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P Borst

Publications and source records attributed to P Borst.

At least 19 recordsLinked to original sources
PubMed1995

A ribosomal DNA promoter replacing the promoter of a telomeric VSG gene expression site can be efficiently switched on and off in T. brucei.

Trypanosoma brucei survives in the mammalian blood-stream by regularly changing its variant surface glycoprotein (VSG) coat. The active VSG gene is located in a telomeric expression site, and coat switching occurs either by replacing the transcribed VSG gene or by changing the expression site that is active. To determine whether VSG expression site control requires promoter-specific sequences, we replaced the active VSG expression site promoter in bloodstream-form T. brucei with a ribosomal DNA (rDNA) promoter. These transformants were fully infective in laboratory animals, and the rDNA promoter, which is normally constitutively active, was efficiently inactivated and reactivated in the context of the VSG gene expression site. As there is no sequence similarity between the VSG expression site promoter and the rDNA promoter, VSG expression site control does not involve sequences specific to the VSG expression site promoter. We conclude that an epigenetic mechanism, such as telomeric silencing, is involved in VSG expression site control in bloodstream-form T. brucei.

Animals↗
PubMed1995

Absence of the mdr1a P-Glycoprotein in mice affects tissue distribution and pharmacokinetics of dexamethasone, digoxin, and cyclosporin A.

We have previously shown that absence of the mouse mdr1a (also called mdr3) P-glycoprotein in mdr1a (-/-) "knockout" mice has a profound effect on the tissue distribution and elimination of vinblastine and ivermectin, and hence on the toxicity of these compounds. We show here that the mouse mdr1a and the human MDR1 P-glycoprotein actively transport ivermectin, dexamethasone, digoxin, and cyclosporin A and, to a lesser extent, morphine across a polarized kidney epithelial cell layer in vitro. Injection of these radio-labeled drugs in mdr1a (-/-) and wild-type mice resulted in markedly (20- to 50-fold) higher levels of radioactivity in mdr1a (-/-) brain for digoxin and cyclosporin A, with more moderate effects for dexamethasone (2- to 3-fold) and morphine (1.7-fold). Digoxin and cyclosporin A were also more slowly eliminated from mdr1a (-/-) mice. Our findings show that P-glycoprotein can be a major determinant for the pharmacology of several medically important drugs other than anti-cancer agents, especially in the blood-brain barrier. These results may explain a range of pharmacological interactions observed between various drugs in patients.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

The P-glycoprotein gene family of Caenorhabditis elegans. Cloning and characterization of genomic and complementary DNA sequences.

P-glycoproteins, encoded by families of evolutionarily conserved genes, can confer a multidrug-resistant phenotype to mammalian tumor cells. To obtain more information on their functions in normal cells we have cloned genomic and complementary DNA sequences of four P-glycoprotein gene homologs of the genetically well-characterized nematode Caenorhabditis elegans, termed pgp-1, pgp-2, pgp-3 and pgp-4, respectively. The genes were physically mapped on chromosome IV (pgp-1), I (pgp-2) and X (pgp-3 and pgp-4). Phenotypic mutants corresponding to these loci have not yet been described. Two of the genes, pgp-1 and pgp-3, were analyzed in detail. They are predicted to encode ATP-binding membrane-spanning proteins of 1321 and 1254 amino acid residues, respectively, with the characteristic features shared by most P-glycoproteins described thus far. Intra-species divergence of P-glycoprotein genes is more pronounced in C. elegans than in mammals. Only 40% of the amino acids of pgp-1 and pgp-3 are identical, in contrast to 77% identity between human MDR1 and MDR3. pgp-1 consists of 14 exons, pgp-3 of 13. The two genes share only one intron position, whereas they share four (pgp-1) and five (pgp-3) intron positions with mammalian P-glycoprotein genes. pgp-1, pgp-2, and pgp-3 are transcribed into low abundance mRNAs in wild-type nematodes. pgp-1 and pgp-3 mRNAs have the trans-spliced leader SL1 at their 5' ends. Arsenite, emetine and actinomycin D drugs did not increase the steady state levels of pgp mRNA, unlike in some mammalian cell types. Heat shock disturbed trans as well as cis-splicing of pgp-1 and led to the accumulation of partially processed pgp-1 RNA. Thus, in C. elegans these genes are not induced in the context of a general stress response, as has been proposed for mammalian P-glycoprotein genes in certain tissues.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

A ribosomal RNA gene promoter at the telomere of a mini-chromosome in Trypanosoma brucei.

The parasitic protozoan Trypanosoma brucei has some hundred mini-chromosomes of 50-150 kb, which mainly consist of telomeric repeats, sub-telomeric repeats and internal 177-bp repeats. Their primary function seems to be to expand the repertoire of non-transcribed sub-telomeric variant surface glycoprotein (VSG) genes. Here we report that two of the smaller mini-chromosomes (55 and 60 kb) contain sequences homologous to the ribosomal RNA gene promoter region. We have targeted by homologous recombination the neomycin phosphotransferase (neo(r)) gene behind the promoter on the 55 kb chromosome and show that this promoter mediates the efficient synthesis of properly trans-spliced and polyadenylated neo mRNA. The resulting high resistance to G418 (a neo analogue) is stable in the absence of drug showing that mitotic segregation of this mini-chromosome is precise. Downstream of the transcription start the wild-type version of the ribosomal promoter is flanked by telomeric repeats. The absence of the sub-telomeric repeats found in other T.brucei chromosome ends suggests that the rDNA-telomeric junction has been formed by de novo addition of telomeric repeats to a broken chromosome end (healing). Our results provide a plausible explanation for the alpha-amanitin-resistant transcription of telomeric repeats in T.brucei reported by Rudenko and Van der Ploeg and they show that trypanosomes can efficiently use RNA polymerase I for the expression of sub-telomeric genes, supporting the notion that the alpha-amanitin-resistant transcription of sub-telomeric VSG genes may also be catalyzed by this enzyme.

Animals↗
PubMed1992

Genetic transfer of non-P-glycoprotein-mediated multidrug resistance (MDR) in somatic cell fusion: dissection of a compound MDR phenotype.

A non-P-glycoprotein-mediated mechanism of multidrug resistance (non-Pgp MDR) has been identified in doxorubicin-selected sublines of the human non-small cell lung carcinoma cell line SW-1573. These sublines are cross-resistant to daunorubicin, VP16-213, Vinca alkaloids, colchicine, gramicidin D, and 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA). They accumulate less drug than the parental cells and their resistance is not due to the MDR1-encoded P-glycoprotein, as the resistant cell lines have lost the low amount of MDR1 mRNA detectable in parental cells. Here we show that the resistant cell lines also contain less topoisomerase II mRNA and enzyme activity than the parental cells. This might contribute to the resistance of these lines to drugs interacting with topoisomerase II, such as doxorubicin, daunorubicin, and VP16-213, but cannot account for the resistance to the other drugs. We have tested whether all properties of the non-Pgp MDR cell lines cosegregate in somatic cell fusions between lethally gamma-irradiated, resistant donor cells and drug-sensitive acceptor cells. Whereas a MDR phenotype with reduced drug accumulation and the loss of MDR1 P-glycoprotein mRNA were cotransferred to the acceptor cells, the decrease in topoisomerase II gene expression was not. We conclude that the MDR phenotype, the reduced drug accumulation, and the loss of MDR1 P-glycoprotein mRNA are genetically linked. They might be due to a single dominant mutation, which does not cause the alteration in topoisomerase II.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

A phosphoglycerate kinase-related gene conserved between Trypanosoma brucei and Crithidia fasciculata.

Trypanosoma brucei and Crithidia fasciculata both contain three different phosphoglycerate kinase (PGK) genes, A, B and C, in a tandem array. The genes B and C encode the major PGKs: the cytosolic and glycosomal PGKs, respectively. The PGK-A genes of both Trypanosomatid species encode open reading frames related to PGK, which have most active site residues conserved, but contain an insert of 80 amino acids at approximately position 80 of the 420 amino acids average PGK sequence. The deduced amino acid sequence of these inserts is conserved between T. brucei and C. fasciculata (48% positional identity), indicating its functional importance. Although we have not been able to demonstrate PGK activity in the PGK-A gene product, we consider it likely that this gene codes for a minor PGK with special function.

Amino Acid Sequence↗
PubMed1991

Efficient production of functional mRNA mediated by RNA polymerase I in Trypanosoma brucei.

The unicellular eukaryote Trypanosoma brucei evades the immune defence of its mammalian host by antigenic variation. The genes for variant-specific surface glycoproteins (VSGs) are expressed within large multicistronic transcription units. Mature messenger RNAs are produced by trans-splicing and polyadenylation. A remarkable feature of the transcription of VSG genes is its insensitivity to the RNA polymerase II inhibitor alpha-amanitin. This has led to the speculation that RNA polymerase I, normally only involved in the transcription of ribosomal RNA genes, also mediates expression of these surface antigen genes. In higher eukaryotes, however, transcripts produced by RNA polymerase I were found to be poor substrates for processing into mature mRNAs. In contrast, we show here that the RNA polymerase I of T. brucei can mediate the efficient production of functional mRNA for neomycin phosphotransferase. This exceptional ability may be related to the unusual way in which pre-mRNAs are capped in trypanosomes. In most eukaryotes, mRNAs are modified at their 5' end by a capping activity associated with RNA polymerase II; in trypanosomes, mRNAs acquire their 5'-cap from capped mini-exon donor RNA by trans-splicing, a process that could be independent of the RNA polymerase producing the pre-mRNA.

Animals↗
PubMed1991

Alpha-amanitin-resistant transcription units in trypanosomes: a comparison of promoter sequences for a VSG gene expression site and for the ribosomal RNA genes.

Transcription of the predominant surface antigen genes in Trypanosoma brucei is unusual in its resistance to the RNA polymerase inhibitor alpha-amanitin, a property typical for rDNA transcription in eukaryotes. Transcription of most other protein-coding genes in trypanosomes is sensitive to alpha-amanitin. To investigate whether RNA polymerase I, the polymerase that transcribes rRNA genes, can give rise to functional mRNAs in trypanosomes, we have fused the putative promoter of the T.brucei rRNA genes to the chloramphenicol acetyl transferase (CAT) gene and determined CAT activity after transient expression of chimeric constructs in procyclic trypanosomes. We show here that the rRNA promoter yields the same high CAT activity as the promoters for the two predominant surface antigen genes of trypanosomes, the Variant-specific Surface Glycoprotein (VSG) gene of bloodstream trypanosomes and the procyclin gene of insect-form trypanosomes, both of which are also transcribed by an alpha-amanitin-insensitive RNA polymerase. RNA polymerase I of trypanosomes seems therefore able to synthesize pre-mRNAs that are effectively processed into translatable mRNAs. Dissection of the promoter segments showed the minimal elements for a VSG gene expression site promoter to be confined to a segment of -60 to +77 bp, overlapping the most 5' putative transcription start sites as determined in vivo by RNase protection experiments. For the ribosomal promoter region a segment of -258 to +200 bp relative to the putative transcription start site was sufficient for maximal CAT activity. There is a precise requirement for specific nucleotides at the rRNA transcription start site. We detect no homology between the sequences required for promoter function of the three alpha-amanitin-resistant transcription units, rRNA, VSG and procyclin (parp) genes. This suggests that the sequence-specific recognition of these promoters either occurs by common factors detecting sequence homologies that escape us, or by separate factors that bind to different DNA sequences but interact with a common alpha-amanitin-resistant RNA polymerase.

Amanitins↗
PubMed1991

Characterization of the human MDR3 P-glycoprotein and its recognition by P-glycoprotein-specific monoclonal antibodies.

We have cloned a human MDR3 complementary DNA, coding for a P-glycoprotein, into a mammalian expression vector and cotransfected it with a selectable marker into drug-sensitive human BRO melanoma cells. With low frequency we obtained stable, MDR3-expressing clones. Immunocytochemical and immunoblotting analysis of these clones using the monoclonal antibody C219 indicated that human MDR3 P-glycoprotein, like human MDR1 P-glycoprotein, was mainly localized in the plasma membrane and probably glycosylated. Although a significant fraction of the cells (5-10%) in one of the MDR3-expressing clones expressed as much P-glycoprotein as a clearly drug-resistant MDR1-transfected clone, we found no resistance against a range of drugs affected by multidrug resistance. The drugs tested included vincristine, colchicine, VP16-213, daunorubicin, doxorubicin, actinomycin D, and gramicidin D. We did not detect enhanced daunorubicin efflux either in any of the MDR3-expressing cells by fluorescence microscopy. Direct selection with vincristine, actinomycin D, gramicidin D, or daunorubicin of BRO cells transfected with expression constructs containing the regular MDR3 complementary DNA, or a complementary DNA representing a major MDR3 splice variant (C(-141)), likewise failed to yield resistant clones. Thus, although human MDR3 P-glycoprotein is highly similar to human MDR1 P-glycoprotein, we found no indications that it can transport drugs. We investigated the cross-reactivity of the monoclonal antibodies C219, C494, JSB-1, HYB-241, and MRK16, recognizing human MDR1 P-glycoprotein, with human MDR3 P-glycoprotein using immunocytochemistry and immunoblotting. Apart from monoclonal antibody C219, none of the monoclonal antibodies showed detectable cross-reactivity with human MDR3 P-glycoprotein. In our hands, monoclonal antibodies MRK16 and HYB-241 were most suitable for sensitive and specific cytochemical detection of human MDR1 P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

A novel DNA nucleotide in Trypanosoma brucei only present in the mammalian phase of the life-cycle.

The existence of an unusual form of DNA modification in the bloodstream form of the African trypanosome Trypanosoma brucei has been inferred from partial resistance to cleavage of nuclear DNA with PstI and PvuII (Bernards et al, 1984; Pays et al, 1984). This putative modification is correlated with the shut-off of telomeric Variant-specific Surface Glycoprotein (VSG) gene expression sites (ESs). The modification only affects inactive VSG genes with a telomeric location, and it is absent in procyclic (insect form) trypanosomes in which no VSG is made at all. Previous attempts to detect unusual nucleosides in T.brucei DNA were unsuccessful, but we now report the detection of two unusual nucleotides, called pdJ and pdV, in T.brucei DNA, using the 32P-postlabeling technique. Nucleotide pdV was present in both bloodstream form and procyclic T.brucei DNA and co-migrated in two different two-dimensional thin layer chromatography (2D-TLC) systems with hydroxymethyldeoxyuridine 5'-monophosphate (pHOMedU). In contrast, nucleotide pdJ was exclusively present in bloodstream form trypanosomal DNA. Levels of pdJ were higher in DNA enriched for telomeric sequences than in total genomic DNA and pdJ was also detected in other Kinetoplastida species exhibiting antigenic variation. Postlabeling and 2D-TLC analyses showed base J to be different from the known eukaryotic unusual DNA bases 5-methylcytosine, N6-methyladenine and hydroxymethyluracil, and also from (glucosylated) hydroxymethylcytosine, uracil, alpha-putrescinylthymine, 5-dihydroxypentyluracil and N6-carbamoylmethyladenine. We conclude that pdJ is a novel eukaryotic DNA nucleotide and that it is probably responsible for the partial resistance to cleavage by PvuII and PstI of inactive telomeric VSG genes. It may therefore be involved in the regulation of ES activity in bloodstream form trypanosomes.

Animals↗
PubMed1991

Antigenic variation in Trypanosoma brucei: a telomeric expression site for variant-specific surface glycoprotein genes with novel features.

African trypanosomes evade the immune response of their host by periodically changing their variant surface glycoprotein (VSG) coat. Each coat is encoded by a separate VSG gene. Expressed genes are in a telomeric expression site (ES) and there are several sites in each trypanosome. To study the transcription control of VSG genes in Trypanosoma brucei we have analyzed an ES, called the dominant ES (DES), that readily switches off and on. The promoter area of the DES is very similar to that of the 221 ES (Zomerdijk et al., 1990). It can be switched off and on in vivo without detectable DNA alterations in the vicinity of the transcription start and it can drive high transient expression of a reporter gene in transfection experiments. However, there are also two major differences between the DES and the 221 ES. First, one version of the DES contains an additional upstream transcription unit overlapping the VSG gene ES promoter. The presence of this upstram transcription is dispensable, however, for the VSG gene ES promoter is active, even if transcription through this start from the upstream promoter is blocked using UV light. Moreover, a second version of the DES present in another trypanosome variant does not produce these upstream transcripts. Secondly, we find that the inactivation of DES transcription in one trypanosome variant is accompanied by DNA alterations in the DES upstream (greater than 2 kb) of the transcription start; reactivation of DES transcription is accompanied by another alteration far upstream. Although we cannot exclude that these DNA rearrangements are incidental, our results raise the possibility that the activity of ES promoters is negatively controlled in cis by far upstream sequences not included in transfection constructs and that alterations in these sequences may lead to (in)activation of the promoter.

Animals↗
PubMed1991

Structure of the human MDR3 gene and physical mapping of the human MDR locus.

Two genes, MDR1 and MDR3, constitute the human P-glycoprotein gene family. To examine the evolutionary relationship between the three known classes of mammalian P-glycoprotein genes, we have cloned the MDR3 gene and compared its structure with that of the human MDR1 and the mouse mdr1 (mdr1b) genes analyzed by other groups. The MDR3 gene contains 28 exons and 27 of these contain coding sequences for the two homologous halves of the protein that correlate with functional domains. This structure is virtually identical to that of the human MDR1 gene and the mouse mdr1 (mdr1b) gene, indicating that the exon/intron structure was fixed before the duplication events that generated different classes of P-glycoproteins, but after the P-glycoproteins diverged from related genes, like the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which has an entirely different exon/intron structure. The four alternatively spliced transcripts of the MDR3 gene arise from alternative splicing of exons 23 and 26. Our analysis of DNA clones covering about 120 kilobases (kb) of the human MDR locus, including the entire MDR3 gene (74 kb) and the intergenic region between both genes (34 kb), combined with pulsed-field gel electrophoresis data shows that the human MDR locus covers about 230 kb. In contrast to the mouse mdr genes, both human genes are transcribed in the same direction (MDR3 located downstream of MDR1). The CpG-rich sequences marking the 5' ends of both genes are hypomethylated to different extents in different cell lines. Hypomethylation roughly correlates with transcriptional activity.

Amino Acid Sequence↗
PubMed1991

Direct and inverted DNA repeats associated with P-glycoprotein gene amplification in drug resistant Leishmania.

The H circle of Leishmania species contains a 30 kb inverted duplication separated by two unique DNA segments, a and b. The corresponding H region of chromosomal DNA has only one copy of the duplicated DNA. We show here that the chromosomal segments a and b are flanked by inverted repeats (198 and 1241 bp) and we discuss how these repeats could lead to formation of H circles from chromosomal DNA. Selection of Leishmania tarentolae for methotrexate resistance indeed resulted in the de novo formation of circles with long inverted duplication, but two mutants selected for arsenite resistance contained new H region plasmids without such duplications. One of these plasmids appears due to a homologous recombination between two P-glycoprotein genes with a high degree of sequence homology. Our results show how the same DNA region in Leishmania may be amplified to give plasmids with or without long inverted duplications and apparently by different mechanisms.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Molecular genetics of antigenic variation.

Antigenic variation is one of the most effective strategies developed by parasites to escape immune destruction. It requires a large wardrobe of surface coats and mechanisms to exchange one coat for an unrelated one. The molecular principles of antigenic variation are now largely known in the bacterial species Borrelia and Neisseria and in the protozoa of the African trypanosome group and these three examples are discussed here by Piet Borst.

Amino Acid Sequence↗