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P Borst

Publications and source records attributed to P Borst.

At least 145 records · Page 8Linked to original sources
PubMed1990

The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei.

The variant-specific surface glycoprotein (VSG) gene 221 of Trypanosoma brucei is transcribed as part of a 60 kb expression site (ES). We have identified the promoter controlling this multigene transcription unit by the use of 221 chromosome-enriched DNA libraries and VSG gene 221 expression site specific transcripts. The start of transcription was determined by hybridization and RNase protection analysis of nascent RNA. The 5' ends of the major transcripts coming from the initiation region map at nucleotide sequences that do not strongly resemble rRNA transcriptional starts even though the transcripts are synthesized by an RNA polymerase highly resistant to alpha-amanitin. The cloned VSG gene 221 ES transcription initiation region promotes high CAT gene expression, when reintroduced by electroporation into T. brucei. We show that the activity of this expression site is controlled at or near transcription initiation in bloodstream trypanosomes. The 221 ES is inactivated without any sequence alteration within 1.4 kb of the transcription start site. This excludes mechanisms of promoter inactivation involving DNA rearrangements in the vicinity of the transcription start site, e.g. promoter inversion or conversion.

Animals↗
PubMed1990

The amplified H circle of methotrexate-resistant leishmania tarentolae contains a novel P-glycoprotein gene.

Acquired resistance to methotrexate in Leishmania species is often associated with the amplification of H circles, 68 kb duplex DNA circles containing a 30 kb inverted repeat. We report here that the H circle of Leishmania tarentolae contains an open reading frame, ltpgpA, that has the attributes of P-glycoproteins (large plasma membrane proteins known to extrude lipophilic drugs from mammalian cells). Although amplification of H circles is associated with proportionally increased levels of a 5.5 kb transcript of the ltpgpA gene, such methotrexate resistant mutants are not cross-resistant to any of the drugs extruded by mammalian multi-drug resistant cells. In Leishmania, ltpgpA is part of a gene family containing at least two other members. Sequences homologous to one of the nucleotide binding sites of ltpgpA are conserved in other kinetoplastida.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Structure of a telomeric expression site for variant specific surface antigens in Trypanosoma brucei.

We have studied the organization of the expression site, in which most chromosome-internal variant-specific surface glycoprotein (VSG) genes of Trypanosoma brucei strain 427 are expressed (the dominant expression site) and compared it to the previously characterized VSG 221 expression site. With the exception of a 500 bp segment and a VSG pseudogene, which are absent from the dominant expression site, overall all major sequence elements of the two sites are organized similarly, as judged from their relative mapping positions by UV inactivation of transcription. Transcription is insensitive to 1 mg alpha-amanitin per ml, a characteristic property of VSG gene expression sites analyzed thus far. The sequence elements of the dominant expression site include at least one other expressed gene of unknown function and homologues of at least two other open reading frames. The large internal duplication of the 60-kb 221 expression site appear to be missing from the dominant site, resulting in a shorter, 40-kb transcription unit. As judged from its relative sensitivity to UV inactivation of transcription, a subsidiary promoter, identified by other methods in the dominant expression site appears fully dependent for its activity on the promoter located 40 kb upstream of the VSG gene. We conclude that all VSG gene expression sites may be similarly organized as large polygenic transcription units.

Animals↗
PubMed1989

Trypanosoma brucei contains two RNA polymerase II largest subunit genes with an altered C-terminal domain.

We have identified and cloned four trypanosomal RNA polymerase largest subunit genes. Here, we present the molecular analysis of two genes, Trp4.8 and Trp5.9. The sequence of these genes shows that they are almost identical to each other and indicates that they encode the largest subunit of RNA polymerase II. Both genes contain a C-terminal extension that is clearly distinct from that of other eukaryotic RNA polymerase II genes, because it lacks the common tandemly repeated heptapeptide sequence and is rich in acidic amino acids. It shares many potential phosphorylation sites, however, with the C-terminal extension of other eukaryotic RNA polymerase II large subunits. The presence of two RNA polymerase II loci suggests that a fourth RNA polymerase could be formed. Interestingly, the fourth gene is only found in species exhibiting antigenic variation.

Amanitins↗
PubMed1989

DNA content and structure of (double) minutes of a methotrexate-resistant cell line.

We have determined the DNA content of intact double minutes (DMs) and of single minutes (SMs) by fluorometry of the individual chromatin bodies in metaphase spreads after staining with Feulgen-Schiff pararosaniline. We find that the intact DMs and SMs of the methotrexate-resistant mouse cell line 3T6R50 contain 4.4 megabase pairs (Mb) and 2.6 Mb DNA respectively, using the DNA content of E. coli (4.7 Mb) as a reference. As the pulsed field gradient gel electrophoresis experiments by van der Bliek et al. (1988) have indicated that the minutes of 3T6R50 cells contain a homogeneous population of 2.5 Mb DNA circles, we conclude that a SM contains one circular double strand DNA molecule of approximately 2.5 Mb, whereas DMs contain two.

Animals↗
PubMed1988

Sequence of mdr3 cDNA encoding a human P-glycoprotein.

We have determined the sequence of the human mdr3 gene using cDNA derived from liver RNA. The mdr3 gene codes for a member of a family of membrane proteins, the P-glycoproteins, overproduced in many multi-drug-resistant (MDR) cell lines. Like its relatives, the protein encoded by mdr3 has a deduced Mr of 140,000, which is presumably increased by glycosylation after synthesis. The sequence consists of two similar halves, each with a series of six hydrophobic segments that may form a membrane channel. The halves also possess nucleotide-binding consensus sequences, which presumably act as ATPases and drive drug transport. The presumed ATPase domains are all but identical to those of the human mdr1 gene product [Chen et al., Cell 47 (1986) 381-389]. We attribute this high level of sequence conservation to the repeated gene conversion that is evident from segments in which mdr1 and mdr3 differ only in a few silent mutations. Divergence between P-glycoprotein family members is greatest at the N terminus and in the 60 amino acid linker connecting the two halves. In the putative trans-membrane domains approx. 80% of the amino acids are conserved between the products of mdr1 and mdr3. Although the function of mdr3 is not yet known, its high homology with mdr1 suggests that it also encodes an efflux pump with broad specificity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

The H circles of Leishmania tarentolae are a unique amplifiable system of oligomeric DNAs associated with drug resistance.

We have induced drug resistance against methotrexate, an inhibitor of dihydrofolate reductase, and CB3717, an inhibitor of thymidylate synthetase in a strain of Leishmania tarentolae. The drug-resistant strains contain extrachromosomal DNA circles of 68 kilobases with a 30-kilobase inverted duplication flanked by 4- and 5 kilobase unique segments. We show that these circles are highly homologous to the drug-induced H circles of L. tropica (1). All three L. tarentolae strains analyzed contain a chromosomal copy of the H region without duplication, but two of the three strains contain extrachromosomal H circles as well, predominantly present as H circle dimers in one strain and as tetramers in the other. After induction of methotrexate resistance, monomeric circles, presumably derived from the oligomers, become the major type of circle. Our results indicate that the H region represents a genomic region that can be copied at very low frequency to yield circles by a precise, but unusual mechanism under natural conditions in wild-type cells. Although superficially analogous to the episomes of bacteria, the system is without precedent in nature.

Animals↗
PubMed1988

Genes amplified and overexpressed in human multidrug-resistant cell lines.

Multidrug resistance (MDR) is associated with overproduction of Mr 170,000 membrane proteins (P-glycoproteins) caused by either gene amplification, transcriptional activation, or both. In rodents the amplified domain comprises genes that encode P-glycoproteins and at least five unrelated genes, one of which encodes the calcium-binding protein sorcin. The amplification and increased expression of these genes always includes one P-glycoprotein-encoding gene (pgp1 in hamsters, homologous to mdr1 in humans). In human MDR cells only elevated mdr1 expression has been shown thusfar, although another P-glycoprotein encoding gene (mdr3, homologous to hamster pgp3) is closely linked. Here we show that the human homolog of the hamster sorcin gene resides on chromosome 7 like the P-glycoprotein-encoding genes. Furthermore, gene classes designated 4, 5, and 6 are coamplified with mdr1 and mdr3 in the human ovarian carcinoma cell line 2780AD, which strongly suggests that the overall structure of the human MDR domain is the same as in rodents. Class 6 was moderately and mdr1 was highly overexpressed in this cell line. Four other human MDR cell lines also have much higher mdr1 overexpression than expected from the relatively low levels (2- to 30-fold) of gene amplification. This contrasts with the results of previous work with rodent MDR cells, in which the increase in P-glycoprotein mRNA levels usually parallels the increase in gene copy number. Although four of the five human MDR cell lines have coamplified mdr3, its expression was undetectable. Our results confirm the central role of the mdr1 (pgp1) gene in MDR and suggest that different cross-resistance patterns are not due to differential expression of different P-glycoprotein genes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

Boundaries of telomere conversion in Trypanosoma brucei.

Active genes for variant-specific surface glycoproteins (VSGs) reside in telomeric expression sites and may be replaced by other VSG genes via telomere conversions. The availability of a complete map of expression site 221 in variant 221a made it possible to determine the boundaries of such conversions and the sequences that are involved. We have analysed five trypanosome populations that arose from variant 221a through replacement of the 221 gene by another VSG gene. In each of these relapsed populations the telomere conversion ends at a different position in the expression site. In the relapsed population, 221aR3, the boundary was found in the coding region of an expression-site-associated gene (ESAG). This ESAG-2 codes for a potential 368-aa protein of unknown function; it contains a N-terminal signal peptide for mediating transfer to the endoplasmic reticulum and six potential N-glycosylation sites. It shares these structural features with the ESAG-1 protein encoded in the same expression site. ESAG-2 is a member of a large gene family which includes non-functional genes. In 221aR3, the partial conversion of ESAG-2 by an ESAG-2-like sequence has disrupted the open reading frame. The two ESAG-2 sequences are similar (92% identity) suggesting that sequence homology between telomeres provides the opportunity for gene conversion.

Amino Acid Sequence↗
PubMed1988

Circular DNA of 3T6R50 double minute chromosomes.

In pulsed field gradient gel electrophoresis (PFGE) the intact deproteinized circular DNA of Mycoplasma (800 kb) and Escherichia coli (4700 kb) remains trapped in the slot. We show here that gamma-irradiation of the DNA in agarose plugs is a convenient method to partially convert these circles into full-length linears, migrating with the expected mobility in PFGE. We have used this method to study the structure of Double Minute chromosomes (DMs) from the methotrexate (MTX)-resistant mouse cell line 3T6R50. Intact deproteinized DM DNA is immobile in these gels, but is converted into a single band of about 2500 kb by either gamma-irradiation, DNaseI in the presence of Mn2+, or restriction enzymes. We conclude that the DM DNA in 3T6R50 cells consists of a homogeneous population of 2500-kb circles.

Animals↗
PubMed1988

Post-transcriptional control of the differential expression of phosphoglycerate kinase genes in Trypanosoma brucei.

The genes for the cytosolic and glycosomal phosphoglycerate kinases (PGK) of Trypanosoma brucei are found in a compact tandem array together with a third PGK-related gene, expressed at low level. Expression of the two PGK genes is differentially regulated in the life cycle of T. brucei: the glycosomal PGK and its mRNA are abundant in the mammalian stage of the cycle but not in the insect stage, whereas the reverse is found for the cytosolic PGK and its mRNA. Nevertheless, our experiments indicate that the mRNAs for both isoenzymes are derived from a common precursor. Nuclease protection experiments using fragments cloned into single-stranded DNA vectors show the presence of low abundance RNA species running through one gene into the next. Indeed minor RNA species larger than the mature mRNAs are visible in overexposed RNA blots. Analysis of nascent RNA in a nuclear run-on assay indicates that the entire PGK gene array is transcribed at an equal rate throughout in both life cycle stages. We conclude that the PGK genes are part of one large multicistronic transcription unit, which is processed to yield the individual mRNAs with concomitant addition of the 5' 35-nucleotide mini-exon sequence, characteristic of all trypanosome mRNAs. It follows that the steady-state levels of the PGK mRNAs are controlled post-transcriptionally.

Animals↗
PubMed1988

Evidence for gene conversion between the phosphoglycerate kinase genes of Trypanosoma brucei.

Trypanosoma brucei contains a tandem array of three genes for phosphoglycerate kinase (PGKase), genes A, B and C, each coding for a different protein. We have compared allelic variants of this gene array and find evidence for gene conversion between the three genes. Near the 3' end, the different alleles and gene B contain a variable sequence that is similar to the corresponding sequence in either gene A or gene C. This sequence is flanked by glycine triplets that are conserved in all PGKases from bacteria to mammals. The triplets are encoded by (GGT)n, resulting in sequences that resemble the recombination-promoting chi-sites of Escherichia coli. Upstream of the variable sequence, there is an area of 800 base-pairs in which genes A, B and C are highly homologous; in all three genes this region ends with a sharp boundary at which gene B again shows segmental homology with both genes A and C. These results suggest that repeated gene conversion events partially erase the differences between genes A, B and C that arise in evolution and suggest that chi-like sequences may act as recombinational hotspots in protozoa such as T. brucei.

Amino Acid Sequence↗