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C Guthrie

Publications and source records attributed to C Guthrie.

At least 109 records · Page 6Linked to original sources
PubMed1980

In vitro thrombogenicity tests of factor IX concentrates. II: effects of phospholipids and heparin.

Measurement of the total phospholipid (and that portion active in coagulation) in factor IX concentrates revealed no correlation with in vitro tests of potential thrombogenicity, except in the case of the recalcification time and the thrombin generation test which may detect coagulant phospholipid as well as the presence of thrombogenic enzymes. This is probably due to separation of the prothrombin complex proteins from most phospholipid during ion-exchange chromatography. Although low levels of phospholipid remain in the final product these are apparently insufficient to effect appreciable activation of factor IX concentrates despite low levels of antithrombin III. Two tests which measure the formation of thrombin and factor Xa after recalcification of concentrates were affected by the addition of exogenous phospholipid. However this is a relative effect such that differences are quantitative rather than qualitative. Heparin addition during production of factor IX concentrate was found to have only minor effects on the results of in vitro thrombogenicity tests of the final product. This was confirmed in the laboratory by incubation of unheparinised products with heparin for periods of up to 6 hr.

Blood Coagulation Tests↗
PubMed1980

Folding up a transfer RNA molecule is not simple.

For much of its history, molecular biology has concerned itself with the implications and consequences of the "Central Dogma" (Crick, 1958): DNA leads to RNA leads to protein. This "pathway" is of course atypical in that it describes, not chemical interconversions, but rather the flow of information from gene to product. It has been only recently appreciated, however, that in those cases where the final gene product is an RNA molecule, this pathway is not simply truncated. To the contrary, in virtually every instance for which sufficient data are now available, it is found that the RNA transcript is subject to a series of reactions in which the primary gene product undergoes nucleotide additions, deletions, and modifications. The set of biosynthetic reactions that intervene between transcription of the gene and production of the mature functional product is collectively referred to as RNA processing. It now appears that the existence of these complex biosynthetic events cannot always be adequately explained by the necessity to overcome otherwise insurmountable topological or energetic constraints: the final product can, at least in some cases, "self-assemble." The genesis of transfer RNA by this indirect route appears to insure the delivery of a functional product, at the right rate, at the right time. We suggest, moreover, that this process is primarily determined by recognition of some of the same structural elements in the precursor that are required for the function of the mature tRNA molecule.

Base Sequence↗
PubMed1979

Rare transfer ribonucleic acid essential for phage growth. Nucleotide sequence comparison of normal and mutant T4 isoleucine-accepting transfer ribonucleic acid.

One of the eight tRNA species coded by bacteriophage T4 is unique in that (1) it is found in a yield lower by three- to fourfold than that of any other tRNA and (2) while dispensable for growth in standard laboratory hosts, it is essential for phage propagation in a natural isolate of Escherichia coli (strain CT439). We report here the nucleotide sequence of this tRNA and of several mutationally altered forms. The molecule is 77 nucleotides in length and has the anticodon N-A-U. Depending on the pairing properties of the "wobble" nucleotide N, this sequence could correspond to one or more of the isoleucine-specific codons (formula: see text) or to the methionine-specific codon A-U-G. Since a T4-specific acceptor activity for isoleucine which is stimulated in ribosome binding by A-U-A but not A-U-U has been reported previously, we infer that we have sequenced a tRNA Ile species which preferentially recognizes A-U-A. Mutant HA1 is unable to grow in CT439; it produces no tRNA Ile. The primary mutational alteration is a transition four residues from the 5'terminus which converts a C.G to a U.G base pair. The consequences of this lesion can be partially reversed by second-site mutations nearby in the acceptor stem. Unexpectedly, the tRNA Ile synthesized in these revertants still retains two unusual structural features found in the wild-type molecule: the opposition of two Up residues in the amino acid acceptor stem and the opposition of an Ap and a Gp residue in the anticodon stem. Implications of these structual anomalies for a possibly unique physiological role of this minor tRNA species are discussed.

Base Sequence↗
PubMed1979

S1 nuclease as a probe for the conformation of a dimeric tRNA precursor.

We have employed S1 nuclease to probe the structure of an intermediate in tRNA biosynthesis available only in radiochemical purity. The dimeric precursor to tRNAGln and tRNALeu from bacteriophage T4 was digested with the single-strand specific nuclease, and the products of the reaction were compared with the S1 digestion products of the mature cognate tRNA'S. Quantitation and sequence analysis of the products revealed that the location and accessibility of S1 cleavage sites in the precursor were substantially identical with those in the mature forms. Based on these conclusions, it is argued that the dimer is comprised of two domains in which the specific features of both secondary and tertiary conformation closely resemble those found in the mature molecules; at the same time we noted small but apparently significant differences in certain regions of the molecule which may reflect signals for various maturation events. Finally, we have determined that the sites of precursor cleavage by RNase P, the endonuclease which generates the mature 5' termini of these tRNAs, were completely inaccessible to S1 digestion.

Anticodon↗
PubMed1979

A precursor to a minor species of yeast tRNASer contains an intervening sequence.

Certain tRNAs in S. cerevisiae (tRNATyr and tRNAPhe) arise via precursor molecules which are mature at the 5' and 3' termini but contain intervening sequences adjacent to the anticodon (Knapp et al., 1978; O'Farrell et al., 1978). In addition to these molecules, precursors to several other tRNAs accumulate in a temperature-sensitive mutant (ts136) at the nonpermissive temperature. We have analyzed one of these species and shown that it is a precursor to a minor species of tRNASer. This precursor is also mature at both termini and contains an intervening sequence of 19 nucleotides adjacent to the hypermodified A residue 3' to the anticodon. The sequence can be arranged in a secondary structure in which the anticodon stem is extended by additional base-pairing, and contains the sites of excision and ligation within two looped regions. Support for this structure was provided by analysis of the products of limited digestion with RNAase T1. recently Piper (1978) reported the isolation of a minor species of tRNASer which decodes UCG. He found this species to be structurally heterogeneous and determined that the less abundant form corresponds to the tRNA which is altered in the recessive lethal SUP-RL1 amber suppressor. Our data now suggest that the more abundant form may be restricted to reading UCA in vivo; thus mutation of the minor species would result in complete loss of UCG-decoding ability and explain the recessive lethality of SUP-RL1. We have shown that the precursor which accumulates in ts136 corresponds exclusively to this minor tRNASerUCG species. Our results suggest that this may be the only gene for tRNASer in yeast which contains an intervening sequence.

Anticodon↗
PubMed1978

The nucleotide sequence of threonine transfer RNA coded by bacteriophage T4.

The nucleotide sequence of a low molecular weight RNA coded by bacteriophage T4 (and previously identified as species alpha) has been determined. The molecule is of particular biological interest for its associated biosynthetic properties. This RNA is 76 nucleotides in length, contains eight modified bases, and can be arranged in a cloverleaf configuration common to tRNAs. The anticodon sequence is UGU, which corresponds to the threonine-specific codons ACA G. The nucleotide sequence was determined primarily by nearest-neighbor analysis of RNA synthesized in vitro using [alpha-32P]nucleoside triphosphates. Using the single-strand specific nuclease S1, two in vivo labeled half-molecules were generated and analysed. This information together with restrictions imposed by nearest-neighbor data, provided a unique linear sequence of nucleotides with the features of secondary structure common to tRNA molecules.

Anticodon↗
PubMed1977

Nucleotide sequence of an arginine transfer ribonucleic acid from bacteriophage T4.

The nucleotide sequence of a phage T4-coded low molecular weight RNA, previously designated polyacrylamide gel band epsilon, has been determined. This RNA can be arranged in the cloverleaf configuration common to tRNAs, with an anticodon sequence, U-C-U, which corresponds to the arginine-specific codons A-G-A and A-G-G; it is therefore assumed to be an arginine tRNA. The complete nucleotide sequence of this RNA species is: pG-U-C-C-C-G-C-U-G-G-U-G-U-A-A-U-Gm2'-G-A-D-A-G-C-A-U-A-C-G-A-U-C-C-U-U-C-U-A-A-G-psi-U-U-G-C-G-G-U-C-C-U-G-G-T-psi-C-G-A-U-C-C-C-A-G-G-G-C-G-G-G-A-U-A-C-C-AOH. The nucleotide sequence was determined by analysis of RNA, uniformly labeled in vivo, according to the conventional techniques. In addition, RNA synthesized in vitro in the presence of alpha-32P-labeled nucleoside triphosphates was analyzed through the use of nearest neighbor sequencing techniques. Although a unique sequence could not be determined by this latter analysis, restrictions on the sequence imposed by nearest neighbor data and secondary structure common to tRNA molecules allowed prediction of the correct nucleotide sequence.

Arginine↗
PubMed1976

A functional requirement for modification of the wobble nucleotide in tha anticodon of a T4 suppressor tRNA.

Temperature-sensitive mutants of E. coli have been isolated which restrict the growth of strains of bacteriophage T4 which are dependent upon the function of a T4-coded amber or ochre suppressor transfer RNA. One such mutant restricts the growth of certain ochre but not amber suppressor-requiring phage. Analysis of the T4 tRNAs synthesized in this host revealed that many nucleotide modifications are significantly reduced. The modifications most strongly affected are located in the anticodon regions of the tRNA'S. The T4 ochre suppressor tRNAs normally contain a modified U residue in the wobble position of the anticodon; it has been possible to correlate tha absence of this specific modification in the mutant host with the restriction of suppressor activity. Furthermore, the extent of this restriction varies dramatically with the site of the nonsense codon, indicating that the modification requirement is strongly influenced by the local context of the mRNA. An analysis of spontaneous revertants of the E. coli ts mutant indicates that temperature sensitivity, restriction of phage suppressor function, and undermodification of tRNA are the consequences of a single genetic lesion. The isolation of a class of partial revertants to temperature insensitivity which have simultaneously become sensitive to streptomycin suggests that the translational requirement for the anticodon modification can be partially overcome by a change in the structure of the ribosome.

Anticodon↗
PubMed1974

Transfer RNA biosynthesis: the nucleotide sequence of a precursor to serine and proline transfer RNAs.

The nucleotide sequence of a transfer RNA precursor molecule coded by bacteriophage T4 has been determined. The molecule is a single polynucleotide chain which contains two transfer RNA species that are destined to recognize serine and proline. The 3' -CCA(OH) termini of both mature transfer RNA species are absent in the precursor molecule; these termini must therefore be added enzymatically at a subsequent stage of maturation. Nucleotide residues unique to the precursor are located at both ends of the molecule and between the two transfer RNA sequences.

Base Sequence↗
PubMed1972

Eight transfer RNAs induced by infection of Escherichia coli with bacteriophage T4.

Bacteriophage T4 induces the synthesis of eight transfer RNAs upon infection of E. coli. The tRNAs are easily detected and resolved into pure species by polyacrylamide gel electrophoresis of RNA labeled with (32)P after T4 infection. Two-dimensional fingerprints of RNase T(1) products derived from individual gel bands give patterns characteristic of single tRNAs. Furthermore, the T(1) digest of each gel band has a single oligonucleotide that contains the minor nucleotides Tp and Psip, a characteristic feature of all known tRNAs. Four larger RNAs are also seen in the polyacrylamide gels. Fingerprint and genetic analyses demonstrate that these molecules are related to the tRNAs, but the exact nature of this relationship is not known.

Base Sequence↗