Optimization of rice .alpha.-amylase production using temperature-sensitive mutants of Saccharomyces cerevisiae for the PHO regulatory system

ABSTRACT The histone H3 amino terminus, but not that of H4, is required to prevent the constitutively bound activator Cha4 from remodeling chromatin and activating transcription at the CHA1 gene in Saccharomyces cerevisiae. Here we show that neither the modifiable lysine residues nor any specific region of the H3 tail is required for repression of CHA1. We then screened for histone H3 mutations that cause derepression of the uninduced CHA1 promoter and identified six mutants, three of which are also temperature-sensitive mutants and four of which exhibit a sin − phenotype. Histone mutant levels were similar to that of wild-type H3, and the mutations did not cause gross alterations in nucleosome structure. One specific and strongly derepressing mutation, H3 A111G, was examined in depth and found to cause a constitutively active chromatin configuration at the uninduced CHA1 promoter as well as at the ADH2 promoter. Transcriptional derepression and altered chromatin structure of the CHA1 promoter depend on the activator Cha4. These results indicate that modest perturbations in distinct regions of the nucleosome can substantially affect the repressive function of chromatin, allowing activation in the absence of a normal inducing signal (at CHA1) or of Swi/Snf (resulting in a sin − phenotype).

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Isolation of temperature-sensitive mutants for mRNA capping enzyme in Saccharomyces cerevisiae

MGG Molecular & General Genetics ◽

10.1007/bf00290360 ◽

1995 ◽

Vol 249 (2) ◽

pp. 147-154 ◽

Cited By ~ 3

Author(s):

Masahiro Yamagishi ◽

Kiyohisa Mizumoto ◽

Akira Ishihama

Keyword(s):

Saccharomyces Cerevisiae ◽

Temperature Sensitive ◽

Temperature Sensitive Mutants ◽

Mrna Capping ◽

Capping Enzyme

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DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.9.2.365-376.1989 ◽

1989 ◽

Vol 9 (2) ◽

pp. 365-376

Author(s):

M E Budd ◽

K D Wittrup ◽

J E Bailey ◽

J L Campbell

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Replication ◽

Dna Polymerase ◽

Temperature Sensitive ◽

Dna Polymerase I ◽

X Ray ◽

Temperature Sensitive Mutants ◽

Polymerase I ◽

Dna Polymerase Ii

We have used a set of seven temperature-sensitive mutants in the DNA polymerase I gene of Saccharomyces cerevisiae to investigate the role of DNA polymerase I in various aspects of DNA synthesis in vivo. Previously, we showed that DNA polymerase I is required for mitotic DNA replication. Here we extend our studies to several stages of meiosis and repair of X-ray-induced damage. We find that sporulation is blocked in all of the DNA polymerase temperature-sensitive mutants and that premeiotic DNA replication does not occur. Commitment to meiotic recombination is only 2% of wild-type levels. Thus, DNA polymerase I is essential for these steps. However, repair of X-ray-induced single-strand breaks is not defective in the DNA polymerase temperature-sensitive mutants, and DNA polymerase I is therefore not essential for repair of such lesions. These results suggest that DNA polymerase II or III or both, the two other nuclear yeast DNA polymerases for which roles have not yet been established, carry out repair in the absence of DNA polymerase I, but that DNA polymerase II and III cannot compensate for loss of DNA polymerase I in meiotic replication and recombination. These results do not, however, rule out essential roles for DNA polymerase II or III or both in addition to that for DNA polymerase I.

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A mutation in the tRNA nucleotidyltransferase gene promotes stabilization of mRNAs in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5778-5784.1992 ◽

1992 ◽

Vol 12 (12) ◽

pp. 5778-5784

Author(s):

S W Peltz ◽

J L Donahue ◽

A Jacobson

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Synthesis ◽

Steady State ◽

Relative Number ◽

Mrna Turnover ◽

Temperature Sensitive ◽

Nonpermissive Temperature ◽

Yeast Saccharomyces Cerevisiae ◽

Temperature Sensitive Mutants ◽

Steady State Levels

To identify trans-acting factors involved in mRNA decay in the yeast Saccharomyces cerevisiae, we have begun to characterize conditional lethal mutants that affect mRNA steady-state levels. A screen of a collection of temperature-sensitive mutants identified ts352, a mutant that accumulated moderately stable and unstable mRNAs after a shift from 23 to 37 degrees C (M. Aebi, G. Kirchner, J.-Y. Chen, U. Vijayraghavan, A. Jacobson, N.C. Martin, and J. Abelson, J. Biol. Chem. 265:16216-16220, 1990). ts352 has a defect in the CCA1 gene, which codes for tRNA nucleotidyltransferase, the enzyme that adds 3' CCA termini to tRNAs (Aebi et al., J. Biol. Chem., 1990). In a shift to the nonpermissive temperature, ts352 (cca1-1) cells rapidly cease protein synthesis, reduce the rates of degradation of the CDC4, TCM1, and PAB1 mRNAs three- to fivefold, and increase the relative number of ribosomes associated with mRNAs and the overall size of polysomes. These results were analogous to those observed for cycloheximide-treated cells and are generally consistent with models that invoke a role for translational elongation in the process of mRNA turnover.

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Localization of Core Spindle Pole Body (SPB) Components during SPB Duplication in Saccharomyces cerevisiae

The Journal of Cell Biology ◽

10.1083/jcb.145.4.809 ◽

1999 ◽

Vol 145 (4) ◽

pp. 809-823 ◽

Cited By ~ 146

Author(s):

Ian R. Adams ◽

John V. Kilmartin

Keyword(s):

Saccharomyces Cerevisiae ◽

Spindle Pole Body ◽

Spindle Pole ◽

Temperature Sensitive ◽

Large Plaque ◽

Temperature Sensitive Mutants ◽

Pole Body ◽

Cytoplasmic Face ◽

Preceding Cell

We have examined the process of spindle pole body (SPB) duplication in Saccharomyces cerevisiae by electron microscopy and found several stages. These include the assembly, probably from the satellite, of a large plaque-like structure, the duplication plaque, on the cytoplasmic face of the half-bridge and its insertion into the nuclear envelope. We analyzed the role of the main SPB components in the formation of these structures by identifying them from an SPB core fraction by mass spectrometry. Temperature-sensitive mutants for two of the components, Spc29p and Nud1p, were prepared to partly define their function. The composition of two of the intermediates in SPB duplication, the satellite and the duplication plaque, was examined by immunoelectron microscopy. Both contain cytoplasmic SPB components showing that duplication has already been partly achieved by the end of the preceding cell cycle when the satellite is formed. We show that by overexpression of SPB components the structure of the satellite can be changed and SPB duplication inhibited by disrupting the attachment of the plaque-like intermediate to the half-bridge. We present a model for SPB duplication where binding of SPB components to either end of the bridge structure ensures two separate SPBs.

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RCC1, a regulator of mitosis, is essential for DNA replication.

Molecular and Cellular Biology ◽

10.1128/mcb.12.8.3337 ◽

1992 ◽

Vol 12 (8) ◽

pp. 3337-3345 ◽

Cited By ~ 50

Author(s):

M Dasso ◽

H Nishitani ◽

S Kornbluth ◽

T Nishimoto ◽

J W Newport

Keyword(s):

Dna Replication ◽

Regulatory System ◽

Sperm Chromatin ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Replication Complex ◽

Temperature Sensitive Mutants ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts

Temperature-sensitive mutants in the RCC1 gene of BHK cells fail to maintain a correct temporal order of the cell cycle and will prematurely condense their chromosomes and enter mitosis at the restrictive temperature without having completed S phase. We have used Xenopus egg extracts to investigate the role that RCC1 plays in interphase nuclear functions and how this role might contribute to the known phenotype of temperature-sensitive RCC1 mutants. By immunodepleting RCC1 protein from egg extracts, we find that it is required for neither chromatin decondensation nor nuclear formation but that it is absolutely required for the replication of added sperm chromatin DNA. Our results further suggest that RCC1 does not participate enzymatically in replication but may be part of a structural complex which is required for the formation or maintenance of the replication machinery. By disrupting the replication complex, the loss of RCC1 might lead directly to disruption of the regulatory system which prevents the initiation of mitosis before the completion of DNA replication.

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CDKA and CDKB kinases from Chlamydomonas reinhardtii are able to complement cdc28 temperature-sensitive mutants of Saccharomyces cerevisiae

PROTOPLASMA ◽

10.1007/s00709-008-0285-z ◽

2008 ◽

Vol 232 (3-4) ◽

pp. 183-191 ◽

Cited By ~ 4

Author(s):

M. Čížková ◽

A. Pichová ◽

M. Vítová ◽

M. Hlavová ◽

J. Hendrychová ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Chlamydomonas Reinhardtii ◽

Temperature Sensitive ◽

Temperature Sensitive Mutants

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Temperature-sensitive mutants of Saccharomyces cerevisiae with an abnormal distribution of ribosomal subunits

Biochemical Society Transactions ◽

10.1042/bst0100092 ◽

1982 ◽

Vol 10 (2) ◽

pp. 92-93

Author(s):

JUDITH A. MITLIN ◽

MICHAEL CANNON ◽

LINDA GRITZ

Keyword(s):

Saccharomyces Cerevisiae ◽

Temperature Sensitive ◽

Ribosomal Subunits ◽

Temperature Sensitive Mutants ◽

Abnormal Distribution

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A NEW METHOD FOR MUTANT SELECTION IN SACCHAROMYCES CEREVISIAE

Genetics ◽

10.1093/genetics/79.2.175 ◽

1975 ◽

Vol 79 (2) ◽

pp. 175-186

Author(s):

Susan A Henry ◽

Bernard Horowitz

Keyword(s):

Saccharomyces Cerevisiae ◽

Fatty Acid ◽

Fold Increase ◽

New Method ◽

Temperature Sensitive ◽

Macromolecular Synthesis ◽

Mutant Selection ◽

Temperature Sensitive Mutants ◽

Selection For ◽

Selection Of

ABSTRACT A new method for the selection of auxotrophic, antibiotic- and temperature-sensitive mutants in Saccharomyces cerevisiae is reported. The technique is based upon the observation that certain fatty acid auxotrophs of yeast die when deprived of fatty acid only under conditions supporting growth. When macromolecular synthesis is blocked, the fatty acid-starved cells survive. By appropriate manipulation of a fatty acid-requiring strain enrichment as great as 75-fold was achieved for certain classes of auxotrophic mutants. An enrichment of approximately 100-fold is possible for some antibiotic-sensitive mutants. Selection for temperature-sensitive mutants, however, resulted in less than a 2-fold increase in the frequency of such mutants, probably because of the heterogeneity of this mutant category. It is likely that only that fraction of temperature-sensitive mutations which rapidly and reversibly blocks macromolecular synthesis is selected by this technique.

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