Linkage analysis of the myosin heavy chain gene in Dictyostelium discoideum using a mutation generated by homologous recombination

Summary A mutation (mhcA1 in strain HMM) created by insertional gene inactivation was used to map the Dictyostelium discoideum myosin heavy chain gene (mhcA) to linkage group IV. Three phenotypic traits associated with this mutation (slow colony growth, inability of the mutant to develop past aggregation, and the presence of five to ten integrated vector copies) cosegregated as expected for the consequences of a single insertional event. This linkage was confirmed using a restriction fragment length polymorphism. The mhcA1 mutation was recessive to wild type and was nonallelic with mutations at the following loci on linkage group IV: aggJ, aggL, couH, minA, phgB and tsgB. This work demonstrates the ability to apply standard techniques developed for D. discoideum parasexual genetic analyses to mutants generated by transformation, which is of particular relevance to analysis of genes for which no classical mutations or restriction fragment length polymorphisms are available.     

Characterization of the biomass of the stems of sweet sorghum

In this study the amount of glucose, sucrose and fructose was determined in the water soluble fraction while cellulose, hemicellulose, pectin and lignin contents were determined in the alcohol insoluble fraction after hydrolysis. Stalks of sweet sorghum (Sorghum vulgare L., var. saccharatum) cv. Vespa, Soave, Roce and MN 1500 at the physiological ripeness stage were used. The results of the analysis of variance with the least significant difference method (LSD, = 0.05) show that cv. Vespa and Roce have a significantly higher total amount of glucose, fructose and sucrose and at the same time, a lower cellulose, pectin, hemicellulose and lignin content then cv. Soave and cv. MN 1500.     

Ascorbic acid as a factor controlling "in vivo" its biosynthetic pathway

The capacity of ascorbic acid biosynthesis in potato tuber tissue is closely correlated with the ascorbic acid content of the cells: the lower the endogenous content of ascorbic acid, the greater its biosynthesis. At the highest level of ascorbic acid found in the cells, the biosynthetic capacity is virtually zero. In these conditions, adding glucose (the first precursor of ascorbic acid) has no effect whatsoever, whereas adding galactono-gamma-lactone (the last precursor) induces a high rate of ascorbic acid synthesis. It is suggested that AA biosynthesis is subject to a regulatory mechanism "in vivo" which controls an initial step in the biosynthetic pathway. The last step in this pathway, catalyzed by galactone oxidase, is never blocked and, moreover, its activity is greater than that of the preceding steps.     

Reaction of Woodward''s reagent K with D-xylose isomerases. Modification of an active site carboxylate residue.

D-Xylose isomerases from Streptomyces violaceoruber, Streptomyces sp., Lactobacillus xylosus, Lactobacillus brevis and Bacillus coagulans were rapidly inactivated by Woodward's reagent K. Second-order rate constants in the absence of ligands, at pH 6.0 and 25 degrees C, were 41, 36, 22, 95 and 26 M-1.min-1 respectively. Spectral analysis at 340 nm revealed that inactivation was correlated with modification of five, six, two, three and six carboxylate residues per monomer respectively. In the presence of protecting ligands, modification of one carboxylate group was prevented. The results support the idea of an active site glutamate or aspartate group that may contribute to the catalytic activity of all these D-xylose isomerases.     

Unexpected influence of ionic strength on branched-pathway interactions between beta-lactamases and beta-halogenopenicillanates.

Ionic strength strongly influenced the turnover/inactivation ratio in the interaction between beta-halogenopenicillanates and some class A beta-lactamases. This suggested the stabilization of a highly charged intermediate by solvation. Those data could be interpreted on the basis of a reaction pathway where an episulphonium ion was transiently formed. The various mechanisms proposed for explaining the formation of the dihydrothiazine chromophore are discussed.     

Larval and adult morphology ofCamallanus anabantis Pearse, 1933 andC. kulasirii (Yeh, 1960) (Nematoda: Camallanidae) from freshwater fishes,with notes on the validity of some related forms

Larvae and adults ofCamallanus anabantis andC. kulasirii, recovered from the West Bengali freshwater fishes,Anabas testudineus andOphicephalus punctatus, respectively, are described on the basis of detailed morphological studies under the light microscope. Larval forms collected fromA. testudineus are deemed to be of the third and fourth stages when compared with those from experimental studies of the life cycle ofC. anabantis. Moreover, the present fourth stage female larvae are similar to the females ofC. pearsei, both morphologically and metrically.C. pearsei is, therefore, believed to represent the fourth stage female larvae ofC. anabantis. Similarly, adult males and females recovered fromO. punctatus closely resembleC. kulasirii andC. fernandoi, respectively. The larval forms from this host are fourth stage and can be distinguished as males and females, but both possess a buccal capsule bearing beaded longitudinal ridges similar to that of adult males. The late fourth stage (just prior to the final moult) female larva is, however, found to possess a buccal capsule transitional between that of the adult male and female and also betweenC. kulasirii andC. fernandoi. C. fernandoi is, therefore, presumed to represent the females ofC. kulasirii. However,C. pearsei andC. fernandoi are regarded, for the present, asspecies inquirendae.     

Mitochondrial genome in animal cells. Structure, organization, and evolution

In the past decade, the development of new DNA, RNA, and protein technologies has greatly incremented the knowledge about the organization and expression of mitochondrial DNA. The complete base sequence of mitochondrial DNA of several animals is known and many data are rapidly accumulating on the mitochondrial genomes of other systems. Here we discuss the results so far obtained that disclosed unexpected features of mitochondrial genetics. Furthermore, mitochondrial DNA has become established as a powerful tool for evolutionary studies in animals. Evidences are presented demonstrating that the evolution of mitochondrial DNA has proceeded in different ways in the various taxonomic groups. Data on heteroplasmic animals, which demonstrate the rapid evolution of mitochondrial DNA, are also presented.     

Induction of NADP-dependent malic dehydrogenase activity in brain of singi fish, heteropneustes fossilis (bloch), by 3,5,3′-triiodothyronine

For elucidation of thyroid hormone-induced responsiveness of fish brain, various doses (0.012, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2 and 4 μg/g) of triiodothyronine (T₃) were injected in Singi fish, Heteropneustes fossilis (Bloch), for 3 consecutive days and the changes in cytosolic NADP-dependent malic enzyme (ME, EC 1.1.1.40) activity in whole brain tissue were determined. Compared to the control, the ME activity increased with lower doses (0.012, 0.025 and 0.05 μg/g) and decreased with higher doses (1, 2 and 4 μg/g) of T₃, showing a biphasic nature of thyroid hormone action. The enzyme activity remained unaltered with 0.1, 0.25 and 0.5 μg of T₃/g in comparison to the control. Immersion of the fishes in cycloheximide-containing medium (0.5 mg/l) inhibited the T₃ (0.025 μg/g)-induced rise in ME activity. On the other hand, the NAD-dependent cytosolic malate dehydrogenase (EC 1.1.1.37) activity and the total protein content of brain cytosol remained unaltered with all doses of T₃ used. The thyroid hormone specificity of cytosolic NADP-dependent malic enzyme in fish brain is thus documented.     

Gene replacement in Dictyostelium: generation of myosin null mutants.

The eukaryotic slime mold Dictyostelium discoideum has a single conventional myosin heavy chain gene (mhcA). The elimination of the mhcA gene was achieved by homologous recombination. Two gene replacement plasmids were constructed, each carrying the G418 resistance gene as a selective marker and flanked by either 0.7 kb of 5' coding sequence and 0.9 kb of 3' coding sequence or 1.5 kb of 5' flanking sequence and 1.1 kb of 3' flanking sequence. Myosin null mutants (mhcA- cells) were obtained after transformation with either of these plasmids. The mhcA- cells are genetically stable and are capable of a variety of motile processes. Our results provide genetic proof that in Dictyostelium the conventional myosin gene is required for growth in suspension, normal cell division and sporogenesis, and illustrate how gene targeting can be used as a tool in Dictyostelium.     

Conformational analysis of PKI(5-22)amide, the active inhibitory fragment of the inhibitor protein of the cyclic AMP-dependent protein kinase.

Fourier-transform i.r. spectroscopy, 1H-n.m.r. spectroscopy and X-ray scattering were used to study the conformation and shape of the peptide PKI(5-22)amide, which contains the active site of the inhibitor protein of the cyclic AMP-dependent protein kinase [Cheng, Van Pattern, Smith & Walsh (1985) Biochem. J. 231, 655-661]. The X-ray-scattering solution studies show that the peptide has a compact structure with Rg 0.9 nm (9.0 A) and a linear maximum dimension of 2.5 nm (25A). Compatible with this, Fourier-transform i.r. and n.m.r. determinations indicate that the peptide contains approx. 26% alpha-helix located in the N-terminal one-third of the molecule. This region contains the phenylalanine residue that is one essential recognition determinant for high-affinity binding to the protein kinase catalytic site.