Genetic and physical mapping of a novel region close to the fragile X site on the human X chromosome

We report the isolation and characterization of a novel DNA marker (1A1) in Xqter in the region of the fragile X. Genetic studies in families segregating for the fragile X syndrome suggest that 1A1 lies between the disease mutation and the distal locus, DXS52. Studies in normal and fragile X families show that 1A1 is tightly linked to DXS52 (Zmax = 17.20; theta max = 0.03) and F8 (Zmax = 7.01; theta max = 0.08). Multipoint mapping of families supports the order Xcen-DXS105-FRAXA-1A1-DXS52-(F8, DXS115)-Xqter. Pulsed-field gel electrophoresis (PFGE) studies demonstrate that 1A1 defines a new region of at least 2 Mb of DNA not physically linked to DXS52 or F8, thus extending the physical map of Xq27-qter to over 4 Mb. Complex partial digestion PFGE patterns, probably due to differing degrees of methylation, are observed with 1A1 in unrelated normal and fragile-X-positive individuals, whereas other distal markers give uniform digestion profiles. Physical data suggest that 1A1 lies in a region less CpG rich than other distal markers in Xq27-qter.     

Vectors permitting visual monitoring of simple transposition events

The construction and use of two novel transposon(Tn)-delivery vectors is described. These vectors carry Inc.W or Inc.N broad-host-range transfer functions cloned next to the narrow-host-range replicon of pBR329. The host specificities of pSLX10 and pSLX23 both complement and extend the host specificities of existing Tn delivery vectors. Plasmids pSLX10 and pSLX23 were shown to transfer at high frequency in intergeneric matings. The lux genes which are present on each vector permit the visual monitoring of transconjugants which have retained a Tn element, but are devoid of plasmid molecules. pSLX10 and pLSX23 were efficiently used to generate a range of auxotrophic mutants in various strains of Pseudomonas as well as to clone genes from Serratia liquefaciens. These vectors may have general applicability to identify and clone genes in a wide range of Gram-negative bacteria.     

Initiation of protein synthesis by internal entry of ribosomes into the 5' nontranslated region of encephalomyocarditis virus RNA in vivo.

Expression vectors that yield mono-, di-, and tricistronic mRNAs upon transfection of COS-1 cells were used to assess the influence of the 5' nontranslated regions (5'NTRs) on translation of reporter genes. A segment of the 5'NTR of encephalomyocarditis virus (EMCV) allowed translation of an adjacent downstream reporter gene (CAT) regardless of its position in the mRNAs. A deletion in the EMCV 5'NTR abolishes this effect. Poliovirus infection completely inhibits translation of the first cistron of a dicistronic mRNA that is preceded by the capped globin 5'NTR, whereas the second cistron preceded by the EMCV 5'NTR is still translated. We conclude that the EMCV 5'NTR contains an internal ribosomal entry site that allows cap-independent initiation of translation. mRNA containing the adenovirus tripartite leader is also resistant to inhibition of translation by poliovirus.     

Characterization of the genome of Pseudomonas aeruginosa bacteriophage phi PLS27 with particular reference to the ends of the DNA.

The DNA of Pseudomonas aeruginosa rough-specific bacteriophage phi PLS27 was studied. The genome size as determined by summing the sizes of restriction fragments was 42.7 kilobase pairs. Of particular interest was the fact that the DNA was insensitive to certain common restriction endonucleases including EcoRI, BamHI, and HindIII. The ends of the phage DNA were cloned and sequenced, revealing direct repeats of 318 nucleotides. The left end of the genome when cloned into the promoter selection vector pKK232-8 exhibited promoter activity in Escherichia coli. Two promoters bearing greater than 70% sequence homology to the plasmid pNM74 TOL operon and PAK pilin promoters were identified.     

Assessment of uncoupling by amiloride analogs.

The amiloride analogs N5-methyl-N5-isobutylamiloride, N5-ethyl-N5-isopropylamiloride, and N5,N5-hexamethyleneamiloride are frequently used to investigate NaH exchange on the premise that they are highly specific inhibitors of the NaH-antiporters. We assessed the relative protonophoric activity of these compounds in reconstituted and native membrane vesicles, using acridine orange fluorescence to measure intravesicular pH. All the compounds tested were found to be potent protonophores at concentrations which are normally used to demonstrate inhibition of NaH exchange. Uncoupling was dependent on both the pH of the assay system and the total amount of lipid present. At the pH optima, which lay in a range from 7.5 to 8.5, these amiloride analogs were more potent uncouplers than the classical protonophore carbonyl cyanide m-chlorophenylhydrazone. Therefore, extreme care must be taken in the interpretation of results obtained using these or similar derivatives of amiloride.     

Receptor expression and oxidase activity in human neutrophils: Regulation by granulocyte-macrophage colony-stimulating factor and dependence upon protein biosynthesis

Incubation of human bloodstream neutrophils with 50 u/ml recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) primed the respiratory burst (as assessed by fMet-Leu-Phe stimulated luminol-dependent chemiluminescence) and resulted in a rapid (within 15 min) upregulation of expression of CD11b and CD18 (as measured by FACS analysis). This rapid priming and modulation of receptor expression was not inhibited by cycloheximide and hence appeared to be independent ofde novo protein biosynthesis. When neutrophils were incubated for up to 5 h in culture, the fluorescence distributions of CD11b and CD18 declined indicating the loss of expression of these receptors as the neutrophils aged, but in rGM-CSF treated suspensions receptor expression was maintained. When neutrophils were incubated in the presence of cycloheximide, they progressively lost their ability to generate reactive oxidants in response to fMet-Leu-Phe so that by 5 h incubation with this inhibitor they could only generate about 25% of the oxidative response stimulated in untreated cells, and the expression of CD16 and CD18 was grossly impaired. Similar effects were observed in rGM-CSF treated suspensions except that cycloheximide required longer incubation times (typically 4–5 h) before impairment of function or receptor expression occurred. These data show thatde novo protein biosynthesis is required for both the maintenance of neutrophil function and also for the continued expression of some plasma membrane receptors.Abbreviations fMet-Leu-Phe N-formylmethionyl-Leucyl-Phenylalanine - rGM-CSF recombinant granulocyte-macrophage colony-stimulating factor - FITC fluorescein isothiocyanate conjugate - Luminol 5-amino-2,3-dihydrophthalazine-1,4-dione     

Quenching of Hoechst 33258 fluorescence in erythroid precursors.

Quenching of the fluorescence of DNA-bound Hoechst 33258 in erythroid precursors was studied by flow cytometry and cytochemistry. This quenching artifact may affect the measurement of ploidy in specific cases. The bone marrow cells of two patients with hemolytic disease and active erythropoiesis contained subpopulations of cells with an apparent hypodiploid DNA content as measured by flow cytometry of paraformaldehyde-fixed cells stained with Hoechst 33258. No aneuploidy was detected in either of the two cases when cells were stained with mithramycin or 7-aminoactinomycin D. Cells exhibiting reduced Hoechst 33258 fluorescence expressed glycophorin A and low amounts of CD36, and were therefore erythroid precursors. In one case studied, the number of cells with reduced Hoechst 33258 fluorescence and glycophorin A expressed agreed well with the number of cells containing nuclear hemoglobin. In the other case, hemoglobin was present in a significant proportion of nucleated cells. Calculated values for the efficiency of resonance energy transfer from Hoechst 33258 to hemoglobin were in accordance with the observed levels of quenching (approximately 10%). However, the results could also be explained by hemoglobin reabsorption of Hoechst 33258 fluorescence. Nuclei stained with Hoechst 33258 showed uniform fluorescence, probably due to extraction of hemoglobin during the isolation procedure.     

Significant extension in northern Australia of the known geographic range of the Shield Shrimp Triops australiensis (Crustacea: Notostraca)

The program BIOCLIM predicts the total geographic distribution of species, based upon the biogeoclimatic characteristics common to the localities at which they are known to occur. Field studies in the Northern Territory have located the Shield Shrimp Triops australiensis at localities substantially north of its known and predicted geographic distribution.