Original Mutant Strain Research Articles

RAPD Analysis on Avermectin High-Producing Strain of Streptomyces Avermilis Irradiated by 12C6+ Ions

Italic textAbstract. Industrial Streptomyces avermitilis were irradiated by 80 MeV/u 12C6+ ions whith were provided by the Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou Heavy Ion Accelerator. The high titers of mutants were obtained with different doses. The original strains and the high titers of mutants were detected and analyzed by Random Amplified Polymorphic DNA technology (RAPD). The results show that bands could be got from amplify the original strain by 4 primers of 28 primers and the high titers of mutants were amplified by the 4 primers. The amplified products were analysed by Jaccard formula. After 10 Gy, 30 Gy, 40 Gy, 45 Gy, 50 Gy, 70 Gy isodose irradiation, the similarity index were 0.63, 0.75, 0.71, 0.8, 0.61, 0.73. It was confirmed that at the DNA level, not only the high titers strains and the original mutant strain’s DNA were different, but also the high titers of the different mutants’ DNA were different. Therefore, ion implantation method changed the different Streptomyces avermitilis genetic material which promote the importance trace of avermectin biosynthesis changed, and different high titers of Streptomyces avermitilis were obtained.

ISOLATION AND CHARACTERIZATION OF A CELL WALL‐DEFECTIVE MUTANT OF CHLAMYDOMONAS MONOICA (CHLOROPHYTA)1

Cell wall–defective strains of Chlamydomonas have played an important role in the development of transformation protocols for introducing exogenous DNA (foreign genes or cloned Chlamydomonas genes) into C. reinhardtii. To promote the development of similar protocols for transformation of the distantly related homothallic species, C. monoica, we used UV mutagenesis to obtain a mutant strain with a defective cell wall. The mutant, cw‐1, was first identified on the basis of irregular colony shape and was subsequently shown to have reduced plating efficiency and increased sensitivity to lysis by a non‐ionic detergent as compared with wild‐type cells. Tetrad analysis of crosses involving the cw‐1 mutant confirmed 2:2 segregation of the cw:cw+ phenotypes, indicating that the wall defect resulted from mutation of a single nuclear gene. The phenotype showed incomplete penetrance and variable expressivity. Although some cells had apparently normal cell walls as viewed by TEM, many cells of the cw‐1 strain had broken cell walls and others were protoplasts completely devoid of a cell wall. Several cw‐1 isolates obtained from crosses involving the original mutant strain showed a marked enhancement of the mutant phenotype and may prove especially useful for future work involving somatic cell fusions or development of transformation protocols.

Kinetic analysis of the interaction between the QutA and QutR transcription-regulating proteins.

The QutR protein is a multidomain repressor protein that interacts with the QutA activator protein. Both proteins are active in the signal transduction pathway that regulates transcription of the quinic acid utilization (qut) gene cluster of the microbial eukaryote Aspergillus nidulans. In the presence of quinate, production of mRNA from the eight genes of the qut pathway is stimulated by the QutA activator protein. The QutR protein plays a key role in signal recognition and transduction, and a deletion analysis has shown that the N-terminal 88 amino acids are sufficient to inactivate QutA function in vivo. Using surface plasmon resonance we show here that the N-terminal 88 amino acids of QutR are able to bind in vitro to a region of QutA that genetic analysis has previously implicated in transcription activation. We further show that increasing the concentration of a full-length (missense) mutant QutR protein in the original mutant strain can restore its repressing function. This is interpreted to mean that the qutR mutation in this strain increases the equilibrium dissociation constant for the interaction between QutA and QutR. We propose a model in which the QutA and QutR proteins are in dynamic equilibrium between bound (transcriptionally inactive) and unbound (transcriptionally active) states.

Isolation and Identification of fxsA, an Escherichia coli Gene that can Suppress F Exclusion of Bacteriophage T7

A selection for mutants of Escherichia coli that survive coexpression of bacteriophage T7 gene10 and plasmid F pifA has allowed the identification of a newly defined genetic locus, fxsA.fxsA is located at 94.1min on the E.coli chromosome; the gene is monocistronic and non-essential for growth. Overexpression of fxsA is necessary for resistance to the toxicity of T7 gene 10 in the presence ofpifA ; the original mutant strain contains a promoter-up mutation, changing a G residue to the “invariant” T in the −10 hexamer of a σ70promoter. This chromosomal mutation causes a 25-fold increase in the level offxsA mRNA. The initiation codon of fxsA is shown to be UUG, and the FxsA protein is then deduced to consist of 158 amino acid residues. A similar mutant selection that demanded cell survival to a challenge of T7 gene 1.2 andpifA also resulted in the isolation of the identical promoter-up mutation that affects expression of fxsA. The increased levels of FxsA resulting from the promoter-up mutation allow phage T7 to avoid exclusion by the F plasmid, presumably by protecting the cell from premature death due to gene 10 or to gene 1.2 expression in the presence of the PifA protein.

Rapid genotyping of mutant mice using dried blood spots for polymerase chain reaction (PCR) analysis

Spontaneous neurologic mutations in the mouse provide powerful tools for the study of mammalian central nervous system development. The study of mouse neurologic mutants has led to a better understanding of the complex mechanisms involved in the development of the nervous system. Because few of these mutations have been identified, molecular probes distinguishing heterozygotes from homozygotes are generally unavailable. Further, most neurologic mouse mutants breed poorly as homozygotes, making it necessary to breed heterozygotes and select homozygous mutant progeny based on phenotype. The requirement for heterozygous breeding and the lack of molecular markers specific for the mutation have hampered developmental studies because the underlying neurologic perturbations occur before the mutant mice can be identified by phenotype. The recent identification and chromosomal assignment of simple sequence repeats (SSRs), repetitive sequences of DNA found at a high density throughout the mouse genome, provide the tools for mapping mutations in the mouse and for subsequent genotyping of potential mutants prior to phenotype onset. The SSRs are useful because these markers are polymorphic (for review see Weber, J.L., Human DNA polymorphisms based on length variations in simple-sequence tandem repeats. In: K.E. Davies and S.M. Tilghman (Eds.), Genetic and Physical Mapping. Genome Analysis, Vol. 1, Cold Spring Harbor Laboratory Press, Plainview, NY, 1990, pp. 159–181 [16]), that is, the size of the individual SSRs differs among strains of mice. Following polymerase chain reaction (PCR) amplification of an SSR and separation of PCR products by polyacrylamide gel electrophoresis, one can easily visualize differences in the size of the PCR product between mouse strains. Many mutations in the mouse arose spontaneously on inbred strains and were subsequently backcrossed onto a different strain. After many generations of congenic backcrosses, the only DNA retained from the original mutant strain is composed of the mutant gene and closely linked regions. Thus, it is possible to cross the mutant strain to a different mouse strain and map the mutation by correlating mutant phenotype to SSRs the same size as the original mutant strain. We have mapped the tottering ( tg), Purkinje cell degeneration ( pcd), and nervous ( nr) mutations using SSRs in backcrossed mouse strains. The SSRs distinguishing mutant from normal strains can then be used to genotype potential mutant pups before the onset of the mutant phenotype. The protocol described below can be adapted to almost any mutation congenically inbred for genotyping. Here we describe a method for selecting primers appropriate for genotyping potential mouse mutants and a rapid protocol for genotype screening. Even with SSRs distinguishing mutant from normal mice, genotyping several mice simultaneously can be a daunting task. This is primarily because the protocols available for preparing DNA for PCR amplification are time-consuming, requiring several purification steps including phenol extractions. Although kits are commercially available for DNA preparation without organic extractions, these kits tend to be expensive. The protocol described is a rapid, inexpensive method of determining the genotype of mice using PCR analysis of dried blood spots. The protocol only requires PCR primers distinguishing among alleles and is therefore ideal for the rapid identification of potential mutants for those mouse mutations which have been mapped using microsatellite markers. The DNA preparation protocol may also be used in rapid screening of potential transgenic mice. © 1997 Elsevier Science B.V. All rights reserved.

The peroxin Pex17p of the yeast Yarrowia lipolytica is associated peripherally with the peroxisomal membrane and is required for the import of a subset of matrix proteins.

PEX genes encode peroxins, which are required for the biogenesis of peroxisomes. The Yarrowia lipolytica PEX17 gene encodes the peroxin Pex17p, which is 671 amino acids in length and has a predicted molecular mass of 75,588 Da. Pex17p is peripherally associated with the peroxisomal membrane. The carboxyl-terminal tripeptide, Gly-Thr-Leu, of Pex17p is not necessary for its targeting to peroxisomes. Synthesis of Pex17p is low in cells grown in glucose-containing medium and increases after the cells are shifted to oleic acid-containing medium. Cells of the pex17-1 mutant, the original mutant strain, and the pex17-KA mutant, a strain in which most of the PEX17 gene is deleted, fail to form normal peroxisomes but instead contain numerous large, multimembraned structures. The import of peroxisomal matrix proteins in these mutants is selectively impaired. This selective import is not a function of the nature of the peroxisomal targeting signal. We suggest a regulatory role for Pex17p in the import of a subset of matrix proteins into peroxisomes.

Characterization of regulatory mutations causing anaerobic derepression of the sodA gene in Escherichia coli K12: cooperation between cis- and trans-acting regulatory loci.

The genetic loci leading to anaerobic derepression of a sodA::lacZ protein fusion in a UV-generated mutant strain (UV14) of Escherichia coli were identified. The mutant (UV14) was found to harbour two altered loci: one is in the trans-regulatory gene fnr (fumarate nitrate reduction) where leucine-129 was changed to glutamine (fnr14), and the second (sodA14) is in the promoter region (cis) of the sodA gene apparently affecting the binding of the Fur (ferric uptake regulation) protein. Introduction of an fnr+ gene into UV14 restored anaerobic repression of sodA::lacZ and restored the ability of the cells to reduce nitrate. However, when either the fnr14 or the sodA14 mutation was introduced into an otherwise wild-type background, only slight anaerobic derepression of sodA was observed. When both the cis- and trans-acting mutations (i.e. sodA14 and fnr14) were combined simultaneously in an otherwise wild-type background, the specific activity of sodA::lacZ expression was comparable to that of the original mutant strain (UV14). Furthermore, a genetically confirmed fur fnr double mutant was also similarly derepressed in anaerobic sodA::lacZ expression. The data presented suggest that the cis-mutation in UV14 (sodA14) affects the Fur-binding site in the sodA promoter, while having no effect on Fnr or Arc mediated repression. Also, a second putative Fnr-binding site that straddles the ribosomal binding-site was identified in the sodA gene.

Myocardial and vascular lesions in the LA/N-corpulent rat.

The LA/N-corpulent (LA/N-cp) rat is a normotensive strain derived from Koltesky's original mutant strain of the spontaneously hypertensive rat (SHR). When homozygous for the cp gene (cp/cp), the rats are hyperphagous, hyperlipidemic, hyperinsulinemic, and obese. The rats have been shown, by scanning electron microscope observation of the arterial system, to develop arterial lesions and occasional occlusive thrombi. These are significantly more frequent in the corpulent rats. Histological examination of the heart has shown the presence of four distinguishable types of lesions: type A, muscle scar or cell dropout with pigment deposition and inflammatory cell accumulation; type B, necrosis of a small number of myocytes with reactive inflammatory cells; type C, nodule of chronic inflammatory cells; type D, muscle scar without chronic inflammatory cells. Complete transverse sections of three regions of the heart were examined from rats 3, 6, and 9 months of age. Rats that were homozygous normal (+/+) showed an absence of all types of cardiac lesions. Male corpulent rats showed frequent lesions of all types with the frequency rising consistently with age. Female corpulent rats showed a similar incidence of types C and D lesions to males, but no type B lesions were found. Type A lesions, the most common in corpulent males, were found at a greatly reduced relative frequency. In some instances, we have found occlusive thrombi in a coronary artery of corpulent rats. The disease process in these animals occurs with a normal low fat, virtually cholesterol-free diet and no experimental manipulation.

Revertants of pleiotropic cross resistance and collateral sensitivity in yeast: A multivariate analysis

Five different selective media (Table 2) were used to isolate revertants of a pleiotropic cross resistant and collaterally sensitive strain of yeast. Of the 2676 revertants isolated, 1542 were visually classified as complete revertants, 238 as dauermodifications and 896 as partial revertants. Growth of the 896 partial revertants was evaluated on 24 diagnostic media (Table 1). Multivariate classification of revertant growth (Fig. 1) arranged the revertants into 9 groups and the media into two major groups (Table 4). The identification of two groups of media was consistent with the notion of a single gene alteration of mitochondrial and plasma membrane function in the original mutant strain. The 9 revertant groups had phenotypes suggesting pseudo-wild type (1 group), pseudo-mutant type (2 groups), loss of collateral sensitivity (2 groups), loss of cross resistance (1 group) and loss of different aspects of the resistance phenotype (3 groups). One of the latter 3 groups had a phenotype similar to strains reported by Guerineau et al. (Biochem. Biophys. Res. Commun. 61, 462) to be deficient in 2 μ DNA. The five media used to select revertants enriched for different groups within the 9 different groups of revertants (Table 5). The multivariate analyses used in this paper would be of general use in the analysis of other complex pleiotropic phenotypes.

Altered mRNA metabolism in ribonuclease III-deficient strains of Escherichia coli.

The metabolism of mRNA from the lactose (lac) operon of Escherichia coli has been studied in ribonuclease (RNase) III-deficient strains (rnc-105). The induction lag for beta-galactosidase from the first gene was twice as long, and enzyme synthesis was reduced 10-fold in one such mutant compared with its isogenic rnc+ sister; in the original mutant strain AB301-105, synthesis of beta-galactosidase was not even detectable, although transduction analysis revealed the presence of a normal lac operon. This defect does not reflect a loss of all lac operon activity galactoside acetyltransferase from the last gene was synthesized even in strain AB301-105 but at a rate several times lower than normal. Hybridization analyses suggested that both the frequency of transcription initiation and the time to transcribe the entire operon are normal in rnc-105 strains. The long induction lag was caused by a longer translation time. This defect led to translational polarity with reduced amounts of distal mRNA to give a population of smaller-sized lac mRNA molecules. All these pleiotropic effects seem to result from RNase III deficiency, since it was possible to select revertants to rnc+ that grew and expressed the lac operon at normal rates. However, the rnc-105 isogenic strains (but not AB301-105) also changed very easily to give a more normal rate of beta-galactosidase synthesis without regaining RNase III activity or a faster growth rate. The basis for this reversion is not known; it may represent a "phenotypic suppression" rather than result from a stable genetic change. Such suppressor effects could account for earlier reports of a noninvolvement of RNase III in mRNA metabolism in deliberately selected lac+ rnc-105 strains. The ribosomes from rnc-105 strains were as competent as ribosomes from rnc+ strains to form translation initiation complexes in vitro. However, per mass, beta-galactosidase mRNA from AB301-105 was at least three times less competent to form initiation complexes than was A19 beta-galactosidase mRNA. RNase III may be important in the normal cell to prepare lac mRNA for translation initiation. A defect at this step could account for all the observed changes in lac expression. A potential target within a secondary structure at the start of the lac mRNA is considered. Expression of many operons may be affected by RNase III activity; gal and trp operon expressions were also abnormal in RNase III- strains.