Search PubMed⌕ Search

Biomedical subjects

P Borst

Publications and source records attributed to P Borst.

At least 217 records · Page 12Linked to original sources
PubMed1984

Discontinuous synthesis of mRNA in trypanosomes.

Many trypanosome mRNAs have the same sequence of 35 nucleotides at their 5' end, encoded by a mini-exon located in 1.35-kb tandemly linked repeats. We have analysed nascent and steady-state mini-exon transcripts to determine how the mini-exon sequence is joined to the main part of trypanosome mRNAs. We show here that steady-state RNA from Trypanosoma brucei contains a transcript of 141 nucleotides that starts at the 5' border of the mini-exon. Isolated nuclei transcribe the segment corresponding to the 141 nucleotide RNA at a high rate; transcription of other areas of the 1.35-kb mini-exon repeat is approximately 750-fold lower. We propose that transcription of protein-coding genes in trypanosomes is discontinuous and involves the 141-nucleotide transcript as an intermediate.

Animals↗
PubMed1984

Deficiency of plasmalogens in the cerebro-hepato-renal (Zellweger) syndrome.

We have analyzed the phospholipid composition of various organs of patients with the cerebro-hepato-renal (Zellweger) syndrome. The phospholipid composition of tissues from controls and patients was very similar except for their plasmalogen contents. In controls about 50% of the phosphatidylethanolamine fraction of brain, heart, kidney and skeletal muscle and about 10% of that fraction in control liver tissue was found to consist of plasmalogen. In control heart muscle, but not in other control tissues about 25% of the phosphatidylcholine fraction consist of plasmalogens. In contrast, plasmalogens were nearly absent in the corresponding tissues of Zellweger patients. The amount of phosphatidylethanolamine plasmalogens in both erythrocytes and fibroblasts of Zellweger patients is lowered significantly compared to control erythrocytes and control fibroblasts respectively, although this reduction is not as dramatic as in brain, heart, kidney, skeletal muscle and liver of patients. Phosphatidylcholine-plasmalogens are only present in low amounts in both controls, heterozygotes and patients. In recent years considerable evidence has accumulated to show that peroxisomes are involved in cellular lipid metabolism. Notably, the key enzymes of ether lipid (plasmalogen) biosynthesis in rodents were recently found to be located in peroxisomes. Since electronmicroscopic studies have shown that peroxisomes are absent in liver and kidney of patients with the cerebro-hepato-renal syndrome, our results suggest that an inability to integrate these key enzymes in a functional peroxisome leads to a severe disturbance in plasmalogen biosynthesis. We propose that the multiple clinical and biochemical defects in Zellweger patients are secondary to a deficiency in peroxisomal function.

Brain Diseases↗
PubMed1984

Two modes of activation of a single surface antigen gene of Trypanosoma brucei.

Several genes for variant antigens in trypanosomes are activated by duplicative translocation to a telomeric expression site. A second--nonduplicative--mode of activation is restricted to telomeric antigen genes. We show here that the single telomeric gene for antigen 221 can be activated in both ways. We also show that gene 221 is split and that the 5' 35 nucleotide sequence, common to all surface antigen mRNAs, is not encoded within 8.5 kb upstream of the 221 coding region. No major rearrangements are observed within 55 kb upstream of the 221 coding region upon nonduplicative activation. Gene inactivation is usually accompanied by deletion of the gene and at least 8.5 kb upstream and may involve conversion by another telomere. These results are not readily explained by a single expression site model. The duplicative gene 221 activation differs from conventional duplicative activation in the extent of the transposed segment, which is larger and may include the entire segment between gene and telomere.

Animals↗
PubMed1984

Chromosome rearrangements in Trypanosoma brucei.

We have studied chromosome rearrangements in T. brucei using pulsed field gradient gel electrophoresis to separate chromosome-sized DNA molecules. We detect size changes in a set of small chromosomes (200-700 kb) at a frequency of 10(-5) to 10(-6) per trypanosome division; this results in a radical difference in the size distribution of these chromosomes in different T. brucei isolates. Several of these chromosome rearrangements can be related to a change in the expression of surface antigen genes. Such rearrangements may be undetectable by standard gel electrophoresis and Southern blot analysis because the DNA segment transferred is too large to detect the breakpoint with the antigen gene probe. We also provide additional evidence for the notion that transcription of protein-coding genes in T. brucei and related flagellates is discontinuous. The possibility that gene rearrangements are essential for all changes in variant surface gene expression remains open.

Animals↗
PubMed1984

Structure of the growing telomeres of Trypanosomes.

We have developed a method for the molecular cloning of DNA adjacent to chromosome ends (telomeres). A recombinant DNA clone obtained from the telomeres of the protozoan Trypanosoma brucei contains large stretches of the repeat (CCCTAA)n. This repeat is flanked by a larger subtelomeric repeat (29 bp in one case). These repeats account for the presence of large DNA stretches not cut by restriction enzymes downstream of telomeric VSG genes. All telomeres analyzed thus far (more than 30) grow by approximately 6 bp per trypanosomal division and contract by occasional large deletions. Our results suggest that growth is due mainly to addition of CCCTAA units.

Animals↗
PubMed1984

Antigenic variation in Trypanosoma brucei analyzed by electrophoretic separation of chromosome-sized DNA molecules.

Pulsed field gradient gel electrophoresis fractionates chromosome-sized DNA molecules from T. brucei. About 60% of the DNA remains in or close to the gel slot (large DNA). There are about three chromosomes of approximately 2 Mb, at least six chromosomes of 200-700 kb, and roughly a hundred mini-chromosomes of 50-150 kb. The basic copy genes for VSGs 118 and 221 reside in large DNA. Their activation by duplicative transposition leads to the appearance of an additional copy in the 2 Mb DNA, showing that activation involves an interchromosomal gene transposition. When gene 221 is activated without duplication, it remains in large DNA, proving that at least two sites for expression of VSG genes exist. In support of this, the mini-exons encoding the 5' 35 nucleotides of VSG messenger RNAs are in large and 2 Mb DNA. The mini-chromosomes hybridize strongly to VSG gene probes and are absent in C. fasciculata. We suggest that their main function is to provide a large pool of telomeric VSG genes.

Animals↗
PubMed1983

Telomere conversion in trypanosomes.

Activation of the gene coding for variant surface glycoprotein (VSG) 118 in Trypanosoma brucei proceeds via a duplicative transposition to a telomeric expression site. The resulting active expression-linked extra copy (ELC) is usually flanked by DNA that lacks sites for most restriction enzymes and that is thought to interfere with the cloning of the ELC as recombinant DNA in Escherichia coli. We have circumvented this problem by cloning an aberrant 118 ELC gene, flanked at the 3'-side by at least 1 kb DNA, that contains restriction enzyme sites. Our analysis shows that this DNA and the 3'-end of the 118 ELC gene are derived from another VSG gene (1.1006) that is permanently located at a telomeric position. We propose that the 3'-end of the 1.1006 gene and (all of) its 3' flanking sequence moved to the expression site by a telomere conversion. Such a telomere conversion can also account for the appearance of an extra copy of the 1.1006 gene detected in a sub-population of our trypanosome strain.

Amino Acid Sequence↗
PubMed1983

Size fractionation of Trypanosoma brucei DNA: localization of the 177-bp repeat satellite DNA and a variant surface glycoprotein gene in a mini-chromosomal DNA fraction.

We have size-fractionated intact DNA from Trypanosoma brucei into a major large DNA fraction (greater than 350S) and minor middle-sized (60-250S) and small (less than 60S) DNA fractions. Large DNA contains the rRNA genes, the basic copy genes for several variant surface glycoproteins (VSGs), including one which lies near a telomer, and the expression-linked copies of the two VSG genes. The middle-sized DNA contains at least one VSG gene, but the hybridization of this fraction with probes for the conserved repetitive sequences that mark the edges of the transposed segments of VSG genes, suggests that it may contain many VSG genes. The 177-bp repeat satellite DNA is also exclusively found in this fraction.

Animals↗
PubMed1983

Characterization of satellite DNA in Trypanosoma brucei and Trypanosoma cruzi.

We have determined the properties of the simple-sequence satellite DNAs from two protozoa, Trypanosoma brucei and Trypanosoma cruzi. The T. brucei satellite DNA contains 29 mol% guanine plus cytosine and is made up of long tandem arrays of a 177 base-pair repeat. Sequence heterogeneity in these repeats is limited and restricted to certain positions as shown by sequence analysis, restriction enzyme digestion and two-dimensional analysis of nucleotides bordering the AluI and HhaI recognition sites in the repeat. The repeat contains two copies of a 19 base-pair sequence differing by a single base-pair substitution and several additional copies of part of this sequence. Sequence variants of the repeat are clustered in the DNA. Satellite DNA is not detectably linked to other DNA and no transcripts of this DNA are found in T. brucei. The T. cruzi satellite DNA repeat is 196 base-pairs long and contains 53 mol% guanine plus cytosine. Direct repetitions longer than eight base-pairs were not observed in the nucleotide sequence of this repeat. The nucleotide sequences of the satellites of T. brucei and T. cruzi are not related. In cell fractionation experiments, the T. brucei and T. cruzi satellite DNAs were recovered from the nuclear fraction. Micrococcal nuclease digestion of nuclear fractions yielded 193 and 197 base-pair nucleosomal oligomers in T. brucei and T. cruzi, respectively; these oligomers contained satellite DNA but not the extranuclear kinetoplast DNA. The 193 base-pair nucleosomal repeat of T. brucei is significantly different from the 177 base-pair satellite repeat. Satellite and nucleosomal repeats are, therefore, not in phase in T. brucei. These satellite DNAs are the first to be observed in protozoa, and we conclude that their properties are similar to those of satellites from animals or plants.

Animals↗
PubMed1983

The transposition unit of variant surface glycoprotein gene 118 of Trypanosoma brucei. Presence of repeated elements at its border and absence of promoter-associated sequences.

At the DNA level, antigenic variation in trypanosomes is brought about by the replacement of one variant surface glycoprotein (VSG) gene by another in an expression site with a strong promoter. In several cases studied, mobilization of a VSG gene for expression involves a duplication-transposition. We have determined the DNA sequence of most of the transposed segment of one such VSG, the VSG 118. At the 3' side, the transposed segment ends within the end of the gene; at the 5' side, the transposed segment is preceded by a putative VSG gene, extending our previous conclusion that VSG genes are tightly clustered. The total length of the transposed segment is about 3.5 X 10(3) base-pairs and 1.8 X 10(3) base-pairs of this codes for the VSG 118 messenger RNA. Near the 5' border of the transposed segment we find five imperfect repeats of about 70 base-pairs that are also present in front of other VSG genes, as shown by hybridization. The termini of three minor VSG 118-specific transcripts map within these repeats. The repeats have the potential to adopt non-B-DNA conformations, and could play a role in the recombination process that exchanges VSG genes in the expression site or, less likely, in pre-mRNA processing. Comparison of the DNA and the mRNA sequence has previously revealed that a terminal exon of 35 nucleotides is spliced onto the main body of the RNA. We show here that these 35 nucleotides are not in the transposed segment and they must, therefore, be contributed by the expression site. This argues persuasively that the transposition activates VSG gene expression by promoter addition rather than by a position effect.

Amino Acid Sequence↗
PubMed1983

Activation of the genes for variant surface glycoproteins 117 and 118 in Trypanosoma brucei.

We have studied the activation of genes for VSGs (variant surface glycoproteins) in Trypanosoma brucei (strain 427) in six independently isolated trypanosome clones; four expressing the gene for VSG 118 and two the gene for VSG 117. In all cases, gene activation is brought about by a duplicative transposition of the gene to an expression site located close to the end of a chromosome. The DNA segments flanking the expression-linked extra gene copy are nearly devoid of restriction enzyme recognition sites and their lengths vary by more than 10,000 base-pairs among different variants. From the correspondence of five upstream restriction sites, we conclude that the same expression site is used in each case. The transposition event does not lead to detectable alterations in the sequence coding for the mature protein. All restriction enzyme recognition sites detected in the basic copy gene are present also in each of the expression-linked copies. This argues against the introduction of mutations by an error-prone polymerase during the synthesis of the expression-linked copy. In five of the six variants, the 3' end of the VSG messenger RNA differs from that of the corresponding basic copy gene by multiple point mutations, insertions and deletions, starting at positions varying from 16 nucleotides upstream to 113 downstream of the last codon of the mature protein. We attribute this end alteration to the recombination process that introduces the gene into the expression site. We confirm that the expression-linked gene copy is more sensitive to DNase I than the corresponding basic copy gene. This appears to be due to its activated state and not to its location near the end of a chromosome, because another basic copy VSG gene permanently located near a chromosome end is not hypersensitive to DNase I. The mature transcripts of the 117 and 118 genes all possess the same 35 nucleotides at their 5' ends and these are not encoded contiguously in the basic gene copies with the remainder of the mRNAs. This extends our previous conclusion, that mature VSG mRNAs are formed by a splicing process in which the 35-nucleotide sequence encoded in the expression site is fused onto the body of the mRNA contributed by the transposed gene.

Animals↗
PubMed1983

The major transcripts of the kinetoplast DNA of Trypanosoma brucei are very small ribosomal RNAs.

The nucleotide sequence has been determined of a 2.2 kb segment of kinetoplast DNA, which encodes the major mitochondrial transcripts (12S and 9S) of Trypanosoma brucei. The sequence shows that the 12S RNA is a large subunit rRNA, although sufficiently unusual for resistance to chloramphenicol to be predicted. The 9S RNA has little homology with other rRNAs, but a possible secondary structure is not unlike that of the 2.5-fold larger E. coli 16S rRNA. We conclude that the 12S RNA (about 1230 nucleotides) and the 9S RNA (about 640 nucleotides) are the smallest homologues of the E. coli 23S and 16S rRNAs yet observed.

Animals↗
PubMed1983

Tandem repetition of the 5' mini-exon of variant surface glycoprotein genes: a multiple promoter for VSG gene transcription?

Activation of some variant surface glycoprotein (VSG) genes involves a duplicative transposition to an expression site, which completes the gene by addition of a mini-exon coding for the 5' 35 nucleotides of VSG mRNAs. Using a 22 nucleotide probe we have found some 200 copies of the mini-exon on a tandemly arranged 1.35 kb repetitive element. This repeat is highly conserved in three trypanosome species. The mini-exon on the repeat is flanked by a 5' splice site that resembles the consensus sequence. We have not found a single mini-exon within 10 kb of the transposed VSG gene exon in the expression site. We propose a model in which the arrays of mini-exon repeats function as a repetitive promoter for efficient transcription of VSG genes.

Animals↗
PubMed1983

Transcription of mitochondrial DNA.

While mitochondrial DNA (mtDNA) is the simplest DNA in nature, coding for rRNAs and tRNAs, results of DNA sequence, and transcript analysis have demonstrated that both the synthesis and processing of mitochondrial RNAs involve remarkably intricate events. At one extreme, genes in animal mtDNAs are tightly packed, both DNA strands are completely transcribed (symmetric transcription), and the appearance of specific mRNAs is entirely dependent on processing at sites signalled by the sequences of the tRNAs, which abut virtually every gene. At the other extreme, gene organization in yeast (Saccharomyces) is anything but compact, with long stretches of AT-rich DNA interspaced between coding sequences and no obvious logic to the order of genes. Transcription is asymmetric and several RNAs are initiated de novo. Nevertheless, extensive RNA processing occurs due largely to the presence of split genes. RNA splicing is complex, is controlled by both mitochondrial and nuclear genes, and in some cases is accompanied by the formation of RNAs that behave as covalently closed circles. The present article reviews current knowledge of mitochondrial transcription and RNA processing in relation to possible mechanisms for the regulation of mitochondrial gene expression.

Animals↗
PubMed1982

An analysis of cosmid clones of nuclear DNA from Trypanosoma brucei shows that the genes for variant surface glycoproteins are clustered in the genome.

Trypanosoma brucei contains more than a hundred genes coding for the different variant surface glycoproteins (VSGs). Activation of some of these genes involves the duplication of the gene (the basic copy or BC) and transposition of the duplicate to an expression site (yielding the expression-linked copy or ELC). We have cloned large fragments of genomic DNA in cosmid vectors in Escherichia coli. Cosmids containing the BCs of genes 117, 118 and 121 were readily obtained, but DNA containing the ELCs was strongly selected against in the cosmid and plasmid cloning systems used. We have analysed the distribution of VSG genes in the genome using probes for the sequences at the edges of the transposed segment which are partially homologous among these genes. In genomic cosmid clone banks, about 9% of all colonies hybridize with probes from the 5'- and 3'-edges of the transposed segment, showing that these sequences are linked in the genome. Moreover, the 117 and 118 BC cosmids contain several additional putative VSG genes in tandem, as deduced from hybridization and sequence analyses. We conclude that the VSG genes are highly clustered and share common sequences at the borders of the transposed segment.

Animals↗