Evidence for transposition of dispersed repetitive DNA families in yeast.

Dispersed repetitive DNA sequences from yeast (Saccharomyces cerevisiae) nuclear DNA have been isolated as molecular hybrids in lambdagt. Related S. cerevisiae strains show marked alterations in the size of the restriction fragments containing these repetitive DNAs. "Ty1" is one such family of repeated sequences in yeast and consists of a 5.6 kilobase (kb) sequence including a noninverted 0.25 kb sequence of another repetitious family, "delta", on each end. There are about 35 copies of Ty1 and at least 100 copies of delta (not always associated with Ty1) in the haploid genome. A few Ty1 elements are tandem and/or circular, but most are disperse and show (along with delta) some sequence divergence between repeat units. Sequence alterations involving Ty1 elements have been found during the continual propagation of a single yeast clone over the course of a month. One region with a large number of delta sequences (SUP4) also shows a high frequency of sequence alterations when different strains are compared. One of the differences between two such strains involves the presence or absence of a Ty1 element. The novel joint is at one inverted pair of delta sequences.     

Amplified fragment length polymorphism fingerprinting of 16 banana cultivars (Musa cvs.)

Banana is one of the most important subtropical crops. The genetic system, however, is relatively unknown and is complicated by specific interhybridization, heterozygosity, and polyploidy, which are common in most clones. These factors make identification of closely related banana cultivars difficult, particularly when sterile. Amplified fragment length polymorphism (AFLP) analysis using eight primer combinations was carried out on 16 banana cultivars. Results showed that AFLP could be used to distinguish the different cultivars by their unique banding patterns. Unique AFLP molecular markers were detected for 12 banana cultivars, which can be used to develop specific probes for identification purposes. The cluster analysis also revealed the need for a link between genotype studies using molecular techniques and the current system of classification of Musa cultivars based purely on morphological traits.     

Establishment of rat brain endothelial cells susceptible to rat cytomegalovirus ALL-03 infection

Endothelial cells have been implicated as key cells in promoting the pathogenesis and spread of cytomegalovirus (CMV) infection. This study describes the isolation and culture of rat brain endothelial cells (RBEC) and further evaluates the infectious potential of a Malaysian rat CMV (RCMV ALL-03) in these cultured cells. Brain tissues were mechanically fragmented, exposed to enzymatic digestion, purified by gradient density centrifugation, and cultured in vitro. Morphological characteristics and expression of von Willebrand factor (factor VIII-related antigen) verified the cells were of endothelial origin. RBEC were found to be permissive to the virus by cytopathic effects with detectable plaques formed within 7 d of infection. This was confirmed by electron microscopy examination which proved the existence of the viral particles in the infected cells. The susceptibility of the virus to these target cells under the experimental conditions described in this report provides a platform for developing a cell-culture-based experimental model for studies of RCMV pathogenesis and allows stimulation of further studies on host cell responses imposed by congenital viral infections.     

Intermediary carbon metabolism of Azospirillum brasilense.

Azospirillum brasilense Sp 7 grew rapidly in AZO medium containing reduced nitrogen and succinate as an energy source, with a doubling time of 43 min. No growth was measured with glucose as the sole carbon source. In contrast, Azospirillum lipoferum Sp 59b could grow in media containing either succinate or glucose with a doubling time of 69 min and 223 min, respectively. Warburg-Barcroft respirometry showed that the rate of oxygen consumption by A. brasilense Sp 7 on glucose medium (0.034 mumol of O2 min-1 mg-1 of cell protein) was only one-quarter of that on succinate medium (0.14 mumol of O2 min-1 mg-1). Radioisotopic labeling showed that very little glucose was assimilated by A. brasilense Sp 7 as compared to succinate. High respiration rates were measured on A. lipoferum Sp 59b with either succinate (0.15 mumol of O2 min-1 mg-1) or glucose (0.13 mumol of O2 min-1 mg-1) as the sole carbon source. The pattern of CO2 evolution from differentially labeled succinate indicated that A. brasilense Sp 7 had a complete tricarboxylic acid cycle. Assimilation of most of the radioactivity from labeled succinate, pyruvate, and acetate into lipids suggested a strong anabolic metabolism and the presence of an active malic enzyme of phosphoenolpyruvate carboxykinase. The distribution of radioactivity from differentially labeled pyruvate showed that gluconeogenesis competed with pyruvate dehydrogenase. Uptake and incorporation of labeled acetate also indicated the presence of a glyoxylate cycle in A. brasilense Sp 7.     

Mutational analysis of the structure and function of opioid receptors

The cloning of the opioid receptors allows the investigation of receptor domains involved in the peptidic and nonpeptidic ligand interaction and activation of the opioid receptors. Receptor chimera studies and mutational analysis of the primary sequences of the opioid receptors have provided insights into the structural domains required for the ligand recognition and receptor activation. In the current review, we examine the current reports on the possible involvement of extracellular domains and transmembrane domains in the high-affinity binding of peptidic and nonpeptidic ligands to the opioid receptor. The structural requirement for the receptors' selectivity toward different ligands is discussed. The receptor domains involved in the activation and subsequent cellular regulation of the receptors' activities as determined by mutational analysis will also be discussed. Finally, the validity of the conclusions based on single amino acid mutations is examined.     

On the necessity of different statistical treatment for Illumina BeadChip and Affymetrix GeneChip data and its significance for biological interpretation

Background  

The original spotted array technology with competitive hybridization of two experimental samples and measuring relative expression levels is increasingly displaced by more accurate platforms that allow determining absolute expression values for a single sample (for example, Affymetrix GeneChip and Illumina BeadChip). Unfortunately, cross-platform comparisons show a disappointingly low concordance between lists of regulated genes between the latter two platforms.     

Cl--dependent and Cl--independent Na+/ HCO3- acid extrusion in cultured rat cerebellar astrocytes

It is still uncertain whether the Na+-dependent Cl--HCO3- exchanger (NCBE) is expressed in mammalian astrocytes. Using fluorescent indicators to monitor the intracellular pH (pHi) and intracellular Na+ or Cl- levels, the NCBE in cultured rat cerebellar astrocytes was examined in detail. In nominally bicarbonate-free (Hepes-buffered) medium, a marked pHi recovery from internal acid load was seen which could be blocked completely by 30 microM HOE 694, a specific Na+-H+ exchanger isoform 1(NHE-1) inhibitor, at a pHi above 6.9. These conditions were therefore used to block NHE activity in CO2/HCO3-buffered media when the NCBE was being studied at pHi above 6.9. After internal acid loading in completely Cl--free bicarbonate-buffered medium (containing HOE 694), the rates of pHi recovery and transient Na+ influx were considerably slowed, and the Cl--dependent acid extrusion was both Na+- and 4,4-diisothiocyano-stilbene-disulphonic acid (DIDS)-sensitive. Moreover, a HCO3-dependent Cl- efflux during internal acid injection was seen. These results suggest that the NCBE is present in astrocytes. Following repetitive internal acid loading by addition of 5% CO2 to internal Cl- depleted cells, a similar rate of pHi recovery was consistently seen, suggesting Cl--independent pHi regulation also occurred in astrocytes. Moreover, this pHi recovery was completely blocked in the absence of sodium or on addition of DIDS, confirming that the Na+-HCO3 cotransporter (NBC) is present. Thus, the present study provides evidence that both the NCBE and NBC play important roles in acid extrusion in cultured mammalian astrocytes.     

MGMT Ile143Val polymorphism,dietary factors and the risk of breast,colorectal and prostate cancer in the European Prospective Investigation into Cancer and Nutrition (EPIC)-Norfolk study

O⁶-Methylguanine-DNA methyltransferase (MGMT) repairs DNA damage caused by alkylating agents including N-nitroso compounds from diet. MGMT Ile143Val polymorphism may lead to less DNA damage repair and increased cancer risk depending on the environmental exposures. We investigated interactions between dietary factors and the MGMT Ile143Val polymorphism in relation to breast, colorectal and prostate cancer risk. There were 276/1498, 273/2984 and 312/1486 cases/controls for the breast, colorectal and prostate cancer studies respectively; all nested within the EPIC-Norfolk study, a prospective cohort of approximately 25,000 men and women aged 40–79. Baseline 7-day food diary data were collected for dietary assessment. MGMT Ile143Val polymorphism was not overall associated with breast, colorectal and prostate cancer risk. There was a significant interaction between this polymorphism and intake of red and processed meat on colorectal cancer risk (P_interaction = 0.04) suggesting an increased risk among carriers of the variant genotype compared to the MGMT Ile143Ile common genotype. A lower colorectal cancer risk was seen with higher intake of vitamin E and carotene among the variant genotype group but not in the common genotype group (P_interaction = 0.009 and P_interaction = 0.005 for vitamin E and carotene, respectively). A higher prostate cancer risk was seen with higher alcohol intake among the variant genotype (OR = 2.08, 95% CI = 1.21–3.57, P_interaction = 0.0009) compared to the common genotype with lower alcohol intake. In this UK population, the MGMT Ile143Val polymorphism was not overall associated with breast, colorectal and prostate cancer risk. There was evidence for this polymorphism playing a role in modulating the risk of prostate cancer in presence of alcohol. For colorectal cancer, the MGMT Ile143Val polymorphism may confer increased or decreased risk depending on the dietary exposure.