Molecular genetics in clinical practice: evolution of a DNA diagnostic service.

The development of a molecular genetics diagnostic service over a three year period was studied in a National Health Service region with a population of three million. Starting from a time when few diagnostic applications were possible, the number of disorders and the overall demand had grown rapidly. Conditions for which molecular genetic diagnosis had been provided included Duchenne and Becker muscular dystrophy, myotonic dystrophy, Huntington''s disease, and cystic fibrosis. Of 405 requests for diagnosis, 151 (37%) related to determination of carrier state, 187 (46%) to determining the feasibility of future prenatal diagnosis, and 67 (17%) were prenatal diagnostic biopsy samples, almost exclusively of first trimester chorion. DNA samples for future diagnostic use with a wide range of genetic disorders had also been banked. The study showed a need for close integration with clinical genetics services to allow satisfactory genetic counselling and interpretation of risks.     

Induction and release of secondary seed dormancy in genetically pure lines of Avena fatua

Induction and release of secondary dormancy in genetically pure dormant (AN-51, Mont 73) and non-dormant (CS-40, SH-430) lines of wild oat ( Avena fatua L.) were studied. These lines differed with regard to the optimal period of anaerobiosis necessary for induction of dormancy, and/or the degree (% of seeds acquiring dormancy) and duration of the dormancy induced. Secondary dormancy could be induced more effectively in the after-ripened seeds of dormant lines than in the non-dormant lines, where only a short-term dormancy could be induced (in 5–7 week-old-seeds). Higher anaerobiosis temperatures were more effective in inducing dormancy in all lines studied. Thus, as with primary dormancy, wild oat biotypes exhibit genetic variability in their secondary dormancy behaviour and factors like temperature can modify the expression of this trait.
The germination stimulants kinetin, isopentenyl adenine, sodium azide, potassium nitrate, ethanol and substituted phthalimides, which break primary dormancy in wild oats, stimulated germination of secondarily dormant seeds (line AN-51). Since these chemicals are structurally diverse, primary and secondary dormancies appear to be similar in part in their regulation.
Salicylhydroxamic acid, an inhibitor of cyanide-insensitive (alternative) respiration, did not inhibit: 1, spontaneous release of secondary dormancy in the line SH-430; and 2, stimulation of germination of secondarily dormant AN-51 seeds by various chemicals (except azide), suggesting that this respiratory pathway is not necessary for the release of induced dormancy.     

Purification of Mitochondrial Glutamate Dehydrogenase from Dark-Grown Soybean Seedlings

Proteins in extracts from cotyledons, hypocotyls, and roots of 5-d-old, dark-grown soybean (Glycine max L. Merr. cv Williams) seedlings were separated by polyacrylamide gel electrophoresis. Three isoforms of glutamate dehydrogenase (GDH) were resolved and visualized in gels stained for GDH activity. Two isoforms with high electrophoretic mobility, GDH1 and GDH2, were in protein extracts from cotyledons and a third isoform with the lowest electrophoretic mobility, GDH3, was identified in protein extracts from root and hypocotyls. Subcellular fractionation of dark-grown soybean tissues demonstrated that GDH3 was associated with intact mitochondria. GDH3 was purified to homogeneity, as determined by native and sodium dodecyl sulfate-polyacrylamide gels. The isoenzyme was composed of a single 42-kD subunit. The pH optima for the reductive amination and the oxidative deamination reactions were 8.0 and 9.3, respectively. At any given pH, GDH activity was 12- to 50-fold higher in the direction of reductive amination than in the direction of the oxidative deamination reaction. GDH3 had a cofactor preference for NAD(H) over NADP(H). The apparent Michaelis constant values for [alpha]-ketoglutarate, ammonium, and NADH at pH 8.0 were 3.6, 35.5, and 0.07 mM, respectively. The apparent Michaelis constant values for glutamate and NAD were 15.8 and 0.10 mM at pH 9.3, respectively. To our knowledge, this is the first biochemical and physical characterization of a purified mitochondrial NAD(H)-dependent GDH isoenzyme from soybean.     

Molecular Cloning and Characterization of a Calmodulin Gene from Arabidopsis thaliana

The calcium binding protein, calmodulin Is involved in regulating various cellular and biochemical processes. A gene tor calmodulin (CaM) has been Isolated from a genomic library of Arabidopsis thaliana constructed in ; EMBL-4 using a heterologous cDNA probe from electric eel. One of the positive clones was characterized and the region containing the calmodulin gene sequences was Identified, excised using appropriate restriction enzymes and subcloned Into a plasmid vector. The genomic clone contains a complete copy of the calmodulin gene. A comparison of the nucleotide sequence of the part of the clone with those of the other plant and animal systems confirms that the clone In fact contains the calmodulin gene sequences. Southern hybridization ulling the calmodulin gene sequences as a probe reveals the presence of more than one copy of the calmodulin gene. The results of this investigation taken together with those Iff the other. indicate that the calmodulin gene belongs to a small mutigene family consisting of atieast four member. In the Arabidopsis genome.     

The novel flightless-I gene brings together two gene families, actin- binding proteins related to gelsolin and leucine-rich-repeat proteins involved in Ras signal transduction

The Drosophila melanogaster gene flightless-I, involved in gastrulation and muscle degeneration, has Caenorhabditis elegans and human homologues. In these highly conserved genes, two previously known gene families have been brought together, families encoding the actin- binding proteins related to gelsolin and the leucine-rich-repeat (LRR) group of proteins involved in protein-protein interactions. Both these gene families exhibit characteristics of molecular changes involving replication slippage and exon shuffling. Phylogenetic analyses of 19 amino acid sequences of 6 related protein types indicate that actin- associated proteins related to gelsolin are monophyletic to a common ancestor and include flightless proteins. Conversely, comparison of 24 amino acid sequences of LRR proteins including the flightless proteins indicates that flightless proteins are members of a structurally related subgroup. Included in the flightless cluster are human and mouse rsp-1 proteins involved in suppressing v-Ras transformation of cells and the membrane-associated yeast (Saccharomyces cerevisae) adenylate cyclase whose analogous LRRs are required for interaction with Ras proteins. There is a strong possibility that ligands for this group could be related and that flightless may have a similar role in Ras signal transduction. It is hypothesized that an ancestral monomeric gelsolin precursor protein has undergone at least four independent gene reorganization events to account for the structural diversity of the extant family of gelsolin-related proteins and that gene duplication and exon shuffling events occurred prior to or at the beginning of multicellular life, resulting in the evolution of some members of the family soon after the appearance of actin-type proteins.      

Mode of Dinitroaniline Herbicide Action: II. CHARACTERIZATION OF [C]ORYZALIN UPTAKE AND BINDING

The intracellular binding of dinitroaniline herbicides was studied in order to analyze the mechanism of their colchicine-like action. When corn root apices (5 millimeters) are incubated in [¹⁴C]oryzalin (a dinitroaniline herbicide), the ¹⁴C is taken up rapidly, reaching a plateau in about 4 hours, which corresponds to the minimum incubation time in oryzalin required to get maximum inhibition of elongation. At 4 hours, the [¹⁴C]oryzalin concentration inside the roots is 35 times higher than that in the incubation medium. Since this accumulation of [¹⁴C]oryzalin is not affected by 1 millimolar sodium azide and there is no metabolism of [¹⁴C]oryzalin under these conditions, the [¹⁴C]oryzalin must be accumulated (bound) in corn root apices by a process not requiring metabolic energy.     

Magnesium-induced inner membrane aggregation in heart mitochondria: prevention and reversal by carboxyatractyloside and bongkrekic acid

Mg(2+) at an optimal concentration of 2mM (ph 6.5) induces large increases (up to 30 percent) in the optical density of bovine heart mitochondria incubated under conditions of low ionic strength (< approx. 0.01). The increases are associated with aggregation (sticking together) of the inner membranes and are little affected by changes in the energy status of the mitochondria. Virtually all of a number of other polyvalent cations tested and Ag(+) induce increases in mitochondrial optical density similar to those induced by Mg(2+), their approximate order of concentration effectiveness in respect to Mg(2+) being: La(3+) > Pb(2+) = Cu(2+) > Cd(2+) > Zn(2+) > Ag(+) > Mn(2+) > Ca(2+) > Mg(2+). With the exception of Mg(2+), all of these cations appear to induce swelling of the mitochondria concomitant with inner membrane aggregation. The inhibitors of the adenine nucleotide transport reaction carboxyatratyloside and bongkrekic acid are capable of preventing and reversing Mg(2+)-induced aggregation at the same low concentration required for complete inhibition of phosphorylating respiration, suggesting that they inhibit the aggregation by binding to the adenine nucleotide carrier. The findings are interpreted to indicate (a) that the inner mitochondrial membrane is normally prevented from aggregating by virtue of its net negative outer surface change, (b) that the cations induce the membrane to aggregate by binding at its outer surface, decreasing the net negative charge, and (c) that carboxyatractyloside and bongkrekic acid inhibit the aggregation by binding to the outer surface of the membrane, increasing the net negative charge.     

On the Estimation of the Population Mean with Known Coefficient of Variation

For the estimation of population mean a class of estimators has been proposed when the coefficient of variation is known and its efficiency is compared with the usual unbiased estimator and the estimators suggested by various researchers. The properties of the proposed class of estimators have been also discussed in the case of normal population.