An assessment of genetic relationships within the genus Digitalis based on PCR-generated RAPD markers

RAPD markers were used to study inter-specific variation among six species of the genus Digitalis: D. obscura, D. lanata, D. grandiflora, D. purpurea, D. thapsi and D. dubia, and the hybrid D. excelsior (D. purpurea×D. grandiflora). A total of 91 highly reproducible bands amplified with four arbitrarily chosen decamer primers were obtained. Homology of the co-emigrating RAPD markers was tested by blot hybridisation and sequencing of selected bands. The application of a range of statistical approaches for RAPD data analysis, including distance and parsimony methods, family clustering and the analysis of molecular variance (AMOVA), indicated that these molecular markers were taxonomically informative in Digitalis. The species relationships revealed were fully consistent with those previously obtained using morphological affinities. The hybrid D. excelsior seems to have stronger affinity to the section Digitalis than to Grandiflorae. This is the first known report of the application of RAPD markers for the study of genetic relationships among species of the genus Digitalis. Received: 20 October 1999 / Accepted: 9 November 1999     

Statistical analysis of yeast genomic downstream sequences reveals putative polyadenylation signals

The study of a few genes has permitted the identification of three elements that constitute a yeast polyadenylation signal: the efficiency element (EE), the positioning element and the actual site for cleavage and polyadenylation. In this paper we perform an analysis of oligonucleotide composition on the sequences located downstream of the stop codon of all yeast genes. Several oligonucleotide families appear over-represented with a high significance (referred to herein as ‘words’). The family with the highest over-representation includes the oligonucleotides shown experimentally to play a role as EEs. The word with the highest score is TATATA, followed, among others, by a series of single-nucleotide variants (TATGTA, TACATA, TAAATA . . .) and one-letter shifts (ATATAT). A position analysis reveals that those words have a high preference to be in 3′ flanks of yeast genes and there they have a very uneven distribution, with a marked peak around 35 bp after the stop codon. Of the predicted ORFs, 85% show one or more of those sequences. Similar results were obtained using a data set of EST sequences. Other clusters of over-represented words are also detected, namely T- and A-rich signals. Using these results and previously known data we propose a general model for the 3′ trailers of yeast mRNAs.     

Removal of N-glycans from cell surface proteins induces apoptosis by reducing intracellular glutathione levels in the rhabdomyosarcoma cell line S4MH

Expression of determined Asn-bound glycans (N-glycans) in cell surface glycoproteins regulates different processes in tumour cell biology. Specific patterns of N-glycosylation are displayed by highly metastatic cells and it has been shown that inhibition of N-glycan processing restrains cell proliferation and induces cell death via apoptosis. However, the mechanisms by which different N-glycosylation states may regulate cell viability and growth are not understood. Since malignant cells express high levels of intracellular glutathione (GSH) and a reduction of intracellular GSH induces cell death via apoptosis, we investigated whether GSH was involved in the induction of apoptosis by removal of cell surface N-glycans. We found that removal of N-glycans from cell surface proteins by treating the rhabdomyosarcoma cell line S4MH with tunicamycin or N-glycosidase resulted in a reduction in intracellular GSH content and cell death via apoptosis. Moreover, GSH depletion caused by the specific inhibitor of GSH synthesis BSO induced apoptosis in S4MH cells. This data indicates that adequate N-glycosylation of cell surface glycoproteins is required for maintenance of intracellular GSH levels that are necessary for cell survival and proliferation.     

Immunological identification of yeast SCO1 protein as a component of the inner mitochondrial membrane

Summary The SCO1 gene of Saccharomyces cerevisiae encodes a 30 kDa protein which is specifically required for a post-translational step in the accumulation of subunits 1 and 2 of cytochrome c oxidase (COXI and COXII). Antibodies directed against a -Gal::SCO1 fusion protein detect SCO1 in the mitochondrial fraction of yeast cells. The SCO1 protein is an integral membrane protein as shown by its resistance to alkaline extraction and by its solubilization properties upon treatment with detergents. Based on the results obtained by isopycnic sucrose gradient centrifugation and by digitonin treatment of mitochondria, SCO1 is a component of the inner mitochondrial membrane. Membrane localization is mediated by a stretch of 17 hydrophobic amino acids in the amino-terminal region of the protein. A truncated SCO1 derivative lacking this segment, is no longer bound to the membrane and simultaneously loses its biological function. The observation that membrane localization of SCO1 is affected in mitochondria of a rho ⁰ strain, hints at the possible involvement of mitochondrially coded components in ensuring proper membrane insertion.     

Human leukemic K562 cells: differential effects of 5-azacytidine on DNA methylation of epsilon-, gamma-globin and 7SL RNA genes

5 Azacytidine ribonucleoside (5 Aza CR), greatly enhances erythroid differentiation of the K562(h) cell line, with a sharp increase of embryonic and fetal globin gene expression. This phenomenon is correlated with the undermethylation of gamma-globin but not of epsilon-globin, as the epsilon-globin gene is already extensively undermethylated before 5AzaCR induction. By contrast no variations in both DNA methylation and expression are observed in 7SL RNA genes.     

Isolation of a cAMP receptor protein from yeast mitochondria (Mr 45000) and comparison with mitochondrial RNA polymerase (Mr 45000)

We have isolated a cAMP-binding protein from highly purified yeast mitochondria by affinity chromatography. It is a lipophilic protein of molecular mass 45 000 Da, which is tightly membrane-bound and localized on the outer surface of the inner membrane. It can be solubilized in active form under mild conditions. The cAMP receptor resembles mitochondrial RNA polymerase prepared as described by Levens et al. [(1981) J. Biol. Chem. 256, 1474] in a surprisingly large number of properties including molecular mass. Comparison of the two proteins revealed that the polypeptide previously considered as RNA polymerase is, in fact, a mitochondrial cAMP receptor protein.     

Genetic aspects of ethanol tolerance and production bySaccharomyces cerevisiae

We developed a method of hybrid selection between homothallic wild-type and heterothallic strains. The hybrids obtained were used to study the heredity of ethanol tolerance and production. Both characters segregated independently, but no ethanol-sensitive strains were able to produce high levels of ethanol. At least four genes are implicated in ethanol tolerance.     

Statistical-mechanical theory of passive transport through partially sieving or leaky membranes

The basic equations for multicomponent transport through partially sieving or leaky membranes are discussed from a statistical-mechanical viewpoint. They have the same mathematical form as the corresponding equations for open membranes, but differ in a discontinuous way from the equations for semipermeable membranes (since a "leak" in a semipermeable membrane constitutes a discontinuous or singular perturbation). Partially sieving membranes can be made to mimic semipermeable behavior through the introduction of characteristic time scales. They may approximate semipermeable behavior at short times, but always deviate at longer times.