Temperature sensitive mutation

Habrobracon containing visible mutations, temperature-sensitive lethals, and other interesting genetic alterations are backcrossed to make the mutations homozygous before putting them in the stock collection (Fig. 2, part... 

Mutation. For industrial appHcations, mutations are induced by x-rays, uv irradiation or chemicals (iiitrosoguanidine, EMS, MMS, etc). Mutant selections based on amino acid or nucleotide base analogue resistance or treatment with Nystatin or 2-deoxyglucose to select auxotrophs or temperature-sensitive mutations are easily carried out. Examples of useful mutants are strains of Candida membranefaciens, which produce L-threonine Hansenu/a anomala, which produces tryptophan or strains of Candida lipolytica that produce citric acid. An auxotrophic mutant of S. cerevisiae that requires leucine for growth has been produced for use in wine fermentations (see also Wine). This yeast produces only minimal quantities of isoamyl alcohol, a fusel oil fraction derived from leucine by the Ehrlich reaction (10,11). A mutant strain of bakers yeast with cold-sensitive metaboHsm shows increased stabiUty and has been marketed in Japan for use in doughs stored in the refrigerator (12). 

Mutations Much of our knowledge of viral reproduction and how it is regulated has depended on the isolation and characterization of virus mutants. Several kinds of mutants have been studied in viruses host-range mutants, plaque-type mutants, temperature-sensitive mutants, nonsense mutants, transposons, and inversions. 

Temperature-sensitive mutations are those which allow a virus to replicate at one temperature and not at another, due to a mutational alteration in a virus protein that renders the protein unstable at moderately high temperatures. For instance, temperature-sensitive mutants are known in which the phage will not be replicated in the host at 43 °C but will at 25 °C, although the host functions at both temperatures. Such mutations are called conditionally lethal, since the virus is unable to reproduce at the higher temperature, but replicates at the lower temperature. 

DNA polymerase III has all the enzymatic activities of DNA polymerase I. A subunit of the enzyme is the product of the dna E gene. Temperature-sensitive mutations of this gene testify to the importance... 

Temperature-sensitive mutations usually arise from a single mutation s effect on the stability of the protein. Temperature-sensitive mutations make the protein just unstable enough to unfold when the normal temperature is raised a few degrees. At normal temperatures (usually 37°C), the protein folds and is stable and active. However, at a slightly higher temperature (usually 40 to 50°C) the protein denatures (melts) and becomes inactive. The reason proteins unfold over such a narrow temperature range is that the folding process is very cooperative—each interaction depends on other interactions that depend on other interactions. 

For a number of temperature-sensitive mutations it is possible to find (or make) a seond mutation in the protein that will suppress the effects... 

The most convincing example that inhibition of PC synthesis can induce apoptosis comes from a cell line with a temperature-sensitive defect in one of the enzymes in the CDP-choline pathway (Cui et al, 1996). The MT58 cell line is a Chinese hamster ovary (CHO) derived cell line with a mutation in CTP phosphocholine cytidylyltransferase (CT), which renders the... 

A successful tool in the early studies of metabolic pathways was blocking the pathway at some specific point. This could be done by the use of either mutants or inhibitors. Schekman et al have isolated a number of yeast mutants with blocks in their secretion pathway (Schekman, 1982). It is not yet known which proteins these mutations affect, but this is clearly a most promising approach for identifying those components involved in transport. In animal cells there are no cellular mutants with blocks in the intracellular transport of protein from the ER to the cell surface. There are, however, genetic diseases which affect the routing of lysosomal enzymes to the lysosomes (Neufeld et al, 1975 Sly and Fischer, 1982). For viruses it has been possible to isolate temperature-sensitive mutants in which a mutation in the viral glycoprotein arrests... 

Molecular studies explained this apparent paradox when the temperature-sensitive G a Ala366Ser mutation of the G a protein was identified. At 32°C, the G a 366Ser mutation results in the constitutive cAMP accumulation that causes the testosterone secretion that is the hallmark of the testotoxicosis phenotype. At 37°C, however, the G a 366Ser mutation results in loss of adenylyl cyclase signaling, causing PHP-Ia. As a result, a single mutation that performs differently in different tissues causes precocious puberty and abnormalities of PTH and TSH (91). 

Zuk D, Belk JP, Jacobson A (1999) Temperature-sensitive mutations in the Sacchammyces cerevisiae MRT4, GRC5, SLA2 and THS1 genes result in defects in mRNA turnover. Genetics 153 35-47... 

The third method uses genetic mutation as the primary tool. This method requires the isolation of conditional mutants, that is, mutants that behave normally under one set of conditions but abnormally under another. Most commonly, temperature-sensitive DNA replication mutants are used. Such mutants have been isolated in E. coli, and they grow normally at a low (permissive) temperature (33°C) but poorly or not at all at a high (nonpermissive) temperature (41°C). Preliminary analysis of the temperature-sensitive step provides clues to the stage of replication affected. For... 

May revert during replication or mutation to a more pathogenic form within the host May induce a disease state in a host with a compromised immune system Temperature sensitive... 

Duronio, R. J., Reed, S. I., and Gordon, J. I. 1992. Mutations of human myristoyl-CoA protein N-myristoyltransferase cause temperature-sensitive myristic acid auxotrophy in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 89 4129—4133. 

Temperature-sensitive mutations, in particular, have been very valuable in helping to define many of the proteins involved in replication. Several of these proteins have already been discussed. Temperature-sensitive mutations take effect at a certain temperature, e.g., 42-47°C, and not at another, e.g., 30°C or less. Mutations that affect replication are called dna mutations. Many that have been identified in E. coli code for various proteins associated with DNA chain growth at the replication fork. For example, the gene dnaG codes for primase (the DnaG protein) which has already been discussed. Some, however, code for proteins involved also or exclusively with the initiation of a cycle of replication at oriC. Examples of these are dnaA, B and C. 

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