Double-strand-break repair and recombination catalyzed by a nuclear extract of Saccharomyces cerevisiae.

An in vitro system for double-strand-break repair and recombination of plasmid substrates catalyzed by extracts prepared from yeast nuclei has been developed. Recombination events that generate crossover products were detected amongst reaction products by Southern blot hybridization, or by the polymerase chain reaction (PCR). The recombination reaction was found to be stimulated by a double-strand break within homologous sequences and proceeded by a mechanism that involved branched DNA intermediates. In addition to pairing events that generate crossovers, the formation of inverted repeats (head-to-head and tail-to-tail joined products) was also detected. Two models are presented which propose that the formation of crossover products and inverted repeats occur by similar mechanisms.     

Fibronectin receptor overexpression and loss of transformed phenotype in a stable variant of the K562 cell line.

A variant of the K562 erythroleukemia cell line, FA-K562, was selected by cycles of adhesion to solid-phase plasma fibronectin (FN). FA-K562 expresses fourfold more cell-surface alpha 5 beta 1 fibronectin receptor (FNR) than parental K562. In addition to expected differences in adhesion to FN, other differences between FA-K562 and K562 implicate this FNR in the regulation of cell growth and morphology. FA-K562 proliferates slowly in liquid culture, its cloning efficiency in soft agar is only approximately 10% compared with approximately 85% for parental K562, and it is nontumorigenic in nude mice. The reduced soft agar growth potential of FA-K562 involves FNR function, because either glycine-arginine-glycine-aspartate-serine (GRGDS) or monoclonal anti-alpha 5 antibody in the agar medium increased cloning efficiency of FA-K562 about fivefold. Morphologically, FN-adherent FA-K562 become fibroblastoid in appearance, assemble filamentous actin, and differ from K562 in vimentin staining intensity and pattern. Soluble GRGDS peptide inhibits both FA-K562 adhesion to FN and the associated cytoskeletal changes. These findings link the alpha 5 beta 1 FNR to both the transformed phenotype and morphology of FA-K562.     

Immunological and Chemical Identification of Intracellular Forms of Adenovirus Type 2 Terminal Protein

Highly purified adenovirus type 2 terminal protein (TP) with an apparent M_r of 55,000 (55K) was prepared in quantities of 10 to 30 μg from guanidine hydrochloride- or sodium dodecyl sulfate-disrupted virions (60 to 120 mg). Guinea pigs were immunized with 14 to 20 injections of TP in amounts of 1 to 2 μg. Antiserum to TP was used to study the intracellular polypeptides related to adenovirus type 2 TP. By immunoprecipitation with anti-TP serum, we identified 80K and 76K polypeptides in the nucleoplasmic and cytoplasmic S100 fractions of [³⁵S]methionine-labeled cells early and late after infection with Ad2. By immunoautoradiographic analysis which eliminates coprecipitation of unrelated proteins, we identified an 80K polypeptide (probably an 80K-76K doublet) in unlabeled, late infected cells, using anti-TP serum and ¹²⁵I-labeled staphylococcal protein A. About two- to threefold-higher levels of the 80K and 76K polypeptides were present in the nucleoplasm than in the S100 fraction, and two- to threefold-higher levels were found in late infected cells than in early infected cells (cycloheximide enhanced, arabinofuranosylcytosine treated). We did not detect the 80K or 76K polypeptide in uninfected cells, indicating that these polypeptides are virus coded. Tryptic peptide map analysis showed that the 80K and 76K polypeptides are very closely related and that they share peptides with the DNA-bound 55K TP. Our data provide the first direct demonstration of intracellular 80K and 76K forms of TP. The intracellular 80K and 76K polypeptides are closely related or identical to the 80K polypeptide that Challberg and co-workers (Proc. Natl. Acad. Sci. U.S.A. 77:5105-5109, 1980) detected at the termini of adenovirus DNA synthesized in vitro and to the 87K polypeptide that Stillman and co-workers (Cell 23:497-508, 1981) translated in vitro. We did not detect the 55K TP in early or late infected cells, consistent with the proposal by Challberg and co-workers that the 80K polypeptide is a precursor to the virion-bound TP and that the conversion of the 80K polypeptide to the 55K TP occurs during virus maturation. The 80K and 76K polypeptides have many more methionine-containing tryptic peptides than does the 55K TP, and most of the tryptic peptides unique to the 80K and 76K polypeptides are very hydrophobic. Thus, the conversion of the 80K and 76K polypeptides to the 55K TP may involve the removal of a specific hydrophobic protein region.     

Effects of Growth Regulators on Polyamine Content and Peroxidase Activity in Hevea brasiliensis Callus

3, 4-dichlorophenoxyacetic acid (3,4-D) and benzylaminopurine(BAP) at 9 µM (control medium) was compared with 4.5,2.25, and 0.45 µM for ability to induce callogenesis andembryogenesis from seed explants of Hevea brasiliensis. Supplyingthese growth regulators at 4.5 µM for 20 d improved embryogenicpotential compared with the control medium (El Hadrami, Carronand d'Auzac, 1991, Annals of Botany 67, 511–515), sustainedputrescine, spermidine and spermine at a higher level throughoutof much of the culture period (40–70 d), and maintainedlow levels of peroxidase activity. In the control medium, poorcallus embryogenesis is considered a consequence of rapid ageingof tissues characterized by (i) acceleration of an early buttransient production of polyamines, which promoted embryogeniccapacity, and (ii) an early peak in peroxidase activity thatwas positively correlated with callus browning, one of the factorslimiting embryogenesis. Somatic embryogenesis, polyamines, peroxidase, Hevea brasiliensis, rubber-tree     

Effect of Photoperiod and Electrical Potentials Applied to Petioles on the Glucose-6-phosphate Dehydrogenase Activity used as Marker of Floral Induction in Shoot Apices of Spinach I

The increase of glucose-6-phosphate dehydrogenase (G-6-PD) activityhas been proposed as an early marker of floral evocation inthe shoot apical meristem of spinach. This induction is obtainedby the transfer of vegetative plants from short days to continuouslight. The exposure of a single leaf to continuous light inducedthe same effect as that produced with the whole plant. In vegetativeconditions (short days), an electrical potential of ten volts(electrical current: 12·5 µA) applied to the petioleof a single leaf induced a weak increase of G-6-PD activityin the shoot apex, while under inductive conditions, this increasewas similar to that of control plants (transferred to continuouslight). Application of an electrical potential to the petioleand the root inhibits the increase linked to the inductive transfer.These results show that an externally applied electrical potentialmay interact with the natural electrical gradients along theshoot and consequently inhibit the normal floral developmentby an as yet unspecified mechanism. Spinacia oleracea, floral induction, interorganic relations, glucose-6-phosphate dehydrogenase, shoot apex, electrical potentials     

Chloroplast DNA phylogeography of the argan tree of Morocco

Polymorphisms in the chloroplast genome of the argan tree (Sapotaceae), an endemic species of south-western Morocco, have been detected by restriction site studies of PCR-amplified fragments. A total of 12 chloroplast DNA (cpDNA) and two mitochondrial DNA (mtDNA) fragments were amplified and digested with a single restriction enzyme ( Hin fI). Polymorphisms were identified in six of the cpDNA fragments, whereas no mtDNA polymorphisms were detected in a survey of 95 individuals from 19 populations encompassing most of the natural range of the species. The cpDNA polymorphisms allowed the identification of 11 haplotypes. Two lineages, one in the south-east and the other in the north-west, divide the range of the argan tree into two distinct areas. The level of genetic differentiation measured at the haplotype level ( G _STc= 0.60) (i.e. with unordered haplotypes) was smaller than when phylogenetic relationships were taken into account ( N _STc= 0.71–0.74) (ordered haplotypes), indicating that population history must be considered in the study of the geographical distribution of cpDNA lineages in this species. If contrasted with the level of nuclear genetic differentiation measured in a previous study with isozymes ( G _STn= 0.25), the results indicate a relatively high level of gene flow by seeds, or conversely a relatively low level of gene flow by pollen, as compared with other tree species. Goats and camels could have played an important role in disseminating the fruits of this tree.     

Physiological Responses of Spring Durum Wheat Cultivars to Early-season Drought in a Mediterranean Environment

The Mediterranean climate of North Africa is characterized byuncertain rainfall immediately after seedling emergence, leadingto drought early in the growing season which depresses durumwheat production. However, there is limited understanding ofthe physiological basis of resistance of spring durum wheatto drought in rainfed Mediterranean regions. The objectivesof this study were to examine differences in some physiologicalcharacters among spring durum wheat cultivars in response toduration of early-season drought, and to determine the relationshipof these characters to drought resistance. In two field experiments(1995 and 1996 growing seasons) and a glasshouse experiment(1996), six spring sown durum wheat cultivars were evaluatedunder four water regimes: well irrigated and three differentwater deficits from emergence until the onset of tillering,mid-tillering or at the end of tillering. Cultivars differedin their response. Decreases in photosynthesis soon after droughtstress was imposed resulted mainly from reduced stomatal conductance.Continued water deficits also reduced mesophyll photosyntheticactivity. Possible factors determining the drought-resistanceof a cultivar are lower sensitivity of CO₂exchange rate, netCO₂uptake to water loss ratio, stomatal resistance, relativewater content and greater osmotic adjustment under stress. Furthermore,there is sufficient intraspecific variation in these physiologicalattributes to suggest their use as selection tools.Copyright1998 Annals of Botany Company Wheat;Triticum durumDesf.; early-season drought; physiological responses.