gene's transcription start site. Transcription repression is greatly enhanced by elevated concentrations of the cognate amino acid. The amino acid effect is caused by direct association of the ligand with the synthetase, which in turn mediates tighter binding to the DNA.

Alanine↗
PubMed1981

Purification and properties of alanine tRNA synthetase from Escherichia coli A tetramer of identical subunits.

Escherichia coli alanine tRNA synthetase has been purified from a strain which carries the gene on a recombinant pBR322 plasmid. Several per cent of the soluble protein can be obtained as alanine tRNA synthetase in the plasmid containing host cell. The enzyme was proven to be an alpha 4 tetramer with a Mr = 380,000 by the following criteria: i) a single band of Mr = 95,000 was found in sodium dodecyl sulfate gel electrophoresis; ii) gel filtration chromatography gives a single peak at a Mr = 360,000 for the native enzyme; iii) dimethyl suberimidate cross-linking indicates a tetrameric structure; iv) a single undecapeptide N-terminal sequence was found which is Ser-Lys-Ser-Thr-Ala-Glu-Ile-Arg-Gln-Ala-Phe. Limited proteolysis generates a fragment of the native enzyme. The fragment has a Mr = 48,000 (one half that of the native subunit) and, in contrast to the native enzyme, oligomerization of the fragment could be not detected either by gel filtration chromatography or by dimethyl suberimidate cross-linking. The fragment is derived from the NH2-terminal half of the native subunit as shown by their common decapeptide NH2-terminal amino acid sequences. The ATP-PPi exchange activity of 1 mol of fragment is identical with that of 1 mol of native subunit, but aminoacylation activity is absent from the fragment. Therefore, in the fragment, the aminoacyl adenylate formation activity is cleanly separated from tRNA aminoacylation and subunit association properties. These results also mean that, with respect to aminoacyl adenylate formation activity, each subunit in the native tetramer acts independently.

Alanine-tRNA Ligase↗
PubMed1981

A DNA fragment with an alpha-phosphorothioate nucleotide at one end is asymmetrically blocked from digestion by exonuclease III and can be replicated in vivo.

2'-Deoxyadenosine 5'-O-(1-thiotriphosphate) (dATP[alpha S]) was introduced into the 3' ends of DNA restriction fragments with Escherichia coli DNA polymerase I to give phosphorothioate internucleotide linkages. Such "capped" 3' ends were found to be resistant to exonuclease III digestion. Moreover, the resistance to digestion is great enough that, under conditions used by us, just one strand of a double helix is digested by exonuclease III when a cap is placed at only one end; when digestion is carried to completion, this results in production of intact single strands. When digestion with exonuclease III is limited and is followed by S1 nuclease treatment, double-stranded DNA fragments asymmetrically shortened from just one side are produced. In this was thousands of nucleotides can be selectively removed from one end of a restriction fragment. In vitro introduction of phosphorothioate linkages into one end of a linearized replicative plasmid, followed by exonuclease III and S1 nuclease treatments, gives rise to truncated forms that, upon circularization by blunt-end ligation, transform E. coli and replicate in vivo.

Base Sequence↗
PubMed1980

Mass spectra of partial protein hydrolysates as a multiple phase check for long polypeptides deduced from DNA sequences: NH2-terminal segment of alanine tRNA synthetase.

A strategy has been developed for rapid and accurate determination of the amino acid sequence of large proteins, such as many of the members of the class of proteins known as aminoacyl tRNA synthetases. This strategy involves combining DNA sequencing of the gene for the protein of interest with gas chromatographic mass spectrometric identification of tetra- and pentapeptides in partial hydrolysates of the entire protein or very large fragments thereof. These peptides are matched to blocks of codons at locations scattered throughout the entire structural gene. Tetra- and pentapeptide sequences are sufficiently long that they are unlikely to be repeated in the protein sequence or to occur in an incorrect reading frame; therefore, they can be placed at unique clusters of codons on the DNA. This procedure rigorously establishes the proper phasing of the DNA throughout the entire length of the structural gene, and the protein sequence is thereby accurately read from the DNA sequence. This approach is being used to determine the amino acid sequence of EScherichia coli alanine tRNA synthetase, a protein that has approximately 900 amino acids. This paper reports the sequence of the first 165 amino acids from the NH2 terminus.

Alanine-tRNA Ligase↗