Comparison of restriction endonucleases and sources of probes for their efficiency in detecting restriction fragment length polymorphisms in lettuce (Lactuca sativa L.)

Summary Five accessions of members of the C group of male sterile maize cytoplasms (BB, C, ES, PR, and RB) in two nuclear backgrounds (A619 and A632) were examined to elucidate the nature of mitochondrial genome diversity within a related group of cytoplasms. Cosmid and plasmid clones carrying single copy and recombinationally active sequences from N and S cytoplasms of maize were used as probes. Although restriction patterns are quite similar, each of the five could be discriminated by evidence of sequence duplication and recombination, deletion of recombinationally active sequences of N, normal cytoplasm, population of mini-circular DNAs, and by restriction patterns. Each member of the group carried a 1,913 bp minicircular mtDNA, while all entries but RB carried a 1,445 bp minicircular mtDNA. Members of the C group clearly are not molecularly identical; evolution of the group included principal genome reorganization involving sequence duplication/deletion events, apparently independent of the cms trait.Cooperative Investigations of Agricultural Research Service, U.S. Department of Agriculture, and Institute of Food and Agricultural Sciences, University of FloridaMention of trademark, proprietary product, or vendor does not constitute a guarantee of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable     

Nucleic acids in mummified plant seeds: screening of twelve specimens by gel-electrophoresis,molecular hybridization and DNA cloning

Summary Twelve seed specimens of varying ages and from different archaeological sites were analyzed for the presence of polymerized DNA and RNA. Amongst the samples tested, one of Vitis vinifera from an archaeological site in Iran (2,000–3,000 B.C.) was found to be completely devoid of nucleic acids. Zea mais seeds of Precolumbial age from Peru (about 800 A.D.) contained depolymerized DNA and RNA. Samples of Vitis vinifera and Rubus sp. from a Lombard archaeological site (800 A.D.) as well as radiocarbon dated seeds from the site of the Spring Sanctuary near Metaponto (I–IV century B.C.) were found to contain polymerized DNA and rRNA bands. However the electrophoretic properties of the rRNAs in one case and hybridization experiments performed with cloned seed DNA in the other, clearly demonstrated that the polymerized nucleic acids were not of plant origin.     

Fertile revertants from S-type male-sterile maize grown in vitro

Summary Plants were regenerated from callus cultures of maize inbred W182BN with the S(USDA) type of cytoplasmic male sterility (cms). Some regenerates from 16 of 18 separate cultures had fertile tassels. Many other regenerates, whose fertility could not be scored accurately because of abnormal plant morphology, produced fertile progeny after pollination with N cytoplasm W182BN. Revertant plants and/or progeny were obtained from all 18 cultures, which included the CA, D, LBN, and S sources of cmsS. More revertants were recovered from cultures maintained as callus for 12 months than from 3–4 month old cultures. Several types of evidence (absence of segregation for fertility after selfing or pollination of revertants with standard W182BN, pollen viability counts, failure of revertants to restore sterile cmsS lines to fertility, mitochondrial DNA analyses) indicated that the reversion to fertility involved cytoplasmic rather than nuclear alterations. All revertants examined lacked the S1 and S2 plasmid-like DNAs characteristic of the mitochondrial genome of sterile cmsS lines. Most callus cultures lost S1 and S2 after 13–20 months in vitro. No revertants were seen among thousands of W182BN cmsS plants grown from seed in the field or among plants from tissue cultures of W182BN with the C or T types of cms. The cytoplasmic revertants recovered from culture may be useful for the molecular analysis of cmsS.     

The sequence of the 3.3-kilobase repetitive element fromDipodomys ordii suggests a mechanism for its amplification and interspersion

Summary DNA from the kangaroo rat,Dipodomys ordii, contains a 3.3-kb, highly repeated sequence that is interspersed throughout the genome in small tandem clusters. One 3.3-kb unit has been cloned into pBR322 and the nucleotide sequence determined. The clone used was shown to be representative of the bulk of such sequences found in the genomic DNA. The sequence contains 10 homologous subunits each ca. 260 bp in length. Comparison of these to one another yielded a 258-bp consensus sequence containing a 35-bp terminal inverted repeat. Two unique stretches also occur. One of these contains a region that could serve as a promoter for RNA polymerase III; the other contains a sequence related to the ARS sequences of yeast. It is proposed that an ancestral sequence similar to the consensus sequence was amplified to 10 or more units, and that, subsequently, two other sequences were inserted. The properties of these insertions may have led to the dispersal of the sequence throughout the genome.     

Mutations in the E1a gene of type 5 adenovirus result in oncogenic transformation of Fischer rat embryo cells

Transformation of a specific clone of Fischer rat embryo (CREF) cells with wild-type 5 adenovirus (Ad5) or the E1a plus E1b transforming gene regions of Ad5 results in epithelioid transformants that grow efficiently in agar but that do not induce tumors when inoculated into nude mice or syngeneic Fischer rats. In contrast, CREF cells transformed by a host-range Ad5 mutant, H5hrl, which contains a single base-pair deletion of nucleotide 1055 in E1a resulting in a 28-kd protein (calculated) in place of the wild-type 51-kd acidic protein, display a cold-sensitive transformation phenotype and an incomplete fibroblastic morphology but surprisingly do induce tumors in nude mice and syngeneic rats. Tumors develop in both types of animals following injection of CREF cells transformed by other cold-sensitive Ad5 E1a mutants (H5dl101 and H5in106), which contain alterations in their 13S mRNA and consequently truncated 289AA proteins. CREF cells transformed with only the E1a gene (0-4.5 m.u.) from H5hrl or H5dl101 also produce tumors in these animals. To directly determine the role of the 13S E1a encoded 289AA protein and the 12S E1a encoded 243AA protein in initiating an oncogenic phenotype in adenovirus-transformed CREF cells, we generated transformed cell lines following infection with the Ad2 mutant pm975, which synthesizes the 289AA E1a protein but not the 243AA protein, and the Ad5 mutant H5dl520 and the Ad2 mutant H2dl1500, which do not produce the 289AA E1a protein but synthesize the normal 243AA E1a protein. All three types of mutant adenovirus-transformed CREF cells induced tumors in nude mice and syngeneic rats. Tumor formation by these mutant adenovirus-transformed CREF cells was not associated with changes in the arrangement of integrated adenovirus DNA or in the expression of adenovirus early genes. These results indicate, therefore, that oncogenic transformation of CREF cells can occur in the presence of a wild-type 13S E1a protein or a wild-type 12S E1a protein when either protein is present alone, but does not occur when both wild-type E1a proteins are present.     

The sesquiterpene lactone hymenoxon acts as a bifunctional alkylating agent

Hymenoxon, a toxic sesquiterpene lactone found in bitterweed, bound deoxyguanosine in a cell free system and formed adducts with guanine residues in cellular DNA. The reactive dialdehyde form of hymenoxon formed stable Schiff base products with deoxyguanosine which were separable from unreacted hymenoxon and deoxynucleosides by reverse phase high pressure liquid chromatography. Hymenoxon adducts which eluted as a single impure peak from the octadecylsilane column separated on amino and diphenyl-bonded phases with 10% methanol. Tritiated nucleoside adducts were isolated and purified from CFW mouse sarcoma cells treated with hymenoxon. Proton nuclear magnetic resonance spectra of purified hymenoxon-deoxyguanosine adducts revealed a loss of signals for hydroxyl groups in the bishemiacetal of hymenoxon. ¹³C-nuclear magnetic resonance spectra revealed that the major adduct has 35 carbon atoms, indicating an interaction of at least two guanine residues per hymenoxon molecule and suggesting that hymenoxon may cross-link DNA. Sedimentation analysis of treated DNA further showed that DNA cross-linking by hymenoxon (30 µg/ml) was equivalent to that of a known cross-linking agent, mitomycin C (7.5 µg/ml). Hymenoxon was more cytotoxic to DNA cross-link repair-deficient Chinese hamster ovary cell mutants than to repair proficient strains. These data combine to indicate that hymenoxon acts as a bifunctional alkylating agent which cross-links DNA in mammalian cells.CHO Chinese hamster ovary - HYM hymenoxon - MMC mitomycin C - NMR nuclear magnetic resonance - PBS phosphate buffered saline     

In Vivo measurement of unscheduled DNA synthesis and S-phase synthesis as an indicator of hepatocarcinogenesis in rodents

Measurement of chemically induced DNA repair as unscheduled DNA synthesis in rodent liver following in vivo treatment is a useful screen for potential hepatocarcinogens. In addition to measurement of unscheduled DNA synthesis, examination of S-phase synthesis provides an indicator of chemically induced cell proliferation in the liver, which may be a basis for hepatic tumor promotion. Several chemicals and classes of chemicals have been examined using these endpoints. The pyrrolizidine alkaloid riddelline is a potent genotoxic agent in vitro, and in vivo studies confirm this response as riddelline induces significant elevations in unscheduled DNA synthesis and S-phase synthesis in rat liver. Conversely, H. C. Blue dyes #1 and #2 are both potent genotoxic agents in vitro but fail to express this genotoxicity in vivo. H. C Blue #1 induces significant increases in S-phase synthesis in B6C3F1 mouse liver, which correlates with the observed carcinogenicity of this compound. Halogenated hydrocarbons likewise fail to induce unscheduled DNA synthesis in vivo, but many of these compounds do increase hepatic cell proliferation in mice, which may be the principal mechanism of hepatocarcinogenesis in this species.Abbreviations BCMEE bis(2-chloro-l-methylethyl)ether - dThd thymidine - HCB1 H.C. Blue #1 - HCB2 H.C. Blue #2 - UDS unscheduled DNA synthesis     

Electrostatic calculations and model-building suggest that DNA bound to CAP is sharply bent

Two observations suggest that DNA, upon binding to E. coli catabolite gene activator protein (CAP), is sharply bent by a total angle of at least 100-150 degrees: (1) The electrostatic potential field of CAP shows regions of positive potential that form a ramp on 3 sides of the protein. (2) The DNA binding site size as determined by DNA ethylation interference with binding, (Majors: "Control of the E. coli Lac Operon at the Molecular Level." Ph.D. Thesis, Harvard University, Cambridge, 1977) and by relative affinities of DNA fragments of various lengths (Liu-Johnson et al.: Cell 47:995-1005, 1986) requires severe bending of the DNA to maintain its favorable electrostatic contact with the protein.     

Aspergillus nomius,a new aflatoxin-producing species related to Aspergillus flavus and Aspergillus tamarii

Aspergillus nomius is described and represents a new aflatoxigenic species phenotypically similar to A. flavus. Strains examined were isolated from insects and agricultural commodities. Separation from A. flavus is based on the presence of indeterminate sclerotia and a lower growth temperature. Comparisons of DNA relatedness show A. nomius to have only relatively recently evolved from A. flavus and A. tamarii.