Nested chromosomal fragmentation in yeast using the meganuclease I-Sce I: a new method for physical mapping of eukaryotic genomes.

We have developed a new method for the physical mapping of genomes and the rapid sorting of genomic libraries which is based on chromosome fragmentation by the meganuclease I-Sce I, the first available member of a new class of endonucleases with very long recognition sequences. I-Sce I allows complete cleavage at a single artificially inserted site in an entire genome. Sites can be inserted by homologous recombination using specific cassettes containing selectable markers or, at random, using transposons. This method has been applied to the physical mapping of chromosome XI (620 kb) of Saccharomyces cerevisi and to the sorting of a cosmid library. Our strategy has potential applications to various genome mapping projects. A set of transgenic yeast strains carrying the I-Sce I sites at various locations along a chromosome defines physical intervals against which new genes, DNA fragments or clones can be mapped directly by simple hybridizations.     

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.     

Studies on NK cell purification by negative selection in human peripheral blood

We have attempted to improve negative selection procedures for the large scale purification of human CD _in3 ^– CD56⁺ NK cells. In a series of experiments, purifications of NK cells from 10⁸ PBMC were performed by T cell depletion using either direct or indirect anti-CD3 labeling and the Magnetic Activated Cell Separation (MACS) procedure. Contaminating CD3⁺ cells were still present using either one of these two different T cell depletion protocols as shown by phenotyping IL-2 supplemented cell cultures on day 12. A second cycle of purification was therefore added. When MACS and Dynabeads were compared as complementary procedures to the first MACS cycle starting with 10⁸ cells, the Dynabeads method was found to be superior to the MACS with regard to the elimination of residual T cells. Starting from 10⁹ PBMC, we showed that this MACS+Dynabeads procedure gave similar satisfactory results when compared to the scaling-up of a previously established two steps procedure using Dynabeads. These two approaches (MACS+Dynabeads and 2 cycles of Dynabeads) have been also tested in a clinical setting to purify NK cells from cancer patients prior toin vitro expansion. The results indicate that the two methods are equivalent with respect to purity and recovery rate; a slight advantage in terms of feasibility was found in favor of 2 cycles of Dynabeads.     

A protein of Halobacterium halobium immunologically related to the v-myc gene product

A 70 kDa protein of Halobacterium halobium cross-reacts with an antiserum directed against the v-myc gene product of the avian myelocytomatosis virus (MC29). This cross-reaction is in agreement with hybridization studies which indicate that H. halobium possesses DNA and RNA sequences homologous to the v-myc gene.     

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.