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J D Dinman

Publications and source records attributed to J D Dinman.

29 records · Page 2Linked to original sources
PubMed1995

Ribosomal frameshifting in yeast viruses.

Proper maintenance of translational reading frame by ribosomes is essential for cell growth and viability. In the last 10 years it has been shown that a number of viruses induce ribosomes to shift reading frame in order to regulate the expression of gene products having enzymatic functions. Studies on ribosomal frameshifting in viruses of yeast have been particularly enlightening. The roles of viral mRNA sequences and secondary structures have been elucidated and a picture of how these interact with host chromosomal gene products is beginning to emerge. The efficiency of ribosomal frameshifting is important for viral particle assembly, and has identified ribosomal frameshifting as a potential target for antiviral agents. The availability of mutants of host chromosomal gene products involved in maintaining the efficiency of ribosomal frameshifting bodes well for the use of yeast in future studies of ribosomal frameshifting.

Amino Acid Sequence↗
PubMed1995

Molecular cloning of a gene expressed during early embryonic development in Onchocerca volvulus.

Little attention has been paid to the reproductive biology of filarial nematode parasites as a possible target for immunological or chemotherapeutic intervention. An interruption of the reproductive process would, in addition to breaking the cycle of transmission, reduce the morbidity associated with certain filarial infections. As part of our efforts to define molecules that have important functions during filarial embryogenesis, antibodies against embryo-associated proteins were used to identify a 6308-bp cDNA sequence (ovt1) from an Onchocerca volvulus cDNA expression library. The ovt1 cDNA contained an open reading frame that coded for 2022 amino acids. The deduced amino acid sequence was highly hydrophilic, alpha-helical in nature and included two leucine zipper domains. OVT1 also contained a single Arg-Gly-Asp (RGD) site. The results of Southern blot analyses demonstrated that an ovt1-like gene occurs in a number of different species of filarial nematodes. In situ hybridization experiments to identify tissues that contain ovt1 transcripts showed that ovt1 was transcribed at high levels in the late morula/early blastocyst stage of embryonic development. Transcripts for ovt1 were also detected in O. volvulus larvae and in the hypodermal cells of adult parasites. Two fragments of ovt1 were expressed as fusion proteins and the fusion proteins were used to produce antibodies in rabbits. Both antibodies recognized a native protein with an apparent molecular mass of 230 kDa in extracts from gravid female O. volvulus. In addition, the antibodies reacted with a restricted number of lower-molecular mass bands which may represent the products of post-transcriptional or post-translational processing. The predicted coiled-coil structure and the sites of transcription suggest that OVT1 may be a component of the extracellular matrix.

Amino Acid Sequence↗
PubMed1995

5 S rRNA is involved in fidelity of translational reading frame.

Chromosomal mutants (maintenance of frame = mof) in which the efficiency of -1 ribosomal frameshifting is increased can be isolated using constructs in which lacZ expression is dependent upon a -1 shift of reading frame. We isolate a new mof mutation, mof9, in Saccharomyces cerevisiae and show that it is complemented by both single and multi-copy 5 S rDNA clones. Two independent insertion mutations in the rDNA locus (rDNA::LEU2 and rDNA::URA3) also display the Mof- phenotype and are also complemented by single and multi-copy 5 S rDNA clones. Mutant 5 S rRNAs expressed from a plasmid as 20-50% of total 5 S rRNA in a wild-type host also induced the Mof- phenotype. The increase in frameshifting is greatest when the lacZ reporter gene is expressed on a high copy, episomal vector. No differences were found in 5 S rRNA copy number or electrophoretic mobilities in mof9 strains. Both mof9 and rDNA::LEU2 increase the efficiency of +1 frameshifting as well but have no effect on readthrough of UAG or UAA termination codons, indicating that not all translational specificity is affected. These data suggest a role for 5 S rRNA in the maintenance of frame in translation.

Base Sequence↗
PubMed1994

Spermidine deficiency increases +1 ribosomal frameshifting efficiency and inhibits Ty1 retrotransposition in Saccharomyces cerevisiae.

Polyamines have been implicated in nucleic acid-related functions and in protein biosynthesis. RNA sequences that specifically direct ribosomes to shift reading frame in the -1 and +1 directions may be used to probe the mechanisms controlling translational fidelity. We examined the effects of spermidine on translational fidelity by an in vivo assay in which changes in beta-galactosidase activity are dependent on yeast retrovirus Ty +1 and yeast double-stranded RNA virus L-A -1 ribosomal frameshifting signals. In spe2 delta mutants of Saccharomyces cerevisiae, which cannot make spermidine as a result of a deletion in the SPE2 gene, there is a marked elevation in +1 but no change in -1 ribosomal frameshifting. The increase in +1 ribosomal frameshifting efficiency is accompanied by a striking decrease in Ty1 retrotransposition.

Adenosylmethionine Decarboxylase↗
PubMed1994

Translational maintenance of frame: mutants of Saccharomyces cerevisiae with altered -1 ribosomal frameshifting efficiencies.

A special site on the (+) strand of the L-A dsRNA virus induces about 2% of ribosomes translating the gag open reading frame to execute a -1 frameshift and thus produce the viral gag-pol fusion protein. Using constructs in which a -1 ribosomal frameshift at this site was necessary for expression of lacZ we isolated chromosomal mutants in which the efficiency of frameshifting was increased. These mutants comprise eight genes, named mof (maintenance of frame). The mof1-1, mof2-1, mof4-1, mof5-1 and mof6-1 strains cannot maintain M1 dsRNA at 30 degrees, but, paradoxically, do not lose L-A. The mof2-1, mof5-1 and mof6-1 strains are temperature sensitive for growth at 37 degrees, and all three show striking cell cycle phenotypes. The mof2-1 strains arrest with mother and daughter cells almost equal in size, mof5-1 arrests with multiple buds and mof6-1 arrests as single large unbudded cells. mof2-1 and mof5-1 strains are also Pet-. The mof mutations show differential effects on various frameshifting signals.

Base Sequence↗
PubMed1994

SPE1 and SPE2: two essential genes in the biosynthesis of polyamines that modulate +1 ribosomal frameshifting in Saccharomyces cerevisiae.

We previously showed that a mutant of Saccharomyces cerevisiae, which cannot make spermidine as a result of a deletion in the SPE2 gene (spe2 delta), exhibits a marked elevation in +1 ribosomal frameshifting efficiency in response to the Ty1 frameshift sequence, CUU AGG C. In the present study, we found that spermidine deprivation alone does not result in increased +1 ribosomal frameshifting efficiency. The high level of +1 ribosomal frameshifting efficiency in spe2 delta cells is the result of the combined effects of both spermidine deprivation and the large increase in the level of intracellular putrescine resulting from the derepression of the gene for ornithine decarboxylase (SPE1) in spermidine-deficient strains.

Enzyme Repression↗
PubMed1993

Yeast MAK3 N-acetyltransferase recognizes the N-terminal four amino acids of the major coat protein (gag) of the L-A double-stranded RNA virus.

The MAK3 gene of Saccharomyces cerevisiae encodes an N-acetyltransferase whose acetylation of the N terminus of the L-A double-stranded RNA virus major coat protein (gag) is necessary for viral assembly. We show that the first 4 amino acids of the L-A gag protein sequence, MLRF, are a portable signal for N-terminal acetylation by MAK3. Amino acids 2, 3, and 4 are each important for acetylation by the MAK3 enzyme. In yeast cells, only three mitochondrial proteins are known to have the MAK3 acetylation signal, suggesting an explanation for the slow growth of mak3 mutants on nonfermentable carbon sources.

Acetylation↗
PubMed1992

Ribosomal frameshifting efficiency and gag/gag-pol ratio are critical for yeast M1 double-stranded RNA virus propagation.

About 1.9% of ribosomes translating the gag open reading frame of the yeast L-A double-stranded RNA virus positive strand undergo a -1 frameshift and continue translating in the pol open reading frame to make a 170-kDa gag-pol fusion protein. The importance of frameshifting efficiency for viral propagation was tested in a system where the M1 (killer toxin-encoding) satellite RNA is supported by a full-length L-A cDNA clone. Either increasing or decreasing the frameshift efficiency more than twofold by alterations in the slippery site disrupted viral propagation. A threefold increase caused by a chromosomal mutation, hsh1 (high shifter), had the same effect. Substituting a +1 ribosomal frameshift site from Ty1 with the correct efficiency also allowed support of M1 propagation. The normal -1 frameshift efficiency is similar to the observed molar ratio in viral particles of the 170-kDa gag-pol protein to the 70-kDa gag gene product, the major coat protein. The results are interpreted in terms of a packaging model for L-A.

Base Sequence↗
PubMed1991

A -1 ribosomal frameshift in a double-stranded RNA virus of yeast forms a gag-pol fusion protein.

The L-A double-stranded RNA (dsRNA) virus of Saccharomyces cerevisiae has two open reading frames (ORFs). ORF1 encodes the 80-kDa major coat protein (gag). ORF2, which is expressed only as a 180-kDa fusion protein with ORF1, encodes a single-stranded RNA-binding domain and has the consensus sequence for RNA-dependent RNA polymerases of (+)-strand and double-stranded RNA viruses (pol). We show that the 180-kDa protein is formed by -1 ribosomal frame-shifting by a mechanism indistinguishable from that of retro-viruses. Analysis of the "slippery site" suggests that a low probability of unpairing of the aminoacyl-tRNA from the 0-frame codon at the ribosomal A site reduces the efficiency of frameshifting more than the reluctance of a given tRNA to have its wobble base mispaired. Frameshifting of L-A requires a pseudoknot structure just downstream of the shift site. The efficiency of the L-A frameshift site is 1.8%, similar to the observed molar ratio in viral particles of the 180-kDa fusion protein to the major coat protein.

Amino Acid Sequence↗
PubMed1990

Onchocerca volvulus: molecular cloning, primary structure, and expression of a microfilarial surface-associated antigen.

Although the filarial nematode parasite Onchocerca volvulus is an important human pathogen in large areas of Africa and Latin America, little is known of the molecular interactions that govern the clinical status of patients with this chronic, debilitating disease. As a step toward defining the parasite molecules important to the immunobiology of host-parasite interactions, we have identified and cloned a major surface-associated antigen expressed by O. volvulus microfilariae. Radiolabeling experiments demonstrated that O. volvulus microfilariae have a limited repertoire of peptides at the surface. Prominent among these labeled peptides is an 18-kDa component. Immunological cross-reactivity between a surface-associated component of Dirofilaria immitis microfilariae and the 18-kDa surface-associated molecule from O. volvulus was exploited in a strategy to clone this potentially important O. volvulus microfilarial antigen. The cross-reacting antibodies were used to immunoscreen O. volvulus cDNA expression libraries. One clone, M2f.e, contained an open reading frame of 495 bp encoding an 18.1-kDa protein (OVMS18). Antibodies produced against the expression product of M2f.e recognized an 18-kDa component in extracts of O. volvulus microfilariae and bound to the surface of intact O. volvulus and D. immitis microfilariae. Southern blot analyses showed that M2f.e-like sequences are present in the genomic DNA of a number of filarial nematode species, but not in DNAs from nonfilarial nematode species.

Amino Acid Sequence↗