A putative GDP–GTP exchange factor is required for development of the excretory cell in Caenorhabditis elegans

The Caenorhabditis elegans excretory cell extends tubular processes, called canals, along the basolateral surface of the epidermis. Mutations in the exc-5 gene cause tubulocystic defects in this canal. Ultrastructural analysis suggests that exc-5 is required for the proper placement of cytoskeletal elements at the apical epithelial surface. exc-5 encodes a protein homologous to guanine nucleotide exchange factors and contains motif architecture similar to that of FGD1, which is responsible for faciogenital dysplasia. exc-5 interacts genetically with mig-2, which encodes Rho GTPase. These results suggest that EXC-5 controls the structural organization of the excretory canal by regulating Rho family GTPase activities.     

Sequential polymerization of flagellin A and flagellin B into Caulobacter flagella

The mode of polymerization of two species of flagellins, flagellin A and flagellin B, in polar flagella of Caulobacter crescentus was examined. By immunological staining we found that 1 to 1.2 μm of the portion of the flagellar filament proximal to the cell was composed of flagellin B, whereas about 5 μm of the distal portion was composed of flagellin A. This result, together with the previous observation that a flagellin B-less mutant cannot form normal flagella but instead forms stubs in spite of their high level of flagellin A synthesis, indicates that flagellin B is very important for the formation of complete flagella and/or for the initiation of filament formation from the hook.     

RNA-Induced Interference with Animal Virus Infection

Some RNAs, including both single- and double-stranded RNAs, when incubated with chick embryo cell culture induce cellular resistance against viruses. Evidence was now obtained indicating that the induction of cellular resistance by RNA depends on the cellular metabolic activity, especially on the synthesis of cellular RNA and protein. Thus, inhibitors of RNA and protein synthesis, actinomycin D and cycloheximide, were found to inhibit the development of an antiviral state when added before, or during the relatively early period of, incubation of the cells with RNA. In the course of induction of cellular resistance, three stages may be distinguished, the priming stage, the developing stage, and the established resistant stage.     

Stopped-flow investigation of the reaction between vitamin E radical and vitamin C in solution

Kinetic study of the reaction between vitamin E radical and vitamin C has been performed. The rates of reaction of vitamin C (ascorbic acid 1, 6-0-stearyl ascorbic acid 2, and 2,6-O-dipalmitoyl ascorbic acid 3) with vitamin E radical (5,7-diisopropyl-tocopheroxyl) in benzene-ethanol (2:1, v/v) solution have been determined spectrophotometrically, using stopped-flow technique. The second-order rate constants obtained are 549 +/- 30 M-1s-1 for 1, 626 +/- 53 M-1s-1 for 2, and 4.84 +/- 1.41 M-1s-1 for 3 at 25.0 degrees C. The result shows that the ascorbic acid ester 2 having a long-alkyl-chain at 6-position is 1.14 times as reactive as the ascorbic acid 1, whereas the ascorbic acid ester 3 substituted at 2-position is only 0.01 times as reactive as the ascorbic acid 1.     

Restriction map and alpha-amylase activity variation among Drosophila mutation accumulation lines

The specific activities of alpha-amylase were measured for two sets of mutation accumulation lines, each set having originated from a different lethal-carrying second chromosome and SM1(Cy) chromosome and having been maintained by a balanced lethal system for about 300 generations. Significant variation was found to have accumulated among lines of both sets. Because of dysgenic crosses in the early generations of mutation accumulation, insertions or deletions of transposable elements in the Amy gene region were suspected of being the cause of this variation. In order to test this possibility, the structural changes in the 14 kb region of these chromosomes that includes the structural genes for alpha-amylase were investigated by restriction map analysis. We found that most part of the activity variation is due to replacements of a chromosomal region of SM1(Cy), including the structural genes for alpha-amylase, by the corresponding regions of the lethal chromosomes. One line also contained an insertion in this region but this line has an intermediate activity value. Thus, insertions of transposable elements into the Amy gene region were not found to be responsible for the new variation observed in alpha-amylase activity. If we remove those lines with structural changes from the analysis, the genetic variance of alpha-amylase specific activity among lines becomes non-significant in both sets of chromosomes.     

Construction and expression of human aldolase A and B expression plasmids in Escherichia coli host

E. coli expression plasmids for human aldolases A and B (EC 4.1.2.13) have been constructed from the pIN-III expression vector and their cDNAs, and expressed in E. coli strain JM83. Enzymatically active forms of human aldolase have been generated in the cells when transfected with either pHAA47, a human aldolase A expression plasmid, or pHAB 141, a human aldolase B expression plasmid. These enzymes are indistinguishable from authentic enzymes with respect to molecular size, amino acid sequences at the NH2- and COOH-terminal regions, the Km for substrate, fructose 1,6-bisphosphate and the activity ratio of fructose 1,6-bisphosphate/fructose 1-phosphate (FDP/F1P), although net electric charge and the Km for FDP of synthetic aldolase B differed from those for a previously reported human liver aldolase B. In addition, both the expressed aldolases A and B complement the temperature-sensitive phenotype of the aldolase mutant of E. coli h8. These data argue that the expressed aldolases are structurally and functionally similar to the authentic human aldolases, and would provide a system for analysis of the structure-function relationship of human aldolases A and B.     

Effects of auxin and gibberellin on conidial germination and elongation of young hyphae in a cyclic 3′:5′ adenosine monophosphate-dependent protein kinase mutant of Neurospora crassa

The possibility that plant growth regulators may relate to a cyclic 3:5 adenosine monophosphate (cAMP)-dependent protein kinase through the control of cAMP level in the conidial germination process of Neurospora crassa was examined using a cAPM-dependent protein kinase mutant (cpk mutant) which is thought to be cAMP-independent because of defect in the regulatory subunit of cAMP-dependent protein kinase. IAA, 2,4-D and GA₃ promoted conidial germination and elongation of young hyphae in the mutant as well as in the wild-type. The result suggests that the effects of auxin and gibberellin on germination and hyphal elongation are not mediated by cAMP.     

The genotoxicity of a variety of aniline derivatives in a DNA repair test with primary cultured rat hepatocytes

The genotoxicity of a variety of aniline derivatives was examined by a DNA repair test with rat hepatocytes. Out of 37 aniline derivatives, 6 chemicals, i.e., 2,4,6-trimethylaniline (mesidine), 2,4-xylidine, 3,5-diaminobenzoic acid, 3,4-diaminochlorobenzene, 2-chloro-4-methylaniline and 4-chloro-N-methylaniline, elicited positive DNA repair responses. The results are in agreement with the bacterial mutagenicities with or without norharman of these compounds. Positive compounds of unknown carcinogenicity in the present assay, i.e., 3,5-diaminobenzoic acid, 2-chloro-4-methylaniline and 4-chloro-N-methylaniline are suspected of being potentially carcinogenic.     

Structure of the Bombyx mori fibroin light-chain-encoding gene: upstream sequence elements common to the light and heavy chain.

Two overlapping genomic clones containing the fibroin light-chain (Fib-L)-encoding gene (Fib-L) were obtained from the cosmid library of the silkworm, Bombyx mori J-139, by hybridization with the Fib-L cDNA clone. Sequencing of the 14.6-kb region revealed that Fib-L was 13472 bp long containing seven exons, and that the gene contained a large first intron which occupied about 60% of the gene. Comparison of restriction patterns of the J-139 Fib-L with those of eight other B. mori breeds producing normal-level fibroin demonstrated that considerable restriction-fragment length polymorphisms were present in regions containing the first intron and the 3′-flanking sequence. However, sizes of the Fib-L mRNA and the Fib-L polypeptide were very similar among the nine breeds tested, suggesting that the exon sequences and the splice signals were all well conserved. 5′-Flanking regions of Fib-L and the fibroin heavy-chain (Fib-H)-encoding gene (Fib-H) compared in this study contained three 18-30-bp sequences of high similarity and many 8-10-bp common elements, six of which coincided with the binding sites of homeodomain proteins. Gel retardation assays with the nuclear extracts of the posterior and middle silk glands suggested that protein factors present in the posterior silk-gland nuclei could bind to a set of those common upstream elements.