Biomedical subjects

N B Murphy

Publications and source records attributed to N B Murphy.

15 recordsLinked to original sources

PubMed1991

The Trypanosoma brucei DNA polymerase alpha core subunit gene is developmentally regulated and linked to a constitutively expressed open reading frame.

As an initial step towards the characterization of replicative DNA polymerases of trypanosomes, we have cloned, sequenced and examined the expression of the Trypanosoma (Trypanozoon) brucei brucei gene that encodes the DNA polymerase alpha catalytic core (pol alpha). The protein sequence contains the six conserved regions that have been recognized previously in eukaryotic and viral replicative DNA polymerases. In addition, we have identified a seventh region which appears to be conserved primarily in alpha-type DNA polymerases. The T.brucei DNA pol alpha core N-terminus is 123 and 129 amino acids smaller than that of the human and yeast homologue, respectively. The gene is separated by 386 bp from an upstream open reading frame (ORF) of 442 codons. Stable transcripts of the upstream sequence are detected in both dividing and non-dividing forms, while pol alpha transcripts are detected principally in dividing forms. Allelic copies of the T.brucei pol alpha region exhibit restriction site polymorphisms; one such sequence polymorphism affects the amino acid sequence of the T.brucei DNA pol alpha core. The T.brucei pol alpha region cross-hybridizes weakly with that of T.(Nannomonas) congolense and T.(Duttonella) vivax.

Amino Acid Sequence↗

PubMed1991

Variation in resistance to isometamidium chloride and diminazene aceturate by clones derived from a stock of Trypanosoma congolense.

Nine clones were derived from a drug-resistant Trypanosoma congolense stock (IL 2856) and characterized in mice for their sensitivity to isometamidium chloride and diminazene aceturate. All clones were derived from the stock without drug selection and expressed high levels of resistance to isometamidium chloride (50% curative dose [CD50] values ranging from 1.5 to 5.1 mg/kg) and intermediate to high levels of resistance to diminazene aceturate (CD50 values ranging from 5.1 to 21.0 mg/kg). By contrast, the isometamidium chloride and diminazene aceturate CD50 values for a drug-sensitive clone, T. congolense IL 1180, were 0.018 mg/kg and 2.3 mg/kg, respectively. For both drugs, there appeared to be significantly different levels in expression of drug resistance amongst the 9 clones derived from IL 2856. Isoenzyme analysis of 7 enzymes showed that all 9 clones expressed the same electrophoretic variants. Thus, all 9 clones were identical for these phenotypic markers. The clone which expressed the highest level of resistance to isometamidium in mice (IL 3270) was transmitted to Boran cattle via the bite of infected Glossina morsitans centralis. IL 3270 produced an infection rate in tsetse of 5.0%. The resulting infections in cattle were shown to be resistant to intramuscular treatment with 2.0 mg/kg isometamidium chloride and 14.0 mg/kg diminazene aceturate. This contrasts with doses of 0.25 mg/kg isometamidium chloride or 3.5 mg/kg diminazene aceturate which are deemed sufficient to cure fully sensitive infections. Finally, 9 clones (subclones) were derived from IL 3270 and characterized in mice for their sensitivity to isometamidium chloride.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Putative genes of a variant-specific antigen gene transcription unit in Trypanosoma brucei.

In a 7-kilobase (kb) sequence upstream from the 5' barren region, the Trypanosoma brucei AnTat 1.3A expression site carries two putative genes, named ESAG 2 and ESAG 3 for expression site-associated genes, as well as a copy of ESAG 1 (D.F. Cully, H.S. Ip, and G.A.M. Cross, Cell 42:173-182, 1985). At least 3 kb of this expression site exhibits a high degree of homology with the silent telomere carrying the AnTat 1.3A basic copy, whose ESAG 1 is interrupted by stop codons. Like the antigen gene, the region containing the ESAGs is transcribed only in the bloodstream forms, although transcription of 5' barren- and ESAG 2-related sequences also occurs in cultured procyclics. Analysis of steady-state and nascent transcripts suggests a continuous transcription of the whole expression site by an RNA polymerase resistant to alpha-amanitin, possibly initiating at a polymerase I-like promoter located about 17 kb upstream from the antigen gene. This polymerase seems prone to becoming inactivated upon incubation of the trypanosomes at low temperature. The putative protein encoded by ESAG 3 may carry a hydrophobic signal peptide, suggesting interaction with a membrane.

Animals↗

PubMed1987

DNA-binding fingers encoded by a trypanosome retroposon.

A Trypanosoma brucei repeated DNA element (TRS-1, for trypanosome repeated sequence), which seems transposable, may encode a 1651 amino acid polypeptide showing homology with reverse transcriptase. This polypeptide would also carry a DNA-binding domain, as suggested by the presence of five DNA-binding "fingers" homologous to those of the transcription factor TFIIIA of Xenopus laevis and retroviral DNA-binding proteins.

Animals↗

PubMed1987

Trypanosoma brucei repeated element with unusual structural and transcriptional properties.

The genome of Trypanosoma brucei contains up to 400 copies of a conserved sequence (TRS, trypanosome repeated sequence). The majority of TRS copies (TRS1) are 5.2 X 10(3) base-pairs (kb) and are flanked by different separate halves of the previously described transposable element RIME (ribosomal mobile element), although a variant copy (TRS2) contains only the central 1.45 kb portion and lacks RIME. TRS1 elements can probably undergo transposition, since they are dispersed in all chromosome size classes and are bordered by direct repeats of about four base-pairs. Some TRS1 elements may contain an open reading frame over almost their entire length (1651 codons), encoding a protein showing homology with reverse transcriptase. TRS probes detect poly(A)+ transcripts of 5 to 9 kb, generated by a polymerase moderately sensitive to alpha-amanitin. Transcription is developmentally regulated. Both TRS and RIME sense transcripts are preferentially synthesized compared to anti-sense transcripts, and are much more abundant in bloodstream forms than in cultured procyclics.

Animals↗

PubMed1987

Expression of the gene for NAD-dependent glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides cloned in Escherichia coli K-12.

The structural gene (zwf) for Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase has been cloned into Escherichia coli on pBR322 by selecting for complementation of a zwf mutation of E. coli which eliminates glucose-6-phosphate dehydrogenase. The gene has been subcloned on a 3.4-kilobase DNA segment. The encoded glucose-6-phosphate dehydrogenase displays the dual nucleotide specificity (reacting with NAD and NADP) of the L. mesenteroides enzyme and has a subunit Mr of 55,000. A second gene encoding protein of subunit Mr 24,000 is also encoded on the 3.4-kilobase DNA segment. The genes have been located by the analysis of deletions and insertions generated in vitro and by transcriptional mapping with a promoterless chloramphenicol acetyl transferase cartridge inserted at different sites in the 3.4-kilobase fragment.

Cloning, Molecular↗

PubMed1977