Ability of a T-antigen transport-defective mutant of simian virus 40 to immortalize primary cells and to complement polyomavirus middle T in tumorigenesis.

The oncogenic potential of polyomavirus in newborn rats could not be expressed by a genome encoding only the middle T antigen but required the presence of one of the other two viral early genes, small T or large T. The tumorigenicity defect could also be complemented by other viral or cellular genes that are known to be implicated in immortalization and establishment functions. The simian virus 40(cT)-3 mutant (R. E. Lanford and J. S. Butel, Cell 37:801-813, 1984), which fails to localize to the nucleus, has the capacity to complement polyomavirus middle T in tumorigenesis and to immortalize primary rat embryo fibroblasts when it was cotransfected in the presence of pSV2-neo. Our data suggested that under the conditions of DNA-mediated tumor induction and cotransfection with a dominant selection marker, the cellular alterations achieved by nonnuclear oncogenes such as polyomavirus small T and simian virus 40(cT)-3 were sufficient to complement polyomavirus middle T in transformation and tumorigenesis.     

Protection against yellow fever in monkeys by immunization with yellow fever virus nonstructural protein NS1.

Immunization of monkeys with yellow fever virus-specified nonstructural protein NS1 resulted in protection against fatal hepatitis as well as marked reduction in the magnitude of viremia after subcutaneous challenge with yellow fever virus. The results may be relevant to the design of possible subunit or recombinant flavivirus vaccines.     

Lack of UV-induced respiration shutoff in a recF strain of Escherichia coli: temperature conditional suppression at 30 degrees C by the sfrA mutation

A mutation in the recF gene of Escherichia coli results in a radiation-sensitive strain. The RecF pathway and the RecBC pathway account for nearly all of the conjugative recombination occuring in E. coli. recBC cells are radiation-sensitive and carry only out a small amount of recombination but these deficiencies are suppressed by an sbcB as recombination is shunted to the RecF pathway. A recBC sbcB recF strain is very radiation-sensitive and is devoid of recombination ability. These deficiencies are suppressed by the srfA mutation; srfA is a recA allele. UV-induced respiration shutoff is a recA⁺, lexA⁺ and recBC⁺ dependent. We report in this paper that respiration does not shutoff in a recF strain at 37 and 30°C. an srfA mutation suppresses this lack of respiration shutoff effect in a recF srfA mutant at 30°C but not at 37°C; no suppression by this mutation occurs at either temperature in a recF recBC sbcB strain. An srfA strain also does not shut off its respiration at 37°C and shows a temperature conditional UV-induced respiration shutoff response at 30°C. The srfA mutation is thought to cause an altered RecA protein to be produced and we suggest that at 37° This altered protein is temperature sensitive. We conclude from the results in this paper that the recF gene product is required for UV-induced respiration shutoff and that the RecA protein plays a special role in the induction process.     

Hypersensitivity of cells from a new chromosomal-breakage syndrome to DNA-damaging agents

Cells derived from a patient with severe chromosomal breakage, immunodeficiency, and growth retardation were found to resemble those from individuals with ataxia telangiectasia (A-T) in terms of their sensitivity to cell killing and the induction of cytogenic abnormalities by X-rays. Their response to other DNA-damaging agents, including 254-nm UV light, mitomycin C, MNNG, and bleomycin was also A-T-like. In contrast to classical A-T, however, X-irradiated cells exhibited a G₁ block after release from density inhibition of growth that was not significantly different from that of normal controls.     

Single amino acid replacements affecting the thermostability of kanamycin nucleotidyltransferase

Summary Amino acid residues of the carboxyl-terminal region of kanamycin nucleotidyltransferase were modified using segment-directed mutagenesis. Six different mutant enzymes with single amino acid replacements were selected out of 59 clones by DNA sequence analyses. The mutant enzymes were purified and it was found that the thermostability of one mutant enzyme was identical to the wild type, whereas the other five were less thermostable at varying degrees. The data suggested that changes in the enzyme thermostability depend not only on the position but also on the species of amino acid residue replaced.     

Cloning and characterization of a linear 2.3 kb mitochondrial plasmid of maize

Summary A linear 2.3 kb DNA molecule found in maize mitochondria was cloned into pUC8. A natural deletion of this plasmid, found in cmsT and some N (fertile) types of maize plants, was mapped to one end of the plasmid. A minor sequence homology to S-2, another linear mitochondrial plasmid, was detected, as well as more significant sequence homology with chloroplast and maize nuclear DNA. Hybridization to teosinte mitochondrial DNA (mtDNA) revealed the presence of part of the maize plasmid in the high molecular weight mtDNA of the maize relatives. RNA dot hybridization indicates that the plasmid is transcribed in mitochondria. The termini of the 2.3 kb linear plasmid contain inverted repeated sequences; of the first 17 nucleotides of the termini, 16 are identical to the terminal inverted repeats of the linear S plasmids found in the mitochondria of cmsS maize plants.     

Macroptilium atropurpureum (siratro) host specificity genes are linked to a nodD-like gene in the broad host range Rhizobium strain NGR234

Summary A 6.7 kb HindIII fragment from the Sym-plasmid of strain NGR234 was found to code a nodD-like gene flanked by two loci which were required for siratro host range. Transfer of the 6.7 kb fragment from NGR234 to R. trifolii strain ANU843 conferred extended host range ability to this strain on siratro plants but not to other plants normally nodulated by strain NGR234. Tn5 mutagenesis of the 6.7 kb fragment showed that insertions located into loci flanking the nodD-like gene abolished the extended host range phenotype. A hybridization probe spanning one of the host specificity loci was shown to hybridize to three specific bands in the NGR234 genome. Complementation and DNA hybridization data showed that the nodD-like gene of strain NGR234 was functionally similar to that in R. trifolii. The introduction to R. trifolii of the 6.7 kb HindIII fragment containing Tn5 insertions located in the nodD-like gene did not abolish the ability to extend the host range of R. trifolii to siratro plants. However, transfer of the 6.7 kb HindIII to R. trifolii derivatives containing Tn5 insertions into either nodA, B or C or other R. trifolii nod genes failed to confer siratro nodulation to these recipients. Reconstruction experiments showed that the 6.7 kb fragment from strain NGR234 and the 14 kb nodulation region of R. trifolii could induce the nodulation of siratro plants when introduced together into Sym-plasmid-cured Rhizobium strains.     

Lipid interconversions in aging Mycoplasma capricolum cultures.

During the progression of Mycoplasma capricolum cultures from the early exponential to the stationary phase of growth, a decrease in the phospholipid-to-protein ratio and increases in both the unsaturated-to-saturated fatty acid ratio and the diphosphatidylglycerol (DPG)-to-phosphatidylglycerol (PG) ratio were found. The freedom of motion of spin-labeled fatty acids incorporated into the membrane remained unchanged throughout the growth cycle. The increase in DPG was almost stoichiometric with the decrease in PG. Furthermore, exogenous PG added to the medium was incorporated by the cells and partially converted to DPG. The DPG that was accumulated upon aging was always more unsaturated than the PG. This accumulation was enhanced in palmitic acid-poor media, but was inhibited even in aged cells when the cells were grown in palmitic acid-rich media, suggesting that the accumulation of DPG upon aging was associated with changes in the fatty acid composition of membrane lipids rather than with the transition of the cells from the exponential- to stationary-growth phase.     

Characterization of a parasporal inclusion body from sporulating, enterotoxin-positive Clostridium perfringens type A.

Inclusion bodies (IB) synthesized during sporulation and enterotoxin formation by Clostridium perfringens NCTC 8239 and 8798 were isolated and characterized. IB were isolated by disruption of sporangia by sonication in the presence of tetrasodium EDTA and phenylmethylsulfonyl fluoride. Fractionation was carried out in a linear gradient of sodium bromide, sucrose, or diatrizoate sodium. Denaturing and reducing agents were necessary to solubilize the IB. An alkylating agent was required to prevent reaggregation of the subunits. Molecular weight, compositional, and serological analyses and peptide mapping revealed strong similarities between the IB subunits and the enterotoxin synthesized during sporulation by C. perfringens. IB appear to represent the structural component where overproduced enterotoxin accumulates intracellularly. Enterotoxin-like subunits in the IB appeared to be held together by noncovalent and disulfide bonds, which were generally resistant to the action of intracellular proteases of C. perfringens, trypsin, or trypsin plus bile salts.