Irradiation induced physiological and morphological variation in Colletotrichum capsici,incitant of anthracnose of chillies

With the aid of radioactive phosphorus and gamma rays, a number of morphological and biochemical mutants ofColletotrichum capsici have been induced and these showed wide range of variability. Biochemical mutants mostly induced by means of p³² irradiation showed deficiency of amino acids or vitamins. Specific deficiency analysis of some of the biochemical mutants revealed requirements for lysine, methionine, glycine or serine and biotin. These biochemical mutants could not be distinguisbed from the wild type in morphological characters when grown on complete or otherwise suitable supplemented media.     

Genetic and biochemical studies of phosphomannose isomerase deficient mutants ofSaccharomyces cerevisiae

Summary Three mannose-negative mutants ofSaccharomyces cerevisiae have been isolated. These mutants showed growth inhibition when mannose was added to a growth medium containing glycerol or fructose. Crosses between wild type mutants showed segregation of 2⁺/2⁻. Crosses between the mutants themselves showed that they were closely linked. Two mutants (XM3 and D2) showed characteristics of allelic structural alteration of phosphomannoseisomerase. Mutant D4 had a deficiency of phosphomannoseisomerase activity, but with a normal thermostability. Revertants from D4 had a normal thermostability.     

Properties of streptomycin dependent non-nodulating mutants of cowpea rhizobia and Bradyrhizobium japonicum

We have isolated and characterized mutants from cowpea rhizobia strains JRW3 and IRC256 and Bradyrhizobium japonicum USDA110, which show dependence on streptomycin (Sm) for growth. In the presence of Sm, the majority of the Sm^D (streptomycin dependent) mutants showed cross-resistance to other aminoglycoside antibiotics and some showed no growth at 37°C and 40°C. When nodulation abilities of Sm^D mutants (derived from all three strains) were examined, most of them (> 91%) showed non-nodulating phenotypes to their respective hosts. Preliminary biochemical and genetic characterization indicated that drug-uptake function was altered in Sm^D mutant, and the wild type strain JRW3 could be transformed to streptomycin dependent by Sm^D DNA.     

Sake brewing characteristics of a new type of cerulenin-resistant sake yeast mutant

Several new types of cerulenin-resistant mutants of sake yeast were isolated. These mutants showed respiratory deficiency and could grow on media containing a higher concentration of antibiotics than could the parent. Sakes brewed by the mutants produced less succinate than by both the parent yeast and the mutants with respiratory deficiency induced by ethidium bromide. In addition, the acidity of these mutants was decreased. Since low acidity is favourable in both sake and wine, these mutants might be applicable for both sake and wine brewing.     

Characterization of sarcoplasmic reticulum Ca ATPase nucleotide binding domain mutants using NMR spectroscopy

Sarcoplasmic reticulum Ca²⁺ ATPase (SERCA) is essential for muscle function by transporting Ca²⁺ from the cytosol into the sarcoplasmic reticulum through ATP hydrolysis. In this report, the effects of substitution mutations on the isolated SERCA-nucleotide binding domain (SERCA-N) were studied using NMR. ¹⁵N–¹H HSQC spectra of substitution mutants at the nucleotide binding site, T441A, R560V, and C561A, showed chemical shift changes, primarily in residues adjacent to the mutation sites, indicating only local effects. Further, the patterns of chemical shift changes upon AMP–PNP binding to these mutants were similar to that of the wild type SERCA-N (WT). In contrast to these nucleotide binding site mutants, a mutant found in patients with Darier’s disease, E412G, showed small but significant chemical shift changes throughout the protein and rapid precipitation. However, the AMP–PNP dissociation constant (∼2.5 mM) was similar to that of WT (∼3.8 mM). These results indicate that the E412G mutant retains its catalytic activity but most likely reduces its stability. Our findings provide molecular insight into previous clinical, physiological, and biochemical observations.     

Significance of Pyruvate Carboxylase in Sugar Metabolism of Agrobacterium tumefaciens

Mutants, which fail to grow on glucose medium but can grow on succinate medium, were isolated by treatment with N-methyl-N′-nitro-N-nitrosoganidine from the wild-type strain of Agrobacterium tumefaciens, and were found to lose growth on several hexoses and three-carbon intermediates. The revertant mutants, which recovered the ability to grow on glucose medium, simultaneously regained the ability to grow on hexoses and three-carbon intermediates. By comparison of biochemical properties of the wild-type, the mutants and the revertant mutants, two mutant strains were characterized to be pyruvate carboxylase-deficient. Then, we concluded that these mutants might be induced by a single mutation at a genetic locus of pyruvate carboxylase and that the deficiency in the enzyme gave a pleiotropic effect on the ability to grow on hexoses and three-carbon intermediates. Some properties of pyruvate carboxylase of this bacterium were also presented.     

Altered mechanoreceptor response in Drosophila bang-sensitive mutants

Bang-sensitive mutants of Drosophila melano gaster (bas ¹, bss^MW1, eas², tko^25t) display seizure followed by paralysis when subjected to mechanical shock. However, no physiological or biochemical defect has been found to be common to all of these mutants. In order to observe the effects of bang-sensitive mutations upon an identified neuron, and to study the nature of mechanically induced paralysis, we examined the response of a mechanosensory neuron in these mutants. In each single mutant and the double mutant bas ¹ bss^MW1, the frequency of action potentials in response to a bristle displacement was reduced. This is the first demonstration of a physiological defect common to several of the bang-sensitive mutations. Adaptation of spike frequency, cumulative adaptation to repeated stimulation (fatigue) and the time course of recovery from adaptation were also examined. Recovery from adaptation to a conditioning stimulus was examined in two mutants (bas ¹ and bss ^MW1), and initial recovery from adaptation was greater in both mutants. Quantification of receptor potentials was complicated by variability inherent in extracellular recording conditions, but examination of the waveform and range of amplitudes did not indicate clear mutant defects. Therefore the differences observed in the spike response may be due to an alteration of the transfer from receptor potentials to action potential production. DNA sequence analysis of tko and eas has indicated that they encode apparently unrelated biochemical products. Our results suggest that these biochemical lesions lead to a common physiological defect in mechanoreceptors. Although this defect does not provide a straightforward explanation for bang sensitivity, the altered cellular process may lead to bang sensitivity through its action in different parts of the nervous system.Abbreviations APA anterior post-alar - ANP anterior notopleural - bas bang-sensitive - bss bang-senseless - eas easily-shocked tko technical knockout     

Lack of mutagenic effect of vinyl chloride monomer in the mammalian spot test

The kinetics of the killing effect of ethanol was studied at 6–30% concentrations. Ploidy of cells, deficiency of the excision-repair system or holding under no-growth conditions did not influence survival.Ethanol at 24% increased, in the strain, the number of respiration-deficient cells from a spontaneous level of 0.4% up to nearly half of all survivors.Genetic analysis showed the mitochondrial nature of induced respiration-deficient mutants (or rho^?).The influence of yeast resistance to some antibiotics was studied on rho^? mutagenesis, both spontaneous and induced by ethanol. Neomycin-resistant strains were characterized by a significantly lower level of these mutations than were neomycin-sensitive strains.     

Dietary thyroid hormone replacement ameliorates hearing deficits in hypothyroid mice

Thyroid hormone (TH) insufficiency causes variable hearing impairment and mental deficiency in humans. Rodents lacking TH have congenital hearing deficiency that has been attributed to physiologic, morphologic, and developmental abnormalities of the auditory system. We examined four genetically defined strains of hypothyroid mice for development of hearing and response to TH replacement initiated during late gestation and continued through six weeks of age. Auditory brain stem response studies showed variable hearing impairment in homozygous mutants of each strain at three weeks of age relative to normal littermates. Mutants from three of the strains still had hearing deficiencies at six weeks of age. TH-enriched diet significantly improved hearing in three-week-old mutants of each strain relative to untreated mutants. Differences in the level of hearing impairment between the Prop1 ^df and Pit1 ^dw mutants, which have defects in the same developmental pathway, were determined to be due to genetic background modifier genes. Further physiologic and morphologic studies in the Cga ^tm1Sac strain indicated that poor hearing was due to cochlear defects. We conclude that TH supplement administered during the critical period of hearing development in mice can prevent deafness associated with congenital hypothyroidism of heterogeneous genetic etiology.     

Genetic differences in seed longevity of various Arabidopsis mutants

Seeds gradually lose their viability during dry storage. The damage that occurs at the biochemical level can alter the seed physiological status and is affected by the storage conditions of the seeds. Although these environmental conditions controlling loss of viability have been investigated frequently, little information is available on the genetics of seed longevity. Using Arabidopsis mutants in defined developmental or biochemical pathways such as those affected in seed coat composition, seed dormancy, hormone function and control of oxidative stress, we tried to gain insight into the genes and mechanisms controlling viability of stored seeds. Mutations like abscisic acid insensitive3 ( abi3 ) as well as abscisic acid deficient1 ( aba1 ) show reduced longevity, which may be partially related to the seed dormancy phenotype of these mutants. Mutants with seed coat alterations, especially aberrant tests shape ( ats ), showed a stronger reduction in germination percentage after storage, indicating the importance of a 'functional' seed coat for seed longevity. A specific emphasis was placed on mutants affected in dealing with Reactive Oxygen Species (ROS). Because several pathways are involved in protection against ROS and because gene redundancy is a common feature in Arabidopsis , 'double' mutants were generated. These 'double' mutants and the corresponding single mutants were subjected to a controlled deterioration test (CDT) and a germination assay on hydrogen peroxide (H₂O₂) after prolonged storage at two relative humidities. CDT and germination on H₂O₂ affected all genotypes, although it appears that other effects like genetic background are more important than the deficiencies in the ROS scavenging pathway. Explanations for this limited effect of mutations affecting ROS scavenging are discussed.     

Isolation and analysis of Kluyveromyces fragilis mutants displaying alcohol dehydrogenase deficiency

Summary ADH deficient mutants of Kluyveromyces fragilis n^o111 were obtained by two methods. Physiological and biochemical characteristics of these mutants suggest that loss of ADH activity is always connected with a decrease in hexose phosphorylation activity. The mutation might have a phenotypic pleiotropic effect.     

Type 1 fimbriation is negatively regulated by cyclic AMP and its receptor proteinvia conjugative plasmid F inEscherichia coli K-12

Expression of fimbriation was studied inEscherichia coli K-12 CA8000 HfrH, and itscya, crp and MS2 resistant mutants. The cells of cya⁺ crp⁺ parent strain were observed to be flagellated bacilli, lacking fimbriae, unable to agglutinate erythrocytes and deficient in ability to produce surface pellicle during growth in stationary culture. The cells ofcya andcrp mutants were observed to be cocci or coccobacilli devoid of flagella, having haemagglutinating activity, fimbriated and capable of producing surface pellicle in stationary cultures. The fimbriation and haemagglutinating activities were lower incya mutants grown with cAMP supplementation. Thecya andcrp mutants produced relatively small, smooth and compact colonies consisting mostly of fimbriated cells, like those of earlier described Fimσ mutants. Thecya ⁺ crp⁺ MS2 resistant mutant produced large sized colonies like those of parent but was deficient in conjugal donor ability. It resembledcya andcrp mutants in haemagglutinating and fimbriation properties. Thecya andcrp mutants have been earlier shown to be deficient in several Tra functions including conjugal donor ability. It is concluded thatEscherichia coli K-12 cells express fimbriation when Tra functions of F-plasmid carried in them are not expressed either due to deficiency of active cAMP-receptor protein complex or mutation in F-plasmid or when F-plasmid is absent.     

Killing of Klebsiella pneumoniae mediated by conjugation with bacteria carrying antibiotic-resistance plasmids of the group N

Escherichia coli K-12 strains carrying any one of a number of different plasmids of the incompatibility group N have been found to kill Klebsiella pneumoniae, M5a1. A simple spot test is described that could be of value in diagnosing the presence of these plasmids. Killing is closely associated with the ability of the N⁺ strains to mate with M5a1 and all conjugation-deficient (Tra^?) mutants were also unable to kill M5a1 (Kil^?1). At a low frequency, some Kil^? mutants could be isolated that showed little or no transfer deficiency. Plasmids of the groups P and W which have been suspected (on other grounds) to specify conjugative systems interrelated in some manner to that specified by N plasmids, also kill M5a1 but to a lesser degree.     

Mutations as possible replication errors in bacteria growing under conditions of thymine deficiency

When Escherichia coli or Bacillus subtilis cells having inhibited thymidylate synthetase activity were incubated for a long time on solid medium supplemented with a limiting concentration of thymine or thymidine (0.1–0.3 μg/ml) most of them became mutants for one or more genetic markers. This “overall mutagenesis” was detected both in Thy^? bacteria and in prototrophs for thymine (Thy⁺) with thymidylate synthetase inhibited by the addition of 5-fluorodeoxyuridine (FUdR) to the growth medium. When thymine (or thymidine) was present in very low amounts (10^?3 μg/ml) or was totally absent, the efficiency of mutagenesis decreased some 100-fold. The solid growth medium is essential because it supports the filamentous cells grown under conditions of limiting thymine.For some of the mutants with identified deficiency their ability to revert under the action of different mutagens was studied. Most efficient was 5-bromouracil (BU). This reversion is the characteristic response of mutations due to AT → GC transitions. In addition to single mutants, many multiple mutants were induced. The repair-defective strain of E. coli pol A1^? and strains Rec A^? and Exr A^?, which are also defective in UV-induced mutagenesis, showed a high level of mutation induction under the conditions described. All these results are in accord with the hypothesis that overall low-thymine mutagenesis reflects the accumulation of replication errors in DNA under the conditions of a precursor deficiency.     

Biochemical studies of H-2K antigens from a group of related mutants. I. Identification of a shared mutation in B6-H-2 bm5 and B6-H-2 bm16

Structural studies of the H-2 gene products from a group of five closely related but independent C57BL/6 H-2 mutant mice were undertaken. Each of the mutants exhibits reciprocal graft rejection with the parent. The group is remarkable, however, because each member of this group can accept skin grafts from any other member. The results of biochemical analysis of the H-2 glycoproteins from two of these related mutants, bm5 and bm16, are presented in this report. Evidence is given that the H-2K molecules from these two mutants are identical to each other based on comparative tryptic peptide mapping profiles with the parent. From partial amino acid sequence analysis, K products of both mutants have at least one common difference from the parental type located at residue number 116. Definitive studies established that in both bm5 and bm16 a tryosine found in the parent molecule is substituted with a phenylalanine in the mutant. These results show that a biochemical difference between the K products of the two mutants and of the parent can be detected, that the mutants appear to be identical with one another even though they arose independently, and that they differ from the other H-2K ^b mutants analyzed.Abbreviations used in this paper B6 C57BL/6Kh - bm5 B6-H-2^bm5 - bm6 B6-H-2 ^bm6 - bm7 B6.C-H-2 ^bm7 - bm9 B6.C-H-2 ^bm9 - bm16 B6-H-2 ^bm16 - D H-2D - K H-2K - MHC major histocompatibility complex     

A Genetic and Biochemical Analysis of the Temperature Sensitive, Normal-Winged Alleles of the Rudimentary Locus of DROSOPHILA MELANOGASTER

The genetic and biochemical characteristics of a particular class of mutants at the rudimentary locus are described. The mutants are pyrimidine auxotrophs, like classical rudimentary alleles, but they are unique in that they do not alter the size or shape of the wing (Falk and Nash 1974b). Aspartate transcarbamylase and dihydroorotase activities have been measured in seven different normal-winged mutants, and the results indicate that these strains are enzymologically "leaky" mutants. Previous studies have shown that three genetic functions (corresponding to the first three enzymes of pyrimidine synthesis) are associated with the rudimentary locus. Four of the seven mutants appear to affect all three of these functions. Each of the four is temperature sensitive, and a biochemical analysis of the temperature sensitivity of one of these mutants, (r)pyr1-3, suggests that a process affecting the synthesis or assembly of these enzymes is altered at high temperatures.     

Subunit composition,biosynthesis, and assembly of the yeast vacuolar proton-translocating ATPase

The yeast vacuole is acidified by a vacuolar proton-translocating ATPase (H⁺-ATPase) that closely resembles the vacuolar H⁺-ATPases of other fungi, animals, and plants. The yeast enzyme is purified as a complex of eight subunits, which include both integral and peripheral membrane proteins. The genes for seven of these subunits have been cloned, and mutant strains lacking each of the subunits (vma mutants) have been constructed. Disruption of any of the subunit genes appears to abolish the function of the vacuolar H⁺-ATPase, supporting the subunit composition derived from biochemical studies. Genetic studies of vacuolar acidification have also revealed an additional set of gene products that are required for vacuolar H⁺-ATPase activity, but may not be part of the final enzyme complex. The biosynthesis, assembly, and targeting of the enzyme is being elucidated by biochemical and cell biological studies of thevma mutants. Initial results suggest that the peripheral and integral membrane subunits may be independently assembled.     

Fructose-bisphosphatase-deficient mutants of the yeast Schizosaccharomyces pombe

We showed that in the yeast Schizosaccharomyces pombe, fructose-bisphosphatase is not subject to catabolite inactivation as it was observed in Saccharomyces cerevisiae. However, this enzyme activity is sensitive to catabolite repression in both yeasts. Two mutants lacking completely fructose-bisphosphatase activity were found. They were unable to grow on glycerol medium. They were still respiratory competent and exhibited the ability to derepress partially malate dehydrogenase activity. In glucose exponential phase culture, the parental strain lacks completely the fructosebisphosphatase activity due to catabolite repression. In these conditions, the growth is slowed down only in the mutants eventhough both mutants and their parental strain lack this enzyme activity. Normal sporulation and poor spore germination were observed for one mutant whereas, only in the presence of glucose, normal sporulation and normal spore germination were observed for the second mutant. Mendelian segregation of glycerol growth was found for the well germinating mutant. It is of nuclear heredity. The two mutations appeared to be closely linked.Abbreviations FBPase Fructose-1,6-bisphosphatase - fbp ^- genetic symbol for FBPase deficiency - glr ^- symbol for inability to grow on glycerol A. M. Colson is Research Associate au Fonds National de la Recherche Scientifique     

Colchicine resistant friend cells: Application to the study of actinomycin D induced erythroid differentiation

Colchicine resistant (Cch^R) mutants have been isolated from Friend erythroeleukemic cells by successive single-step selections. Measurements of the rate of uptake of [³H]-colchicine into whole cells, and the binding of [³H]-colchicine to cytoplasmic extracts, suggest that these mutants are colchicine-resistant due to a reduced membrane permeability to colchicine, rather than an altered intracellular colchicine-binding target. Consistent with this conclusion is the observation that non-toxic concentrations of Tween–80, a non-ionic detergent, potentiated colchicine uptake into mutant cells. In addition, these Friend cell mutants, like Cch^R mutants of other cell types, are cross-resistant to a variety of unrelated drugs, including daunomycin, puromycin, emetine, and actinomycin D. A comparison of the dose-response curves for the induction of Friend cell differentiation by actinomycin D of both wild-type and two Cch^R cells suggests that actinomycin D permeation is required for its effects on Friend cell differentiation. Potentiation of actinomycin D uptake by Tween–80 significantly lowered the concentration of drug required to induce hemoglobin synthesis in the Cch^R cells, but had no significant effect on either actinomycin D induction of Cch^S cells or DMSO induction of both Cch^S and Cch^R cells. In common with other chemical inducers of Friend cell differentiation, the addition of actinomycin D results in an early decrease in ⁸⁶ RbCl uptake, although this effect on transport occurred 14 hours later than that observed with DMSO.     

Diphtheria toxin resistance in human lymphoblast lines

Stable mutants (Dip^r), highly resistant to diphtheria toxin have been selected from a sensitive human lymphoblast line. A second human lymphoblast line, HH-4 (and its derivative TK6-1) were found to be highly resistant to diphtheria toxin without any previous selection, suggesting the presence of the Dip^r allele in the human population. The resistance of protein synthesis in extracts of mutant cells to diphtheria toxin indicates that the genetic lesion in the resistant lines examined involved an alteration in the protein synthesis. In comparison to sensitive cells, the mutant cell extracts contained reduced (30–40%) levels of ADP-ribosylatable elongation factor-2 activity suggesting that the lesion presumably affects elongation factor-2 in such cells. The biochemical phenotype of these mutants appears similar to that of the Dip^rIIb class of mutants of Chinese hamster cells (4,6) which behave codominantly in hybrids.