Isolation Of Temperature-sensitive Mutants Research Articles

Systematic analysis of essential yeast TAFs in genome-wide transcription and preinitiation complex assembly.

The general transcription factor TFIID is composed of the TATA box binding protein (TBP) and a set of conserved TBP-associated factors (TAFs). Here we report the completion of genome-wide expression profiling analyses of yeast strains bearing temperature-sensitive mutations in each of the 13 essential TAFs. The percentage of the yeast genome dependent on each TAF ranges from 3% (TAF2) to 59-61% (TAF9). Approximately 84% of yeast genes are dependent upon one or more TAFs and 16% of yeast genes are TAF independent. In addition, this complete analysis defines three distinct classes of yeast promoters whose transcriptional requirements for TAFs differ substantially. Using this collection of temperature-sensitive mutants, we show that in all cases the transcriptional dependence for a TAF can be explained by a requirement for TBP recruitment and assembly of the preinitiation complex (PIC). Unexpectedly, these assembly experiments reveal that TAF11 and TAF13 appear to provide the critical functional contacts with TBP during PIC assembly. Collectively, our results confirm and extend the proposal that individual TAFs have selective transcriptional roles and distinct functions.

Isolation of temperature sensitive mutants and their mapping in Agrobacterium tumefaciens.

The first temperature sensitive mutants of plant pathogenic bacterium Agrobacterium tumefaciens were isolated by Tn5 mutagenesis. Several genes responsible for mutants were identified and mapped on the circular and linear chromosomes in A. tumefaciens MAFF301001.

Ore2, a mutation affecting proline biosynthesis in the yeast Saccharomyces cerevisiae, leads to a cdc phenotype.

We report here the isolation of temperature-sensitive mutants of the yeast Saccharomyces cerevisiae which exhibit cdc phenotypes. The recessive mutations defined four complementation groups, named ore1, ore2, ore3 and ore4. At the non-permissive temperature, strains bearing these mutations arrested in the G1 phase of the cell cycle. The wild-type allele of the gene altered in ore2 mutants was cloned. The nucleotide sequence of a fragment which can complement the mutation showed the presence of an open reading frame capable of encoding a protein with 286 amino acid residues. The deduced amino acid sequence showed 25% identity with that of the Escherichia coli delta 1-pyrroline-5-carboxylate reductase, an enzyme of the pathway for the biosynthesis of proline. The ore2 mutants, correspondingly, were found to be capable of growing at the non-permissive temperature on a synthetic medium supplemented with proline. In addition, the chromosomal location of the gene and its restriction map were compatible with those previously reported for the PRO3 gene which encodes the S. cerevisiae delta 1-pyrroline-5-carboxylate reductase.

Selection for Tn10 tet repressor binding to tet operator in Escherichia coli: isolation of temperature-sensitive mutants and combinatorial mutagenesis in the DNA binding motif.

We have constructed a genetic assay which selects positively for a functional interaction between Tet repressor and its cognate operator in Escherichia coli. In this strain Tet repressor blocks expression of lacI and lacZ. This leads to derepression of a lacPO controlled galK gene. The strain can be selected by growth on galactose as the sole carbon source and screened for the beta-galactosidase phenotype. These features allow the identification of one candidate among 10(8) false clones on a single plate. The assay was applied to select mutants with a ts DNA binding phenotype and to screen oligonucleotide generated Tet repressor mutants. Analysis of these mutations revealed that they affect DNA and inducer binding and possibly the dimerization domains. These mutations are located at residues 21, 48, 49, 89 and at the C terminus of the protein (193), respectively.

Isolation of temperature sensitive mutants of Autographa californica nuclear polyhedrosis virus: Phenotype characterization of baculovirus mutants defective in very late gene expression

Temperature sensitive mutants of Autographa californica nuclear polyhedrosis virus were isolated following exposure of parental replicating virus to 5-bromo-2′-deoxyuridine. Two mutants designated ts317 and ts538 were characterized in detail. Both mutants synthesized viral DNA but were defective in infectious extracellular virus production at the nonpermissive temperature suggesting defects in late gene regulation. Pulse-labeling experiments indicated that a switch to late protein synthesis occurred but this synthesis was not maintained with either mutant since all protein synthesis was gradually inhibited by 20 hr postinfection at 33°. Western blots probed with antisera directed against either extracellular virus proteins or very late proteins showed a reduction in late structural proteins, a 10-fold reduction in polyhedrin, and an absence of p10 at the nonpermissive temperature. Northern blots of total intracellular RNA showed that specific late and very late transcripts were synthesized in greatly reduced amounts at the nonpermissive temperature suggesting the absence of very late proteins was a result of inhibition of virus-specific transcription. The genetic analyses indicate that the mutations lie in two closely linked genes, both important in regulating the induction of very late transcription and translation.

Mutational Analysis of the Mitochondrial Rieske Iron-Sulfur Protein of Saccharomyces cerevisiae: I. Construction of a RIP1 deletion strain and isolation of temperature-sensitive mutants

Abstract A protocol has been devised to permit mutational analysis of the Rieske iron-sulfur protein of the mitochondrial cytochrome bc1 complex of Saccharomyces cerevisiae. The gene for this iron-sulfur protein (RIP1) has recently been cloned and sequenced (Beckmann, J. D., Ljungdahl, P. O., Lopez, J. L., and Trumpower, B. L. (1987) J. Biol. Chem. 262, 8901-8909). We have constructed a stable yeast deletion strain, JPJ1, in which the chromosomal copy of RIP1 was displaced by the yeast LEU2 gene by homologous recombination. A linear DNA fragment containing the LEU2 gene was inserted at the breakpoints of an 800-base pair deletion of the iron-sulfur protein gene and used to transform a leu- yeast strain. Leu+ transformants were obtained which were unable to grow on nonfermentable carbon sources. Southern analysis of the transformant, JPJ1, confirmed that the chromosomal copy of the RIP1 gene was deleted and replaced by the LEU2 gene. The genotype of JPJ1 was confirmed by genetic crosses. JPJ1 cannot grow on nonfermentable carbon sources but can be complemented to respiratory competence and transformed by yeast vectors containing the wild type RIP1 gene. The ability to complement strain JPJ1 with episomally encoded iron-sulfur protein provided the basis of a selection protocol by which mutagenized plasmids containing the RIP1 gene were assayed for mutations affecting respiratory growth. Five mutants of RIP1 were identified by their ability to complement JPJ1 to temperature-sensitive respiratory growth. DNA sequence analysis demonstrated that temperature-sensitive respiratory growth resulted from single point mutations within the protein coding region of RIP1. These mutations altered a single amino acid residue in each case. Mutations were dispersed throughout the terminal two-thirds of the protein. Each mutation was recessive and did not affect fermentative growth on dextrose. However, each mutation exerted unique temperature-sensitive growth characteristics on media containing the nonfermentable carbon source glycerol.

13] Isolation of temperature-sensitive mutants

This chapter describes isolation and characterization of mutants that are defective in biochemical, cellular, or developmental processes provide a powerful tool in the analysis of the regulation of these processes at the molecular and cellular levels. A special group is the conditional mutants—usually, temperature sensitive. Temperature-sensitive ( ts ), mainly heat-sensitive, mutants have been isolated in various organisms and also in cultured cell lines. Such mutants have been proven very useful in physiological studies of mammalian cells grown in culture. As a result, all those ts mutants that lose viability rapidly when they become arrested at the high temperature are lost. To recover all potential ts mutants, any selection step that involves exposure of cells to the restrictive temperature is avoided. Screening of conditional mutants by replica plating is a well-known procedure in microorganisms. However, applying similar techniques for mammalian cells is much harder because of technical problems.

Isolation and characterization of temperature sensitive mutants of Broadhaven virus, a Kemerovo group orbivirus (family, Reoviridae).

Eighteen stable temperature sensitive (ts) mutants of Broadhaven virus were isolated without the aid of mutagens. Spontaneous mutants were detected using 41 degrees C as the nonpermissive temperature and 36 degrees C as the permissive temperature. High frequency pairwise recombination defined five recombination groups. Four mutants belonged to group I, three to group II, six to group III, two to group IV, and two to group V. ts 7 was a possible double mutant representing lesions corresponding to those of groups III and V mutants. This is the first reported isolation of temperature sensitive mutants of a tick-borne orbivirus.

Avian sarcoma viruses, protein kinases and cell transformation.

The first RNA tumour virus to be isolated and identified as such was the Rous sarcoma virus (RSV), which causes the transformation of cells in culture as well as fibrosarcomas when injected into suitable host animals (for reviews see Hanafusa (1977) and Bishop (1978)). The genome of RSV has been studied intensively for the past 10-12 years, and it has been shown that the virus itself carries a gene responsible for malignant transformation. This gene, denotedsrcfor sarcoma, was identified genetically through the isolation of temperature-sensitive mutants that were conditional for cell transformation in culture. These mutants are able to transform cells at a temperature of 35 °C, the permissive temperature, but are unable to transform cells morphologically at 41 °C, the non-permissive temperature. The existence of such temperature sensitive mutants implied that the product of the viral transforming gene, in RSV, was a protein (Kawai & Hanafusa 1971). In addition to temperature-sensitive mutants, non-conditional mutants were isolated that had deletions of the src gene. These mutants are unable to transform cells in culture or to cause fibrosarcomas under most conditions. About 4 years ago, the product of the src gene was identified as a phosphoprotein (Mt= 60000); this protein was denoted pp60src(Purchioet al.1978). The RSV genome and the expression of the src gene is illustrated in figure 1.

Genetic Analysis of a Baculovirus, Autographa californica Nuclear Polyhedrosis Virus I. Isolation of Temperature-Sensitive Mutants and Assortment into Complementation Groups

Temperature-sensitive (ts) mutants were isolated from the baculovirus Autographa californica (alfalfa looper) MNPV, grown in Spodoptera frugiperda (fall armyworm) cells in the presence of N-methyl-N'-nitro-N-nitrosoguanidine. Of 567 plaque isolates screened, 27 were temperature sensitive (ts), representing a mutation frequency of 4.8%. Ten ts mutants were studied in detail: six failed to yield nonoccluded virus at 33 degrees C (NOV mutants), whereas the other four produced nonoccluded virus but were restricted in formation of polyhedra at 33 degrees C (Poly mutants). One of the six NOV mutants failed to synthesize viral DNA. Reversion and leak frequencies were determined, and the mutants were assorted into complementation groups based on the yield of polyhedrin synthesis in cells coinfected with pairs of mutants at 33 degrees C, as measured by radioimmunoassay. For NOV mutants, complementation indexes were also based on virus yield and were consistent with those based on polyhedrin synthesis. Nine mutants were assorted into five complementation groups. One mutant remained unclassified.

Analysis of a method for selecting temperature-sensitive mutants of BHK cells.

A procedure for the isolation of temperature sensitive mutants of BHK cells is described. Mutagenized cells were synchronized in G1 by serum starvation at 33 degrees, and, following release, shifted to 37.5 degrees in the presence of FUdR, to kill cells entering the DNA synthetic phase. Eight independently mutagenized series of cells were carried through four cycles of this selection, and surviving cells were tested for ability to grow at 33 degrees and 39.5 degrees after each cycle. The results suggest that such procedure is effective in enriching for non-leaky ts mutants and favors the isolation of mutants which at 39.5 degrees are inhibited in the processes necessary for DNA synthesis and lose viability rapidly. The effectiveness of repeated selection cycles and the general characteristics of the ts mutants isolated by this method were also evaluated.

Isolation of temperature-sensitive mutants from murine leukemic cells (L5178Y)

Two temperature-sensitive mutants (ts1 and ts3) have been isolated from murine leukemic cells, L5178Y, after mutagenesis and cytosine arabinoside selection. Both ts1 and ts3 grew normally at the permissive temperature (33 °C) but not at the non-permissive temperature (39 °C). Consistent results were obtained with the growth patterns in suspension culture as well as the plating efficiencies in soft agar. Temperature shift experiments showed that the mutant cells remained viable after extended exposure to the non-permissive temperature. Labeling studies with radioactive precursors indicated that the synthesis of DNA, but not of RNA or protein, was affected in these mutants at 39 °C. The defective function of ts3 cells was substantially corrected by supplementing alanine, hypoxanthine, and pyruvate.

Genetic Studies with Herpes Simplex Virus Type 1. The Isolation of Temperature-sensitive Mutants, their Arrangement into Complementation Groups and Recombination Analysis Leading to a Linkage Map

SUMMARY Nine 5-bromodeoxyuridine-induced temperature-sensitive (ts) mutants were isolated from stocks of a syncytial plaque type mutant (syn) itself derived from the non-syncytial wild-type (syn +) of the glasgow strain 17 of herpes simplex virus type 1. The nine ts mutants have been assigned to eight cistrons on the basis of different complementation tests. Non-syncytial revertants isolated from the original ts syn mutants were used in reciprocal three-factor crosses of the type: tsX syn × tsY syn + and tsX syn + × tsY syn. With recombination data from these crosses a linkage map has been constructed. The provisional map which locates nine cistrons is linear and spans about 25 recombination units. The regular occurrence of plaques with mixed syn/syn + morphology in the progeny from crosses is reported.

Isolation of temperature-sensitive mutants and the construction of a preliminary genetic map for influenza virus.

SUMMARY Sixteen ts mutants were isolated from influenza virus type A0, strain wsn, after treatment with either fluorouracil or hydroxylamine. Cells doubly infected with several pairs of ts mutants yielded a high proportion of wild-type virus, probably because of genetic recombination. Recombination frequencies with particular pairs of mutants were very variable, even in replicate tubes of the same experiment, until infected cells were treated with receptor-destroying enzyme (rde) after adsorption of virus. Day-to-day variation between experiments could not be eliminated, but was allowed for by including a standard cross in all experiments, and relating recombination frequencies to the values obtained in this cross. Standardized recombination frequencies were then reproducible and characteristic of each pair of mutants. The recombination frequencies thus obtained allowed certain of the mutants to be placed in a sequence with additivity between the intervals, suggesting that influenza virus may have a linear genetic map.