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G R Fink

Publications and source records attributed to G R Fink.

At least 145 records · Page 8Linked to original sources
PubMed1994

nup1 mutants exhibit pleiotropic defects in nuclear pore complex function.

The NUP1 gene of Saccharomyces cerevisiae encodes one member of a family of nuclear pore complex proteins (nucleoporins) conserved from yeast to vertebrates. We have used mutational analysis to investigate the function of Nup1p. Deletion of either the amino- or carboxy-terminal domain confers a lethal phenotype, but partial truncations at either end affect growth to varying extents. Amino-terminal truncation causes mislocalization and degradation of the mutant protein, suggesting that this domain is required for targeting Nup1p to the nuclear pore complex. Carboxy-terminal mutants are stable but do not have wild-type function, and confer a temperature sensitive phenotype. Both import of nuclear proteins and export of poly(A) RNA are defective at the nonpermissive temperature. In addition, nup1 mutant cells become multinucleate at all temperatures, a phenotype suggestive of a defect in nuclear migration. Tubulin staining revealed that the mitotic spindle appears to be oriented randomly with respect to the bud, in spite of the presence of apparently normal cytoplasmic microtubules connecting one spindle pole body to the bud tip. EM analysis showed that the nuclear envelope forms long projections extending into the cytoplasm, which appear to have detached from the bulk of the nucleus. Our results suggest that Nup1p may be required to retain the structural integrity between the nuclear envelope and an underlying nuclear scaffold, and that this connection is required to allow reorientation of the nucleus in response to cytoskeletal forces.

Base Sequence↗
PubMed1994

A role for autophosphorylation revealed by activated alleles of FUS3, the yeast MAP kinase homolog.

We have isolated dominant gain-of-function (gf) mutations in FUS3, a Saccharomyces cerevisiae mitogen-activated protein (MAP) kinase homolog, that constitutively activate the yeast mating signal transduction pathway and confer hypersensitivity to mating pheromone. Surprisingly, the phenotypes of dominant FUS3gf mutations require the two protein kinases, STE7 and STE11. FUS3gf kinases are hyperphosphorylated in yeast independently of STE7. Consistent with this, FUS3gf kinases expressed in Escherichia coli exhibit an increased ability to autophosphorylate on tyrosine in vivo. FUS3gf mutations suppress the signal transduction defect of a severely catalytically impaired allele of STE7. This finding suggests that the tyrosine-phosphorylated form of FUS3 is a better substrate for activation by STE7. Furthermore, these results imply that the degree of autophosphorylation of a MAP kinase determines its threshold of sensitivity to upstream signals.

Alleles↗
PubMed1994

Symmetric cell division in pseudohyphae of the yeast Saccharomyces cerevisiae.

Laboratory strains of Saccharomyces cerevisiae are dimorphic; in response to nitrogen starvation they switch from a yeast form (YF) to a filamentous pseudohyphal (PH) form. Time-lapse video microscopy of dividing cells reveals that YF and PH cells differ in their cell cycles and budding polarity. The YF cell cycle is controlled at the G1/S transition by the cell-size checkpoint Start. YF cells divide asymmetrically, producing small daughters from full-sized mothers. As a result, mothers and daughters bud asynchronously. Mothers bud immediately but daughters grow in G1 until they achieve a critical cell size. By contrast, PH cells divide symmetrically, restricting mitosis until the bud grows to the size of the mother. Thus, mother and daughter bud synchronously in the next cycle, without a G1 delay before Start. YF and PH cells also exhibit distinct bud-site selection patterns. YF cells are bipolar, producing their second and subsequent buds at either pole. PH cells are unipolar, producing their second and subsequent buds only from the end opposite the junction with their mother. We propose that in PH cells a G2 cell-size checkpoint delays mitosis until bud size reaches that of the mother cell. We conclude that yeast and PH forms are distinct cell types each with a unique cell cycle, budding pattern, and cell shape.

Actins↗
PubMed1994

PET and functional testing in temporal lobe epilepsy.

Thirty-seven patients with severe temporal lobe epilepsy were studied interictally with [18F]fluorodeoxyglucose-PET in each of three conditions: resting, during emotional speech, and while performing a visual recognition task. In the resting state, each patient exhibited regional hypometabolism in agreement with his epileptic EEG focus, but that area was typically very large. The zone of maximum dysfunction was significantly better demarcated on activated scans showing an increase in whole-brain metabolism averaging 18%. Concurrently, the midtemporal-focus metabolic contrast was improved by 27%. This effect was more consistently produced by the speech paradigm that also induced significant amygdala recruitment and revealed the individual hemispheric speech dominance. Psychological trait factors played a role only for unspecific global activation.

Adolescent↗
PubMed1994

Induction of pseudohyphal growth by overexpression of PHD1, a Saccharomyces cerevisiae gene related to transcriptional regulators of fungal development.

When starved for nitrogen, MATa/MAT alpha cells of the budding yeast Saccharomyces cerevisiae undergo a dimorphic transition to pseudohyphal growth. A visual genetic screen, called PHD (pseudohyphal determinant), for S. cerevisiae pseudohyphal growth mutants was developed. The PHD screen was used to identify seven S. cerevisiae genes that when overexpressed in MATa/MAT alpha cells growing on nitrogen starvation medium cause precocious and unusually vigorous pseudohyphal growth. PHD1, a gene whose overexpression induced invasive pseudohyphal growth on a nutritionally rich medium, was characterized. PHD1 maps to chromosome XI and is predicted to encode a 366-amino-acid protein. PHD1 has a SWI4- and MBP1-like DNA binding motif that is 73% identical over 100 amino acids to a region of Aspergillus nidulans StuA. StuA regulates two pseudohyphal growth-like cell divisions during conidiophore morphogenesis. Epitope-tagged PHD1 was localized to the nucleus by indirect immunofluorescence. These facts suggest that PHD1 may function as a transcriptional regulatory protein. Overexpression of PHD1 in wild-type haploid strains does not induce pseudohyphal growth. Interestingly, PHD1 overexpression enhances pseudohyphal growth in a haploid strain that has the diploid polar budding pattern because of a mutation in the BUD4 gene. In addition, wild-type diploid strains lacking PHD1 undergo pseudohyphal growth when starved for nitrogen. The possible functions of PHD1 in pseudohyphal growth and the uses of the PHD screen to identify morphogenetic regulatory genes from heterologous organisms are discussed.

Amino Acid Sequence↗
PubMed1994

Left hemispheric neuronal heterotopia: a PET, MRI, EEG, and neuropsychological investigation of a university student.

A 21-year-old left-handed medical student had a prominent unilateral cerebral cortical malformation due to an ontogenetic migration disorder. We performed neuropsychological studies, EEG, T1- and T2-weighted and proton-density MRI, and positron emission tomography (PET) (under both the resting condition and neuropsychological activation). Neuropsychological testing revealed normal intelligence and generally normal memory functioning but selective deficits in tests of verbal fluency and spatial-figural relationships. Proton-density and T2-weighted MRI revealed extensive left cortical heterotopia that included parts of the Wernicke area. PET under the resting condition revealed a small interhemispheric difference with slightly reduced glucose metabolism in the left temporoparietal cortical zone. An activation PET (with the patient performing a verbal fluency test) resulted in a normal overall increase in metabolism but marked deviations in cortical areas. The highest activity changes were in the Broca and Wernicke areas of the right hemisphere, and there was very little activation in those regions of the left hemisphere that were expected to respond well to the activation--the temporal, parietal, and temporo-occipital cortical zones. We conclude that there can be large compensations for unilateral heterotopia.

Adult↗
PubMed1994

Ca2+ transport in Saccharomyces cerevisiae.

Cytosolic free Ca2+ is maintained at submicromolar levels in budding yeast by the activity of Ca2+ pumps and antiporters. We have recently identified the structural genes for two Ca2+ pumps, PMC1 [correction of PCM1] and PMR1, which are required for Ca2+ sequestration into the vacuole and secretory organelles, respectively. The function of either Ca2+ pump is sufficient for yeast viability, but deletion of both genes is lethal because of elevation of cytosolic [Ca2+] and activation of calcineurin, a Ca(2+)- and calmodulin-dependent protein phosphatase. Calcineurin activation decreases Ca2+ sequestration in the vacuole by a putative Ca2+ antiporter and may also increase Ca2+ pump activity. These regulatory processes can affect the ability of yeast strains to tolerate high extracellular [Ca2+]. We propose a model in which the cellular response to changes in the environmental levels of Ca2+ is mediated by calmodulin and calcineurin which, in turn, modulate the various types of Ca2+ transporters.

Animals↗
PubMed1993

Elements of the yeast pheromone response pathway required for filamentous growth of diploids.

Transmission of an external signal from receptors to downstream targets is often mediated by a conserved set of protein kinases that act in sequence (a kinase cascade). In haploid strains of Saccharomyces cerevisiae, a signal initiated by peptide pheromones is transmitted through this kinase cascade to a transcription factor STE12, which is required for the expression of many mating-specific genes. Here it was shown that in diploids some of the same kinases and STE12 are required for filamentous growth, but the pheromone receptors and guanosine triphosphate-binding protein are not required for filament formation. Thus, a similar kinase cascade is activated by different signals in haploids and diploids and mediates different developmental outcomes in the two cell types.

Fungal Proteins↗
PubMed1993

Arabidopsis thaliana auxotrophs reveal a tryptophan-independent biosynthetic pathway for indole-3-acetic acid.

We used tryptophan auxotrophs of the dicot Arabidopsis thaliana (wall cress) to determine whether tryptophan has the capacity to serve as a precursor to the auxin, indole-3-acetic acid (IAA). Quantitative gas chromatography-selected ion monitoring-mass spectrometry (GC-SIM-MS) revealed that the trp2-1 mutant, which is defective in the conversion of indole to tryptophan, accumulated amide- and ester-linked IAA at levels 38-fold and 19-fold, respectively, above those of the wild type. Tryptophan and free IAA were isolated from the trp2-1 mutant grown in the presence of [15N]anthranilate and [2H5]tryptophan, and the relative 15N and 2H5 enrichments of tryptophan and IAA were determined via GC-SIM-MS. The 15N enrichment of tryptophan, 13% +/- 4%, was less than the 15N enrichment of the IAA pool, 39% +/- 4%; therefore, IAA biosynthesis occurs via a tryptophan-independent pathway. The amount of 2H5 incorporated by the plant into IAA from tryptophan (9% +/- 4%) was low and only slightly above the level of spontaneous, nonenzymatic conversion of [2H5]tryptophan to [2H5]IAA. These results show that the dicot Arabidopsis is similar to the monocot Zea mays in that the major route of IAA biosynthesis does not occur through tryptophan.

Arabidopsis↗
PubMed1993

The rRNA-encoding DNA array has an altered structure in topoisomerase I mutants of Saccharomyces cerevisiae.

All the chromosomes from isogenic TOP1 and top1 strains have similar mobility on pulsed-field gels except for chromosome XII, which fails to migrate into the gels in top1 mutants. Chromosome XII contains the tandem repeats of rRNA-encoding DNA (rDNA). When a segment of chromosome XII containing only rDNA is transferred to chromosome III by a recombination event, chromosome III fails to enter a pulsed-field gel in extracts from top1 strains, indicating that the aberrant migration of chromosome XII in top1 mutants is caused by the presence of rDNA. Failure of chromosome XII to migrate into a pulsed-field gel occurs only in preparations from exponentially growing top1 cultures and not in preparations from stationary-phase top1 cultures. rDNA from a top1 strain does enter the gel if it is cut with an enzyme (Pst I) that cuts the tandem rDNA array into single 9-kb repeat units, indicating that more than a single repeat unit is required to maintain the aberrant structure.

Amino Acid Sequence↗
PubMed1993

Cerebral glucose metabolism as a predictor of recovery from aphasia in ischemic stroke.

OBJECTIVE: The recovery of language function seen in poststroke aphasia is significantly related to the severity of the initial neurologic deficit. However, a great deal of variability still remains in the improvement that is actually achieved. To detect predictors that will help in explaining this variability, positron emission tomography (PET) and fluorodeoxyglucose F 18 (18-F-fluorodeoxyglucose) were used and the results were analyzed by stepwise regression. DESIGN: Using PET imaging after injection of fluorodeoxyglucose F 18, the regional changes in glucose metabolism in 26 patients at a period of 12 to 18 days following an ischemic stroke involving the left middle cerebral artery were examined. A second PET examination was performed on 17 of our 26 patients who were able to perform speech activation exercises. All patients received an initial and a 4-month follow-up evaluation of language performance. SETTING: During the two PET studies and the first language assessment, the patients were hospitalized in a neurologic clinic. The follow-up evaluation of language performance was performed when the patients were ambulatory. PATIENTS: Twenty-six patients (10 women, 16 men; aged 38 to 77 years; mean +/- SD, 60 +/- 9.2 years) were selected in the study. Their aphasias were of various types and of varying severity ranging from mild impairment to severe global aphasia. MAIN OUTCOME MEASURES: For the stepwise regression analysis of variables, the following variables were analyzed in resting and activation PET to explain residual variance from the first to the second Token Test: regional cerebral metabolic rate for glucose of infarct and mirror region, left and right cerebral and cerebellar hemispheres, left and right Broca's area, left and right Wernicke's area, and left and right temporoparietal cortex. RESULTS: As was expected, early and late Token Tests exhibit a high correlation (.85). The stepwise regression analysis shows that only the left cerebral hemisphere glucose value of the resting PET had significant effect on the residual variance of the Token Test regression. Regional metabolic rates during speech activation had the largest contribution to a significant recovery from aphasia. The infarct area and its corresponding mirror region, the left Broca's area, and the entire left cerebral hemisphere accounted for 80% of the residual variance. CONCLUSIONS: These results emphasize not only the application of PET activation studies in the prediction of a tissue's potential reserve capacity but also the importance of left hemisphere integrity in the recovery of functional language.

Adult↗
PubMed1993

Effect of propentofylline on regional cerebral glucose metabolism in acute ischemic stroke.

In a randomized double-blind placebo-controlled study in 30 patients with acute ischemic stroke, the effect of the adenosine uptake blocker propentofylline on regional brain glucose metabolism (rCMRglu) was investigated using repeated positron emission tomography (PET) with 2-[18F]fluoro-2-deoxy-D-glucose (FDG). Treatment was initiated within 48 h after onset of symptoms. The clinical course was followed for 3 months. In the propentofylline group, after 14 days rCMRglu was increased in the infarct by 37.3% and was practically unchanged in other brain regions, whereas in the control group glucose metabolism had decreased in all regions (1.4-13.4%). These differences were significant between the two groups [Analysis of variance (ANOVA) p = 0.005]. Although there was a trend toward greater clinical improvement in the propentofylline-treated patients, this did not reach statistical significance. The results correspond to experimental data showing that propentofylline improves energy metabolism in cerebral ischemia. A clinical trial is needed to determine whether this new therapeutic principle can be successfully used in acute human stroke.

Acute Disease↗
PubMed1993

NUP2, a novel yeast nucleoporin, has functional overlap with other proteins of the nuclear pore complex.

We have isolated a new gene, NUP2, that encodes a constituent of the yeast-nuclear pore complex (NPC). The NUP2 protein sequence shares a central repetitive domain with NSP1 and NUP1, the two previously characterized yeast nucleoporins. Like NUP1 and NSP1, NUP2 localizes to discrete spots in the nuclear envelope, as determined by indirect immunofluorescence. Although the sequence similarity among these three nucleoporins suggests that they have a similar role in the nuclear pore complex, NUP2, in contrast to NSP1 and NUP1, is not required for growth. Some combinations of mutant alleles of NUP1, NSP1, and NUP2 display "synthetic lethal" relationships that provide evidence for functional interaction between these NPC components. This genetic evidence of overlapping function suggests that the nucleoporins act in concert, perhaps participating in the same step of the recognition or transit of macromolecules through the NPC.

Amino Acid Sequence↗
PubMed1993

A novel mutation in DNA topoisomerase I of yeast causes DNA damage and RAD9-dependent cell cycle arrest.

DNA topoisomerases, enzymes that alter the superhelicity of DNA, have been implicated in such critical cellular functions as transcription, DNA replication, and recombination. In the yeast Saccharomyces cerevisiae, a null mutation in the gene encoding topoisomerase I (TOP1) causes elevated levels of mitotic recombination in the ribosomal DNA (rDNA), but has little effect on growth. We have isolated a missense mutation in TOP1 that causes mitotic hyper-recombination not only in the rDNA, but also at other loci, in addition to causing a number of other unexpected phenotypes. This topoisomerase I mutation (top1-103) causes slow growth, constitutive expression of DNA damage-inducible genes, and inviability in the absence of the double-strand break repair system. Overexpression of top1-103 causes RAD9-dependent cell cycle arrest in G2. We show that the Top1-103 enzyme nicks DNA in vitro, suggesting that it damages DNA directly. We propose that Top1-103 mimics the action of wild-type topoisomerase I in the presence of the anti-tumor drug, camptothecin.

Alleles↗
PubMed1993

Mutations in POL1 increase the mitotic instability of tandem inverted repeats in Saccharomyces cerevisiae.

Tandem inverted repeats (TIRs or hairpins) of 30 and 80 base-pair unit lengths are unstable mitotically in yeast (Saccharomyces cerevisiae). TIR instability results from deletions that remove part or all of the presumed hairpin structure from the chromosome. At least one deletion endpoint is always at or near the base of the hairpin, and almost all of the repaired junctions occur within short direct sequence repeats of 4 to 9 base pairs. The frequency of this event, which we call "hairpin excision," is influenced by chromosomal position, length of the inverted repeats, and the distance separating the repeat units; increasing the distance between the inverted repeats as little as 25 base pairs increases their chromosomal stability. The frequency of excision is not affected by representative rad mutations, but is influenced by mutations in certain genes affecting DNA synthesis. In particular, mutations in POL1/CDC17, the gene that encodes the large subunit of DNA polymerase I, increase the frequency of hairpin deletions significantly, implicating this protein in the normal maintainance of genomic TIRs.

Base Sequence↗
PubMed1993

Suppressors of trp1 fluorescence identify a new arabidopsis gene, TRP4, encoding the anthranilate synthase beta subunit.

Suppressors of the blue fluorescence phenotype of the Arabidopsis trp1-100 mutant can be used to identify mutations in genes involved in plant tryptophan biosynthesis. Two recessive suppressor mutations define a new gene, TRP4. The trp4 mutant and the trp1-100 mutant are morphologically normal and grow without tryptophan, whereas the trp4; trp1-100 double mutant requires tryptophan for growth. The trp4; trp1-100 double mutant does not segregate at expected frequencies in genetic crosses because of a female-specific defect in transmission of the double mutant genotype, suggesting a role for the tryptophan pathway in female gametophyte development. Genetic and biochemical evidence shows that trp4 mutants are defective in a gene encoding the beta subunit of anthranilate synthase (AS). Arabidopsis AS beta subunit genes were isolated by complementation of an Escherichia coli anthranilate synthase mutation. The trp4 mutation cosegregates with one of the genes, ASB1, located on chromosome 1. Sequence analysis of the ASB1 gene from trp4-1 and trp4-2 plants revealed different single base pair substitutions relative to the wild type. Anthranilate synthase alpha and beta subunit genes are regulated coordinately in response to bacterial pathogen infiltration.

Amino Acid Sequence↗
PubMed1992

SHR3: a novel component of the secretory pathway specifically required for localization of amino acid permeases in yeast.

Mutations in SHR3 block amino acid uptake into yeast by reducing the levels of multiple amino acid permeases within the plasma membrane. SHR3 is a novel integral membrane protein component of the endoplasmic reticulum (ER). shr3 null mutants specifically accumulate amino acid permeases in the ER; other plasma membrane proteins, secretory proteins, and vacuolar proteins are processed and targeted correctly. Our findings suggest that SHR3 interacts with a structural domain shared by amino acid permeases, an interaction required for permease-specific processing and transport from the ER. Even in the presence of excess amino acids, shr3 mutants exhibit starvation responses. shr3 mutants constitutively express elevated levels of GCN4, and mutant shr3/shr3 diploids undergo dimorphic transitions that result in filamentous growth at enhanced frequencies.

Amino Acid Sequence↗