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.

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.

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 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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Three small RNAs within the 10 kb trypanosome rRNA transcription unit are analogous to domain VII of other eukaryotic 28S rRNAs.

We have localized the six ribosomal RNAs (rRNAs) which encode the 28S rRNA region of Trypanosoma brucei. These six rRNAs include two large rRNAs, 28S alpha (approx. 1840 nt) and 28S beta (approx.

A 22-kd protein (sorcin/V19) encoded by an amplified gene in multidrug-resistant cells, is homologous to the calcium-binding light chain of calpain.

We have previously shown that at least five linked genes are co-amplified and overexpressed in the multi-drug resistant (MDR) Chinese hamster ovary cell line CHRC5. We show here that one of these genes (class 4) codes for a small phosphorylated, cytosolic protein, sorcin/V19, known to be overproduced by many MDR cell lines. The class 4 gene codes for a nested set of mRNAs, varying in size between 1000 and 2500 nucleotides.

Differential amplification and disproportionate expression of five genes in three multidrug-resistant Chinese hamster lung cell lines.

At least five linked genes are amplified in the multidrug-resistant Chinese hamster ovary cell line CHRC5, selected with colchicine (A. M. Van der Bliek, T.

The common 5' terminal sequence on trypanosome mRNAs: a target for anti-messenger oligodeoxynucleotides.

Several mature mRNAs of Trypanosoma brucei were previously shown to have a common 5' terminal sequence of 35 nucleotides (nt) encoded by a separate mini-exon. To verify whether all trypanosome mRNAs contain this mini-exon sequence at their 5' end, we have tested oligodeoxynucleotides complementary to different parts of the 35 nt leader sequence for their ability to inhibit translation of total trypanosome mRNA. All oligomers tested inhibited translation of trypanosome mRNAs in a wheat germ extract.

Rapid change of the repertoire of variant surface glycoprotein genes in trypanosomes by gene duplication and deletion.

To study the evolution of the variant surface glycoprotein (VSG) repertoire of trypanosomes we have analysed the DNA region surrounding the VSG 118 gene in different trypanosome strains. We find a remarkable degree of variation in this area. Downstream from the 118 gene a 5.

Characterization of the gene for the microbody (glycosomal) triosephosphate isomerase of Trypanosoma brucei.

To determine how microbody enzymes enter microbodies, we are studying the genes for glycosomal (microbody) enzymes in Trypanosoma brucei. Here we present our results for triosephosphate isomerase (TIM), which is found exclusively in the glycosome. We found a single TIM gene without introns, having one major polyadenylated transcript of 1500 nucleotides with a long untranslated tail of approximately 600 nucleotides.