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D F Wirth

Publications and source records attributed to D F Wirth.

97 records · Page 6Linked to original sources
PubMed1979

Requirements for the insertion of the Sindbis envelope glycoproteins into the endoplasmic reticulum membrane.

Previous work has shown that the Sindbis structural proteins, core, the internal protein, and PE2 and E1, the integral membrane glycoproteins are synthesized as a polyprotein from a 26S mRNA; core PE2 and E1 are derived by proteolytic cleavage of a nascent chain. Newly synthesized core protein remains on the cytoplasmic side of the endoplasmic reticulum while newly synthesized PE2 and E1 are inserted into the lipid bilayer, presumably via their amino-termini. PE2 and E1 are glycosylated as nascent chains. Here, we examine a temperature-sensitive mutant of Sindbis virus which fails to cleave the structural proteins, resulting in the production of a polyprotein of 130,000 mol wt in which the amino-termini of PE2 and E1 are internal to the protein. Although the envelope sequences are present in this protein, it is not inserted into the endoplasmic reticulum bilayer, but remains on the cytoplasmic side as does the core protein in cells infected with wild-type Sindbis virus. We have also examined the fate of PE2 and E1 in cells treated with tunicamycin, an inhibitor of glycosylation. Unglycosylated PE2 and E1 are inserted normally into the lipid bilayer as are the glycosylated proteins. These results are consistent with the notion that a specific amino-terminal sequence is required for the proper insertion of membrane proteins into the endoplasmic reticulum bilayer, but that glycosylation is not required for this insertion.

Animals↗
PubMed1979

Structure of the murine leukemia virus envelope glycoprotein precursor.

The glycosylated env gene precurosr (Pr80env) of Moloney murine leukemia virus has been isolated by selective immunoprecipitation. Use of the drug tunicamycin to inhibit nascent glycosylation or specific cleavage with endoglycosidase H demonstrated that the precursor contained an apoprotein with a molecular weight of 60,000. The finished virion glycoprotein (gp70) was largely resistant to the action of endoglycosidase H. Chromatography of the glycopeptides of Pr80env in conjunction with endoglycosidase H digestion studies suggested that the precursor contained two distinct major glycosylation sites. Analysis of partial proteolytic cleavage fragments of Pr80env before and after endoglycosidase H treatment placed the two glycosylation sites within a 30,000-dalton region of the apoprotein sequence. Kinetic experiments showed that carbohydrate processing as well as proteolytic cleavage are late steps in the maturation of Pr80env.

Apoproteins↗
PubMed1977

How a single Sindbis virus mRNA directs the synthesis of one soluble protein and two integral membrane glycoproteins.

Previous work has shown that the 26S RNA found in Sindbis-infected chicken embryo fibroblasts encodes the three viral structural proteins, one internal protein, core, and two membrane glycoproteins, E1 and E2. This mRNA has one initiation site; core, E1, and E2 are derived by proteolytic cleavage. Here we show that during infection, the 26S RNA is found mainly in membrane-bound polysomes which synthesize all three virion structural proteins. These polysomes are released from the membrane upon treatment with puromycin and high salt. Newly synthesized core protein is localized on the cytoplasmic side of endoplasmic reticulum membranes, while newly synthesized envelope proteins are sequestered by the lipid bilayer. These results suggest that the nascent glycoproteins, presumably their amino termini, are of major importance in directing the binding of polysomes containing 26S mRNA to endoplasmic reticulum membranes and the subsequent transfer of glycoproteins into the bilayer.

Capsid↗
PubMedDate not supplied

Control of tubulin gene expression in the parasitic protozoan Leishmania enriettii.

The life cycle of parasitic protozoa of the genus Leishmania consists of two morphologically distinct forms: (1) amastigotes, the form of the parasite that resides inside macrophages of the mammalian host, which are non-motile and possess only a residual flagellum; and (2) promastigotes, the extracellular forms that live in the gut of the sandfly vector, which are highly motile and possess a single prominent flagellum. During the developmental transformation of amastigotes to promastigotes, the biosynthesis of alpha- and beta-tubulin proteins is dramatically increased, presumably to provide sufficient tubulin for synthesis and maintenance of the flagellum. We show here that the level of alpha- and beta-tubulin mRNA in Leishmania enriettii promastigotes is significantly greater than that in amastigotes, as determined by both Northern blot analysis and by in vitro translation of cellular RNA. These results show that the regulated expression of the tubulin genes is controlled at the level of mRNA accumulation in L. enriettii , by contrast with Leishmania mexicana, in which the control of gene expression has been reported to be at the level of translation.

Animals↗
PubMedDate not supplied

A cloned DNA fragment for identification of Mycobacterium tuberculosis.

The identification of Mycobacterium tuberculosis is a lengthy process. Attempts were made to develop a more rapid, specific, and sensitive assay for identifying tubercle bacilli in biologic specimens by differentially screening plasmid libraries of Sau3AI-digested M. tuberculosis H37Rv (ATCC 25618) DNA with homologous and heterologous DNA for clones that hybridized strongly with M. tuberculosis DNA alone. Three clones, pMTB1, pMTb2, and pMTb3, were selected for further study. Southern analysis indicated that these clones reacted strongly with DNA from strains of M. tuberculosis isolated in different parts of the world, weakly with DNA from other mycobacterial species, and not at all with Escherichia coli or human DNA. Smaller fragments of mycobacterial DNA contained in plasmid pMTb3 were subcloned into pBR322 (pMTb4) or pUC12 (pMTb5). These recombinant plasmids hybridized with DNA from M. tuberculosis, Mycobacterium bovis, and Mycobacterium bovis BCG (bacille Calmette-Guérin) Montreal and may provide the reagents needed for the development of new methods for rapid diagnosis of M. tuberculosis infections.

Blotting, Southern↗
PubMedDate not supplied

Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase of isolates from the Amazon region of Brazil.

Since the late 1970s pyrimethamine-sulfadoxine (PS; FansidarTM Hoffman-LaRoche, Basel) has been used as first line therapy for uncomplicated malaria in the Amazon basin. Unfortunately, resistance has developed over the last ten years in many regions of the Amazon and PS is no longer recommended for use in Brazil. In vitro resistance to pyrimethamine and cycloguanil (the active metabolite of proguanil) is caused by specific point mutations in Plasmodium falciparum dihydrofolate reductase (DHFR), and in vitro resistance to sulfadoxine has been associated with mutations in dihydropteroate synthase (DHPS). In association with a proguanil-sulfamethoxazole clinical trial in Brazil, we performed a nested mutation-specific polymerase chain reaction to measure the prevalence of DHFR mutations at codons 50, 51, 59, 108 and 164 and DHPS mutations at codons 436, 437, 540, 581 and 613 at three sites in the Brazilian Amazon. Samples from two isolated towns showed a high degree of homogeneity, with the DHFR Arg-50/Ile-51/Asn-108 and DHPS Gly-437/Glu-540/Gly-581 mutant genotype accounting for all infections in Peixoto de Azevedo (n = 15) and 60% of infections in Apiacás (n = 10), State of Mato Grosso. The remaining infections in Apiacás differed from this predominant genotype only by the addition of the Bolivia repeat at codon 30 and the Leu-164 mutation in DHFR. By contrast, 17 samples from Porto Velho, capital city of the State of Rondônia, with much in- and out-migration, showed a wide variety of DHFR and DHPS genotypes.

Adult↗