Induction of DNA amplification in the Bacillus subtilis chromosome.

A system allowing the induction of DNA amplification in Bacillus subtilis was developed, based on a thermosensitive plasmid, pE194, stably integrated in the bacterial chromosome. An amplification unit, comprising an antibiotic resistance marker flanked by directly repeated sequences, was placed next to the integrated plasmid. Activation of pE194 replication led to DNA amplification. Two different amplification processes appeared to take place: one increased the copy number of all sequences in the vicinity of the integrated plasmid and was possibly of the onion skin type, while the other increased the copy number of the amplification unit only and generated long arrays of amplification units. These arrays were purified and shown to consist mainly of directly repeated amplification units but to also contain non-linear regions, such as replication forks and recombination intermediates. They were attached to the chromosome at one end only, and were, in general, not stably inherited, which suggests that they are early amplification intermediates. Longer arrays were detected before the shorter ones during amplification. When the parental amplification unit contained repeats which differed by a restriction site the arrays which derived thereof contained in a majority of cases only a single type of repeat. We propose that the amplified DNA is generated by rolling circle replication, and that such a process might underlie a number of amplification events.     

Preparative resolution of some 5,5-hydantoin derivatives via formation of diastereoisomeric salts

Several preparative resolutions of 5,5-disubstituted hydantoins have been achieved via fractional crystallization of diastereoisomeric salts. The process can be extended by making use of the difference between the variation of solubilities of the hydantoins and their salts with α-methylbenzylamine as a function of the alkalinity of the medium. Optimization for each resolution procedure involves a refinement of the excess amount of base needed. © 1992 Wiley-Liss, Inc.     

Cytostatic product(s) released by activated macrophages, unrelated to interleukin 1, tumor necrosis factor alpha, and interferon-alpha/beta

Murine peritoneal macrophages activated for cytotoxicity by trehalose dimycolate in vivo and lipopolysaccharide in vitro released cytostatic factor(s) against EMT6 target cells, in 8-hr conditioned medium (CM). The cytostatic factor(s) completely blocked DNA synthesis by EMT6 cells within 16 hr. Other cell lines are less sensitive (P815 and R-L929) or resistant (KB and HT29) to the cytostatic effect of CM. The anti-proliferative activity of CM had a MW greater than 10,000 Da, as judged by ultrafiltration. It was destroyed by proteases and strongly inhibited by P815 cell product(s). Conditioned media from nonactivated macrophages were not cytostatic against EMT6 cells. No relationship was found between cytostatic factor(s) in CM and interleukin 1 (IL-1), tumor necrosis factor alpha (TNF-alpha), and interferon-alpha/beta (IFN-alpha/beta): the growth of EMT6 cells was unaffected by Hu.r.IL-is and Hu.r.TNF-alpha and was only slightly inhibited by IFN-alpha/beta. Furthermore, cytostatic CM contained low levels of TNF and IFN activities. Finally, antibodies raised against murine IFN-alpha/beta had no effect on the cytostatic activity of CM.     

Regeneration of NAD(+) cofactor by photosensitized electron transfer in an immobilized alcohol dehydrogenase system

The irradiation with visible light of a photosensitizer dye like methylene blue was used to regenerate by electron transfer the oxidized form of a pyridine nucleotide coenzyme (NAD(+)). The process has been studied on a common enzymatic reaction: ethanol oxidation by alcohol-NAD(+) oxidoreductase immobilized on polyacrylamide gel or porous glass balls. In the experimental conditions used, the initial NAD(+) recycling rates were 2.33 x 10(4) cycles/h (polyacrylamide) and 3 x 10(4) cycles/h (glass balls). A total number of 49.5 x 10(4) cycles was obtained for 13 runs of 2 h. The enzyme immobilization strongly increased its stability: after 28 days at 20 degrees C, the residual activity was 25% of the initial value.     

Multiple transformation of plant cells by Agrobacterium may be responsible for the complex organization of T-DNA in crown gall and hairy root

Summary Inoculation of carrot discs and Lotus corniculatus plantlets with mixtures of different Agrobacterium rhizogenes or of A. rhizogenes and A. tumefaciens or with Agrobacterium strains harboring both an Ri and a modified Ti plasmid resulted in frequent multiple (pluribacterial) transformation of cells, as revealed by the mixed opine-type of hairy roots arising from them. Multiple transformation may account for the presence of dispersed T-DNA inserts in crown gall and hairy root lines. A plant genetic engineering strategy based on segregation of T-DNA inserts in the progeny of multiple transformants is proposed.     

Huntington's disease. Contribution of clinical and epidemiological data to genetic counseling

From a regional survey (northwestern part of France 3,9 millions population) and from recent publications, the authors described clinical and epidemiologic aspects useful for genetic counselling. Besides the chronic chorea, the other clinical features justified the denomination "Huntington's disease". Misdiagnosis is frequent especially if the familial disorder is unknown. A prevalence of 50 X 10(-6) corresponds to 2750 choreics and 14.000 at risk descendants in France. Interfamilial heterogeneity is described from mean age of death which is 54.2 for the whole study: one third of families of late onset, around fifty (mean age of death: 64.6), one third of early onset, around thirty (mean age of death: 44.6). The 17 juvenile and infantile cases of the survey belong to this last group. The influence of gene-transmitters sex is more obvious if two generations are taken in account with a preponderance of fathers and grand-fathers in the early onset group and, to a lesser degree, of mothers and grand-mothers in the late onset group. The authors criticize the preclinical detection tests and, from attitudes of families, underline the ethical problems for the future predictive tests by genetic markers. The role of lay organisations is all the more important.     

Study of stereoisomers of N6-phenylisopropyladenosine on the secretion of pancreatic glucagon

This work was designed to characterize the adenosine receptor (A1 or A2) involved in glucagon secretion. The most potent adenosine analogues on A1 receptors are the N6 substituted compounds, among them N6-phenylisopropyladenosine (PIA); furthermore L-PIA is 50 to 100 times more potent than D-PIA on the A1 receptor, whereas it is 3 to 5 times more potent on the A2 receptor; thus the A1 receptor shows a much higher stereoselectivity. The effects of L-PIA and D-PIA were studied on glucagon secretion from the isolated perfused rat pancreas. 1) L-PIA at 1.65 microM induced a transient glucagon secretion which was not greater than that induced by the same concentration of adenosine. 2) D-PIA at a 3 fold higher concentration (4.95 microM) elicited a secretion of glucagon comparable to that induced by L-PIA 1.65 microM; thus the involved receptor does not present a high stereoselectivity for L-PIA. These results support the fact that the receptor involved in glucagon secretion is not of the A1 type.