Pigmentation changes in Brevibecteria induced by mutagenic treatment

Inducible pigmentation changes were observed in pigmented strains of Brevibacterium sp. M27 and B. flavum treated with N-methyl-N'-nitro-N-nitrosoguanidine. The highest frequency of induction was reached already at a survival of 30-40% with the maximal yield of 6-10%. As compared with the initial yellow colour, three new pigmentation types, viz. white, pink and orange, were observed. The yellow pigmented parent strains are most resistant to the lethal effects of UV radiation. By selecting pigmented mutants of all types on media containing antibiotics it was possible to obtain strains that were resistant either to tetracycline or to streptomycin. Auxotrophic pigmented mutants were also isolated. In multiple mutant strains of Brevibacterium sp. M27 a number of strainsexhibited a changed L-lysine production. In some strains the production was variable, whereasother strains did not produce L-lysine at all and stains with a limited production of other amino acids were also detected.     

Evaluation of the risk in autosomal dominant diseases

A lot of traps and difficulties complicate the estimation of a genetic risk in the autosomal dominant diseases. The authors recapitulate the notions of mutation, penetrance and variability and illustrate by some examples the part of each of them, isolated or associated together. The increasing of molecular biology allows to resolve some of these problems, but generate new dangers which are analysed and illustrated.     

Marfan disease

After reviewing the main features of the Marfan syndrome (musculoskeletal, ocular, cardiovascular, pulmonary abnormalities), its autosomal dominant inheritance with high penetrance but variable phenotype and presence of "soft" conditions preventing an easy diagnosis, the authors report their own data relevant to 73 probands: ratio of each clinical manifestation, state of 34% of familial cases and display of a paternal age effect in the sporadic cases. The pathogenic defect is unknown as like the location of the gene. The difficulties of the genetic counseling are then approached: unpredictability of the severity and of the prognosis in the unborn children of an affected patient, benefit of the echocardiography in the management of people at risk.     

Saprophytic production of Clavine alkaloids and activity of Hydroxymethylglutaryl-CoA reductase

In submerged Claviceps cultures the activity of hydroxymethylglutaryl-CoA reductase preceded the increase of alkaloid production and of sterol content. During the first alkaloid phase, cell mevalonate was involved in the biosynthesis of both alkaloids and steroids. In the second production phase, it was predominantly used for alkaloid synthesis. Hydroxymethylglutaryl-CoA reductase appears to be a suitable target for physiological manipulation to increase clavine alkaloid yields.     

On the Divergence of Alleles in Nested Subsamples from Finite Populations

Within-population variation at the DNA level will rarely be studied by sequencing of loci of randomly chosen individuals. Instead, individuals will usually be chosen for sequencing based on some knowledge of their genotype. Data collected in this way require new sampling theory. Motivated by these observations, we have examined the sampling properties of a finite population model with two mutation processes and with no selection or recombination. One mutation process generates new alleles according to an infinite-alleles model, and the other generates polymorphisms at sites according to an infinite-sites model. A sample of n genes is considered. The stationary distribution of the number of segregating sites in a subsample from one of the allelic classes in the sample conditional on the allelic configuration of the sample is studied. A recursive scheme is developed to compute the moments of this distribution, and it is shown that the distribution is functionally independent of the number of additional alleles in the sample and their respective frequencies in the sample. For the case in which the sample contains only two alleles, the distribution of the number of segregating sites in a subsample containing both alleles conditional on the sample frequencies of the alleles is studied. The results are applied to the analysis of DNA sequences of two alleles found at the Adh locus of Drosophila melanogaster. No significant departure from the neutral model is detected.     

High CO(2) Requiring Mutant of Anacystis nidulans R(2)

Some physiological characteristics of a mutant (E₁) of Anacystis nidulans R₂, incapable of growing at air level of CO₂, are described. E₁ is capable of accumulating inorganic carbon (C_i) internally as efficiently as the wild type (R₂). The apparent photosynthetic affinity for C_i in E₁, however, is some 1000 times lower than that of R₂. The kinetic parameters of ribulose 1,5-bisphosphate carboxylase/oxygenase from E₁ are similar to those observed in R₂. The mutant appears to be defective in its ability to utilize the intracellular C_i pool for photosynthesis and depends on extracellular supply of Ci in the form of CO₂. The very high apparent photosynthetic K_m (CO₂) of the mutant indicate a large diffusion resistance for CO₂. Data obtained here are used to calculate the permeability coefficient for CO₂ between the bulk medium and the carboxylation site of cyanobacteria.     

Carbodiimide inactivation of Na,K-ATPase, via intramolecular cross-link formation, is due to inhibition of phosphorylation

We have recently shown that inactivation of renal Na,K-ATPase by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide occurs via an intramolecular cross-link formed between an activated carboxyl group and an endogenous nucleophile (Pedemonte, C.H., and Kaplan, J.H. (1986) J. Biol. Chem. 261, 3632-3639). The modified enzyme shows the same level of Rb+ binding as untreated enzyme: 3.16 and 2.93 ATP-sensitive mumol of Rb+ binding/mumol of phosphoenzyme, respectively. Thus, the Rb+ binding site and the transition accomplished by low affinity nucleotide binding which accelerates de-occlusion are not greatly affected by the carbodiimide inactivation. 1 mM K+ reduces the ADP binding to the high affinity nucleotide binding site to the same extent in normal and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-treated enzyme and Na+ counteracts this effect. Thus, the competition between Na+ and K+ ions for binding to the free enzyme are also largely unaltered by the modification. Phosphorylation from ATP (microM) in the presence of Na+ and Mg2+ ions and from inorganic phosphate in the presence of Mg2+ ions (in the absence or presence of ouabain) is greatly inhibited (85%) following carbodiimide treatment. The extent of inhibition of phosphorylation quantitatively correlates with the residual Na,K-ATPase activity (15%). Consequently, the rate of inactivation by carbodiimide is reduced when a greater proportion of the enzyme is in the phosphorylated form. Fluoroscein isothiocyanate, which inhibits the Na,K-ATPase by covalently modifying a lysine residue close to the high affinity binding site for ATP in the alpha-subunit does not bind to the carbodiimide-inactivated enzyme. Since high affinity nucleotide binding is only partially inhibited by the modification produced by the carbodiimide this suggests that the lysine residue to which fluoroscein isothiocyanate binds is not specifically required for competent nucleotide binding.     

Superior resolution of gamma-crystallins from microdissected eye lens by cation-exchange high-performance liquid chromatography

A novel procedure is presented for the rapid quantitative analysis of eye lens gamma-crystallins and beta s-crystallin by ion-exchange high-performance liquid chromatography on Synchropak CM300. At least six different gamma-crystallin gene products can be resolved from the soluble fraction of calf lens extract. This method is applicable to the analysis of microsections from individual lenses, and can be used to rapidly characterize spatial variations in gamma-crystallin composition which occur with aging and cataractogenesis.     

Isolation and reconstitution of the n-butylmalonate-sensitive dicarboxylate transporter from rat liver mitochondria

The mitochondrial dicarboxylate carrier has been substantially purified from rat liver mitoplasts by extraction with Triton X-114 in the presence of cardiolipin followed by chromatography on hydroxylapatite. Upon incorporation of the hydroxylapatite eluate into phospholipid vesicles, an n-butylmalonate-sensitive malonate/malate exchange has been demonstrated. This exchange activity is enhanced 226-fold relative to the starting material (i.e. detergent-extracted mitoplasts). Silver-stained sodium dodecyl sulfate-polyacrylamide gradient gels verify the high purity of this fraction relative to the starting material. Nonetheless, the banding pattern indicates that several protein species are still present. As isolated, the dicarboxylate transporter is rather unstable but can be stabilized either by the addition of 10% ethylene glycol and subsequent storage at -20 degrees C or by incorporation into phospholipid vesicles in the presence of malate followed by freezing in liquid nitrogen. Such proteoliposomes catalyze a [14C]malonate uptake which is characterized by a first order rate constant of 1.02 min-1 and a t 1/2 of 41 s. This uptake can be inhibited by dicarboxylates (e.g. succinate, malate, unlabeled malonate) but not by either alpha-ketoglutarate or by tricarboxylates (e.g. citrate, threo-Ds-isocitrate). Furthermore, the reconstituted malonate transport is dependent on internal malate and can be inhibited by n-butylmalonate, mersalyl, p-chloromercuribenzoate, and Pi, but not by N-ethylmaleimide. It is concluded that this highly purified fraction contains a reconstitutively active dicarboxylate transporter which, based on its substrate specificity and inhibitor sensitivity, appears to be identical to the native dicarboxylate transport system found in intact rat liver mitochondria.     

Demonstration of separate phosphotyrosyl- and phosphoseryl- histone phosphatase activities in the plasma membranes of a human astrocytoma

A plasma membrane preparation from a human astrocytoma contained p-nitrophenyl phosphate (pNPP), phosphotyrosyl histone, and phosphoseryl histone hydrolysis activities. The pNPPase and phosphotyrosyl histone phosphatase activities were inhibited by vanadate, whereas the phosphoseryl histone phosphatase activity was not; the latter activity was inhibited by pyrophosphate and nucleoside di- and triphosphates. When the membranes were solubilized by Triton X-100 and the solubilized proteins were subjected to column chromatography on DEAE-Sephadex, Sepharose 6B-C1, and wheat germ agglutinin-Sepharose 4B columns, the pNPPase activity from the phosphoseryl histone phosphatase activity. The results from column chromatography also indicated that there may be multiple phosphotyrosyl and phosphoseryl protein phosphatases in the plasma membranes.