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I Roditi

Publications and source records attributed to I Roditi.

48 records · Page 3Linked to original sources
PubMed1990

Trypanosoma brucei: constitutive activity of the VSG and procyclin gene promoters.

The variant surface glycoprotein (VSG) and procyclin are the major surface proteins of the bloodstream and procyclic stages, respectively, of Trypanosoma brucei. The promoter regions of the VSG and procyclin gene transcription units could be mapped thanks to the specific enrichment of initial transcripts that occurs following UV irradiation. Whereas the VSG gene is 45 kb distant from its promoter, procyclin genes are located immediately downstream. We show, by run-on assays on isolated nuclei and by cDNA analysis, that transcription occurs from both promoters in bloodstream as well as in procyclic forms. It is inferred that the control of the stage-specific expression of VSG and procyclin genes is not effected at the level of transcription initiation, but most probably by interfering with the elongation and stability of the specific transcripts.

Animals↗
PubMed1989

Duplication and transcription of procyclin genes in Trypanosoma brucei.

The genes encoding procyclin, the major glycoprotein expressed on the surface of procyclic forms of Trypanosoma brucei, comprise a multigene family. It has previously been demonstrated that procyclin genes in cloned trypanosome strains from Kenya and Uganda show restriction fragment polymorphisms. A detailed study of the Kenyan strain 227 has revealed that procyclin genes are arranged in tandem at 3 distinct loci (Pro A, B and C) and that the polymorphism is due to the duplication of 1.3 kb in the Pro A locus, which has generated an additional procyclin gene. Northern blot analysis has shown that at least 2 loci are transcribed and that a minimum of 3 procyclin genes are expressed within a cloned line. The transcription of procyclin genes is resistant to 1 mg ml-1 alpha-amanitin, whereas that of the 5' flanking gene in the Pro A locus is sensitive. This observation suggests that the two genes form part of separate transcription units with a promoter between them.

Amanitins↗
PubMed1989

Expression of Trypanosoma brucei procyclin as a fusion protein in Escherichia coli.

Procyclin, a glycoprotein surface antigen of procyclic forms of Trypanosoma brucei, was expressed in Escherichia coli as a cro-beta-galactosidase fusion protein. Antibodies produced in rabbits immunised with gel-purified fusion protein bound to the surface of living procyclic culture forms in indirect immunofluorescence assays and were able to immunoprecipitate procyclin from lysates of trypanosomes biosynthetically labelled with tritiated proline. In addition, the antibodies recognised synthetic peptides corresponding to three different regions of the procyclin molecule, including a glutamic acid-proline dipeptide repeat. The results indicate that T. brucei procyclin expressed as a fusion protein is immunogenic and antigenically intact. In contrast, no rabbit antibodies could be produced against a 16-amino-acid synthetic peptide consisting of the dipeptide repeat, even when the peptide was coupled to carrier proteins.

Animals↗
PubMed1989

Procyclin gene expression and loss of the variant surface glycoprotein during differentiation of Trypanosoma brucei.

In the mammalian host, the unicellular flagellate Trypanosoma brucei is covered by a dense surface coat that consists of a single species of macromolecule, the membrane form of the variant surface glycoprotein (mfVSG). After uptake by the insect vector, the tsetse fly, bloodstream-form trypanosomes differentiate to procyclic forms in the fly midgut. Differentiation is characterized by the loss of the mfVSG coat and the acquisition of a new surface glycoprotein, procyclin. In this study, the change in surface glycoprotein composition during differentiation was investigated in vitro. After triggering differentiation, a rapid increase in procyclin-specific mRNA was observed. In contrast, there was a lag of several hours before procyclin could be detected. Procyclin was incorporated and uniformly distributed in the surface coat. The VSG coat was subsequently shed. For a single cell, it took 12-16 h to express a maximum level of procyclin at the surface while the loss of the VSG coat required approximately 4 h. The data are discussed in terms of the possible molecular arrangement of mfVSG and procyclin at the cell surface. Molecular modeling data suggest that a (Asp-Pro)2 (Glu-Pro)22-29 repeat in procyclin assumes a cylindrical shape 14-18 nm in length and 0.9 nm in diameter. This extended shape would enable procyclin to interdigitate between the mfVSG molecules during differentiation, exposing epitopes beyond the 12-15-nm-thick VSG coat.

Amino Acid Sequence↗
PubMed1987

The structure and transcription of an element interspersed between tandem arrays of mini-exon donor RNA genes in Trypanosoma brucei.

Messenger RNAs in Trypanosoma brucei brucei have the same 35 bases at the 5' end. These 35 bases are not encoded contiguously in the genome, but are donated from a 140 base RNA (the mini-exon donor RNA). The mini-exon donor RNA (medRNA) is encoded by 1.35 kbp genes that occur in tandem repeats. A DNA element that is associated with mini-exon donor RNA medRNA genes has been identified and characterised by restriction enzyme mapping and partial sequencing. This element (the medRNA gene associated element: MAE) varies in length between 5.5 and 7 kbp. There are between 20 and 40 copies of the element per haploid genome. In clones of genomic DNA MAEs occurred between two medRNA gene arrays and on both sides of a medRNA gene array. The MAEs were always in the same orientation with respect to the medRNA genes. It is proposed that in the genome MAEs are interspersed between tandem arrays of medRNA genes. The transcription of the element has been investigated. Low levels of a 70-80 base transcript derived from a small part of MAE were detected in steady state RNA. Nuclear run off transcript studies indicated that MAEs were transcribed at high levels and that they possibly contain at least one start and stop of transcription.

Animals↗
PubMed1983

Sequence analysis of fowl plague virus mutant ts47 reveals a nonsense mutation in the NS1 gene.

A mutant of fowl plague virus, ts47, induces the synthesis in infected cells of a truncated NS1 polypeptide at both permissive and restrictive temperatures. Nucleotide sequence analysis of the segment coding for the NS1 polypeptide, segment 8, indicates that this aberration is due to a nonsense mutation. This mutation occurs in the region of the NS1 gene which overlaps with the NS2 gene and there is a corresponding amino acid substitution in the NS2 polypeptide. While it is not clear which polypeptide is responsible for the thermal instability of ts47, the loss of the COOH-terminal 28 amino acid residues from the NS1 polypeptide does not affect replication of the virus at permissive temperatures.

Base Sequence↗
PubMedDate not supplied

The repetitive microtubule-associated proteins MARP-1 and MARP-2 of Trypanosoma brucei.

The microtubular membrane skeleton of Trypanosoma brucei contains two closely related, repetitive, high-molecular-weight microtubule-associated proteins, MARP-1 and MARP-2 (MARP for Microtubule-Associated Repetitive Proteins). Their structure is unusual in that they consist of tandemly arranged, strongly conserved 38-amino-acid repeat units over almost their entire length of about 320 kDa. Their nonrepetitive N and C ends are comparatively short. The predicted amino acid sequences reveal a gradient of similarity between MARP-1 and MARP-2 which increases from the N-terminus (no significant similarity) through the repeat domain (50% similarity) to the C-terminus (94.5% similarity). Transfection of mammalian cell lines with recombinant fragments of MARP-2 demonstrate that the nonrepetitive C-terminus of MARP-2 binds specifically to microtubules. This C-terminus does not show sequence similarity with any other microtubule-associated proteins and thus appears to represent a novel type of microtubule-binding domain.

Amino Acid Sequence↗
PubMedDate not supplied

Molecular characterization of glycosomal NAD(+)-dependent glycerol 3-phosphate dehydrogenase from Trypanosoma brucei rhodesiense.

The primary structure of a 38-kDa protein isolated from membrane preparations of African trypanosomes was determined by protein and DNA sequencing. Searching of the protein database with the trypanosome translated amino acid sequence identified glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) from various prokaryotic and eukaryotic organisms as the optimal scoring protein. Surprisingly, the eukaryotic trypanosome enzyme showed the highest degree of sequence identity with the corresponding enzyme from the prokaryote Escherichia coli. The trypanosome molecule was expressed in Escherichia coli and found to be enzymatically active, thus confirming the identity of the molecule as an NAD(+)-dependent glycerol 3-phosphate dehydrogenase. A monoclonal antibody specific for the 38-kDa protein was used to localize the enzyme to glycosomes. Immunoblotting showed that the monoclonal antibody bound to a 38-kDa protein in African trypanosomes but not in T. cruzi, Leishmania or Crithidia. The enzyme has a pI of 9.1, a net charge of +17 and contains the peroxisome-like targeting tripeptide SKM at its C-terminus, all characteristic of glycosomal enzymes. Amino acids predicted to be involved in the NAD(+)-dependent glycerol 3-phosphate dehydrogenase active site have diverged from those of the mammalian enzyme. Kinetic analyses of the trypanosome GPD and GPD from rabbit muscle showed that the Km values of the two enzymes are different. The data suggest that the trypanosome protein may be a candidate target for rational drug design.

Amino Acid Sequence↗
PubMedDate not supplied

Expression of a polypeptide containing a dipeptide repeat is confined to the insect stage of Trypanosoma brucei.

The protozoan parasite Trypanosoma brucei is transmitted between mammalian hosts by the tsetse fly (Glossina spp.). Trypanosomes ingested by the fly undergo a number of changes in the insect midgut during differentiation to procyclic forms. These include the loss of the variant specific glycoprotein (VSG) coat and the appearance of a common set of procyclic surface antigens. In order to investigate genes other than VSG genes which are expressed only at certain stages of the life cycle, the first cDNA specific to procyclic culture form trypanosomes (equivalent to the stage found in the insect midgut) has been characterized. The encoded polypeptide shows several characteristics of membrane proteins, but its most striking feature is the presence of a repetitive amino-acid sequence in which there are 22 tandem repeats of the dipeptide-Glu-Pro-. Related genes are also found in other trypanosome species and in leishmania. This gene shows many similarities to a number of surface antigen genes described in malaria and, more recently, Trypanosoma cruzi. This is the first example of a repetitive sequence in a parasite protein which is present only in the insect vector, and which therefore cannot be implicated in the mammalian host immune response.

Amino Acid Sequence↗