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P Borst

Publications and source records attributed to P Borst.

At least 199 records · Page 11Linked to original sources
PubMed1985

Mature mRNAs of Trypanosoma brucei possess a 5' cap acquired by discontinuous RNA synthesis.

Mature mRNAs of Trypanosoma brucei have a common 5' terminal sequence of 35 nucleotides. This is acquired by an unknown mechanism from the 5' end of a separately transcribed precursor RNA of about 140 nt called the mini-exon-derived RNA or medRNA. We have investigated the nature of the 5' ends of mature mRNAs and of the medRNA by chemical decapping and enzymic recapping. We infer that a 5' cap is present on both of these RNAs and conclude that the mini-exon-derived RNA donates its 5' cap along with the mini-exon sequence to the pre-mRNA. Using nuclear run-on experiments we show that medRNA synthesis is much more sensitive to alpha-amanitin than 5S RNA synthesis and only slightly less sensitive than tubulin gene transcription. This result, together with the presence of a cap at the 5' end of the medRNA indicates that the mini-exon is transcribed by an RNA polymerase II type enzyme. Our experiments also confirm the existence of a second minor medRNA of about 125 nt and show the presence of other small capped RNAs possibly analogous to the small nuclear RNAs of other organisms.

Amanitins↗
PubMed1985

Further characterization of the extremely small mitochondrial ribosomal RNAs from trypanosomes: a detailed comparison of the 9S and 12S RNAs from Crithidia fasciculata and Trypanosoma brucei with rRNAs from other organisms.

We have determined the nucleotide sequence of a maxi-circle segment from the insect trypanosome Crithidia fasciculata mitochondrial DNA, on which the genes for the major maxicircle transcripts of 9S and 12S are localized. The 5'-terminal sequences of these RNAs were determined by wandering spot analysis. The map coordinates of the 9S and 12S RNAs from Trypanosoma brucei were adjusted with respect to a previous report with the aid of primer extension analysis with reverse transcriptase. This approach allowed us to align the corresponding genes from both organisms which show an overall sequence homology of 77%. The 9S and 12S RNA genes from the two trypanosome species contain sequences, closely related to some of the regions that are universally conserved among ribosomal RNAs from members of the three primary kingdoms and their organelles, even though the overall level of sequence homology is extremely low. These universal sequences occur at positions in the 9S and 12S RNAs that are analogous to those occupied by their counterparts in authentic ribosomal RNAs. The characteristic secondary structure elements flanking these universal sequences in genuine ribosomal RNAs can also be formed in the trypanosomal 9S and 12S RNAs. These results provide unequivocal evidence for a ribosomal function of the 9S and 12S RNAs of trypanosomal mitochondria, notwithstanding their extremely small size (estimated to be 612 and 1141 nucleotides in C. fasciculata, 611 and 1150 nucleotides in T. brucei) and their unusual base composition (83% A+U).

Animals↗
PubMed1985

Trypanosome variant surface glycoprotein genes expressed early in infection.

We have studied further the genes for trypanosomal variant surface glycoproteins expressed during a chronic infection of rabbits with Trypanosoma brucei, strain 427. We show that there are three closely related chromosomal-internal isogenes for VSG 121; expression of one of these genes is accompanied by the duplicate transposition of the gene to a telomeric expression site, also used by other chromosome-internal VSG genes. The 3' end of the 121 gene is replaced during transposition with another sequence, also found in the VSG mRNAs of two other variants. We infer that an incoming VSG gene duplicate recombines with the resident gene in the expression site and may exchange ends in this process. The extra expression-linked copy of the 121 gene is lost when another gene enters the expression site. However, when the telomeric VSG gene 221 is activated without duplication the extra 121 gene copy is inactivated without detectable alterations in or around the gene. We have also analysed the VSG genes expressed very early when trypanosomes are introduced into rats or tissue culture. The five genes identified in 24 independent switching events were all found to be telomeric genes and we calculate that the telomeric 1.8 gene has a 50% chance of being activated in this trypanosome strain when the trypanosome switches the VSG that is synthesized. We argue that the preferential expression of telomeric VSG genes is due to two factors: first, some telomeric genes reside in an inactive expression site, that can be reactivated; second, telomeric genes can enter an active expression site by a duplicative telomere conversion and this process occurs more frequently than the duplicative transposition of chromosome-internal genes to an expression site.

Amino Acid Sequence↗
PubMed1985

Trypanosoma brucei: a surface antigen mRNA is discontinuously transcribed from two distinct chromosomes.

The mRNAs for variant surface glycoproteins (VSGs) and many other proteins in Trypanosoma brucei start with the same sequence of 35 nucleotides, encoded by a separate mini-exon. There are approximately 200 mini-exon genes per trypanosome and these are highly clustered on large chromosomes. We have found two trypanosome variants that express a VSG gene located on a small, 225-kb chromosome. Each gene yields a mRNA containing the 35-nucleotide sequence even though the 225-kb chromosome does not contain a complete mini-exon gene. These results provide a strong support for the hypothesis that transcription of protein-coding genes in trypanosomes is discontinuous.

Animals↗
PubMed1985

Further analysis of intraspecific variation in Trypanosoma brucei using restriction site polymorphisms in the maxi-circle of kinetoplast DNA.

We have compared the maxi-circle kinetoplast DNA of 21 Trypanosoma brucei sp. stocks by analysis of restriction sites for nine restriction endonucleases. The analysis shows most of these stocks to have a maxi-circle sequence similar to that of 11 previously analysed stocks, with a difference of less than 3% between any two stocks. However, seven stocks stand out from the rest with at least two sites lost or gained for six of the nine restriction enzymes used. These seven distinctive stocks fall into two groups with some shared and some unique polymorphisms. One group had already been designated the kiboko group on the basis of isoenzyme patterns, but the relationship between nuclear markers and maxi-circle type is less clear-cut for the other group, designated sindo. Both groups seem to be in a wild animal-tsetse fly transmission cycle, with occasional infections in domestic stock, and may be reproductively isolated from the main T. brucei sp. population. The existence of the kiboko and sindo sub-groups shows that the maxi-circle is not shielded from evolutionary change. The lack of difference observed between the maxi-circles of the majority of T. brucei sp. stocks, including the gambiense and rhodesiense variants, must therefore reflect their close homology. Two geographical trends occur in T. brucei as a whole: (a) a trend in maxi-circle size, with increasing length of the variable region from West to East Africa, and (b) a greater frequency of certain restriction enzyme polymorphisms in East African stocks as compared to West African stocks.

Animals↗
PubMed1985

ADP-ribosyl transferase activity in Trypanosoma brucei.

Nuclear adenosine diphosphoribosyl transferase (ADPRT) catalyses the covalent modification of chromatin proteins by (ADP-ribose)n. This activity, which is entirely dependent on DNA containing strand breaks, is required for efficient DNA excision repair possibly because it regulates DNA ligation. ADPRT activity is also required for cytodifferentiation in a number of different cell types. We report here the presence of ADPRT activity in the blood-stream form of Trypanosoma brucei and its activation by DNA strand breaks formed by exposure to, either exogenously supplied deoxyribonuclease I, or treatment with the methylating agent, dimethylsulphate. 3-Aminobenzamide, but not its chemical analogue 3-aminobenzoic acid, is a competitive inhibitor of ADPRT activity in T. brucei. Intact trypanosomes are readily permeable to this competitive inhibitor of ADPRT activity.

Animals↗
PubMed1985

Trypanosomes of subgenus Trypanozoon are diploid for housekeeping genes.

The ploidy of trypanosomes has until now remained undetermined, although isoenzyme studies and direct measurements of DNA content and complexity suggest diploidy. Direct cytogenetic analysis is not possible, because the chromosomes do not condense at any stage of the cell cycle. We now present evidence from analysis of restriction site polymorphisms in and around three glycolytic enzyme genes (phosphoglycerate kinase, triosephosphate isomerase, glyceraldehyde phosphate dehydrogenase) and the tubulin gene cluster, that trypanosomes of subgenus Trypanozoon are diploid for these housekeeping genes. This result is still compatible with the single copy nature of variant surface glycoprotein (VSG) genes in Trypanozoon, if different VSG genes are present in corresponding positions on paired chromosomes. Using pulse field gradient gel electrophoresis, we show that the genes for the three glycolytic enzymes are all located in very large DNA molecules, but the gene for triosephosphate isomerase is in another fraction from the genes for the other two enzymes. Since all three enzymes are located in glycosomes, which are trypanosome microbodies, the genes for glycosomal enzymes are not all clustered in one chromosomal segment of the trypanosome genome.

Animals↗
PubMed1985

Kinetoplast DNA from Trypanosoma vivax and T. congolense.

We have analysed kinetoplast DNA (kDNA) of the African trypanosomes Trypanosoma vivax and T. congolense. The maxi-circles from these organisms resemble those of T. brucei in size, but only to a limited extent in sequence as judged from restriction enzyme digests and DNA X DNA hybridization. The kDNA networks of T. vivax have three distinguishing features: they contain the highest maxi-circle concentration of any kDNA (at least twice that of T. brucei); they contain the smallest mini-circles (465 bp) yet found thus far and the width of the kDNA nucleoid in thin sections is correspondingly small (55 nm against 91 nm for T. brucei); they contain a substantial fraction of mini-circle dimers.

Animals↗
PubMed1985

Two simultaneously active VSG gene transcription units in a single Trypanosoma brucei variant.

Trypanosomes can change their surface coat either by slotting a different surface antigen gene copy into an active (telomeric) expression site or by activating a new VSG gene expression site and inactivating the old one. How expression sites are activated or inactivated is not clear. We report an exceptional trypanosome variant in which the inactivation of a surface antigen gene is accompanied by a 30 kb DNA insertion 5' of the gene. Transcription of the region upstream of the insertion continues unaltered and retains the characteristic insensitivity to alpha-amanitin of VSG gene transcription units, showing that the expression site is still active. The expressed VSG gene in this trypanosome variant resides in another telomere. Hence, two VSG gene transcription units can be simultaneously active. This argues against a single mobile activating element controlling VSG gene transcription and favors a stochastic model of telomere activation/inactivation.

Animals↗
PubMed1985

Structure and transcription of a telomeric surface antigen gene of Trypanosoma brucei.

The gene encoding variant surface glycoprotein 221 in Trypanosoma brucei is located adjacent to a chromosome end and can be activated with or without a concomitant gene duplication. To test whether transcription initiates within the cloned segment of the 221 gene, we analyzed nascent and stable transcripts. We show here that the 221 coding region and 8.5 kilobases of adjacent upstream DNA are transcribed into nascent RNA at a similar rate when gene 221 is activated without duplication. Since only part of this transcribed upstream segment is transferred with the coding region to another telomere upon duplicative activation of gene 221, we infer that initiation of variant surface glycoprotein gene transcription occurs outside the gene segment that moves into an expression site by gene conversion. Our analysis shows that part of the variant surface glycoprotein 221 transcription unit consists of an unusual 3.5-kilobase tandem array of ca. 50 repeat segments and that a rearrangement in this array accompanies the nonduplicative activation of gene 221. A variant surface glycoprotein pseudogene is located within the transcription unit of gene 221, and we discuss models that account for this unusual situation.

Animals↗
PubMed1984

Alpha-amanitin-insensitive transcription of variant surface glycoprotein genes provides further evidence for discontinuous transcription in trypanosomes.

Many, if not all, mRNAs in T.brucei start with the same sequence of 35 nucleotides, separately encoded in clustered so-called mini-exon repeats. From these mini-exon repeats a 141-nt precursor RNA with the 35-nt sequence at its 5' end is transcribed. Indirect evidence suggests that this RNA is linked in a second step to pre-mRNA transcripts. We have studied the sensitivity of RNA synthesis to alpha-amanitin in isolated trypanosome nuclei. Transcription of several protein coding genes is almost completely inhibited by a concentration of 5 micrograms alpha-amanitin per ml, whereas strong inhibition of mini-exon transcription is achieved with 200 micrograms alpha-amanitin per ml. In contrast, transcription of genes for variant surface glycoproteins (VSGs) is not inhibited by 1000 micrograms alpha-amanitin per ml, as is transcription of the genes for the major rRNAs. Since the mature VSG mRNAs start with the 35-nt sequence, our results provide additional evidence that the 35-nt sequence and the main part of VSG mRNA are produced from independent transcription units, these are transcribed by (partly) different RNA polymerases.

Amanitins↗
PubMed1984

Chromosomes of kinetoplastida.

We have compared chromosome-sized DNA molecules (molecular karyotypes) of five genera (nine species) of kinetoplastida after cell lysis and deproteinization of DNA in agarose blocks and size fractionation of the intact DNA molecules by pulsed field gradient (PFG) gel electrophoresis. With the possible exception of Trypanosoma vivax and Crithidia fasciculata, all species have at least 20 chromosomes. There are large differences between species in molecular karyotype and in the chromosomal distribution of the genes for alpha- and beta-tubulin, rRNA and the common mini-exon sequence of kinetoplastid mRNAs. In all cases, the rRNA genes are in DNA that is larger than 500 kb. Whereas T. brucei has approximately 100 mini-chromosomes of 50-150 kb, only few are found in T. equiperdum; T. vivax has no DNA smaller than 2000 kb. As all three species exhibit antigenic variation, small chromosomes with telomeric variant surface glycoprotein genes cannot be vital to the mechanism of antigenic variation. The apparent plasticity of kinetoplastid genome composition makes PFG gel electrophoresis a potentially useful tool for taxonomic studies.

Animals↗
PubMed1984

Comparison of the genes coding for the common 5' terminal sequence of messenger RNAs in three trypanosome species.

Messenger RNAs of Trypanosoma brucei share a common 5' terminal sequence of 35 nucleotides, encoded by a mini-exon located in 1.35-kb tandemly linked repeats. We show here that sequences, almost identical to the mini-exon of T. brucei, are present in mRNAs from members of two other kinetoplastid subgenera: Trypanosoma vivax and Trypanosoma cruzi. As in T. brucei, these mini-exons are encoded by small tandemly linked repeat elements. We have determined and compared the nucleotide sequences of the mini-exon repeats from T. brucei, T. vivax and T. cruzi. This analysis shows that the mini-exon, its immediate flanking sequences and a T-rich stretch downstream are conserved, but little else. Our data establish the generality of the novel transcription system, that was first found in T. brucei and that yields mRNAs with common, repeat-encoded, 5' termini.

Animals↗
PubMed1984

Modification of telomeric DNA in Trypanosoma brucei; a role in antigenic variation?

Expression of surface antigen genes in Trypanosoma brucei occurs at expression sites located near telomeres. Since only one antigen is produced at a time, a mechanism must exist to prevent the simultaneous activity of multiple expression sites. Here we report that PstI and PvuII restriction sites in silent telomeric antigen genes are partially uncleavable , presumably as a consequence of DNA modification. The modification, which is absent in transcribed genes but returns after gene inactivation, may be specific for telomeric DNA because (1) it is not detected in non-telomeric genes; (2) modification is highest close to the telomere; (3) the level of modification in a telomeric gene is influenced by the size of the telomeric DNA segment downstream. Whether telomere modification is cause or consequence of antigen gene switch-off remains to be determined.

Animals↗
PubMed1984

Many trypanosome messenger RNAs share a common 5' terminal sequence.

The mRNAs for different variant surface antigens of Trypanosoma brucei start with the same 35 nucleotides. This sequence is encoded by a separate mini-exon, located in a 1.35-kb repetitive element. We have reported that trypanosomes contain many transcripts that hybridize to mini-exon probes, even if they do not make the surface antigens. We show here that these transcripts have the mini-exon sequence at their 5' end; they do not contain other sequences from the mini-exon repeat element and are polyadenylated. We have cloned DNA complementary to trypanosome mRNAs and randomly selected 17 clones containing mini-exon sequences. Thirteen of these are derived from different genes that do not code for surface antigens. We conclude that the mini-exon sequence is a common element at the 5' end of many trypanosome mRNAs. As the 200 genes for mini-exons are highly clustered, linkage of the mini-exon sequence to the remainder of most mRNAs may require discontinuous transcription.

Amino Acid Sequence↗
PubMed1984

DNA circles with cruciforms from Isospora (Toxoplasma) gondii.

We have isolated a closed circular duplex DNA fraction from the unicellular parasite Isospora (Toxoplasma) gondii and examined the purified DNA by electron microscopy. A major part of this circular DNA consists of 12-micron circles containing a cruciform with 0.5-micron tails. We also found 23-micron circles with the properties expected of head-to-tail dimers of the 12-micron circles. Some of these dimers have two cruciforms with 0.4-micron tails, some have one cruciform with 0.8-micron tails. When ethidium bromide was diffused into the DNA solution, circles with tails were replaced by twisted circles without tails. Direct mixing of the DNA with high ethidium bromide concentrations (5 micrograms/ml) gave rise to highly twisted circles with tails. This proves that the tailed circles are covalently continuous and indicates that ethidium bromide blocks branch migration. The 0.5-micron tails are part of a 1.7-micron palindrome, which was visualized by spreading denatured DNA under snap-back conditions. We argue that the cruciform is not present in vivo and that the 12-micron circles may represent the mitochondrial DNA of Toxoplasma.

DNA, Bacterial↗
PubMed1984

The inactivation and reactivation of an expression-linked gene copy for a variant surface glycoprotein in Trypanosoma brucei.

We have previously shown that the gene for variant surface glycoprotein 118 of Trypanosoma brucei (strain 427) is activated by a duplicative transposition to a telomeric expression site. In chronically-infected animals, this expression-linked copy is lost when the 118 gene is replaced at the expression site by another variant surface glycoprotein gene. We show here that expression of the 118 gene can also be switched off without loss of the extra expression-linked copy. In two variants, called 1.8b and 1.8c, we find expression of the variant surface glycoprotein 1.8 gene, notwithstanding the continued presence of the 118 expression-linked copy. The 1.8 gene activated has a telomeric location, like the 118 expression-linked copy. In variant 1.8b, activation is accompanied by duplication of the 1.8 gene, resulting in an extra telomeric gene copy; in variant 1.8c it is not. Variants 1.8b and 1.8c both switch back preferentially to expression of the 118 gene. The 5'-flanking regions of the active, inactive and reactivated versions of the 118 expression-linked copy are indistinguishable by restriction mapping up to 28 kb. We conclude that there are at least two separate telomeric expression sites in our T. brucei strain. How these are switched on and off is unclear. The ability to retain expression-linked copies in inactive form may allow the trypanosome to re-programme the order in which variant surface glycoprotein genes are expressed.

Amino Acid Sequence↗