Glutamate Oxidation by Soybean Cotyledon and Leaf Mitochondria

Mitochondria purified from cotyledons of soybean seedlings fiveto ten days old have the capacity to rapidly oxidize glutamate(measured as glutamate dependent oxygen consumption). This capacitywas greatest at ten days after planting but was very low priorto emergence of cotyledons from the vermiculite and during senescence.Solubilized glutamate dehydrogenase activity, on the other hand,was substantial at two days after planting, peaked at sevendays, then declined and rose again during senescence. It issuggested that mitochondrial glutamate oxidation plays a rolein reserve mobilization and amino acid metabolism during seedlinggrowth. Leaf mitochondria and those from senescing cotyledonscould not sustain rapid rates of glutamate oxidation despiteready oxidation of other substrates and high solubilized glutamatedehydrogenase activity, suggesting an alternative role for theenzyme in these tissues. Possible controlling factors are discussed. ² Present address, Garvan Institute, Darlinghurst, N. S. W.,Australia. ³ Permanent address, Department de Biologia Vegetal, Facultatde Biologia, Universitat de Barcelona, Barcelona, Spain. (Received May 6, 1988; Accepted August 3, 1988)     

Rapid detection of alpha-1-antitrypsin deficiency by analysis of a PCR-induced TaqI restriction site

Summary A single base substitution is responsible for the PI-Z mutation in alpha-1-antitrypsin (AAT) deficiency. The Z mutation, which is in exon V of the AAT gene, was analysed directly using a primer designed with a single base substitution in the DNA sequence. During the polymerase chain reaction with this primer, a restriction enzyme site was created in the exon-V-amplified DNA sequence; this site was present in the normal allele (M form) but absent in the Z form. Here, the design of the primer and the application of the designer primer for prenatal diagnosis of chorion villus samples (CVS) for AAT deficiency is described. The method provides a simple rapid means of prenatal diagnosis of AAT deficiency within a day of the collection of the CVS. The detection of the nucleotide base change in AAT deficiency at the Z mutation site provides the opportunity for accurate prenatal diagnosis where no tissue is available from an AAT-affected individual.     

Oxidation of NADH by plant mitochondria: Kinetics and effects of calcium ions

The kinetics of NADH oxidation by the outer membrane electron transport system of intact beetroot (Beta vulgaris L.) mitochondria were investigated. Very different values for V_max and the K_m for NADH were obtained when either antimycin A-insensitive NADH-cytochrome c activity (V_max= 31 ± 2.5 nmol cytochrome c (mg protein)^?1 min^?1; K_m= 3.1 ± 0.8 μM) or antimycin A-insensitive NADH-ferricyanide activity (V_max= 1.7 ± 0.7 μmol ferricyanide (mg protein)^?1 min^?1; K_m= 83 ± 20 μM) were measured. As ferricyanide is believed to accept electrons closer to the NADH binding site than cytochrome c, it was concluded that 83 ± 20 μM NADH represented a more accurate estimate of the binding affinity of the outer membrane dehydrogenase for NADH. The low K_m determined with NADH-cytochrome c activity may be due to a limitation in electron flow through the components of the outer membrane electron transport chain. The K_m for NADH of the externally-facing inner membrane NADH dehydrogenase of pea leaf (Pisum sativum L. cv. Massey Gem) mitochondria was 26.7 ± 4.3 μM when oxygen was the electron acceptor. At an NADH concentration at which the inner membrane dehydrogenase should predominate, the Ca²⁺ chelator, ethyleneglycol-(β-aminoethylether)-N,N,-tetraacetic acid (EGTA), inhibited the oxidation of NADH through to oxygen and to the ubiquinone-10 analogues, duroquinone and ubiquinone-1, but had no effect on the antimycin A-insensitive ferricyanide reduction. It is concluded that the site of action of Ca²⁺ involves the interaction of the enzyme with ubiquinone and not with NADH.     

Genetic Analysis of a Kindred With X-linked Mental Handicap and Retinitis Pigmentosa

A kindred is described in which X-linked nonspecific mental handicap segregates together with retinitis pigmentosa. Carrier females are mentally normal but may show signs of the X-linked retinitis pigmentosa carrier state and become symptomatic in their later years. Analysis of polymorphic DNA markers at nine loci on the short arm of the X chromosome shows that no crossing-over occurs between the disease and Xp11 markers DXS255, TIMP, DXS426, MAOA, and DXS228. The 90% confidence limits show that the locus is in the Xp21-q21 region. Haplotype analysis is consistent with the causal gene being located proximal to the Xp21 loci DXS538 and 5'-dystrophin on the short arm of the X chromosome. The posterior probability of linkage to the RP2 region of the X chromosome short arm (Xp11.4-p11.23) is .727, suggesting the possibility of a contiguous-gene-deletion syndrome. No cytogenetic abnormality has been identified.     

A biometric study of some hybridizing Crataegus populations in Denmark

Crataegus curvisepala Lindm., C. laevigata (Poiret) DC. and C. monogyna Jacq. (Rosaceae) form hybrid complexes in Denmark due to introgression. C. palmstruchii Lindm. seems to be variously introgressed individuals of C. laevigata. C. eremitagensis Raunk., C. raavadensis Raunk. and C. schumacheri Raunk. apparently belong to C. curvisepala x laevigata. The delimitation between C. curvisepala x laevigata and C. laevigata x monogyna is discussed.     

Mutation spectrum in gamma-irradiated shuttle vector replicated in ataxia-telangiectasia lymphoblasts.

Cells from ataxia-telangiectasia (AT) patients are hypersensitive to the lethal effects of ionizing radiation. To assess radiation mutagenesis in these cells, the SV40-based shuttle vector, pZ189, was used to analyze gamma-ray-induced mutations following the plasmid's replication in AT lymphoblasts. Progenies from the AT line GM2783 exposed to 50 Gy showed a mutation frequency of 7.6 x 10(-3), 63-fold over background; surviving plasmids were 3.4% of control. Both values were essentially the same as those of irradiated plasmids replicated in a normal lymphoblast line, GM606. In addition, pZ189 exposed to 25 Gy of gamma radiation and replicated in another normal lymphoblast line and in cells of two additional AT lymphoblast lines showed similar mutation frequencies and percentages of surviving plasmids. Qualitative comparison of plasmid mutations from AT and normal cells showed no significant differences, indicating that the damaged DNA was repaired with similar fidelity in AT and normal cells. These studies suggest that there is no correlation between the enhanced sensitivity of AT cells to killing by ionizing radiation and gamma-radiation-induced mutagenesis of plasmid DNA processed in these cells.     

Identification of the replication-associated protein binding domain within the intergenic region of tomato leaf curl geminivirus.

The geminiviral replication-associated protein (Rep) is the only viral protein required for viral DNA replication. Tomato leaf curl virus (TLCV) Rep was expressed in Escherichia coli as a histidine-tagged fusion protein and purified to homogeneity in non-denaturing form. The fusion protein was used in in vitro binding experiments to identify the Rep-binding elements within the origin of replication of TLCV. Electrophoretic mobility shift assays demonstrated that the Rep binds specifically to a 120 bp fragment within the TLCV intergenic region. Fine resolution of the binding regions within the 120 bp fragment, using DNase I footprinting, demonstrated two footprints covering the sequences GCAATTGGTGTCTCTCAA and TGAATCGGTGTCTGGGG containing a direct repeat of the motif GGTGTCT (underlined). Our results suggest that the repeated motif is involved in virus-specific Rep-binding, but may not constitute the entire binding element. This is the first demonstration of geminivirus sequence elements involved in Rep-binding by direct protein-DNA interaction assays.     

Genetic dissection of a TIR‐NB‐LRR locus from the wild North American grapevine species Muscadinia rotundifolia identifies paralogous genes conferring resistance to major fungal and oomycete pathogens in cultivated grapevine

The most economically important diseases of grapevine cultivation worldwide are caused by the fungal pathogen powdery mildew (Erysiphe necator syn. Uncinula necator) and the oomycete pathogen downy mildew (Plasmopara viticola). Currently, grapegrowers rely heavily on the use of agrochemicals to minimize the potentially devastating impact of these pathogens on grape yield and quality. The wild North American grapevine species Muscadinia rotundifolia was recognized as early as 1889 to be resistant to both powdery and downy mildew. We have now mapped resistance to these two mildew pathogens in M. rotundifolia to a single locus on chromosome 12 that contains a family of seven TIR‐NB‐LRR genes. We further demonstrate that two highly homologous (86% amino acid identity) members of this gene family confer strong resistance to these unrelated pathogens following genetic transformation into susceptible Vitis vinifera winegrape cultivars. These two genes, designated r esistance to P lasmopara v iticola (MrRPV1) are the first resistance genes to be cloned from a grapevine species. Both MrRUN1 and MrRPV1 were found to confer resistance to multiple powdery and downy mildew isolates from France, North America and Australia; however, a single powdery mildew isolate collected from the south‐eastern region of North America, to which M. rotundifolia is native, was capable of breaking MrRUN1‐mediated resistance. Comparisons of gene organization and coding sequences between M. rotundifolia and the cultivated grapevine V. vinifera at the MrRUN1/MrRPV1 locus revealed a high level of synteny, suggesting that the TIR‐NB‐LRR genes at this locus share a common ancestor.