Molecular cloning of the human DNA excision repair gene ERCC-6.

The UV-sensitive, nucleotide excision repair-deficient Chinese hamster mutant cell line UV61 was used to identify and clone a correcting human gene, ERCC-6. UV61, belonging to rodent complementation group 6, is only moderately UV sensitive in comparison with mutant lines in groups 1 to 5. It harbors a deficiency in the repair of UV-induced cyclobutane pyrimidine dimers but permits apparently normal repair of (6-4) photoproducts. Genomic (HeLa) DNA transfections of UV61 resulted, with a very low efficiency, in six primary and four secondary UV-resistant transformants having regained wild-type UV survival. Southern blot analysis revealed that five primary and only one secondary transformant retained human sequences. The latter line was used to clone the entire 115-kb human insert. Coinheritance analysis demonstrated that five of the other transformants harbored a 100-kb segment of the cloned human insert. Since it is extremely unlikely that six transformants all retain the same stretch of human DNA by coincidence, we conclude that the ERCC-6 gene resides within this region and probably covers most of it. The large size of the gene explains the extremely low transfection frequency and makes the gene one of the largest cloned by genomic DNA transfection. Four transformants did not retain the correcting ERCC-6 gene and presumably have reverted to the UV-resistant phenotype. One of these appeared to have amplified an endogenous, mutated CHO ERCC-6 allele, indicating that the UV61 mutation is leaky and can be overcome by gene amplification.     

The organization of genes in yeast mitochondrial DNA

Summary We have fractionated fragments of yeast mtDNA, obtained with restriction endonucleases, on poly(U)-Sephadex columns using the procedure of Flavell and Van den Berg (FEBS Letters (1975) 58, 90–93). The poly(U) forms a triple helix with (dA·dT) clusters in duplex DNA and fractionates DNA fragments on the basis of the length and number of clusters contained in them.mtDNA fragments obtained with endonucleases PstI, BamHI, HindII, HindII+III, EcoRI, HapII and HhaI were separated by poly(U)-Sephadex in three groups: fragments not retained by the column in 2M LiCl, fragments partially retained and fragments (nearly) completely bound in 2 M LiCl and only eluted by 0.1 M LiCl. The separation obtained is adequate for analytical fractionation of fragments and it can be used for the preparative isolation of firmly-bound fragments.In mtDNA digests made with endonuclease HapII, which gives about 70 separable fragments under our conditions, only about 10% of the fragments were firmly bound to poly(U)-Sephadex. This shows that the number of (dA·dT) clusters long enough to result in binding is limited in yeast mtDNA and its suggests that large fragments are bound by only one or a few clusters.Corresponding segments of the physical map of the mtDNAs from Saccharomyces carlsbergensis and Saccharomyces cerevisiae strains JS1-3D and KL14-4A were bound to the column, showing that the (dA·dT) clusters responsible for binding are conserved in the evolution of mtDNA. However, one 3,000 bp insert, only present on KL14-4A mtDNA, causes the loss of a binding site, another long insert introduces a new binding site.Fragments firmly bound to the columns are clustered in one quadrant of the physical map of these three mtDNAs. This quadrant also contains the large insertions present in KL14-4A mtDNA and absent from S. carlsbergensis mtDNA. The possible relation between (dA·dT) clusters and insertions is discussed.Abbreviation bp base pairs     

Regional localization of a beta-galactosidase locus on human chromosome 22.

Human white blood cells with an X/22 translocation [46, XX, t(X;22)(q23;q13)] were fused with Chinese hamster cells. The isolated hybrids were analyzed for human chromosomes and 21 enzyme markers. An electrophoretic technique for studying the beta-galactosidase isoenzymes in man-Chinese hamster hybrid cells was developed. Immunological studies showed that the beta-galactosidase marker studied in these hybrids did contain immunological determinants of human origin. Furthermore the results provided evidence that a locus for beta-galactosidase is situated on chromosome 22 distal to the breakpoint in q13.     

Nuclear magnetic resonance of E. coli ribosomes and viruses.

¹H Nuclear Magnetic Resonance spectra of a number of viruses and E. Coli ribosomes revealed that experimental values of the linewidth (πT₂)^?1 (< 320 Hz) and T₁ (< 1 sec) of the observable nuclei are too small to be accounted for by the system's molecular weight. The nuclei therefore must be internally mobile. From ¹³C Nuclear Magnetic Resonance spectra of 12% ¹³C enriched E. Coli ribosomes, it follows that 30% of the 5000 $\text{CH}{}_2\text{CH}{}_3$ groups, 10–20% of the 225 Phe residues, 20% of the δ-Arg and β-Lys carbons, ～ 100 nucleotides and a number of C_α carbons are internally mobile. It is demonstrated that ¹³C Nuclear Magnetic Resonance can fruitfully be applied to intact ribosomes.     

Environmental impact and bioremediation of seleniferous soils and sediments

Selenium concentrations in the soil environment are directly linked to its transfer in the food chain, eventually causing either deficiency or toxicity associated with several physiological dysfunctions in animals and humans. Selenium bioavailability depends on its speciation in the soil environment, which is mainly influenced by the prevailing pH, redox potential, and organic matter content of the soil. The selenium cycle in the environment is primarily mediated through chemical and biological selenium transformations. Interactions of selenium with microorganisms and plants in the soil environment have been studied in order to understand the underlying interplay of selenium conversions and to develop environmental technologies for efficient bioremediation of seleniferous soils. In situ approaches such as phytoremediation, soil amendment with organic matter and biovolatilization are promising for remediation of seleniferous soils. Ex situ remediation of contaminated soils by soil washing with benign leaching agents is widely considered for removing heavy metal pollutants. However, it has not been applied until now for remediation of seleniferous soils. Washing of seleniferous soils with benign leaching agents and further treatment of Se-bearing leachates in bioreactors through microbial reduction will be advantageous as it is aimed at removal as well as recovery of selenium for potential re-use for agricultural and industrial applications. This review summarizes the impact of selenium deficiency and toxicity on ecosystems in selenium deficient and seleniferous regions across the globe, and recent research in the field of bioremediation of seleniferous soils.     

The role of mobile consumers in lake nutrient cycles: a brief review

Spawning habitats of anchovy Engraulis encrasicolus and sardinella Sardinella aurita were investigated in the Gulf of Gabès by means of two surveys carried out during the summer of 2005 and 2009. The spatial patterns of early developmental stages were analyzed through Lloyd’s patchiness index, single quotient analyses, and principal component analyses. The results showed that Gulf of Gabès is an important area for anchovy and sardinella spawning. Within this area, anchovy and sardinella showed preference for the warmest waters to spawn. The main spawning areas for anchovy were located in the inner parts of the Gulf, and a secondary spawning area was observed offshore. Contrastingly, the main spawning ground of sardinella was found in the center of the Gulf. In both years, early larvae of anchovy showed a greater degree of aggregation than that of sardinella. The developmental stages of both species showed low spatial overlap, indicating that the spawning habitats of these species are spatially differentiated in the Gulf of Gabès.     

Identification of the acrasin of Dictyostelium minutum as a derivative of folic acid

The acrasin of the slime mold Dictyostelium minutum was isolated from aggregating cells and purified. The compound was species specific and more active in the aggregative than in the vegetative stage. Three observations strongly suggest a structural relationship between the acrasin and folic acid. (1) Folic acid inhibited acrasin degradation by D. minutum. (2) Methotrexate, an antagonist of chemotaxis towards folic acid, also inhibited the response to the acrasin. (3) The chemotactic response to an excess of folic acid was delayed. The response was also delayed to simultaneously tested low amounts of a related compound, but not to unrelated compounds (Van Haastert, 1982). The response to the acrasin was observed to be delayed by excess of folic acid. The acrasinase was identified as a folic acid C9-N10 splitting enzyme. Based on chromatographic properties and biological activity of the acrasin and folate derivatives, the chemical structure of the acrasin is discussed.     

Hypersensitivity to ionizing radiation, in vitro, in a new chromosomal breakage disorder, the Nijmegen Breakage Syndrome

The Nijmegen Breakage Syndrome (NBS) is a new chromosomal instability disorder different from ataxia telangiectasia (AT) and other chromosome-breakage syndromes. Cells from an NBS patient appeared hypersensitive to X-irradiation. X-rays induced significantly more chromosomal damage in NBS lymphocytes and fibroblasts than in normal cells. The difference was most pronounced after irradiation in G2. Further, NBS fibroblasts were more readily killed by X-rays than normal fibroblasts. In addition, the DNA synthesis in NBS cells was more resistant to X-rays and bleomycin than that in normal cells. The reaction of NBS cells to X-rays and bleomycin was similar to that of cells from patients with ataxia telangiectasia. Our results indicate that NBS and AT, which also have similar chromosomal characteristics, must be closely related.