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P Borst

Publications and source records attributed to P Borst.

At least 235 records · Page 13Linked to original sources
PubMed1982

RNA splicing is required to make the messenger RNA for a variant surface antigen in trypanosomes.

The expression of the gene for variant surface glycoprotein (VSG) 118 in Trypanosoma brucei is activated by transposing a DNA segment containing the gene and 1-2 kb in front of it to an expression site elsewhere in the genome. By S1 nuclease protection and RNA blotting experiments we show here the presence of several minor transcripts in trypanosomes synthesizing VSG 118, one of which covers the entire transposed segment. Comparison of the sequence of the 5' terminal segment of VSG 118 messenger RNA (mRNA), determined by primed reverse transcription, and the corresponding region of the 118 VSG gene, shows that the 5' terminal 34 nucleotides of the mRNA are not encoded in the 118 VSG gene contiguous with the remainder of the mRNA. We conclude that synthesis of a VSG mRNA involves splicing of a much longer primary transcript, which may start outside the transposed segment.

Animals↗
PubMed1982

Characterization of the expression-linked gene copies of variant surface glycoprotein 118 in two independently isolated clones of Trypanosoma brucei.

We have analysed the gene for variant surface glycoprotein 118 in eight independent clones of Trypanosoma brucei, two of which express the 118 gene. Expression of this gene is strictly coupled to the presence of an extra copy of the gene. In both clones the expression-linked copy is transposed to the same (or a very similar) expression site elsewhere in the genome, but the length of the sequences flanking the transposed segment in the expression site differs markedly. By means of S1 nuclease protection experiments we demonstrate that the 3'-ends of the messenger RNAs for variant surface glycoproteins 118a and 118b are different, in agreement with the hypothesis that the generation of an expression-linked copy involves a recombination between the 3' segment of the basic gene copy and a homologous region present in the expression site.

Animals↗
PubMed1982

Characterization of the DNA duplication-transposition that controls the expression of two genes for variant surface glycoproteins in Trypanosoma brucei.

The genome of Trypanosoma brucei carries over a hundred genes coding for different variants of the major surface glycoprotein. Activation of some of these genes is accompanied by a duplication and transposition of the gene (the basic copy) to another region in the genome where it is transcribed. We present here physical maps of the basic and transposition-activated genes for two surface glycoproteins of Trypanosoma brucei, stock 427. In both cases the transposed segment starts 1-2 kb in front of the coding region and ends within the 3'-terminal region of the gene. The DNA segments flanking both transposed genes are indistinguishable and share a 6-kb stretch upstream and a 8-kb stretch downstream of the transposed segment not cut by several restriction endonucleases. The 5' borders of the two transposed segments are homologous and contain sequences present in many copies in the genome. A different repeated sequence has previously been found at the 3' edge of the transposed segment. The replicative transposition may, therefore, involve a unidirectional gene conversion initiated by base pairing between the edges of the transposed sequence and a single expression site elsewhere in the genome.

Animals↗
PubMed1982

On the DNA content and ploidy of trypanosomes.

We have determined the nuclear and kinetoplast DNA content of two trypanosomatids by quantitative absorption and fluorescence cytophotometry of individual Feulgen-pararosaniline stained cells. For the insect trypanosomatid Crithidia fasciculata we find nuclear and kinetoplast DNA contents of 0.095 and 0.032 pg per non-replicating cell. For the African trypanosome Trypanosoma brucei these values are 0.097 and 0.004 pg. A sub-population of T. brucei cells with two kinetoplasts and one nucleus was found to contain 0.181 pg/nucleus. The DNA values of bloodstream form T. brucei and the procyclic culture from were not significantly different. In DNA-DNA renaturation experiments the haploid amount of DNA in T. brucei was previously found to be 0.041 pg/nucleus (Borst, P., Fase-Fowler, F., Frasch, A.C.C., Hoeijmakers, J.H.J. and Weijers, P.J. (1980) Mol. Biochem. Parasitol. 1,221-246). Our data, therefore, indicate that T. brucei is diploid. No sub-population of haploid cells was observed in T. brucei grown in rats or in culture.

Animals↗
PubMed1982

Rapid evolution of genes coding for variant surface glycoproteins in trypanosomes.

We have used cloned DNA complementary to the messenger RNAs (mRNAs) for different variant surface glycoproteins (VSGs) of Trypanosoma brucei, stock 427, to study the degree of conservation of the corresponding nuclear genes in related trypanosome stocks. Conservation of restriction endonuclease cleavage sites in and around these genes were assessed by hybridization of the complementary DNA (cDNA) probes to nuclear DNA blots of these stocks. One of the genes (117) was found essentially unaltered in 11 out of 12 stocks. A second gene (118) was absent in five stocks. In the seven stocks that contained it, four forms of this 118 gene could be distinguished that differ by loss/gain of several restriction sites. A third gene (221) was only present in T. brucei 427 and in none of 11 other stocks. We conclude that a sub-set of the genes for the variant antigens evolves at a very high rate and we favour the hypothesis that this is due to local hypermutagenesis.

Animals↗
PubMed1981

Subcellular compartmentation of glycolytic intermediates in Trypanosoma brucei.

In cell-fractionation experiments most of the glycolytic enzymes in bloodstream forms of Trypanosoma brucei are recovered in a microbody, called the glycosome [Opperdoes, F. R. and Borst, P. (1977) FEBS Lett. 80, 360-364]. To see whether this compartmentation of glycolytic enzymes is accompanied by compartmentation of metabolites we have pulse-labelled intact T. brucei with [U-14C]glucose and followed the incorporation of radioactivity into glycolytic intermediates separated by anion-exchange chromatography. The kinetics of incorporation provide direct evidence for the existence of two pools of glycolytic intermediates. One pool is completely labelled within 15 s and represents 20-30% of total cellular metabolites. Radioactively labelled pyruvate is already produced after 15 s. Since this pool is directly involved in the glycolytic flux, we conclude that it is present in the glycosome. The second pool which represents 70-80% of the total appears not to be directly involved in glycolysis. Its content equilibrates relatively slowly with the glycosomal pool. It probably represents the cytosol. Incorporation of radioactivity into the total glycerol 3-phosphate pool is more rapid than for the other metabolites studied. This indicates rapid mixing of the glycosomal and cell-sap pools of glycerol 3-phosphate, as required for the extra-glycosomal oxidation of glycerol 3-phosphate by the mitochondrial oxidase. In the presence of 1 mM salicylhydroxamic acid, which mimics anaerobiosis, the trypanosome produces equimolar amounts of pyruvate and glycerol. Labelling of glycerol and glycerol 3-phosphate proceeds at identical rates but at all times the specific activity of glycerol is less than that of glycerol 3-phosphate. This is compatible with our earlier proposal that glycerol is made from glycerol 3-phosphate by glycerol kinase. We conclude that glycolysis in trypanosomes takes place in the glycosome and that the membrane of this organelle is poorly permeable to most glycolytic intermediates.

Animals↗
PubMed1981

Activation of trypanosome surface glycoprotein genes involves a duplication-transposition leading to an altered 3' end.

Expression of the genes for variant surface glycoproteins 117 and 118 in Trypanosoma brucei is accompanied by the appearance of an extra copy of these genes, the expression-linked copy, which differs in the surrounding restriction enzyme sites from the corresponding basic copy of the genes. We present direct evidence that the expression-linked copy is the one used for messenger RNA synthesis. By S1-nuclease-protection experiments we show that cloned basic-copy genes contain the nucleotide sequence of the corresponding messenger RNA except for the last 100 to 150 nucleotides before the poly(A) tail. Comparison of the 3'-terminal sequence of the 117 basic-copy gene and the 117 complementary DNA shows that this region differs by multiple point mutations, insertions and deletions, the differences starting within the coding sequence. Genomic blots demonstrate that a Bsp I site in the 3'-terminal part of the 118 complementary DNA is present in the expression-linked copy but not in the basic-copy gene. We conclude that expression-linked copies are the active genes, and that the generation of expression-linked copies involves a duplication--transposition in which the 3' end of the gene is replaced.

Amino Acid Sequence↗
PubMed1981

Quantitation of genetic differences between Trypanosoma brucei gambiense, rhodesiense and brucei by restriction enzyme analysis of kinetoplast DNA.

We have compared a total of 44 recognition sites for 12 restriction endonucleases on the 20 kilobase pair maxi-circle of kinetoplast DNA from nine Trypanosoma brucei stocks, four which are known to be infective to man (tow 'gambiense' and two 'rhodesiense' variants). In addition to five polymorphic sites, these DNAs differ in the size of a 5 kilo-base pair region which is cleaved only by one of the restriction enzymes tested and which varies in size over 1.5 kilo-base pairs. Our analysis shows that the maxi-circle sequences of these stocks are very similar, the maximal calculated difference between any two being 3%. A relatively large difference was found between a rhodesiense stock from uganda and one from Zambia, confirming the distinction between northern and southern East African rhodesiense stocks found by analysis of enzyme polymorphisms (Gibson et al. (1980) Adv. Parasitol. 18, 175-246). The gambiense variants could not be identified by unique restriction site polymorphisms, but contained the smallest maxi-circle found thus far in T. Brucei. Our results indicate that T. brucei stocks infective and not infective to man are so closely related as to preclude their differentiation by analysis of kinetoplast DNA. This analysis is useful, however, in providing quantitative information about relatedness of stocks.

Animals↗