scholarly journals The dominant PNM2- mutation which eliminates the psi factor of Saccharomyces cerevisiae is the result of a missense mutation in the SUP35 gene.

Genetics ◽  
1994 ◽  
Vol 137 (3) ◽  
pp. 659-670 ◽  
Author(s):  
S M Doel ◽  
S J McCready ◽  
C R Nierras ◽  
B S Cox
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Suppressor Gene ◽  
Chain Termination ◽  
Major Band ◽  
Minor Band ◽  
Terminal Domain ◽  
Translation Fidelity ◽  
Extrachromosomal Element ◽  
A Minor ◽  
Sup35 Gene

Abstract The PNM2- mutation of Saccharomyces cerevisiae eliminates the extrachromosomal element psi. PNM2 is closely linked to the omnipotent suppressor gene SUP35 (also previously identified as SUP2, SUF12, SAL3 and GST1). We cloned PNM2- and showed that PNM2 and SUP35 are the same gene. We sequenced the PNM2- mutant allele and found a single G-->A transition within the N-terminal domain of the protein. We tested the effects of various constructs of SUP35 and PNM2- on psi inheritance and on allosuppressor and antisuppressor functions of the gene. We found that the C-terminal domain of SUP35 protein (SUP35p) could be independently expressed; expression produced dominant antisuppression. Disruption of the N-terminal domain of PNM2- destroyed the ability to eliminate psi. These results imply that the domains of SUP35p act in an antagonistic manner: the N-terminal domain decreases chain-termination fidelity, while the C-terminal domain imposes fidelity. Two transcripts were observed for SUP35, a major band at 2.4 kb and a minor band at 1.3 kb; the minor band corresponds to 3' sequences only. We propose a model for the function of SUP35, in which comparative levels of N- and C-terminal domains of SUP35p at the ribosome modulate translation fidelity.

scholarly journals The SUP35 omnipotent suppressor gene is involved in the maintenance of the non-Mendelian determinant [psi+] in the yeast Saccharomyces cerevisiae.

Genetics ◽  
1994 ◽  
Vol 137 (3) ◽  
pp. 671-676 ◽  
Author(s):  
M D Ter-Avanesyan ◽  
A R Dagkesamanskaya ◽  
V V Kushnirov ◽  
V N Smirnov
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Elongation Factor ◽  
Suppressor Gene ◽  
Terminal Region ◽  
Recessive Trait ◽  
Yeast Saccharomyces Cerevisiae ◽  
Haploid Strain ◽  
Suppressor Effect ◽  
Sup35 Gene

Abstract The SUP35 gene of yeast Saccharomyces cerevisiae encodes a 76.5-kD ribosome-associated protein (Sup35p), the C-terminal part of which exhibits a high degree of similarity to EF-1 alpha elongation factor, while its N-terminal region is unique. Mutations in or overexpression of the SUP35 gene can generate an omnipotent suppressor effect. In the present study the SUP35 wild-type gene was replaced with deletion alleles generated in vitro that encode Sup35p lacking all or a part of the unique N-terminal region. These 5'-deletion alleles lead, in a haploid strain, simultaneously to an antisuppressor effect and to loss of the non-Mendelian determinant [psi+]. The antisuppressor effect is dominant while the elimination of the [psi+] determinant is a recessive trait. A set of the plasmid-borne deletion alleles of the SUP35 gene was tested for the ability to maintain [psi+]. It was shown that the first 114 amino acids of Sup35p are sufficient to maintain the [psi+] determinant. We propose that the Sup35p serves as a trans-acting factor required for the maintenance of [psi+].

scholarly journals Interaction of aldolase with actin-containing filaments. Structural studies

1980 ◽  
Vol 186 (1) ◽  
pp. 99-104 ◽  
Author(s):  
M Stewart ◽  
D J Morton ◽  
F M Clarke
Keyword(s):  
Structural Changes ◽  
Preceding Paper ◽  
Ultrastructural Changes ◽  
Cross Linking ◽  
Fructose Bisphosphate ◽  
Major Band ◽  
Minor Band ◽  
Computer Methods ◽  
Fructose Bisphosphate Aldolase ◽  
A Minor

Electron micrographs of the paracrystals formed when fructose bisphosphate aldolase (EC 4.1.2.13) is added to actin-containing filaments were analysed by computer methods so that ultrastructural changes could be correlated with the various stoicheiometries of binding determined in the preceding paper [Walsh, Winzor, Clarke, Masters & Morton (1980) Biochem. J. 186, 89-98]. Paracrystals formed with aldolase and either F-actin or F-actin-tropomyosin have a single light transverse band every 38 nm, which is due to aldolase molecules cross-linking the filaments. In contrast, the paracrystals formed between aldolase and F-actin-tropomyosin-troponin filaments show two transverse bands every 38 nm: a major band, interpreted as aldolase binding to troponin, and a minor band, interpreted as aldolase cross-linking the filaments. The intensity of the minor band varies with Ca2+ concentration, being greatest when the Ca2+ concentration is low. A model for the different paracrystal structures which relates the various patterns and binding stoicheiometries to structural changes in the actin-containing filaments is proposed.

Erythrina Cristagalli Lectin-reactive α-fetoprotein-E2: A Marker of Hepatocellular Carcinoma and Other Malignancies

1998 ◽  
Vol 13 (1) ◽  
pp. 24-29 ◽  
Author(s):  
M. Kamei ◽  
A. Misawa ◽  
J. Arai ◽  
K. Kamakura ◽  
K. Taketa
Keyword(s):  
Hepatocellular Carcinoma ◽  
Chronic Hepatitis ◽  
Yolk Sac ◽  
Antibody Affinity ◽  
Major Band ◽  
Minor Band ◽  
Affinity Electrophoresis ◽  
Hepatocellular Carcinomas ◽  
Yolk Sac Tumors ◽  
A Minor

A newly isolated lectin Erythrina cristagalli (ECL) was tested for separation of human α-fetoprotein (AFP) glycoforms by affinity electrophoresis at 0.5 mg/ml and separated AFP bands were detected by antibody-affinity blotting. Three AFP bands, AFP-E1, AFP-E2 and AFP-E3 in order of increasing affinity, were obtained. Sera from control patients with chronic hepatitis and cirrhosis gave a major band of AFP-E1 and a minor or trace band of AFP-E2 (3.4±2.3%), while those from patients with mostly advanced hepatocellular carcinomas had increased proportions of AFP-E2 band (16.6±10.2%). With a cutoff level of 8% (mean+2SD of AFP-E2 for controls), the sensitivity for hepatocellular carcinoma was 72% at a specificity of 100%. Gastrointestinal tumors had much higher percentages of AFP-E2 and occasionally positive AFP-E3. Most of the yolk sac tumors examined showed AFP-E3 in addition to AFP-E2, although AFP-E3 was a minor band. Thus, AFP-E2 is potentially a clinically useful marker for differentiation of increased AFP in hepatocellular carcinoma and other malignancies from that in precancerous chronic hepatitis or cirrhosis.

scholarly journals Reduction of Sulphite to Sulphide Catalysed by Desulfoviridin From Desulfovibrio gigas

1974 ◽  
Vol 27 (1) ◽  
pp. 7 ◽  
Author(s):  
HE Jones ◽  
GW Skyring
Keyword(s):  
Column Chromatography ◽  
Gel Electrophoresis ◽  
Major Band ◽  
Minor Band ◽  
Desulfovibrio Gigas ◽  
A Minor

Desulfoviridin from D. gigas was partially purified by column chromatography and further purified by gel electrophoresis into a major band preparation and a minor band preparation. All partially purified and electrophoretically purified preparations catalysed the methylviologen-linked reduction of sulphite to sulphide, but stoichiometric reduction to sulphide was not demonstrated with the major band preparation. Desulfoviridin did not catalyse the reduction of thiosulphate or trithionate.

scholarly journals Extrachromosomal elements cause a reduced division potential in nib 1 strains of Saccharomyces cerevisiae.

Genetics ◽  
1989 ◽  
Vol 122 (4) ◽  
pp. 749-757
Author(s):  
R Sweeney ◽  
V A Zakian
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Open Reading Frames ◽  
Simultaneous Presence ◽  
Extrachromosomal Elements ◽  
Severe Reduction ◽  
Extrachromosomal Element ◽  
Generalized Sensitivity ◽  
Reading Frames

Abstract The nib 1 allele of yeast confers a sensitivity to an endogenous plasmid, 2 mu DNA, in that nib 1 strains bearing 2 mu DNA (cir+) exhibit a reduction in division potential. In the present study, the reduction in division potential characteristic of nib 1 cir+ strains is shown to be dependent on the simultaneous presence of both the A and the D open reading frames of 2 mu DNA as well as on the presence of an unidentified extrachromosomal element other than 2 mu DNA. Furthermore, in nib 1 strains, an uncharacterized extrachromosomal element can cause a less severe reduction of division potential in the absence of intact 2 mu DNA. Thus, the nib 1 allele may confer a generalized sensitivity to extrachromosomal elements.

Anti-Cdc25 antibodies inhibit guanyl nucleotide-dependent adenylyl cyclase of Saccharomyces cerevisiae and cross-react with a 150-kilodalton mammalian protein

1992 ◽  
Vol 12 (6) ◽  
pp. 2653-2661
Author(s):  
E Gross ◽  
I Marbach ◽  
D Engelberg ◽  
M Segal ◽  
G Simchen ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Protein ◽  
Adenylyl Cyclase ◽  
Gene Product ◽  
Cyclase Activity ◽  
Yeast Saccharomyces Cerevisiae ◽  
Terminal Domain ◽  
Yeast Homolog ◽  
Cdc25 Protein ◽  
Beta Galactosidase

The CDC25 gene product of the yeast Saccharomyces cerevisiae has been shown to be a positive regulator of the Ras protein. The high degree of homology between yeast RAS and the mammalian proto-oncogene ras suggests a possible resemblance between the mammalian regulator of Ras and the regulator of the yeast Ras (Cdc25). On the basis of this assumption, we have raised antibodies against the conserved C-terminal domain of the Cdc25 protein in order to identify its mammalian homologs. Anti-Cdc25 antibodies raised against a beta-galactosidase-Cdc25 fusion protein were purified by immunoaffinity chromatography and were shown by immunoblotting to specifically recognize the Cdc25 portion of the antigen and a truncated Cdc25 protein, also expressed in bacteria. These antibodies were shown both by immunoblotting and by immunoprecipitation to recognize the CDC25 gene product in wild-type strains and in strains overexpressing Cdc25. The anti-Cdc25 antibodies potently inhibited the guanyl nucleotide-dependent and, approximately 3-fold less potently, the Mn(2+)-dependent adenylyl cyclase activity in S. cerevisiae. The anti-Cdc25 antibodies do not inhibit cyclase activity in a strain harboring RAS2Val-19 and lacking the CDC25 gene product. These results support the view that Cdc25, Ras2, and Cdc35/Cyr1 proteins are associated in a complex. Using these antibodies, we were able to define the conditions to completely solubilize the Cdc25 protein. The results suggest that the Cdc25 protein is tightly associated with the membrane but is not an intrinsic membrane protein, since only EDTA at pH 12 can solubilize the protein. The anti-Cdc25 antibodies strongly cross-reacted with the C-terminal domain of the Cdc25 yeast homolog, Sdc25. Most interestingly, these antibodies also cross-reacted with mammalian proteins of approximately 150 kDa from various tissues of several species of animals. These interactions were specifically blocked by the beta-galactosidase-Cdc25 fusion protein.

The C-terminal domain of Saccharomyces cerevisiae DNA topoisomerase II

1994 ◽  
Vol 14 (5) ◽  
pp. 3197-3207
Author(s):  
P R Caron ◽  
P Watt ◽  
J C Wang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Localization ◽  
Topoisomerase Ii ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
Mutant Proteins ◽  
Terminal Domain ◽  
Catalytically Active

A set of carboxy-terminal deletion mutants of Saccharomyces cerevisiae DNA topoisomerase II were constructed for studying the functions of the carboxyl domain in vitro and in vivo. The wild-type yeast enzyme is a homodimer with 1,429 amino acid residues in each of the two polypeptides; truncation of the C terminus to Ile-1220 has little effect on the function of the enzyme in vitro or in vivo, whereas truncations extending beyond Gln-1138 yield completely inactive proteins. Several mutant enzymes with C termini in between these two residues were found to be catalytically active but unable to complement a top2-4 temperature-sensitive mutation. Immunomicroscopy results suggest that the removal of a nuclear localization signal in the C-terminal domain is likely to contribute to the physiological dysfunction of these proteins; the ability of these mutant proteins to relax supercoiled DNA in vivo shows, however, that at least some of the mutant proteins are present in the nuclei in a catalytically active form. In contrast to the ability of the catalytically active mutant proteins to relax supercoiled intracellular DNA, all mutants that do not complement the temperature-dependent lethality and high frequency of chromosomal nondisjunction of top2-4 were found to lack decatenation activity in vivo. The plausible roles of the DNA topoisomerase II C-terminal domain, in addition to providing a signal for nuclear localization, are discussed in the light of these results.

scholarly journals Interaction with Tap42 Is Required for the Essential Function of Sit4 and Type 2A Phosphatases

2003 ◽  
Vol 14 (11) ◽  
pp. 4342-4351 ◽  
Author(s):  
Huamin Wang ◽  
Xiaodong Wang ◽  
Yu Jiang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Amino Acid Substitution ◽  
Binding Domain ◽  
Charge Amino Acid ◽  
Catalytic Subunits ◽  
Regulatory Subunits ◽  
Terminal Domain ◽  
Major Form ◽  
Essential Function

In Saccharomyces cerevisiae, Pph21 and Pph22 are the two catalytic subunits of type 2A phosphatase (PP2Ac), and Sit4 is a major form of 2A-like phosphatase. The function of these phosphatases requires their association with different regulatory subunits. In addition to the conventional regulatory subunits, namely, the A and B subunits for Pph21/22 and the Sap proteins for Sit4, these phosphatases have been found to associate with a protein termed Tap42. In this study, we demonstrated that Sit4 and PP2Ac interact with Tap42 via an N-terminal domain that is conserved in all type 2A and 2A-like phosphatases. We found that the Sit4 phosphatase in the sit4-102 strain contains a reverse-of-charge amino acid substitution within its Tap42 binding domain and is defective for formation of the Tap42-Sit4 complex. Our results suggest that the interaction with Tap42 is required for the activity as well as for the essential function of Sit4 and PP2Ac. In addition, we showed that Tap42 is able to interact with two other 2A-like phosphatases, Pph3 and Ppg1.

Immunoisolation of Kex2p-containing organelles from yeast demonstrates colocalisation of three processing proteinases to a single Golgi compartment

1993 ◽  
Vol 106 (3) ◽  
pp. 815-822
Author(s):  
N.J. Bryant ◽  
A. Boyd
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Secretory Pathway ◽  
High Yield ◽  
Trans Golgi Network ◽  
Processing Enzymes ◽  
Terminal Domain ◽  
Golgi Compartment ◽  
Functional Equivalent ◽  
Golgi Network

One of the Golgi compartments of Saccharomyces cerevisiae is defined by the presence of a specific endoproteinase, Kex2p, which cleaves precursor polypeptides at pairs of basic residues. We have used antibodies directed against the cytoplasmically disposed C-terminal domain of Kex2p to develop an immuno-affinity procedure for the isolation of Kex2p-containing organelles. The method gives a high yield of sealed organelles that are essentially free of contamination from other secretory pathway organelles while being significantly enriched for two other late Golgi enzymes, dipeptidylaminopeptidase A and the Kex1 carboxypeptidase. Our findings provide clear evidence for a single yeast Golgi compartment containing all three late-processing enzymes, which is likely to be the functional equivalent in yeast of the mammalian trans-Golgi network.

Isolation of the SUP45 omnipotent suppressor gene of Saccharomyces cerevisiae and characterization of its gene product

1985 ◽  
Vol 5 (4) ◽  
pp. 816-822
Author(s):  
H J Himmelfarb ◽  
E Maicas ◽  
J D Friesen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Protein ◽  
Blot Hybridization ◽  
Single Copy ◽  
Suppressor Gene ◽  
Yeast Cells ◽  
In Vitro Translation ◽  
Nonsense Suppression ◽  
Synthesis Rate

The Saccharomyces cerevisiae SUP45+ gene has been isolated from a genomic clone library by genetic complementation of paromomycin sensitivity, which is a property of a mutant strain carrying the sup45-2 allele. This plasmid complements all phenotypes associated with the sup45-2 mutation, including nonsense suppression, temperature sensitivity, osmotic sensitivity, and paromomycin sensitivity. Genetic mapping with a URA3+-marked derivative of the complementing plasmid that was integrated into the chromosome by homologous recombination demonstrated that the complementing fragment contained the SUP45+ gene and not an unlinked suppressor. The SUP45+ gene is present as a single copy in the haploid genome and is essential for viability. In vitro translation of the hybrid-selected SUP45+ transcript yielded a protein of Mr = 54,000, which is larger than any known ribosomal protein. RNA blot hybridization analysis showed that the steady-state level of the SUP45+ transcript is less than 10% of that for ribosomal protein L3 or rp59 transcripts. When yeast cells are subjected to a mild heat shock, the synthesis rate of the SUP45+ transcript was transiently reduced, approximately in parallel with ribosomal protein transcripts. Our data suggest that the SUP45+ gene does not encode a ribosomal protein. We speculate that it codes for a translation-related function whose precise nature is not yet known.

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