Chloroplast and cytoplasmic low-molecular-weight ribonucleic acid components of the leaf of Vicia faba L

1. A method for the extraction of plant nucleic acids and their separation on methylated-serum-albumin-kieselguhr columns is described. It is demonstrated that the characteristics of the elution profiles of material from the same source are consistently reproducible. 2. Major dissimilarities were found in the elution profiles of nucleic acids from root and from leaves of Vicia faba L. These dissimilarities were confirmed by polyacrylamide-gel electrophoresis. 3. Four distinct types of low-molecular-weight RNA were demonstrated to be present in leaves, clearly distinguished by their behaviour when chromatographed on methylated-serum-albumin-kieselguhr columns. (a) Both cytoplasmic and chloroplast ribosomes contained a low-molecular-weight RNA, and these components were distinct from each other. (b) The chloroplast possessed a unique ;soluble' RNA (i.e. RNA that is not precipitated by centrifugal forces that sediment ribosomes) which was not present in the rest of the cell. (c) A soluble component, probably transfer RNA, was found in both the chloroplasts and in the cytoplasm. 4. The components distinguishable by methylated-serum-albumin-kieselguhr column chromatography could not be distinguished by sucrose-density-gradient centrifugation.     

Radiation Survival of Food Pathogens in Complex Media

When 15 bacterial species representing genera associated with food-borne diseases were irradiated individually, all except Escherichia coli and Streptococcus faecalis showed typical linear dose-survival curves in Hartsell's broth. The minimal lethal dose (MLD) for the organisms tested ranged from 3.0 x 10(5) to 6.0 x 10(5) rad. Salmonella paratyphi B, S. wichita, S. typhi, E. coli, and S. faecalis were found to be the least sensitive to radiation. In commercially canned crabmeat the survival curves of S. typhi, S. paratyphi B, and S. wichita exhibited to varying degrees an initial linear death decline with increasing radiation doses, followed by a distinct tailing effect caused by survival of low numbers at the higher doses. The above species of Salmonella were further individually subjected to gamma-radiation in various dilutions of crabmeat. The "tailing effect" gradually disappeared, with the dose-survival curve tending to become linear as the concentration of the crabmeat decreased.     

Cloned mouse melanocyte lines carrying the germline mutations albino and brown: complementation in culture

We have established two new immortal lines of mouse melanocytes, melan-b and melan-c, from mice homozygous for the brown (b) and albino (c) mutations respectively. Both lines were derived through differentiation in vitro of embryonic epidermal melanoblasts. The brown melanocytes are visibly brown by light microscopy, and centrifuged cell suspensions form brown pellets. The albino melanocytes form white pellets and contain abundant unpigmented premelanosomes as shown by transmission electron microscopy. Like normal, non-immortal melanocytes and like the immortal black melanocyte line melan-a, both lines show little or no growth in a standard, serum-supplemented medium, but proliferate well in the presence of 12-o-tetradecanoyl phorbol-13-acetate (TPA). Sustained growth of the albino cells also requires either keratinocyte feeder cells or 2-mercaptoethanol (2-ME). The modal chromosome numbers are 39 for melan-b and 40 (diploid) for melan-c. Neither line is tumorigenic in nude mice. Heterokaryons between the two lines can be constructed and form wild-type, black pigment. Melanocyte lines can now be reproducibly generated from mice of different strains, and provide tools for molecular studies of germline coat-colour mutations. These two lines provide elegant means to study the developmentally controlled expression of the two complementary genes, B and C, with black melanin pigment as a readily detectable natural marker.     

Glycolipids and transmembrane signaling: antibodies to galactocerebroside cause an influx of calcium in oligodendrocytes

This is the first study to provide evidence that one function for the surface glycolipid galactocerebroside (GalC) is participation in the opening of Ca2+ channels in oligodendroglia in culture. This glycolipid is a unique differentiation marker for myelin-producing cells; antibodies to GalC have been shown to markedly alter oligodendroglial morphology via disruption of microtubules (Dyer, C. A., and J. A. Benjamins. 1988. J. Neurosci. 8:4307-4318). This study demonstrates that extracellular EGTA blocks anti-GalC-induced disassembly of microtubules in oligodendroglial membrane sheets, demonstrating that an influx of extracellular Ca2+ mediates the cytoskeletal changes. The Ca2+ influx was examined directly by loading oligodendroglia with the fluorescent dye Indo-1 in defined medium, and measuring changes in Ca2+ in individual cells with a laser cytometer. Upon addition of anti-GalC IgG, a marked sustained increase in intracellular Ca2+ occurred in 80% of the oligodendroglia observed. EGTA blocked the increase, indicating the increase is due to an influx of extracellular Ca2+, and not due to release from intracellular stores. The effect is specific, since Ca2+ levels remain normal in oligodendroglia treated with nonimmune IgG; astrocytes do not respond to the anti-GalC. The Ca2+ response in oligodendrocytes is dependent on concentration of antibody and GalC on the oligodendroglial membrane surface. The Ca2+ influx is not mediated by voltage-sensitive Ca2+ channels: it is not blocked by cadmium, and depolarization with K+ does not mimic the response. The kinetics of the response suggest that second messenger-mediated opening of Ca2+ channels is involved.     

Wheat phosphoglycerate kinase: evidence for recombination between the genes for the chloroplastic and cytosolic enzymes.

We have isolated and sequenced cDNA clones containing the entire coding region of both the chloroplast and cytosolic versions of phosphoglycerate kinase from wheat. Comparison of these sequences reveals a higher than expected level of similarity between the nucleic acids and encoded proteins. Analysis of this data in relation to that for phosphoglycerate kinase sequences of mammals, prokaryotes and yeasts suggests that the wheat genes have recombined. This has resulted in the chloroplast and cytosolic kinases being more similar to each other than would be expected if the chloroplast enzyme had evolved directly from that of a prokaryotic progenitor.