Biomedical subjects

P Borst

Publications and source records attributed to P Borst.

At least 163 records · Page 9Linked to original sources

PubMed1988

The tissue dependent expression of hamster P-glycoprotein genes.

Using a sensitive RNase protection assay, we have measured the levels of P-glycoprotein mRNA in fourteen different Chinese hamster tissues and in three cell lines to determine whether a relationship exists between the level of P-glycoprotein expression and the occurrence of primary and acquired multidrug resistance (MDR) in cancer. P-Glycoprotein mRNA was detected in all tissues, suggesting that the protein is a normal constituent of the cell. High levels of P-glycoprotein mRNA were found in oesophagus, testis and uterus. Intermediate levels in brain, lung and ovary, and a very low level in the adrenal gland, bladder, bone marrow, heart, kidney, liver and spleen. There is no obvious correlation between this expression pattern and the occurrence of primary or acquired MDR.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1988

The topogenic signal of the glycosomal (microbody) phosphoglycerate kinase of Crithidia fasciculata resides in a carboxy-terminal extension.

To determine how microbody proteins enter microbodies, we have previously compared the genes for the cytosolic and glycosomal (microbody) phosphoglycerate kinases (PGKs) of Trypanosoma brucei and found the microbody enzyme to differ from other PGKs and the cytosolic form in two respects: a high net positive charge and a C-terminal extension of 20 amino acids (Osinga et al., 1985). Here we present the comparison of the genes for the cytosolic and glycosomal PGKs of Crithidia fasciculata, another kinetoplastid organism. The amino acid sequences of the two Crithidia isoenzymes are virtually identical, except for a C-terminal extension of 38 amino acids. We conclude that this extension must direct the glycosomal PGK to the glycosome. The extensions of the Crithidia and Trypanosoma enzymes are both rich in small hydrophobic and hydroxyl amino acids.

Amino Acid Sequence↗

PubMed1988

Nucleoside analysis of DNA from Trypanosoma brucei and Trypanosoma equiperdum.

We have digested trypanosome DNA with a combination of pancreatic DNase I, nuclease P1 and bovine alkaline phosphatase and fractionated the resulting nucleosides on a Supelcosil LC-18-S column by high pressure liquid chromatography. We find less than 0.1% unusual nucleosides, both in Trypanosoma brucei and in a Trypanosoma equiperdum stock, in contrast to a previous report of an unusual nucleoside replacing dC at 1.3% of total nucleosides in T. equiperdum. Our results agree with previous suggestions that the modification of inactive telomeric expression sites for variant-specific surface glycoprotein genes in T. brucei only affects a very small fraction of the total DNA.

Animals↗

PubMed1987

Controlled turnover and 3' trimming of the trans splicing precursor of Trypanosoma brucei.

The maturation of mRNAs in Trypanosoma brucei involves a novel step, in which a short capped sequence is spliced in trans onto the 5' end of nascent mRNAs from a 140-nucleotide precursor. This precursor is called the mini-exon-derived RNA or medRNA. We have used drugs and ultraviolet irradiation as inhibitors to probe the synthesis and processing of medRNA in vivo. Inhibition of RNA synthesis by chloroquine shows that the half-life of medRNA is about 4 minutes. Despite this high turnover, only limited accumulation of medRNA could be achieved following a block in the synthesis of high molecular weight splice acceptor substrates by UV irradiation. This implies that there is a constraint on the steady-state levels of medRNA and that excess medRNA is degraded in the cell. A 3' shortened version of medRNA accumulates upon a block in normal medRNA processing by UV irradiation or upon treatment of the cells with actinomycin D or novobiocin but was shown not to participate in trans splicing, making it a likely candidate for an in vivo degradation intermediate.

Animals↗

PubMed1987

The anatomy and transcription of a telomeric expression site for variant-specific surface antigens in T. brucei.

The variant specific surface glycoprotein (VSG) genes of T. brucei are expressed in telomeric expression sites. We have determined the structure of the active site in trypanosome variant 221a, which contains VSG gene 221, by analysis of cloned DNA segments that represent 65 kb of the 5'-flanking region of the VSG gene. In nuclear run-on experiments, 57 kb of adjacent sequences are cotranscribed with the VSG gene at approximately similar rates and in the alpha-amanitin-resistant manner characteristic of VSG genes. Besides the VSG mRNA, this expression site yields at least seven stable RNAs, suggesting that it is a multicistronic transcription unit. Our results also show that insertion of a transcriptional terminator is not the general mechanism of switching off expression sites.

Animals↗

PubMed1987

Inactivation of transcription by UV irradiation of T. brucei provides evidence for a multicistronic transcription unit including a VSG gene.

We have used inactivation of transcription by UV irradiation to map transcription units in trypanosomes. The relative inactivation rate of the transcription of mini-exon, 5S, and rRNA genes was inversely proportional to the previously estimated lengths of these transcription units. The telomeric transcription unit containing the gene for variant-specific surface glycoprotein (VSG) 221 was inactivated as a single unit of 60 kb. This long transcription unit comprises at least one other protein-coding gene and yields seven other stable mRNAs. These data thus provide evidence for a multicistronic transcription unit for cellular genes in a eukaryote.

Animals↗

PubMed1987

Trypanosoma brucei variant-specific glycoprotein gene chromatin is sensitive to single-strand-specific endonuclease digestion.

Active variant surface glycoprotein (VSG) gene chromatin is preferentially digested by the restriction enzyme HinfI in nuclei of bloodstream variants of Trypanosoma brucei. HinfI sensitivity of VSG gene chromatin is not observed in nuclei of relapse variants in which the VSG gene has been inactivated in situ. Active VSG gene chromatin is preferentially degraded by the single-strand-specific endonucleases S1 and Bal31. This sensitivity is not the result of pre-existing single-strand breaks or a detectably altered nucleosomal organization. Trypanosome nuclei in which the run-on transcription of VSG genes has been specifically shut down have been used to show that Hinfl and Bal31 sensitivity is not dependent upon continued transcription of the VSG gene. The presence of single-stranded DNA regions within VSG gene chromatin is consistent with a model in which VSG genes are activated by increased torsional stress.

Animals↗

PubMed1987

Chromosomal localization of three genes coamplified in the multidrug-resistant CHRC5 Chinese hamster ovary cell line.

At least five gene classes are amplified in the multidrug-resistant CHO cell line CHRC5. Protein products have been identified for two classes; class 2 codes for the large membrane P-glycoprotein, whereas class 4 encodes the small cytoplasmic calcium-binding protein sorcin (V19). By DNA analysis we have shown previously that these five genes are linked in two groups: class 1 + 2 + 3; and class 4 + 5. By use of in situ hybridization with complementary DNAs derived from the resistant cell line we demonstrate here that genes from both linkage groups are amplified and situated together in each of two different chromosomal regions of the resistant Chinese hamster cell line. The positions of the amplicons correspond to cytogenetically identified homogeneously staining regions in an altered 7q+ chromosome and in a rearranged Z-7 [t(3;4)] chromosome. The native genes were mapped both in the CHRC5 line and in a normal diploid Chinese hamster cell strain, CHNF 86. We confirm the position of the class 2 gene on 1q26 and we show that class 4 and 5 genes are located in the same region of 1q. We conclude that the gene classes 2, 4, and 5 are closely juxtaposed in the normal Chinese hamster genome and comprise one amplicon in resistant cells. Our results are compatible with the hypothesis that multidrug resistance is due to overexpression of P-glycoprotein genes and that the other genes amplified in the CHRC5 line are coamplified because they happen to lie close to the P-glycoprotein genes.

Animals↗

PubMed1987

RNA end-labeling and RNA ligase activities can produce a circular rRNA in whole cell extracts from trypanosomes.

We have found two enzymatic activities in whole cell extracts from Trypanosoma brucei; an end-labeling reaction involving a single uridine at the 3' end of ribosomal RNAs (rRNAs) and an RNA ligase activity joining 5' monophosphates to 3' hydroxyl groups. The RNA ligase acts upon one of the small rRNAs (180 nucleotides) from the trypanosome ribosomal repeat unit, forming a circular RNA. The specific circularization of this small rRNA is probably dependent on the secondary structure of the molecule and is not detectable in vivo.

Animals↗

PubMed1987

Coincident multiple activations of the same surface antigen gene in Trypanosoma brucei.

Trypanosomes with a coat of variant surface glycoprotein (VSG) 118, consistently appear around day 20 when a rabbit is infected with Trypanosoma brucei strain 427. There is a single chromosome-internal gene for VSG 118 and this is activated by duplicative transposition to a telomeric expression site. We show here that the expression-linked extra copy of VSG gene 118 in a day 18 population of a chronic infection is heterogeneous, and we infer that the population is not monoclonal but is the result of multiple independent activations of the 118 gene. We show that the heterogeneity of expression-linked extra copies is also present in other trypanosome populations expressing chromosome-internal VSG genes. We present a model for the timing of VSG gene activation during chronic infection that emphasizes two features: the relative activation and inactivation frequencies of different expression sites, and the degree of homology of the sequences flanking VSG genes with expression sites.

Antigens, Protozoan↗

PubMed1987

Programmed gene rearrangements altering gene expression.

Programmed gene rearrangements are used in nature to to alter gene copy number (gene amplification and deletion), to create diversity by reassorting gene segments (as in the formation of mammalian immunoglobulin genes), or to control the expression of a set of genes that code for the same function (such as surface antigens). Two major mechanisms for expression control are DNA inversion and DNA transposition. In DNA inversion a DNA segment flips around and is rejoined by site-specific recombination, disconnecting or connecting a gene to sequences required for its expression. In DNA transposition a gene moves into an expression site where it displaces its predecessor by gene conversion. Gene rearrangements altering gene expression have mainly been found in some unicellular organisms. They allow a fraction of the organisms to preadapt to sudden changes in environment, that is, to alter properties such as surface antigens in the absence of an inducing stimulus. The antigenic variation that helps the causative agents of African trypanosomiasis, gonorrhea, and relapsing fever to elude host defense is controlled in this way.

Animals↗

PubMed1987

Stable introduction of exogenous DNA into Trypanosoma brucei.

The lack of a homologous transformation system for trypanosomes is a serious handicap to the study of gene expression in these protozoans. Attempts to develop such a system have been complicated by the lack of suitable homologous vectors and ignorance of the requirements for mRNA synthesis which is discontinuous in trypanosomes. We have found that Trypanosoma congolense, a close relative of T. brucei, contains exceptionally small chromosomes, which can be isolated whole and distinguished from those of T. brucei by the presence of a unique satellite DNA. We show here that mini-chromosomes from T. congolense can be introduced into T. brucei by electroporation and detected by hybridisation with T. congolense satellite DNA. The introduced DNA can survive through several generations in the absence of any selective pressure. These results provide the basis for the development of a transformation system for trypanosomes.

Animals↗

PubMed1987

The human mdr3 gene encodes a novel P-glycoprotein homologue and gives rise to alternatively spliced mRNAs in liver.

We have found cDNAs corresponding to a novel human P-glycoprotein gene in liver cDNA banks. The sequence of the 3' part of this cDNA reveals a domainal organization of the derived protein similar to that of the known P-glycoproteins and an 80% amino acid homology with the product of the human mdr1 gene (Chen et al., 1986). The new gene lies within 500 kb from mdr1 as determined by pulsed field gradient gel electrophoresis and is designated mdr3, as it appears to correspond to the third of the three P-glycoprotein genes mapped in the hamster multidrug resistance domain. mdr3 yields a transcript of 4100 nucleotides, 400 nucleotides less than the mdr1 transcript; the difference is accounted for by the shorter 3'-untranslated region of the mdr3 mRNA. Our cDNAs provide evidence for alternative splicing of mdr3 pre-mRNAs. One alternative is an insert of seven amino acids between the two major blocks of the nucleotide binding site and another is a deletion of 43 or 47 amino acids covering the putative transmembrane segment 5a. We speculate that these alternatives superimposed on differential expression of P-glycoprotein homologues could provide an explanation for the large variation in cross-resistance patterns observed in cell lines selected for multidrug resistance with different cytostatic drugs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1987

Common elements on the surface of glycolytic enzymes from Trypanosoma brucei may serve as topogenic signals for import into glycosomes.

In Trypanosoma brucei, a major pathogenic protozoan parasite of Central Africa, a number of glycolytic enzymes present in the cytosol of other organisms are uniquely segregated in a microbody-like organelle, the glycosome, which they are believed to reach post-translationally after being synthesized by free ribosomes in the cytosol. In a search for possible topogenic signals responsible for import into glycosomes we have compared the amino acid sequences of four glycosomal enzymes: triosephosphate isomerase (TIM), glyceraldehyde-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK) and aldolase (ALDO), with each other and with their cytosolic counterparts. Each of these enzymes contains a marked excess of positive charges, distributed in two or more clusters along the polypeptide chain. Modelling of the three-dimensional structures of TIM, PGK and GAPDH using the known structural coordinates of homologous enzymes from other organisms indicates that all three may have in common two 'hot spots' about 40 A apart, which themselves include a pair of basic amino acid residues separated by a distance of about 7 A. The sequence of glycosomal ALDO, for which no three-dimensional information is available, is compatible with the presence of the same configuration on the surface of this enzyme. We propose that this feature plays an essential role in the import of enzymes into glycosomes.

Amino Acid Sequence↗

PubMed1987

In vivo labelling of intermediates in the discontinuous synthesis of mRNAs in Trypanosoma brucei.

Discontinuous mRNA synthesis in trypanosomes is thought to involve a 140-nucleotide precursor, called the mini-exon-derived RNA or medRNA, which contributes its 5' 35 nucleotides to the 5' end of nascent mRNAs. We used in vivo labelling of RNA to show that medRNA has a half-life of less than 6 min, whereas putative high mol. wt intermediates containing the 3' part of the medRNA have an average half-life of less than 1 min. This eliminates priming of pre-mRNA synthesis by intact medRNA as the main mode of discontinuous mRNA synthesis. Potential intermediates of 35 and 105 nucleotides were labelled in parallel with medRNA, but their significance could not be assessed in RNA preparations containing medRNA, as they are also produced by artefactual cleavage of medRNA. We show, however, that high mol. wt RNA, free of medRNA, can release medRNA segments upon a debranching treatment. These results are consistent with a trans splicing mechanism involving short-lived forked intermediates, analogous to lariats in cis splicing systems.

Adenosine↗

PubMed1987

Structure of amplified DNA, analyzed by pulsed field gradient gel electrophoresis.

Pulsed field gradient electrophoresis allows the separation of large DNA molecules up to 2,000 kilobases (kb) in length and has the potential to close the resolution gap between standard electrophoresis of DNA molecules (smaller than 50 kb) and standard cytogenetics (larger than 2,000 kb). We have analysed the amplified DNA in four cell lines containing double minute chromosomes (DMs) and two lines containing homogeneously staining regions. The cells were immobilized in agarose blocks, lysed, deproteinized, and the liberated DNA was digested in situ with various restriction endonucleases. Following electrophoretic separation by pulsed field gel electrophoresis, the DNA in the gel was analysed by Southern blotting with appropriate probes for the amplified DNA. We find that the DNA in intact DMs is larger than 1,500 kb. Our results are also compatible with the notion that the DNA in DMs is circular, but this remains to be proven. The amplified segment of wild-type DNA covers more than 550 kb in all lines and possibly up to 2,500 kb in some. We confirm that the repeat unit is heterogeneous in some of the amplicons. In two cell lines, however, with low degrees of gene amplification, we find no evidence for heterogeneity of the repeats up to 750 (Y1-DM) and 800 kb (3T6-R50), respectively. We propose that amplicons start out long and homogeneous and that the heterogeneity in the repeat arises through truncation during further amplification events in which cells with shorter repeats have a selective advantage. Even if the repeats are heterogeneous, however, pulsed field gradient gels can be useful to establish linkage of genes over relatively short chromosomal distances (up to 1,000 kb). We discuss some of the promises and pitfalls of pulsed field gel electrophoresis in the analysis of amplified DNA.

Adrenal Cortex Neoplasms↗

PubMed1987

Kinetoplast DNA of Trypanosoma evansi.

We show here that the kinetoplast DNA (kDNA) networks from six Trypanosoma evansi strains differ from those of T. brucei by their lack of maxi-circles and absence of mini-circle sequence heterogeneity. The lack of maxi-circles is sufficient to account for the inability of T. evansi to multiply in tsetse flies, since this requires functional mitochondria containing maxi-circle gene products. Judged by restriction enzyme analysis, five of the six T. evansi strains contain mini-circles that differ less than 4% in sequence. This type A mini-circle is found in strains from East Africa, West Africa and South America. Another strain from East Africa contains a very different mini-circle (type B), which shows about the same degree of hybridization to type A mini-circles as to a mini-circle from T. brucei. We propose that the pronounced sequence heterogeneity of the mini-circles of T. brucei has arisen by recombination of strains that had diverged for long periods of time in reproductive isolation. We further propose that the homogeneous mini-circles of T. evansi (and T. equiperdum) reflect the inability of species to mate. This proposal implies that mini-circle heterogeneity indicates (infrequent) genetic exchange and that all kinetoplastid flagellates with heterogeneous mini-circles exchange DNA.

Animals↗

PubMed1987

Platelet-activating factor: mediator of the third pathway of platelet aggregation? A study in three patients with deficient platelet-activating factor synthesis.

Thrombin, collagen, and Ca2+-ionophore A23187 aggregate platelets in the presence of inhibitors of the first (ADP-mediated) and second (cyclooxygenase-dependent) pathway of platelet activation. This aggregation, via a third pathway, was hypothesized to be mediated by the alkoxyether lipid platelet-activating factor (PAF). We recently demonstrated virtual absence of plasmalogen-type alkoxyether lipids and deficiency in key enzymes of their biosynthesis in Zellweger patients. We hypothesized that PAF synthesis might also be impaired. We report two Zellweger patients with an undetectable A23187-induced PAF synthesis of leukocytes (patients, less than 3 pmol PAF/10(8) granulocytes (PMN); four age-matched controls, 249-2,757 pmol PAF/10(8) PMN; five adult controls, 291-5,433 pmol PAF/10(8) PMN). In a third patient, residual PAF synthesis was detected. However in all patients the thrombin-induced third mechanism of platelet aggregation was present. We therefore conclude that PAF may not be the mediator of the third pathway.

Abnormalities, Multiple↗