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D Y Thomas

Publications and source records attributed to D Y Thomas.

152 records · Page 9Linked to original sources
PubMed1973

Mitochondrial genetic analysis by zygote cell lineages in Saccharomyces cerevisiae.

Yeast strains were constructed carrying multiple mitochondrial markers conferring resistance to the inhibitors erythromycin, chloramphenicol, paromomycin and oligomycin. A pedigree analysis of two crosses was made by micromanipulating buds from zygotes. The first few daughter buds isolated from the zygotes sometimes gave rise to diploid clones which had a mixture of mitochondrial types. All possible classes of mitochondrial parental and recombinant types were found although they never appeared all together as the progeny from a single zygote. It was inferred that multiple recombination events took place in zygotes and in some of the buds derived from them. After removal of the first four or so daughter buds, subsequent buds from the zygote carried one mitochondrial type only. In cross I in which three markers were analyzed this was most frequently one of the parental types. In cross II (involving four mitochondrial markers) the later buds from the zygotes were frequently of recombinant mitochondrial type.

Chloramphenicol↗
PubMedDate not supplied

Synthesis of human insulin gene. VIII. Construction of expression vectors for fused proinsulin production in Escherichia coli.

We have constructed two families of plasmids suitable for the cloning of genes and for directing the synthesis of large amounts of fused proteins in Escherichia coli. The plasmids include the E. coli lac promoter and a portion of the coding sequence for beta-galactosidase, which can code for approx. 590 or 450 amino acids. The truncated beta-galactosidase gene ends with a poly-linker region at the 3' end, which can be cleaved by any one of the eight common restriction enzymes and joined to the gene coding for any desired protein. Each family includes three plasmids that enable fusion to be made in all three of the translational reading frames. We have cloned a synthetic human proinsulin gene into these plasmids, and 30% of the total E. coli protein was represented by the 590 amino acid-long truncated beta-galactosidase fused to proinsulin. The yield of proinsulin in this system is more than twice the amount produced by using a 1007 amino acid-long beta-galactosidase gene for fusion.

Base Sequence↗
PubMedDate not supplied

Mutagenesis of Ste18, a putative G gamma subunit in the Saccharomyces cerevisiae pheromone response pathway.

The yeast STE18 gene product has sequence and functional similarity to the gamma subunits of G proteins. The cloned STE18 gene was subjected to a saturation mutagenesis using doped oligonucleotides. The populations of mutant genes were screened for two classes of STE18 mutations, those that allowed for increased mating of a strain containing a defective STE4 gene (compensators) and those that inhibited mating even in the presence of a functional STE18 gene (dominant negatives). Three amino acid substitutions that enhanced mating in a specific STE4 (G beta) point mutant background were identified. These compensatory mutations were allele specific and had no detectable phenotype of their own; they may define residues that mediate an association between the G beta and G gamma subunits or in the association of the G beta gamma subunit with other components of the signalling pathway. Several dominant negative mutations were also identified, including two C terminal truncations. These mutant proteins were unable to function in signal transduction by themselves, but they prevented signal transduction mediated by pheromone, as well as the constitutive signalling which is present in cells defective in the GPA1 (G alpha) gene. These mutant proteins may sequester G beta or some other component of the signalling machinery in a nonfunctional complex.

Fungal Proteins↗
PubMedDate not supplied

A conserved Gbeta binding (GBB) sequence motif in Ste20p/PAK family protein kinases.

Serine/threonine protein kinases of the Ste20p/PAK family are highly conserved from yeast to man. These protein kinases have been implicated in the signaling from heterotrimeric G proteins to mitogen-activated protein (MAP) kinase cascades and to cytoskeletal components such as myosin-I. In the yeast Saccharomyces cerevisiae, Ste20p is involved in transmitting the mating-pheromone signal from the betagamma-subunits of a heterotrimeric G protein to a downstream MAP kinase cascade. We have previously shown that binding of the G-protein beta-subunit (Gbeta) to a short binding site in the non-catalytic carboxy-terminal region of Ste20p is essential fortransmitting the pheromone signal. In this study, we searched protein sequence databases for sequences that are similar to the Gbeta binding site in Ste20p. We identified a sequence motif with the consensus sequence S S L phi P L I/V x phi phi beta (x: any residue; phi: A, I, L, S, or T; beta: basic residues) that is solely present in members of Ste20p/PAK family protein kinases. We propose that this sequence motif, which we have designated GBB (Gbeta binding) motif, is specifically responsible for binding of Gbeta to Ste20p/PAK protein kinases in response to activation of heterotrimeric G protein coupled receptors. Thus, the GBB motif is a novel type of signaling domain that serves to link protein kinases of the Ste20p/PAK family to G protein coupled receptors.

Amino Acid Sequence↗