Previously, we showed that the yeast Saccharomyces cerevisiae cold-sensitive mutation tcp1-1 confers growth arrest concomitant with cytoskeletal disorganization and disruption of microtubule-mediated ...processes. We have identified two new recessive mutations, tcp1-2 and tcp1-3, that confer heat- and cold-sensitive growth. Cells carrying tcp1 alleles were analyzed after exposure to the appropriate restrictive temperatures by cell viability tests, differential contrast microscopy, fluorescent, and immunofluorescent microscopy of DNA, tubulin, and actin and by determining the DNA content per cell. All three mutations conferred unique phenotypes indicative of cytoskeletal dysfunction. A causal relationship between loss of Tcp1p function and the development of cytoskeletal abnormalities was established by double mutant analyses. Novel phenotypes indicative of allele-specific genetic interactions were observed when tcp1-1 was combined in the same strain with tub1-1, tub2-402, act1-1, and act1-4, but not with other tubulin or actin mutations or with mutations in other genes affecting the cytoskeleton. Also, overproduction of wild-type Tcp1p partially suppressed growth defects conferred by act1-1 and act1-4. Furthermore, Tcp1p was localized to the cytoplasm and the cell cortex. Based on our results, we propose that Tcp1p is required for normal development and function of actin and microtubules either through direct or indirect interaction with the major cytoskeletal components.
In Saccharomyces cerevisiae, Upf3p is required for nonsense-mediated mRNA decay (NMD). Although localized primarily in the cytoplasm, Upf3p contains three sequence elements that resemble nuclear ...localization signals (NLSs) and two sequence elements that resemble nuclear export signals (NESs). We found that a cytoplasmic reporter protein localized to the nucleus when fused to any one of the three NLS-like sequences of Upf3p. A nuclear reporter protein localized to the cytoplasm when fused to one of the NES-like sequences (NES-A). We present evidence that NES-A functions to signal the export of Upf3p from the nucleus. Combined alanine substitutions in the NES-A element caused a re-distribution of Upf3p to a subnuclear location identified as the nucleolus and conferred an Nmd- phenotype. Single mutations in NES-A failed to affect the distribution of Upf3p and were Nmd+. When an NES element from HIV-1 Rev was inserted near the C terminus of a mutant Upf3p containing multiple mutations in NES-A, the cytoplasmic distribution typical of wild-type Upf3p was restored but the cells remained phenotypically Nmd-. These results suggest that NES-A is a functional nuclear export signal. Combined mutations in NES-A may cause multiple defects in protein function leading to an Nmd- phenotype even when export is restored.
A mutational analysis of the eukaryotic elongation factor EF-1 alpha indicates that this protein functions to limit the frequency of errors during genetic code translation. We found that both amino ...acid misincorporation and reading frame errors are controlled by EF-1 alpha. In order to examine the function of this protein, the TEF2 gene, which encodes EF-1 alpha in Saccharomyces cerevisiae, was mutagenized in vitro with hydroxylamine. Sixteen independent TEF2 alleles were isolated by their ability to suppress frameshift mutations. DNA sequence analysis identified eight different sites in the EF-1 alpha protein that elevate the frequency of mistranslation when mutated. These sites are located in two different regions of the protein. Amino acid substitutions located in or near the GTP-binding and hydrolysis domain of the protein cause suppression of frameshift and nonsense mutations. These mutations may effect mistranslation by altering the binding or hydrolysis of GTP. Amino acid substitutions located adjacent to a putative aminoacyl-tRNA binding region also suppress frameshift and nonsense mutations. These mutations may alter the binding of aminoacyl-tRNA by EF-1 alpha. The identification of frameshift and nonsense suppressor mutations in EF-1 alpha indicates a role for this protein in limiting amino acid misincorporation and reading frame errors. We suggest that these types of errors are controlled by a common mechanism or closely related mechanisms.
Yeast casein kinase I homologues: an essential gene pair Robinson, L.C; Hubbard, E.J.A; Graves, P.R ...
Proceedings of the National Academy of Sciences - PNAS,
1992, 19920101, 1992-01-01, 1992-Jan-01, 1992-01-00, Letnik:
89, Številka:
1
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We report the isolation of an essential pair of Saccharomyces cerevisiae genes that encode protein kinase homologues. The two genes were independently isolated as dosage-dependent suppressors. ...Increased dosage of YCK1 suppressed defects caused by reduced SNF1 protein kinase activity, and increased dosage of YCK2 relieved sensitivity of wild-type cells to salt stress. The two genes function identically in the two growth assays, and loss of function of either gene alone has no discernible effect on growth. However, loss of function of both genes results in inviability. The two predicted protein products share 77% overall amino acid identity and contain sequence elements conserved among protein kinases. Partial sequence obtained for rabbit casein kinase I shares 64% identity with the two yeast gene products. Moreover, an increase in casein kinase I activity is observed in extracts from cells overexpressing YCK2. Thus YCK1 and YCK2 appear to encode casein kinase I homologues.
Salivary gland disease in dogs and cats: 245 cases (1985-1988) Spangler, W.L. (California Veterinay Diagnostics, Inc., West Sacramento, CA); Culbertson, M.R
Journal of the American Veterinary Medical Association,
02/1991, Letnik:
198, Številka:
3
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Diagnostic pathology records spanning 41 months (July 1985 through November 1988) were searched for diagnoses of salivary gland disease in dogs and cats. Review of 87,392 records from that period ...revealed 245 cases (0.3%) in which salivary gland tissue had been evaluated. During that period, salivary gland tissue was submitted to the laboratory almost twice as often from dogs (160 cases) as from cats (85 cases). On the basis of histologic examination, 89% of salivary gland submissions from small animal practices were allotted to 1 of 5 major categories: malignant neoplasms (30%; 74/245), sialadenitis (26%; 64/245), normal salivary gland (16%; 40/245), sialocele (9%; 21/245), and salivary gland infarction (8%; 20/245). The remaining 11% of submissions included various degenerative or fibrotic lesions, ductal ectasia, sialolithiasis, edema, benign neoplasia, and secondary salivary involvement with systemic or cervical lymphosarcoma or with fibrosarcoma in the head and neck.
The CTF13 gene codes for a subunit of the kinetochore in Saccharomyces cerevisiae. The temperature-sensitive mutation ctf13-30, which confers reduced fidelity of chromosome transmission, is a G --> A ...transition causing an amino acid substitution of Lys for Glu146. Strains carrying one chromosomal copy of ctf13-30 fail to grow at the restrictive temperature, whereas a haploid strain carrying two copies of ctf13-30 can grow. Four genes, UPF1, UPF2, UPF3, and ICK1, were represented among extragenic suppressors of ctf13-30. The UPF genes encode proteins that promote rapid decay of pre-mRNAs and mRNAs containing a premature stop codon. Suppressor mutations in these genes restore kinetochore function by causing increased accumulation of ctf13-30 mRNA. They also cause increased accumulation of CYH2 pre-mRNA, which is a natural target of UPF-mediated decay. Mutations in ICK1 restore kinetochore function but have no effect on ctf13-30 mRNA or CYH2 pre-mRNA accumulation. Most importantly, loss of UPF1 function causes increased accumulation of wild-type CTF13 mRNA but has no effect on the mRNA half-life. We propose that UPF-mediated decay modulates the mRNA level of one or more factors involved in CTF13 mRNA expression.
Nonsense-mediated mRNA decay (NMD) performs two functions in eukaryotes, one in controlling the expression level of a substantial subset of genes and the other in RNA surveillance. In the vast ...majority of genes, nonsense mutations render the corresponding transcripts prone to surveillance and subject to rapid degradation by NMD. To examine whether some classes of nonsense transcripts escape surveillance, we asked whether NMD acts on mRNAs that undergo subcellular localization prior to translation. In Saccharomyces cerevisiae, wild-type ASH1 mRNA is one of several dozen transcripts that are exported from the mother-cell nucleus during mitotic anaphase, transported to the bud tip on actin cables, anchored at the bud tip, and translated. Although repressed during transport, translation is a prerequisite for NMD. We found that ash1 nonsense mutations affect transport and/or anchoring independently of NMD. The nonsense transcripts respond to NMD in a manner dependent on the position of the mutation. Maximal sensitivity to NMD occurs when transport and translational repression are simultaneously impaired. Overall, our results suggest a model in which ash1 mRNAs are insensitive to NMD while translation is repressed during transport but become sensitive once repression is relieved.
Silicon vertex tracker: a fast precise tracking trigger for CDF Ashmanskas, W; Bardi, A; Bari, M ...
Nuclear instruments & methods in physics research. Section A, Accelerators, spectrometers, detectors and associated equipment,
06/2000, Letnik:
447, Številka:
1
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The Silicon Vertex Tracker (SVT), currently being built for the CDF II experiment, is a hardware device that reconstructs 2-D tracks online using measurements from the Silicon Vertex Detector (SVXII) ...and the Central Outer Tracker (COT). The precise measurement of the impact parameter of the SVT tracks will allow, for the first time in a hadron collider environment, to trigger on events containing B hadrons that are very important for many studies, such as CP violation in the b sector and searching for new heavy particles decaying to b
b
̄
. In this report we describe the overall architecture, algorithms and the hardware implementation of the SVT.
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