Sequence and expression of the kettin gene in Drosophila melanogaster and Caenorhabditis elegans

Kettin is a large modular protein associated with thin filaments in the Z-disc region of insect muscles. The sequence of a 21.3 kb contig of the Drosophila gene has been determined. The corresponding protein sequence has 35 immunoglobulin-like (Ig) domains which are separated by shorter linker sequences, except near the N and C termini of the molecule where linker sequences are short or missing. This confirms a model in which each Ig domain binds to an actin protomer. The Drosophila kettin gene is at 62C 1-3 on the third chromosome. Two P-element insertions, l(3)j1D7 and l(3)rL182 are in the kettin gene, and complementation tests showed that existing l(3)dre8 mutations are in the same gene. The RNA was detected in wild-type Drosophila embryos at stage 11, first in the gut invagination region of the mesoderm, and by stage 13 in both visceral and somatic mesoderm. Somatic mesoderm expression became segmental at stage 13. RNA expression was greatly reduced in embryos of P-element homozygotes but normal in heterozygotes. The structure of the flight muscle in all the heterozygous mutants was normal, including the myofibril-cuticle connections, and they were able to fly. Kettin sequence homologous to the Drosophila protein, was identified in the Caenorhabditis elegans genome database. The RNA was detected in pharyngeal, body wall and anal depressor muscles of larvae and adult worms, as well as in the male gonad. Antibody to insect kettin labelled the pharyngeal, body wall, anal depressor and proximal gonadal muscles in adult worms. Body wall muscles were labelled in an obliquely striated pattern consistent with the Z-disc localisation in insect muscle. The relationship of kettin to D-titin, which has been assigned to the same chromosomal locus in Drosophila, is discussed.     

Conservation,diversification and expansion of C2H2 zinc finger proteins in the Arabidopsis thaliana genome

Background  

The classical C2H2 zinc finger domain is involved in a wide range of functions and can bind to DNA, RNA and proteins. The comparison of zinc finger proteins in several eukaryotes has shown that there is a lot of lineage specific diversification and expansion. Although the number of characterized plant proteins that carry the classical C2H2 zinc finger motifs is growing, a systematic classification and analysis of a plant genome zinc finger gene set is lacking.     

Polyphosphate/ATP-dependent NAD kinase of Corynebacterium glutamicum: biochemical properties and impact of ppnK overexpression on lysine production

Nicotinamide adenine dinucleotide phosphate (NADP) is synthesized by phosphorylation of either oxidized or reduced nicotinamide adenine dinucleotide (NAD/NADH). Here, the cg1601/ppnK gene product from Corynebacterium glutamicum genome was purified from recombinant Escherichia coli and enzymatic characterization revealed its activity as a polyphosphate (PolyP)/ATP-dependent NAD kinase (PPNK). PPNK from C. glutamicum was shown to be active as homotetramer accepting PolyP, ATP, and even ADP for phosphorylation of NAD. The catalytic efficiency with ATP as phosphate donor for phosphorylation of NAD was higher than with PolyP. With respect to the chain length of PolyP, PPNK was active with short-chain PolyPs. PPNK activity was independent of bivalent cations when using ATP, but was enhanced by manganese and in particular by magnesium ions. When using PolyP, PPNK required bivalent cations, preferably manganese ions, for activity. PPNK was inhibited by NADP and NADH at concentrations below millimolar. Overexpression of ppnK in C. glutamicum wild type slightly reduced growth and ppnK overexpression in the lysine producing strain DM1729 resulted in a lysine product yield on glucose of 0.136 ± 0.006 mol lysine (mol glucose)⁻¹, which was 12% higher than that of the empty vector control strain.     

Nutritional characteristics of two marine thioplocas determined by autoradiography

Autoradiographic evidence showed strong incorporation by two marine thioplocas of ¹⁴C from acetate, amino acids, carbonate, glucose and glycinc and of ³H from thymidine. Methane, methanol and glutamate were not incorporated. Since the data suggested that sulfide was needed for strong incorporation, a mixotrophic nutrition was indicated.     

A mitochondrial superoxide signal triggers increased longevity in Caenorhabditis elegans

The nuo-6 and isp-1 genes of C. elegans encode, respectively, subunits of complex I and III of the mitochondrial respiratory chain. Partial loss-of-function mutations in these genes decrease electron transport and greatly increase the longevity of C. elegans by a mechanism that is distinct from that induced by reducing their level of expression by RNAi. Electron transport is a major source of the superoxide anion (O^{⋅ –}), which in turn generates several types of toxic reactive oxygen species (ROS), and aging is accompanied by increased oxidative stress, which is an imbalance between the generation and detoxification of ROS. These observations have suggested that the longevity of such mitochondrial mutants might result from a reduction in ROS generation, which would be consistent with the mitochondrial oxidative stress theory of aging. It is difficult to measure ROS directly in living animals, and this has held back progress in determining their function in aging. Here we have adapted a technique of flow cytometry to directly measure ROS levels in isolated mitochondria to show that the generation of superoxide is elevated in the nuo-6 and isp-1 mitochondrial mutants, although overall ROS levels are not, and oxidative stress is low. Furthermore, we show that this elevation is necessary and sufficient to increase longevity, as it is abolished by the antioxidants NAC and vitamin C, and phenocopied by mild treatment with the prooxidant paraquat. Furthermore, the absence of effect of NAC and the additivity of the effect of paraquat on a variety of long- and short-lived mutants suggest that the pathway triggered by mitochondrial superoxide is distinct from previously studied mechanisms, including insulin signaling, dietary restriction, ubiquinone deficiency, the hypoxic response, and hormesis. These findings are not consistent with the mitochondrial oxidative stress theory of aging. Instead they show that increased superoxide generation acts as a signal in young mutant animals to trigger changes of gene expression that prevent or attenuate the effects of subsequent aging. We propose that superoxide is generated as a protective signal in response to molecular damage sustained during wild-type aging as well. This model provides a new explanation for the well-documented correlation between ROS and the aged phenotype as a gradual increase of molecular damage during aging would trigger a gradually stronger ROS response.