Multipoint genetic mapping of the Xq26-q28 region in families with fragile X mental retardation and in normal families reveals tight linkage of markers in q26–q27

Summary The q26–q28 region of the human X chromosome contains several important disease loci, including the locus for the fragile X mental retardation syndrome. We have characterized new polymorphic DNA markers useful for the genetic mapping of this region. They include a new BclI restriction fragment length polymorphism (RFLP) detected by the probe St14-1 (DXS52) and which may therefore be of diagnostic use in hemophilia A families. A linkage analysis was performed in fragile X families and in large normal families from the Centre d'Etude du Polymorphisme Humain (CEPH) by using seven polymorphic loci located in Xq26-q28. This multipoint linkage study allowed us to establish the order centromere-DXS100-DXS86-DXS144-DXS51-F9-FRAX-(DXS52-DXS15). Together with other studies, our results define a cluster of nine loci that are located in Xq26-q27 and map within a 10 to 15 centimorgan region. This contrasts with the paucity of markers (other than the fragile X locus) between the F9 gene in q27 and the G6PD cluster in q28, which are separated by about 30% recombination.     

The presence of an inhibitor of benzylamine oxidase in human blood plasma.

The presence of an inhibitor of benzylamine oxidase in human blood plasma was observed. It may be removed by use of either a DEAE-cellulose column or a Sephadex G-200 column.     

Regulation of the 3 beta-hydroxysteroid dehydrogenase activity in tissue fragments and microsomes from human term placenta: kinetic analysis and inhibition by steroids

The effects of 50 microM of progesterone (P4), estradiol (E2), estrone (E1), estriol (E3), dehydroepiandrosterone (DHIA), androstenedione (delta 4) and testosterone (T) on the bioconversion of [3H]pregnenolone (6 nM) to [3H]P4 were investigated by incubating 200 mg of tissue fragments as well as equivalent aliquots of microsomes from human term placenta during 30 min. All the steroids assayed, except E3, significantly inhibited the [3H]P4 formation in a microsome incubation system with respect to the control assay (P less than 0.001). Conversely in a tissue incubation system. P4, E1 as well as E3 had no effect on [3H]pregnenolone bioconversion while E2 slightly decreased the [3H]P4 formation (P less than 0.05) compared with the control. A significant inhibition was observed in this system with the other steroids (P less than 0.001). To investigate these apparent different results of inhibition-noninhibition of the same steroids irrespective of the system of incubation used, the effects of P4, E2 and T on 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) activity were studied in tissue fragments and microsomes in kinetic terms. The results found indicate that these steroids inhibited in a competitive fashion the 3 beta-HSD activity in both systems. The different Ki values found in tissue fragments and microsomes respectively for P4 (1.8 microM vs 0.5 microM), E2 (2.3 microM vs 0.6 microM) and T (0.25 microM vs 0.3 microM) explain the bioconversion results obtained in presence of 50 microM of the same steroids. These results include inhibition of [3H]P4 formation by T in tissue fragments as well as in microsomes whereas P4 and E2 inhibited the [3H]P4 formation only in microsomes. Furthermore, the comparison of these Ki values with the available data of intraplacental and circulating concentrations of the same steroids in human term pregnancy suggest that only P4 would be expected to cause marked 3 beta-HSD inhibition in physiological conditions.     

Trisomy 21 mosaicism in two subjects from two generations.

In the course of a chromosome fragility investigation on the cancer prone hereditary disorder xeroderma pigmentosum, a low proportion of cells with a 47,XY,+21 karyotype was found in lymphocyte cultures of a patient not showing any Down syndrome symptom. The presence of trisomy 21 mosaicism was demonstrated also in peripheral blood of the healthy father and confirmed by "chromosome painting" that allowed a rapid detection of chromosomes 21 on metaphase cells and interphase nuclei. The trisomic cell line was not detected in fibroblast cultures. The analysis of chromosome 21 heteromorphism indicated that in both subjects the mosaic could result from either a diploid or an aneuploid zygote. Since in the trisomic cell line of the father and the son the extra chromosome 21 seems to be the same, a predisposition toward mitotic errors (non-disjunction or anaphase lagging) may be postulated, leading to the recurrent gain or loss of a specific chromosome 21. In order to test the hypothesis of an abnormal mitotic behaviour of the chromosome 21, we investigated the centromere separation index and the DNA restriction pattern in Southern blots probed with satellite DNA sequences specific for chromosome 21 centromere. Both the approaches did not reveal any peculiar feature that may account for the genetically determined proneness to mitotic error observed in the family.     

Abrupt modifications of phospholipid bilayer properties at critical cholesterol concentrations.

The fluorescence generalized polarization (GP) of 2-dimethylamino-6-lauroylnaphthalene (Laurdan) reveals different effects of cholesterol on the phase behavior of phospholipid bilayers. Phospholipid vesicles composed of gel, liquid-crystalline, and coexisting domains of the two phases have been studied at temperatures from 1 to 65 degrees C, without cholesterol and with cholesterol concentrations of 3-50 mol %. Laurdan GP measurements show the general effect of cholesterol of increasing the molecular dynamics of the gel and of decreasing the molecular dynamics of the liquid-crystalline phase. In the liquid-crystalline phase, the increased order yields Laurdan GP values close to those obtained in the gel phase. At cholesterol concentrations > 15 mol % a phase transition cannot be detected. Using the wavelength dependence of the excitation and emission GP spectra we determine that differences between the two phospholipid phases cannot be detected. In particular, in vesicles composed of coexisting gel and liquid-crystalline phases the GP wavelength dependence characteristic of coexisting domains cannot be observed at cholesterol concentrations > or = 15 mol %. Cholesterol causes the decrease in both the polarity and the dipolar relaxation effects on the neighborhood of the fluorescent naphthalene moiety of Laurdan. Probably because of a cholesterol-induced increase in the bilayer packing, these effects do not occur continuously with the increase of cholesterol concentration in the bilayer. Cholesterol concentrations inducing higher Laurdan GP values have been determined at about 5, 10, 15, 30, and 45 mol % with respect to phospholipids. We propose that the formation of ordered molecular microdomains at critical cholesterol concentrations can explain the occurrence of the observed discontinuities.     

DNA fingerprint analysis for the detection of induced mutations in mammalian cells in culture

A mutation assay in cultured mammalian cells based on the direct analysis of minisatellite DNA was developed. Band pattern variations reflect DNA alterations ranging from single base changes to complex rearrangements. By DNA fingerprinting a large number of autosomal loci throughout the human genome can be simultaneously checked, therefore minimizing the size of the samples of cell colonies to be scored in the absence of phenotypic selection. For the mutation assay chinese hamster cells (V79) were treated with Nitrosoguanidine and 14 independent colonies were isolated and expanded. DNA fingerprints were obtained after digestion of the DNA extracted from each clone with bothHinfI andHae III, and hybridisation with both 33.15 and 33.6 probes. Twelve colonies from untreated cells were also analysed. Several differences in the band pattern of treated colonies were observed when compared with untreated cells; digestion withHae III and hybridisation with 33.15 probe allowed the detection of the highest frequency of induced variants. The results suggest that minisatellite sequences are hypermutable sites that can be used to monitor the mutagenic potential of chemical agents directly at the DNA level, without phenotypic selection. Moreover, with the method herein decribed, it is possible to distinguish between true mutations and epimutations, such as those caused by changes in DNA methylation.