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Biomedical subjects

I J Purvis

Publications and source records attributed to I J Purvis.

13 recordsLinked to original sources
PubMed1996

Conservation of synteny between the genome of the pufferfish (Fugu rubripes) and the region on human chromosome 14 (14q24.3) associated with familial Alzheimer disease (AD3 locus)

The genome of the pufferfish (Fugu rubripes) (400 Mb) is approximately 7.5 times smaller than the human genome, but it has a similar gene repertoire to that of man. If regions of the two genomes exhibited conservation of gene order (i.e., were syntenic), it should be possible to reduce dramatically the effort required for identification of candidate genes in human disease loci by sequencing syntenic regions of the compact Fugu genome. We have demonstrated that three genes (dihydrolipoamide succinyltransferase, S31iii125, and S20i15), which are linked to FOS in the familial Alzheimer disease focus (AD3) on human chromosome 14, have homologues in the Fugu genome adjacent to Fugu cFOS. The relative gene order of cFOS, S31iii125, and S20i15 was the same in both genomes, but in Fugu these three genes lay within a 12.4-kb region, compared to >600 kb in the human AD3 locus. These results demonstrate the conservation of synteny between the genomes of Fugu and man and highlight the utility of this approach for sequence-based identification of genes in human disease loci.

Acyltransferases↗
PubMed1993

Enzymatic production of optically pure (2'R-cis)-2'-deoxy-3'-thiacytidine (3TC, lamivudine): a potent anti-HIV agent.

Although equipotent in terms of antiviral activity, the two enantiomers of 2'-deoxy-3'-thiacytidine (BCH 189) differ markedly in their cytotoxicity. (2'R-cis)-2'-deoxy-3'-thiacytidine (3TC) is substantially less toxic than its optical antipode, and is undergoing development for the therapy of HIV infection. Cytidine deaminase from Escherichia coli is shown here to deaminate 2'-deoxy-3'-thiacytidine enantioselectively to leave 3TC essentially optically pure. This reaction has been used to develop a process for production of 3TC in multikilogram amounts. The production of cytidine deaminase was enhanced by strain improvement, fermentation development, and finally by cloning and overexpression of the gene. The enzyme was immobilized on Eupergit-C, which allowed it to be reused many times. The biotransformation conditions were optimized so that the best use could be made of the catalyst. A robust scaleable product isolation process was developed to yield the crystalline product. Overall, yields through the resolution process of 76% were obtained. All aspects of this process are capable of substantial further scaleup with only minor modifications.

Antiviral Agents↗
PubMed1991

An androgen-inducible expression system for Saccharomyces cerevisiae.

A novel controllable expression system for Saccharomyces cerevisiae has been developed. Expression of the gene encoding the human androgen receptor, from a strong yeast promoter, results in transactivation of a hybrid promoter carrying androgen-responsive sequences such that a target gene may be expressed in an androgen-dependent manner. By selection of an appropriate combination of androgen receptor level, target-gene copy number and concentration of the androgenic ligand, dihydrotestosterone, the expression level can be set within a 1400-fold range with no detectable effect on normal cell growth.

Androgens↗
PubMed1989

The yeast pyruvate kinase gene does not contain a string of non-preferred codons: revised nucleotide sequence.

The sequence of the gene encoding pyruvate kinase from Saccharomyces cerevisiae was re-determined because of failures with oligonucleotide-directed mutagenesis experiments involving a region thought to contain a string of five contiguous non-preferred codons. This region was found to be difficult to sequence and was shown to have three extra bases when compared with the published sequence [(1983) J. Biol. Chem. 258, 2193-2201]. The revised sequence demonstrates that the yeast pyruvate kinase gene does not have a cluster of non-preferred codons, and that it therefore is not an example of the class of genes which possibly exhibit translational control by the presence of non-preferred codons.

Amino Acid Sequence↗
PubMed1988

Messenger RNA degradation in Saccharomyces cerevisiae.

The analysis of 17 functional mRNAs and two recombinant mRNAs in the yeast Saccharomyces cerevisiae suggests that the length of an mRNA influences its half-life in this organism. The mRNAs are clearly divisible into two populations when their lengths and half-lives are compared. Differences in ribosome loading amongst the mRNAs cannot account for this division into relatively stable and unstable populations. Also, specific mRNAs seem to be destabilized to differing extents when their translation is disrupted by N-terminus-proximal stop codons. The analysis of a mutant mRNA, generated by the fusion of the yeast PYK1 and URA3 genes, suggests that a destabilizing element exists within the URA3 sequence. The presence of such elements within relatively unstable mRNAs might account for the division between the yeast mRNA populations. On the basis of these, and other previously published observations, a model is proposed for a general pathway of mRNA degradation in yeast. This model may be relevant to other eukaryotic systems. Also, only a minor extension to the model is required to explain how the stability of some eukaryotic mRNAs might be regulated.

Cloning, Molecular↗
PubMed1987

The effects of alterations within the 3' untranslated region of the pyruvate kinase messenger RNA upon its stability and translation in Saccharomyces cerevisiae.

A 53 basepair deletion was constructed within the 3' untranslated region (3' UTR) of the yeast pyruvate kinase (PYK) gene borne upon a centromeric plasmid. Various modular assemblies of the pUC13 polylinker DNA (single unit = 44 bp) were used to replace the deleted region, and the effects of these modifications upon both transcript stability and translation ascertained in yeast. The use of a differential probing stratagem, based on the hybridisation of specific oligonucleotides to either pUC13 polylinker or unaltered PYK 3' UTR sequences, allowed for discrimination between mutant (plasmid borne) and wild-type (chromosomal) PYK transcripts. In no construct was there any significant alteration in mRNA stability, but translation of the PYK mRNA was severely curtailed by truncation of the 3' UTR or the presence of a strong hairpin-loop structure in the 3' UTR. A specific mutation in the N-terminal coding sequences, which created a premature termination codon in both a 3' 'tagged' PYK plasmid and a PYK/LacZ fusion gene, aborted the translation of a majority of their transcripts but left their chemical half-lives unaltered. This observation is at variance with some previously published data (Losson & Lacroute (1979) Proc Natl Acad Sci USA 76, 5134; Pelsey & Lacroute (1984) Curr Genet 8, 277), but is consistent with our own earlier observation that there is no obvious link between ribosome loading and mRNA stability in yeast (Santiago et al. (1986) Nucleic Acids Res 14, 8347). Possible reasons for this disparity are discussed.

Fungal Proteins↗
PubMed1987

Translation and stability of an Escherichia coli beta-galactosidase mRNA expressed under the control of pyruvate kinase sequences in Saccharomyces cerevisiae.

Plasmids were assembled in which the coding region of the pyruvate kinase (PYK) gene of Saccharomyces cerevisiae was replaced by that of the B-galactosidase (LacZ) gene from Escherichia coli. Analysis of the resultant, chimaeric transcripts from low copy number, centromeric plasmids indicated that this substitution caused a dramatic reduction in the steady-state level of the messenger RNA (mRNA). This fluctuation cannot be wholly accounted for by the 2-fold decrease in mRNA stability observed. This is consistent with the existence of a transcriptional Downstream Activation Site (DAS) within the PYK coding region, analogous to the DAS reported within the yeast phosphoglycerate kinase gene (PGK; Kingsman, S M et al. (1985) Biotech. Gen. Eng. Rev. 3, 377). At these low levels of heterologous gene expression, comparison of the distribution of PYK and PYK/LacZ transcripts across polysome gradients revealed no significant effect mediated by their striking disparity in codon usage. Nevertheless, upon increasing B-galactosidase mRNA levels, via manipulation of plasmid copy number, a distinct decline in ribosome loading was observed for the heterologous PYK/LacZ transcript which was not mirrored by either endogenous PYK transcripts or other yeast mRNAs of high (Ribosomal protein 1) or moderate (Actin) codon bias. However, high levels of the PYK/LacZ mRNA did affect the translation of an endogenous mRNA with poor codon bias (TRP2). The possible basis for this phenomenon is discussed.

Bacterial Proteins↗
PubMed1987

Messenger RNA stability in Saccharomyces cerevisiae: the influence of translation and poly(A) tail length.

A comparison between the half-lives of 10 specific yeast mRNAs and their distribution within polysomes (fractionated on sucrose density gradients) was used to test the relationship between mRNA translation and degradation in the eukaryote Saccharomyces cerevisiae. Although the mRNAs vary in their distribution across the same polysome gradients, there is no obvious correlation between the stability of an mRNA and the number of ribosomes it carries in vivo. This suggests that ribosomal protection against nucleolytic attack is not a major factor in determining the stability of an mRNA in yeast. The relative lengths of the poly(A) tails of 9 yeast mRNAs were analysed using thermal elution from poly(U)-Sepharose. No dramatic differences in poly(A) tail length were observed amongst the mRNAs which could account for their wide ranging half-lives. Minor differences were consistent with shortening of the poly(A) tail as an mRNA ages.

Half-Life↗
PubMed1987

The efficiency of folding of some proteins is increased by controlled rates of translation in vivo. A hypothesis.

We propose that the way in which some proteins fold is affected by the rates at which regions of their polypeptide chains are translated in vivo. Furthermore, we suggest that their gene sequences have evolved to control the rate of translational elongation such that the synthesis of defined portions of their polypeptide chains is separated temporally. We stress that many proteins are capable of folding efficiently into their native conformations without the help of differential translation rates. For these proteins the amino acid sequence does indeed contain all the information needed for the polypeptide chain to fold correctly (even in vitro, after denaturation). However, other proteins clearly do not fold efficiently into their native conformation in vitro. We argue that the efficiency of folding of these problematic proteins in vivo may be improved by controlled synthesis of the nascent polypeptide.

Codon↗
PubMed1986

The relationship between mRNA stability and length in Saccharomyces cerevisiae.

A rapid and convenient procedure has been developed for the measurement of mRNA half-life in S.cerevisiae using the transcriptional inhibitor, 1,10-phenanthroline. A range of half-lives from 6.6 +/- 0.67 minutes to over 100 minutes, relative to the stability of the 18S rRNA control, has been obtained for fifteen mRNAs. They include the pyruvate kinase and actin mRNAs, as well as 13 randomly picked mRNAs of unknown function. The mRNAs clearly fall into two populations when their lengths and half-lives are analysed; one population is considerably more stable than the other when mRNAs of similar length are compared. Also, within each population, there is an inverse relationship between mRNA length and half-life. These results suggest that mRNA length and at least one additional factor strongly influence mRNA stability in yeast.

Cloning, Molecular↗
PubMed1983

Isolation and characterisation of DraI, a type II restriction endonuclease recognising a sequence containing only A:T basepairs, and inhibition of its activity by uv irradiation of substrate DNA.

A type II restriction endonuclease, DraI, isolated from Deinococcus radiophilus ATCC 27603 recognises the palindromic hexanucleotide sequence (formula; see text) and cleaves it, as indicated by the arrows, to produce blunt-ended fragments. The yield of enzyme is 100 to 1000 times that of the only other known type II restriction endonuclease that recognises a sequence composed solely of A:T basepairs, the isoschizomer AhaIII (1). Ultraviolet irradiation of the DNA substrate at relatively low doses inhibits the activity of DraI by "protecting" the recognition sequence and this may be exploited to give control of partial digestion of DNA by DraI.

Adenine↗