PubMed Health⌕ Search

Biomedical subjects

K D Stuart

Publications and source records attributed to K D Stuart.

At least 37 records · Page 2Linked to original sources
PubMed1993

The alkane-inducible Candida maltosa ALI1 gene product is an NADH:ubiquinone oxidoreductase subunit homologue.

The ALI1 gene product in Candida maltosa was previously shown to be essential for n-alkane assimilation, possibly as a transcription factor [Hwang et al., Gene 106 (1991) 61-69]. We show that the predicted sequence is highly homologous to a subunit of respiratory complex I from another fungus, Neurospora crassa, and from Bos taurus. The predicted protein contains a motif conserved in this subunit from mitochondria, chloroplasts and bacteria. It also contains an N-terminal sequence that suggests a mitochondrial (mt) localization and a role for mt respiration in n-alkane assimilation.

Alkanes↗
PubMed1993

Structural organization of the maxicircle variable region of Trypanosoma brucei: identification of potential replication origins and topoisomerase II binding sites.

The maxicircle of the parasitic protozoan Trypanosoma brucei, one component of the mitochondrial genome, has size differences among isolates that localize to the variable region (VR) between the ND5 and 12S rRNA genes. We present here the nucleotide sequence of this entire region, thus completing the sequence of the maxicircle genome. We also find heterogeneously sized transcripts from throughout most of the VR. The VR has three distinct sections, each with characteristic repeated sequences. The repeated sequences in two sections are short and highly reiterated; the intraspecies size variation occurs within this region. The third section contains non-repetitive sequences and a large duplication immediately upstream of the 12S rRNA gene. Two repeat units within section I contain a sequence that has homology to the DNA replication origin of minicircles. This region also contains sequences with homology to topoisomerase II binding and cleavage sites. These findings suggest a role for the VR in DNA replication of the maxicircle.

Animals↗
PubMed1993

Extensive editing of CR2 maxicircle transcripts of Trypanosoma brucei predicts a protein with homology to a subunit of NADH dehydrogenase.

Several genes of the Trypanosoma brucei mitochondrial genome (the maxicircle) encode mRNAs that are so extensively altered by RNA editing that the gene cannot be identified by analysis of the DNA sequence. The 322-nucleotide preedited RNA of one of these genes, CR2, is converted into a 647-nucleotide transcript by the addition of 345 uridines and the deletion of 20 genomically encoded uridines. The fully edited transcript has an open reading frame that predicts a 194-amino-acid protein. This protein, which we name ND9 (NADH dehydrogenase subunit 9), has homology to a subunit of NADH dehydrogenase (respiratory complex I). Seven guide RNAs that can specify edited CR2 sequence have been identified. Steady-state levels of unedited ND9 transcripts are greater in bloodstream than in procyclic forms, but edited ND9 mRNA is present in similar abundance in both life cycle stages.

Amino Acid Sequence↗
PubMed1992

Molecular organization of Leishmania RNA virus 1.

The complete 5284-nucleotide sequence of the double-stranded RNA genome of Leishmania RNA virus 1 (LRV1) was determined and contains three open reading frames (ORFs) on the plus (+) (mRNA) strand. The predicted amino acid sequence of ORF3 has motifs characteristic of viral RNA-dependent RNA polymerases. ORF2, which may encode the major viral coat protein, overlaps ORF3 by 71 nucleotides, suggesting a +1 translational frameshift to produce a gag-pol type of fusion protein. Two alternative models for the frameshift are presented. The 5' splice leader sequence of kinetoplastid mRNAs is not in LRV1 RNA. This suggests that the 450-base region at the 5' end of the LRV1 (+)-strand, which contains ORF1 and is highly conserved among viral strains, does not encode protein but has a role in initiation of translation and/or RNA stability. The similarity of LRV1 genomic organization, replication cycle, and RNA-dependent RNA polymerase sequence to those of the yeast virus ScV L-A suggests a common ancestral origin. The possibility that LRV1 affects pathogenesis in leishmaniasis is intriguing.

Amino Acid Sequence↗
PubMed1992

A multicopy, extrachromosomal DNA in Leishmania infantum contains two inverted repeats of the 27.5-kilobase LD1 sequence and encodes numerous transcripts.

Leishmania DNA 1 (LD1) is a 27.5-kb sequence that occurs as an inverted repeat in a 55-kb multicopy, circular DNA in Leishmania infantum ITMAP263. The sequence is also found with a different genomic organization, possibly a tandem array, within a 1.5-Mb chromosome in all Leishmania isolates. About 26 stable transcripts of LD1 sequence, ranging from 0.6 to 15 kb, are found in ITMAP263. Transcripts were detected from both strands of the entire LD1 sequence, but the inverted repeat nature of the circular molecule prevented determination of whether transcription proceeded in one or both directions. Nine abundant transcripts (0.6-8.4 kb) from adjacent regions on the same strand of the repeat unit may represent mature mRNAs. One of these transcripts was shown to contain the 39-nucleotide spliced leader sequence characteristic of the 5' termini of trypanosomatid mRNAs. Several transcripts from the other strand of the repeat unit are also abundant and contain sequence complementary to some of the putative mRNAs. Less abundant, larger transcripts that span sequences encoding abundant mRNAs are also present, suggesting that transcription of LD1 is polycistronic.

Animals↗
PubMed1989

A novel telomeric gene conversion in Trypanosoma brucei.

Gene conversion is one mechanism of antigenic variation in Trypanosoma brucei. Variant surface glycoprotein (VSG) genes are duplicated by this process to telomeric locations from which they may be expressed. We examined four independent antigenic switches in which the IsTaR 1.1 minichromosomal VSG gene is duplicated to a large chromosome where it is expressed. An unusual feature of three of these telomeric gene conversions is that the distance between the VSG gene and the end of the chromosome is identical for both the basic and duplicated copies following the antigenic switch. This suggests that the gene conversion is initiated 5' to the VSG gene and extends to the end of the telomere. The data also suggest that events other than simple nucleotide addition account for telomeric growth.

Animals↗
PubMed1989

Trypanosoma brucei: frequent loss of a telomeric variant surface glycoprotein gene.

We have observed the loss of an inactive telomeric variant surface glycoprotein (VSG) gene that is located on a minichromosome in Trypanosoma brucei. If this is due to gene conversion, it is the third "silent" gene conversion (i.e., one that does not produce an antigenic switch) detected in 19 antigenic switches of the IsTaR 1 serodeme. This is surprisingly frequent since the immune response cannot select against the inactive gene. We estimate that 10(-1) to 10(-3) telomeric VSG gene conversions occur per generation, which is at least 100 times more frequent than antigenic switching. Since all three "silent" gene conversions involved an IsTat 5 VSG gene, the frequency may vary among telomeric VSG genes. However, the high gene conversion frequency for the 5 VSG gene does not ensure a higher antigenic switch frequency than other telomeric VSG genes for which we have probes. These results suggest that gene conversion rapidly alters the repertoire of telomeric VSG genes, possibly including those on minichromosomes, producing a continual variation in the VSG genes that are more likely to be expressed.

Animals↗
PubMed1988

Multiple events associated with antigenic switching in Trypanosoma brucei.

The genomic environments of six unrelated variant surface glycoprotein (VSG) gene families in the IsTaR 1 serodeme of Trypanosoma brucei were examined by Southern blot analysis of DNA resolved by conventional and pulse field gradient electrophoresis. The genomic locations of these gene family members were characterized in variants arising from 19 independent antigenic switches of which 13 represent single relapses. We have identified 9 distinct telomeric sites containing VSG genes on 7 different chromosomes ranging in size from approximately 65 kilobase pairs to greater than 3 megabase pairs. VSG genes are expressed in at least 6 of these telomeric sites. All of the observed antigenic switches can be explained by two types of processes: gene conversion and telomeric activation, with almost half (9/19) the result of combinations of these processes. The implications of these observations are discussed with particular reference to the frequencies of occurrence of each process.

Animals↗
PubMed1988

Changes in telomere length associated with antigenic variation in Trypanosoma brucei.

In the IsTaR 1 serodeme, we have identified variant surface glycoprotein (VSG) genes in nine different telomeric sites. We have measured the distance from the 3' end of these VSG genes to the end of the chromosome (the 'telomere length') in 20 variant antigen types (VATs) of the serodeme. Analyses of the changes in telomere length during 19 antigenic switches involving eight telomeric sites indicate a median increase in telomere length of 0.6 kilobase pairs during each switch. This may be accounted for by the 6-10 bp increase in telomere length per generation associated with DNA replication described by others. The changes in telomere lengths do not form a normal distribution since a substantial fraction show unusually large increases in telomere length or decreases in telomere length during an antigenic switch. These changes are probably caused by recombinations 3' to the VSG gene. No significant differences were detected in the behavior of telomeres at each of the eight different telomeric sites, nor were changes in telomere lengths significantly different between different antigenic switches. However, it was found that those telomeres where transcription was activated during the antigenic switch showed a significantly greater increase in telomere length than those telomeres not involved in regulation of VSG gene expression. Conversely, there was a strong correlation between transcriptional inactivation of a telomeric expression site and a decrease in telomere length. These findings suggest that processes (possibly genomic recombinations) 3' to the VSG gene coding region may be associated with a change in the transcriptional status of the VSG gene.

Animals↗
PubMed1988

Variant specific transcripts from the co-transposed segments of variant surface glycoprotein genes in Trypanosoma brucei.

We examined steady-state transcripts from the sequences immediately upstream of the 1 and 11 variant surface glycoprotein (VSG) genes in Trypanosoma brucei of the IsTaR 1 serodeme. These sequences, the co-transposed segment, differ in size and sequence, and produce a variety of variant-specific polyadenylated transcripts. Most, if not all, of these transcripts, some of which may be precursors, contain the spliced leader (SL). Variant specific transcription extends from 5' of the co-transposed segment to 100-400 nucleotides downstream of the major VSG mRNA polyadenylation site. The 1 VSG mRNA has at least 2, and possibly 3, SL addition sites; the major site is 120 nucleotides downstream of the polyadenylation site identified by sequencing a cDNA from the co-transposed segment. Two general models for the production of the co-transposed segment transcripts are discussed.

Animals↗
PubMed1982

Localization of kinetoplast DNA maxicircle transcripts in bloodstream and procyclic form Trypanosoma brucei.

Over 80% of the maxicircle and numerous minicircles of Trypanosoma brucei kinetoplast DNA have been cloned. The uncloned maxicircle segment contains few restriction endonuclease cleavage sites, varies in size among strains, and may be unstable in conventional cloning systems. cDNA prepared to bloodstream or procyclic trypomastigote RNA hybridized to all but one maxicircle segment, but did not hybridize to minicircles. Fourteen maxicircle transcripts were detected in RNA from both bloodstream and procyclic trypomastigotes. The coding sequences for these transcripts were localized and account for most of the maxicircle. One region of the maxicircle, which borders the variable region, was not found to be transcribed. We conclude that the maxicircle is largely but not completely transcribed in both bloodstream and procyclic trypomastigotes, whereas minicircle transcription is minimal or absent in these stages. Qualitative transcriptional differences which could account for mitochondrial respiratory differences between the bloodstream and procyclic trypomastigotes were not observed.

Chromosome Mapping↗
PubMed1971

Evidence for the retention of kinetoplast DNA in an acriflavine-induced dyskinetoplastic strain of Trypanosoma brucei which replicates the altered central element of the kinetoplast.

A pleomorphic dyskinetoplastic strain of Trypanosoma brucei was produced by repeated acriflavine treatment. No kinetoplastic cells reappeared after 2 yr of maintenance in the absence of acriflavine. These dyskinetoplastic cells retained and therefore replicated the central element of the kinetoplast. This element was present in the "condensed" state typical of acriflavine-treated cells rather than the normal fibrillar state. Whole-cell DNA extracted from both normal and dyskinetoplastic strains revealed three bands upon isopycnic sedimentation, and there was no detectable alteration in buoyant density of any of these DNA components in the dyskinetoplastic strain. It seems likely that the dyskinetoplastic strain has retained its kinetoplast DNA but in an altered state.

Acridines↗