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Biomedical subjects

W A Whalen

Publications and source records attributed to W A Whalen.

16 recordsLinked to original sources
PubMed1999

Regulation of mRNA export by nutritional status in fission yeast.

We have isolated a mutation in nup184(nup184-1) that is synthetically lethal with the mRNA export defective rae1-167 mutation in Schizosaccharomyces pombe. The consequence of the synthetic lethality is a defect in mRNA export. The predicted Nup184p is similar to Nup188p of Saccharomyces cerevisiae, and a Nup184p-GFP fusion localizes to the nuclear periphery in a punctate pattern. The Deltanup184 null mutant is viable and also is synthetically lethal with rae1-167. In a rae1(+) background, both the nup184-1 and Deltanup184 mutations confer sensitivity to growth in nutrient-rich medium (YES) that is accompanied by nuclear poly(A)+ RNA accumulation. Removal of the cAMP-dependent protein kinase, Pka1p, relieved the growth and mRNA export defects of nup184 mutants when grown in nutrient-rich medium. The activation of Pka1p is necessary, but not sufficient, to cause the severe poly(A)+ RNA export defects when nup184 mutant cells are incubated in YES, suggesting nutritional status can also regulate poly(A)+ RNA export. Our results suggest that the regulation of poly(A)+ RNA export by Pka1p kinase appears to be indirect, via a translation-dependent step, but post-translationally in response to YES.

Cell Nucleus↗
PubMed1997

The human RAE1 gene is a functional homologue of Schizosaccharomyces pombe rae1 gene involved in nuclear export of Poly(A)+ RNA.

A Schizosaccharomyces pombe temperature-sensitive mutant, rae1-1, was previously identified by us as being defective in nuclear export of Poly(A)+ RNA when grown at restrictive temperature. Here, we report the isolation of the human homologue of the S. pombe rae1 gene. The RAE1 genes are highly conserved in evolution in both structure and function. The human RAE1 cDNA, when expressed from the CMV-promoter, can suppress partially the temperature sensitivity of the rae1-1 mutant. This is also reflected by increased Poly(A)+ RNA export at a restrictive temperature. An epitope tagged human Rae1p localizes to both the nucleus and the cytoplasm in transiently transfected HeLa cells. We discuss the potential role of Rae1p in nuclear cytoplasmic trafficking in yeast and higher eukaryotic cells.

Biological Transport↗
PubMed1997

Advancement through mitosis requires rae1 gene function in fission yeast.

Growth of the rae1-1 mutant of Schizosaccharomyces pombe at restrictive temperature results in accumulation of poly(A)+ RNA in the nucleus and a cell cycle arrest at the G2/M boundary. We demonstrate here that rae1 function is required for a process other than mRNA export which is essential for advancement through mitosis. Cells lacking rae1 function arrest with elevated Cdc2p kinase levels at a step before the formation of a mitotic spindle and without separation of the spindle pole bodies. Rae1p was localized to the nuclear periphery, consistent with a role in nucleocytoplasmic trafficking, which could include protein import. We propose a model where rae1 functions in cell cycle progression through trafficking of proteins required for mitosis.

CDC2 Protein Kinase↗
PubMed1997

Npp106p, a Schizosaccharomyces pombe nucleoporin similar to Saccharomyces cerevisiae Nic96p, functionally interacts with Rae1p in mRNA export.

To identify components of the mRNA export machinery in Schizosaccharomyces pombe, a screen was developed to identify mutations that were synthetically lethal with the conditional mRNA export allele rae1-167. Mutations defining three complementation groups were isolated, and here we report the characterization of npp106 (for nuclear pore protein of 106 kDa). This gene encodes a predicted protein that has significant similarity to the Nic96p nucleoporin of Saccharomyces cerevisiae. Consistent with Npp106p being a nucleoporin, a functional green fluorescent protein (GFP)-tagged Npp106p localized to the nuclear periphery. In contrast to NIC96, the npp106 gene is not essential. Moreover, a delta npp106 mutant did not show cytoplasmic mislocalization of a simian virus 40 nuclear localization signal-GFP-LacZ reporter protein, and a fraction of cells had accumulation of poly(A)+ RNA in the nucleus. A consequence of the synthetic lethality between rae1-167 and npp106-1 was the accumulation of poly(A)+ RNA in the nucleus when cells were grown under synthetic lethal conditions. In addition to npp106-1, which is a nonsense mutation that truncates the protein at amino acid 292, the delta npp106 mutation was synthetically lethal with rae1-167, suggesting that the synthetic lethality is a consequence of the loss of a function of npp106. We further demonstrate that a region between amino acids 74 and 348 of Npp106p is required for complementation of the synthetic lethality. These results uncover a potential direct or indirect involvement of Npp106p in mRNA export.

Amino Acid Sequence↗
PubMed1990

Action of an RNA site at a distance: role of the nut genetic signal in transcription antitermination by phage-lambda N gene product.

The N gene product of Escherichia coli phage lambda is a transcriptional activator that captures the host RNA polymerase and modifies it to a termination-resistant form, permitting gene expression in two large polycistronic operons of the phage genome. Antitermination in vitro requires at least one host factor called NusA, which directly binds the N protein as well as RNA polymerase, and also a transcribed cis-acting site known as nut, within which lies the hypothesized N-recognition signal, boxB. BoxB is an interrupted palindrome capable of forming a hairpin in the mRNA. Inhibition studies with complementary DNA oligonucleotides provide evidence for a direct role of the boxB hairpin in antitermination. Kinetic studies of transcript elongation reveal that the boxB hairpin does not induce an appreciable pause to hold polymerase captive for engagement by N and NusA. Moreover, the efficiency of antitermination remains virtually the same whether N and NusA are added early, prior to nut site transcription, or added later, after the polymerase has already transcribed past the nut site. After transcription of the nut site, RNA polymerase remains susceptible to modification by N and NusA for an appreciable amount of time and distance, and the nut site DNA becomes dispensable for this modification. These results lead to the hypothesis that the boxB RNA hairpin acts in a manner analogous to the DNA enhancers, binding N and mediating a productive polymerase-NusA-N interaction by mRNA looping.

Bacterial Proteins↗
PubMed1985

beta-Chloro-L-alanine inhibition of the Escherichia coli alanine-valine transaminase.

beta-Chloro-L-alanine, an amino acid analog which inhibits a number of enzymes, reversibly inhibited the Escherichia coli K-12 alanine-valine transaminase, transaminase C. This inhibition, along with the inhibition of transaminase B, accounted for the isoleucine-plus-valine requirement of E. coli in the presence of beta-chloro-L-alanine.

Alanine↗
PubMed1984

Gratuitous repression of avtA in Escherichia coli and Salmonella typhimurium.

avtA , which encodes transaminase C (alanine-valine transaminase), is repressed by excess-L-alanine or L-leucine, and also by limitation for any of a number of amino acids in Escherichia coli and Salmonella typhimurium. Amino acid limitation causes repression by promoting the accumulation of L-alanine or L-leucine or both. avtA is also repressed by L-alpha-aminobutyric acid and other nonprotein amino acids which are structurally similar to L-alanine. We hypothesize that L-alanine and L-alpha-aminobutyric acid, whose syntheses are catalyzed by transaminase C, are the true corepressors of avtA . Repression by structural analogs of the true corepressors is termed gratuitous repression.

Alanine↗
PubMed1983

Transductional instability of Tn5-induced mutations: generalized and specialized transduction of Tn5 by bacteriophage P1.

Generalized transduction is commonly used to move transposon-induced mutations among bacterial strains by selecting for inheritance of a transposon-encoded resistance determinant. Although complete cotransduction of the resistance determinant and the chromosomal mutation might be expected, it is often found that when Tn5(Kan) insertion mutations are transduced by bacteriophage P1 most of the nonmutant kanamycin-resistant transductants area due to specialized transduction of Tn5. Such P1::Tn5 specialized transducing phage are not found when a mutant Tn5 element lacking a functional transposase is employed.

Coliphages↗
PubMed1983

Role of alanine-valine transaminase in Salmonella typhimurium and analysis of an avtA::Tn5 mutant.

In Salmonella typhimurium, as in Escherichia coli, mutations in avtA, the gene encoding the alanine-valine transaminase (transaminase C), are silent unless they are combined with mutations involved in isoleucine-valine biosynthesis. avtA is repressed by leucine or alanine but not by valine. Transaminase C is found at reduced levels upon starvation for any one of several amino acids. We hypothesize that this is due to repression of avtA by the elevated alanine and leucine pools found in amino acid-starved cells.

Culture Media↗
PubMed1982

Analysis of an avtA::Mu d1(Ap lac) mutant: metabolic role of transaminase C.

Escherichia coli can synthesize alpha-ketoisovalerate, the precursor of valine, leucine, and pantothenate, by three routes: anabolically via dihydroxyacid dehydrase and catabolically via both the branched-chain amino acid transaminase (transaminase B) and the alanine-valine transaminase (transaminase C). An E. coli K-12 mutant devoid of transaminase C (avtA) was isolated by mutagenizing an isoleucine-requiring strain devoid of transaminase B (ilvE::Tn5) with Mu d1(Ap lac) and selecting for valine-requiring derivatives which were ampicillin resistant, Lac+, able to crossfeed an ilvD mutant, and unable to grow on alpha-ketoisovalerate in place of valine. Strains defective in one, two, or all three alpha-ketoisovalerate metabolic enzymes were constructed, and their properties were analyzed. The data indicated that avtA is the structural gene for transaminase C, that transaminase C is a single enzyme species, and that the sole pathway for pantothenate biosynthesis is from alpha-ketoisovalerate. The data further showed that isoelectric inhibits the transaminase B-catalyzed deamination of valine in vivo.

Aminobutyrates↗