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G A Cross

Publications and source records attributed to G A Cross.

At least 127 records · Page 7Linked to original sources
PubMed1981

Activation of trypanosome surface glycoprotein genes involves a duplication-transposition leading to an altered 3' end.

Expression of the genes for variant surface glycoproteins 117 and 118 in Trypanosoma brucei is accompanied by the appearance of an extra copy of these genes, the expression-linked copy, which differs in the surrounding restriction enzyme sites from the corresponding basic copy of the genes. We present direct evidence that the expression-linked copy is the one used for messenger RNA synthesis. By S1-nuclease-protection experiments we show that cloned basic-copy genes contain the nucleotide sequence of the corresponding messenger RNA except for the last 100 to 150 nucleotides before the poly(A) tail. Comparison of the 3'-terminal sequence of the 117 basic-copy gene and the 117 complementary DNA shows that this region differs by multiple point mutations, insertions and deletions, the differences starting within the coding sequence. Genomic blots demonstrate that a Bsp I site in the 3'-terminal part of the 118 complementary DNA is present in the expression-linked copy but not in the basic-copy gene. We conclude that expression-linked copies are the active genes, and that the generation of expression-linked copies involves a duplication--transposition in which the 3' end of the gene is replaced.

Amino Acid Sequence↗
PubMed1981

Glycopeptides from variant surface glycoproteins of Trypanosoma Brucei. C-terminal location of antigenically cross-reacting carbohydrate moieties.

Glycopeptides have been purified after trypsin or pronase cleavage of variant surface glycoproteins from five antigenic variants of one clone of Trypanosoma brucei. Each peptide has been characterised by its amino acid and sugar composition and partial or complete amino acid sequence. The peptides were assayed in an indirect precipitation radioimmunoassay for inhibition of precipitation of one variant surface glycoprotein by a heterologous antiserum raised against another variant. Two classes of glycopeptide were obtained, those which showed complete inhibition and those which had no inhibitory activity. The non-cross-reacting glycopeptides were all derived from internal sites in the polypeptide chain and had only mannose and glucosamine as the constituents of the glycosyl group with one exception which also contained galactose. The carbohydrate moieties involved in the immunological cross-reaction were found to be attached at or very close to the C-terminus of the protein and contained galactose, mannose and glucosamine. Considerable variation in the size and composition of these glycosyl moieties occurred between the variant glycoproteins and, in two cases, within individual glycoproteins. The immunological cross-reactivity was constant despite this oligosaccharide heterogeneity. There was significant amino acid sequence homology between glycopeptides from certain variants in contrast to the absence of homology observed previously for the N-terminal sequence of intact surface glycoproteins. Some C-terminal glycopeptide sequences suggest the occurrence of proteolytic processing subsequent to glycosylation and prior to variant surface glycoprotein isolation. The terminal amino acid (Asx or Ser) of all isolated variant surface glycoproteins was glycosylated.

Amino Acid Sequence↗
PubMed1980

A variant surface glycoprotein of Trypanosoma brucei synthesized with a C-terminal hydrophobic 'tail' absent from purified glycoprotein.

Sequential expression of variant surface glycoproteins (VSGs) enables the parasitic protozoan Trypanosoma brucei to evade the immune response of its mammalian hosts. Studies of several VSGs, which have been isolated as soluble molecules following disruption of cells in the absence of detergent, have indicated extensive amino acid diversity and the absence of a hydrophobic segment which might serve to anchor the carboxy terminus to the membrane. The carboxy-terminal tryptic peptides of six VSGs have recently been characterized and shown to be glycosylated. Three of these VSGs terminated with a glycosylated aspartate or asparagine residue (Asx), suggesting that the VSG was cleaved following synthesis and glycosylation and before characterization. We present here nucleotide sequence data which suggest that the primary translation product of one VSG gene contains a hydrophobic tail at the carboxy terminus which is not found on the isolated, mature glycoprotein. The data also predict that the glycosylated residue is aspartic acid rather than the anticipated asparagine.

Amino Acid Sequence↗
PubMed1980

Protective monoclonal antibodies recognising stage-specific merozoite antigens of a rodent malaria parasite.

Immunity to malaria is mediated, at least in part, by antibody. Resistance to infection has been passively transferred with immune serum or its immunoglobulin fraction in human, simian and rodent malaria. However, because of the structural and antigenic complexity of the malaria parasites, it has proved difficult to identify and characterise those parasite antigens against which protective antibody is directed. We have produced several hybrid cell lines secreting monoclonal antibodies against the rodent malaria parasite, Plasmodium yoelii, and we now report that, of the antibodies tested, only those specific for antigens exclusive to the merozoite were protective in passive transfer experiments. Other anti-P. yoelii monoclonal antibodies, apparently recognising antigens in the membrane of the infected erythrocyte, were not protective on passive transfer. The protective monoclonal antibodies should be useful in the isolation of the important antigens of this parasite.

Animals↗
PubMed1980

Novel expression-linked copies of the genes for variant surface antigens in trypanosomes.

Pathogenic African trypanosomes evade the immune system of their mammalian hosts by the sequential expression of alternative cell-surface glycoproteins (reviewed in refs 1,2). Variant surface glycoproteins (VSGs) purified from cloned variants of Trypanosoma brucei have similar molecular weights (about 60,000), but differ in amino acid composition, N-terminal amino acid sequence and C-terminal structure. We have cloned DNA complementary to the messenger RNA's for four immunologically distinct VSGs and hybridised these complementary DNAs (cDNAs) with restriction digests of T. brucei nuclear DNA, fractionated by gel electrophoresis and transferred to nitrocellulose strips. Each cDNA recognises a unique set of fragments and this basic set is present unaltered in the nuclear DNAs from the four variants. In addition, each probe recognises an extra fragment only in nuclear DNA isolated from cells expressing the VSG corresponding to the cDNA probe. We infer that activation of a VSG gene involves the production of an expression-linked copy of that gene.

Animals↗
PubMed1980

The isolation of plasmids containing DNA complementary to messenger RNA for variant surface glycoproteins of Trypanosoma brucei.

We have isolated poly(A)+ RNA from four antigenic variants (117, 118, 121, 221) of one clone of Trypanosoma brucei. Translation of these poly(A)+ RNAs in a rabbit reticulocyte lysate gave rise to proteins that could be precipitated with antisera against homologous variant surface glycoprotein, the protein responsible for antigenic variation in trypanosomes. From the electrophoretic mobility of these in vitro products in sodium dodecyl sulphate (SDS) gels we infer that variant surface glycoproteins (VSGs) are made as pre-proteins, which require trimming to yield mature VSGs. The total translation products from the four poly(A)+ RNAs produced a complex set of bands on SDS gels, which only differed in the region where the variant pre-glycoproteins migrated. The only detectable variation in the messenger RNA populations of these variants is, therefore, in the messenger RNA for variant pre-glycoproteins. We have made duplex DNA copies of these poly(A)+ RNAs, linked the complementary DNA to plasmid pBR322 by GC tailing and cloned this recombinant DNA in Escherichia coli. Colony hybridization with complementary DNA made on poly(A)+ RNA showed that 7--10% of the colonies contained DNA that hybridized only with the homologous probe. Plasmid DNA was isolated from ten such colonies (two or three of each variant complementary DNA), bound to diazobenzyloxymethyl-cellulose (DBM) paper and used to select complementary messenger RNA from total poly(A)+ RNA by hybridization. In eight cases the RNA recovered from the filter gave variant pre-glycoprotein as the predominant product of in vitro translation. Poly(A)+ RNA from each of the variants only hydridized to the homologous complementary DNA in filter hybridizations. Each trypanosome variant, therefore, contains no detectable messenger RNAs for the three heterologous variant-specific glycoproteins tested. We conclude from this lack of cross-hybridization that antigenic diversity in trypanosomes, unlike antibody diversity in mammals, does not involve the linkage of a repertoire of genes for the variable N-terminal half to a single gene for the C-terminal half of the VSGs.

Animals↗
PubMed1980

Antigenic variation in clones of animal-infective Trypanosoma brucei derived and maintained in vitro.

Eighteen clones of variable antigen type 052 of Trypanosoma brucei stock S.427 were derived and maintained as animal-infective bloodstream forms in vitro for up to 60 days of cultivation. The antigenic composition of such clones was monitored weekly by immunofluorescent analysis of viable trypanosomes, using antisera raised to isolated variant-specific surface glycoproteins of both 052 and variable antigen type (221) which consistently appeared in the first relapse population of type 052 in vitro. The appearance of new variants was detected in 9 of the 18 clones 18--46 days following initiation of the clone and variable antigen type 221 was found in all 9 clones. On one or more occasions in 8 of such clones, viable trypanosomes were found which did not react with either antiserum but were mouse-infective on the 4 occasions tested and probably represent other variable antigen types. The process of antigen variation in vitro appears to resemble the process in vivo except that new variant types are detected earlier in vivo. This possibly results from different growth rates of the trypanosomes in vivo and in vitro, together with the fact that elimination of the initial variant population by the host's immune response facilitates the detection of newly arising variable antigen types in vivo.

Animals↗
PubMed1980

In vitro cloning of animal-infective bloodstream forms of Trypanosoma brucei.

Clones of animal-infective bloodstream forms of Trypanosoma brucei (stocks S.427 and LUMP 227) were made by transferring a single organism from bloodstream-form cultures into each well of Microtest II Tissue Culture Plates containing bovine fibroblast-like feeder cells. When the number of trypanosomes increased to 10(2)--10(3)/well on days 4--16, they were transferred into plastic T-25 culture flasks also containing feeder cells and fresh medium. Cultures were thereafter maintained by partially replacing the trypanosome suspension with the same volume of fresh medium (diluting the density to 2--5 x 15(5) trypanosomes/ml) every 24 h. Sub-cultivations could be made by transferring 1--2 ml of the trypanosome suspension to a new culture flask at 4--5 day intervals. A total of 42 clones in the 3 series TC221, TC52 and TC227, carrying variable antigen types (VATs) 221, 052 and ILTat 1.4, respectively, have been established. Average population doubling times for clones of TC221, TC52 and TC227 were 8.7, 14.5 and 15.5 h respectively. Of 35 populations examined, 34 clones retained the original specificity of their VATs for at least 8--32 days from cloning. One cloned population of TC52 consisted of 99.8% VAT 052 and 0.2% VAT 221 at the time when the first VAT test was made on day 18.

Animals↗
PubMed1980

An introduction to antigenic variation in trypanosomes.

The salivarian trypanosomes have a unique capacity for antigenic variation at the cell surface. This phenomenon is their primary mechanism for evasion of the host's immune response. Variation is mediated through alternate expression of an extensive repertoire of variant surface glycoproteins (VSGs). Extensive amino acid sequence diversity is responsible for the antigenic diversity of VSGs. All the isolated VSGs of Trypanosoma brucei studied also contain an immunologically cross-reacting glycosyl side chain at the C-terminus, which probably represents a recognition site for proteolytic processing of the hydrophobic putative membrane-binding tail present on the synthesized molecule but not so far found on purified VSGs.

Amino Acid Sequence↗
PubMed1980

The genes for variant antigens in trypanosomes.

We have studied the mechanism of antigenic variation by using DNA complementary to the messenger RNAs for four variant surface glycoproteins of Trypanosoma brucei. Pure complementary DNAs were obtained by cloning as recombinant DNA in Escherichia coli. Using these complementary DNAs as hybridization probes, we have analyzed the genes for these variant surface glycoproteins. The results provide new information on the origin and evolution of antigenic variation, and on the mechanism involved in switching from one antigenic type to another.

Animals↗
PubMed1979

Selective cleavage of variant surface glycoproteins from Trypanosoma brucei.

Two conformationally distinct regions were revealed by tryptic cleavage of six undenatured variant surface glycoproteins purified from clones of Trypanosoma brucei. Within 5 min, the native glycoproteins (65,000 mol.wt.) were cleaved, yielding a large N-terminal fragment (48,000-55,000 mol.wt. depending on the variant) together with one or more C-terminal fragments. After 30-60 min incubation, further breakdown of the large fragment occurred in some variants. The ultimate large product (40,000-52,000 mol.wt.) was very resistant to further degradation by trypsin (in the absence of denaturation). The distinction between N-terminal and C-terminal domains may be significant in relation to the organization and function of these glycoproteins on the trypanosome surface.

Amino Acids↗
PubMed1977

The synthesis of a variant-specific antigen by Trypanosoma brucei in vitro.

A variant-specific surface antigen from a cloned population of Trypanosoma brucei S42 has been isolated and partially characterized. [35S]L-methionine was found to be incorporated into this material by cells incubated in vitro in a chemically defined medium. Incorporation of [35S]L-methionine was inhibited by cycloheximide and puromycin at concentrations which are known to specifically inhibit protein synthesis in other systems. The rate of synthesis of the variant-specific antigen in vitro has been estimated to be about 8% of the rate in vivo. Newly synthesized [35S]L-methionine-labelled variant-specific antigen was incorporated into the surface coat.

Animals↗
PubMed1977

Threonine catabolism in Trypanosoma brucei.

L-Threonine is catabolized by Trypanosoma brucei to give equimolar quantities of glycine and acetate. The pathway, which involves the two enzymes L-threonine dehydrogenase (EC 1.1.1.103) and aminoacetone synthase (acetyl-CoA:glycine C-acetyltransferase, EC 2.3.1.29) and subsequent hydrolysis of the acetyl-CoA, is most active in cultured trypanosomes but is also present in bloodstream forms. L-Threonine dehydrogenase from both culture and bloodstream forms of trypanosomes has an apparent molecular weight of between 28 000 and 38 000, and is sensitive to a wide range of sulphydryl reagents.

Acetone↗
PubMed1977

Isolation of mitochondria and mitochondrial RNA from Crithidia fasciculata.

Two methods were used to isolate mitochondria from Crithidia fasciculata. In the first method, cells were weakened by exposure to hypotonic conditions and then disrupted by blending; mitochondria were subsequently isolated using disodium 3,5-diacetoamido-2,4,6-triiodobenzoate gradients. In the second, cells were treated with digitonin before disruption; mitochondria were purified by differential centrifugation. Both preparations were examined with the electron microscope and were also shown to possess several characteristic biochemical properties of mitochondria. Kinetoplast DNA was present in the mitochondria, uncontaminated by nuclear DNA. Analysis by polyacrylamide gel electrophoresis showed two RNA components of molecular weights of 0-47 X 10(6) and 0-22 X 10(6), in addition to cytoplasmic RNA contamination. Four mitochondrial components with sedimentation coefficients of 14-6S, 11-4S, 10-1S and 9-9S were identified on sucrose density gradients. Ethidium bromide abolished the incorporation of [5-3H]uridine into the presumed mitochondrial RNA.

Cell Fractionation↗