Sperm aging in the male after sexual rest: Contribution to chromosome anomalies

The chromosome complement was studied in first-cleavage metaphases of mouse zygotes resulting from sperm aged in the male physiologically, after sexual rest. Females were inseminated by control males mating at 3-day intervals while experimentals mated to males that had had a sexual rest of 14 or more days. A total of 1954 eggs were collected 33–35 h post-HCG from 101 superovulated females mated to 42 controls and 43 experimental males. The fertilization rate was similar in both groups, being 84% and 85%, respectively. G-banded or Q-banded chromosomes were analyzed in 301 (68.3%) controls and 392 (49%) experimental first-cleavage metaphases. The overall rate of chromosome anomalies in controls was 4.45% as compared to 10.94% in experimentals, a highly significant difference. In the experimental group compared to controls, the frequency of trisomy, triploidy, structural rearrangements, and tetraploidy increased from 3.9% to 6.9%, 0% to 1.6%, 0.8% to 2.8%, and 0% to 1.3%, respectively. The genomic source of origin of the abnormalities was determined on the basis of differential condensation of the genomes. In the experimentals, grossly unbalanced sperm (diploids, disomics, double disomics, and those with large fragments) fertilized significantly more oocytes compared to controls. Our results implicate an advantage either in numbers or fertilizing capability for chromosomally abnormal sperm in a physiologically aged population.     

Biotransformations Of Fluoroaromatic Compounds: Accumulation Of Hydroxylated Products From 3-Fluorophthalic Acid Using Mutant Strains Of Pseudomonas Testosteroni

Biotransformations of 3-fluorophthalic acid have been investigated using blocked mutants of Pseudomonas testosteroni that are defective in the metabolism of phthalic acid (benzene-1,2-dicar-boxyfic acid). Mutant strains were grown with L-glutamic acid in the presence of 3-fluorophthalic acid as inducer of phthalic acid catabolic enzymes. Products that accumulated in the medium were isolated, purified and identified as the fluoroanalogues of those produced from phthalic acid by the same strains. The previously undescribed fluorochemicals cis-3-fluoro-4,5-dihydro-4,5-dihydroxyphthalic acid (VI) and 3-fluoro-4,5-dihydroxyphthalic acid (VII) have been obtained by biotransformation of 3-fluorophthalic acid, and 3-fluoro-5-hydroxyphthalic acid (X) from (VI) by freeze drying. In addition, samples of 2-fluoro-3,4-dihydroxybenzoic acid (2-fluoroprotocatechuic acid, VIII) and 3-fluoro-4,Sdi-hydroxybenzoic acid (5-fluoroprotocatechuic acid, IX) were obtained with a mutant deficient in the ring-fission enzyme, showing that the fluorine substituent in their precursor substrate (VII) is not recognized by the decarboxylase of the pathway, which shows no preference for which carboxyl group is removed. These studies of 3-fluorophthalic acid catabolism demonstrate the opportunities available for the production of novel fluorochemicals in reasonable yields by microbial transformations.     

Murine “housekeeping” enzyme (genetic locus:Idh-1) is regulated in an allele-specific manner

The murine “housekeeping” enzyme, cytosolic NADP-isocitrate dehydrogenase (E.C. 1.1.1.42) (genetic locus:Idh-1), exhibited a complex pattern of allele-specific expression. Protein electrophoresis on cellulose-acetate gels and determination of relative enzymatic activity by means of densitometry revealed that in heart tissue (but not liver tissue) of certain hybrid crosses the AA-homodimer was underrepresented relative to total enzymatic activity, and the degree of underrepresentation changed during development. In mixtures of homozygous tissue extracts of heart tissue (but not liver tissue) the AA-homodimer was underrepresented relative to the BB-homodimer. Relative activity of allelic isozymes varied as a function of tissue (heart versus liver), age, and the parental source of the Idh-1^a allele, but did not vary as a function of sex. Allele-specific expression was also exhibited in kidney tissue of the same animals. In adult male kidney tissue extracts from heterozygotes, the AA-hornodimer was underrepresented relative to total enzymatic activity; in adult female kidney tissue extracts from heterozygotes, a more codominant phenotype was observed. Tissue extracts from immature hybrid animals exhibited a phenotype midway between the adult male and adult female phenotypes. Tissue extracts from castrated males exhibited a phenotype equivalent to that seen in females. Relative activity of allelic isozymes in kidney varied as a function of age and sex, but did not vary as a function of the parental source of the Idh-1^a allele. While cytosolic NADP-IDH is a “housekeeping” enzyme, expressed in multiple tissues of the mouse, differences in the relative intensities of allelic isozyme bands provide evidence for tissue- and stage-specific regulatory variation.     

Genetic relationship between o6 and pro-1 mutants in maize

Summary This paper reports that the opaque-6 (o6) mutation of maize, which causes seedling lethality and interferes in the endosperm with the synthesis of zeins and b-32 protein, is a proline requiring mutant functionally allelic to proline-1 (pro-1). Furthermore, immunological studies on the b-32 content of ten independently originated o6 and pro-1 alleles demonstrated that four alleles contain an apparently normal b-32 protein while the others are either devoid of it or contain trace amounts of cross-reacting proteins of lower molecular weight.     

Growth in vitro of arrested embryos from lethal mutants ofArabidopsis thaliana

Summary Seventeen embryo-lethal mutants ofArabidopsis thaliana with lethal phases ranging from the globular to mature cotyledon stages of development were analyzed by culturing arrested embryos on nutrient media designed to promote either callus formation or the completion of embryo development and the recovery of homozygous mutant plants. Enriched media supplemented with vitamins, amino acids, and nucleosides were used to identify potential auxotrophic mutants. Wild-type embryos produced extensive callus on basal and enriched media supplemented with 2,4-D and kinetin. Numerous roots developed when wildtype callus was grown in the presence of NAA and kinetin. Mutant embryos arrested prior to the heart stage of development formed only a slight amount of callus on basal and enriched media. Arrested embryos from mutants 122G-E and 112A-2A reached a later stage of development and gave the most interesting responses in culture. 122G-E mutant embryos failed to grow on basal media but produced extensive callus and homozygous mutant plants on enriched media. The specific nutrient required for growth of this mutant remains to be determined. Arrested embryos from mutant 112A-2A developed into abnormal plants without roots when placed in culture. Mutant callus also failed to form roots on a variety of root-inducing media. Expression of this mutant gene therefore disrupts development of the root apical meristem during both embryogenesis in vivo and organogenesis in vitro.     

Nicotiana tabacum mutants with chloroplast encoded streptomycin resistance and pigment deficiency

Summary Callus ofNicotiana tabacum SRI, a mutant with maternally inherited streptomycin resistance, was induced from leaf sections. Callus pieces were mutagenised with N-ethyl-N-nitrosourea and inoculated onto a shoot-induction medium on which calli are normally green. White callus sectors were observed in the mutagenised cultures, and white and variegated shoots were regenerated from these sectored calli. The SR1-A10 line regenerated a chimeric shoot with white leaf margins. The chimeric shoot was grafted onto a normal green rootstock, grown into a flowering plant in the greenhouse, and crosses were made. The SRI-A15 line was crossed using flowers formed on albino plants grown in sterile culture. Pigment deficiency was maternally inherited in both lines. Physical mapping of the chloroplast genome of the SR1-A15 mutant by SalI, PstI and BamHI restriction endonucleases did not reveal any difference between the SR1-A15 and the parental SRI chloroplast genomes.     

Shoot regeneration of mesophyll protoplasts transformed by Agrobacterium tumefaciens,not achievable with untransformed protoplasts

Summary Alternative methods for shoot regeneration in protoplast derived cultures were developed in Nicotiana paniculata and Physalis minima. In both species protoplast derived callus is not regeneratable to shoots by conventional methods, e.g. hormone treatment. Leaf discs and stem segments of N. paniculata and P. minima were incubated with Agrobacterium tumefaciens shooter strains harbouring pGV 2215 or pGV 2298 or wildtype strain B6S3. After 36 h of co-incubation protoplasts were prepared. (Leaf disc and stem segment cloning). Co-cultivation experiments were also undertaken with protoplasts of both species. Transformed clones, characterized by their hormone independent growth and octopine production, could be isolated after about two months. Transformation frequencies of leaf disc and stem segment cloning and co-cultivation experiments varied from 5×10^–3 to 5×10^–5. After about one year of cultivation on hormone-free culture medium, shoots could be recovered from colonies of N. paniculata, transformed by the strain harbouring pGV 2298. In protoplast derived colonies of P. minima, shoot induction was obtained only after transformation by bacteria carrying pGV 2215. This demonstrates the importance of the particular shooter mutant, as well as the response of the host plant. Transformed shoots of P. minima produced octopine, whereas octopine production in transformed shoots and callus of N. paniculata was undetectable after one year of cultivation, though T-DNA was still present in the plant genome. Transformed shoots of N. paniculata and P. minima do not produce any roots. Shoots of N. paniculata have an especially tumerous phenotype. Shoots of both species were successfully grafted to normal donor plants of N. tabacum.Abbreviations B5-h Gamborg medium without hormones (Gamborg 1968) - V47 protoplast medium (Binding 1974) - D2a protoplast medium (Li et al. 1980) - MS-h Murashige and Skoog medium without hormones (Murashige and Skoog 1962) Dedicated to Professor Dr. G. Melchers in occasion of his 80th birthday     

Phycobilisomes of wild type and pigment mutants of the cyanobacterium Synechocystis PCC 6803

Mutants affected in their pigment content and in the structure of their phycobilosomes (PBS) were isolated in the cyanobacterium Synechocystis PCC 6803 by enriching a population with the inhibitor p-hydroxymercuribenzoate. Three of these mutants, PMB 2, PMB 10 and PMB 11, with original phenotypes, are described. Applying several criteria of analysis (77K absorption and fluorescence, protein electrophoretic patterns, electron microscopy), it was possible to assign the component polypeptides to each substructure of the phycobilisome. The model structure obtained fits with those described in other species PMB 10 and PMB 11, completely lacking PC, are the first source of pure PBS cores available, in which no contamination by residual PC can be feared, and are thus particularly interesting for further biochemical studies. The capacity of genetic transformation of Synechocystis PCC 6803 by chromosomal DNA makes this system very convenient for the analysis of the regulation of synthesis of the PBS constituents.Abbreviations PSI, PSII photosystems I, II - PBS phycobilisomes - PC phycocyanin - APC allophycocyanin - APC-B alophycocyanin B - PE phycoerythrin - PEC phycoerythrocyanin - WT wind type - Chl chlorophyll Present address: Service de Physiologie Microbienne Institut Pasteur, 28, rue du Docteur Roux, F-75724 Paris Cedex 15, France     

Accumulation and secretion of exoglucanase activity in yeast secretory mutants

Representative conditional yeast secretory mutants, blocked in transport of secretory and plasma membrane proteins from the endoplasmic reticulum (sec 18), from the Golgi body (sec 7) and in transport of secretory vesicles (sec 1), accumulated exoglucanase, a constitutive yeast activity, when incubated at the restrictive temperature (37°C). Different proportions of the accumulated activity were released by mutant cells under permissive conditions. The presence or absence of cycloheximide during the secretion period made no differences in the results. More than 90% of the internal activity was bound to membrane in wild type cells. However, only the soluble pool underwent changes during the accumulation or secretion periods. The bulk of secretory invertase accumulated by sec 1 was also soluble. By contrast sec 7 and sec 18 accumulated membrane-bound as well as soluble invertase forms and both were secreted in similar proportions in each mutant. More than 90% of the accumulated invertase was secreted at the permissive temperature in sec 18 cells. That percentage was significantly lower for exoglucanase (<65%). Concomitantly, invertase accumulated by this mutant exited from the cells with a lower half time (t 1/2=150 min). These results may be interpreted assuming that exoglucanase is exported by a passive flow of the soluble pool.Non-standard abbreviations p-NPG p-nitrophenyl--d-glucopyranoside - Con A concanavalin A - Tris tris(hydroxymethyl)-amino-methane     

Ethanol-sensitive mutants of Saccharomyces cerevisiae

Saccharomyces cerevisiae mutants unable to grow at ethanol concentrations at which the wild type strain S288C does grow, have been isolated. Some of them show additional phenotypic alterations in colony size, temperature sensitivity and viability in ethanol, which cosegregate with the growth sensitivity in ethanol. 21 selected monogenic ethanol-sensitive mutants define 20 complementation groups, denominated ETA1 to ETA20, which indicates that there is a high number of genes involved in the ethanol tolerance/sensitivity mechanism.Out of 21 selected monogenic mutants, 20 are not altered in the glycolytic pathway since, when maintained in glucosesupplemented medium, they can produce as much ethanol as the wild type and at about the same velocity. Nor do any of the mutants seem to be altered in the lipid biosynthetic pathway since, whether grown in the absence or in the presence of ethanol, their concentration of fatty acids and ergosterol is similar to that of the wild type under the same conditions. Therefore growth sensitivity to ethanol does not seem necessarily to be related to carbohydrate or lipid metabolism.Non-common abbreviations YP yeast extract peptone medium - YPD yeast extract peptone dextrose agar or medium - YPG yeast extract peptone glycerol agar - YPDE yeast extract peptone dextrose ethanol agar or medium - SD yeast nitrogen base dextrose agar - SPO yeast extract potassium acetate glucose agar - PD parental ditype - NPD non-parental ditype - TT tetratype