Genetic suppression and phenotypic masking of a Myxococcus xanthus frzF- defect

An insertion of transposon Tn5-lac, ω4519, generates a lacZ fusion with a Myxococcus xanthus promoter expressed during both vegetative growth and development. Sequence analysis of the junction of ω4519 with M. xanthus DNA shows that the insertion is in frzF, a homologue of cheR from Salmonella typhimurium. When frzF^- (or frzCD^-) cells are starved for nutrients at modest densities, they aggregate to form a radial pattern and produce fewer than 1% of the wild-type complement of spores. At higher densities, frzF::ω519 cells form‘frizzy’aggregates and produce 80–90% of the wild-type complement of spores. In contrast, when cells with both a frzF^- (or frzCD^-) and an sgtA1 mutation are allowed to develop at either low or high cell densities, they produce frizzy aggregates containing a near wild-type complement of heat-resistant spores. In addition to suppressing the density dependence of fruiting-body morphogenesis, the sglA1 mutation also suppresses the sporulation defect caused by two different frzF^- mutations and a frzCD^- mutation. In contrast, a mutation in a different S motility gene, sglG1, does not suppress the frz^- mutations. Thus, the suppression of frz^- mutations by sgl^- mutations is allele-specific, and depends on the sgl allele, but not the frz allele. Because the phenotypes of frz^- mutations have been determined in a (suppressing) sglA1 genetic background, the frz genes may play more central roles in development than initially recognized.     

Single Cl- channels activated by Ca2+ in Drosophila S2 cells are mediated by bestrophins

Mutations in human bestrophin-1 (VMD2) are genetically linked to several forms of retinal degeneration but the underlying mechanisms are unknown. Bestrophin-1 (hBest1) has been proposed to be a Cl(-) channel involved in ion and fluid transport by the retinal pigment epithelium (RPE). To date, however, bestrophin currents have only been described in overexpression systems and not in any native cells. To test whether bestrophins function as Ca(2+)-activated Cl(-) (CaC) channels physiologically, we used interfering RNA (RNAi) in the Drosophila S2 cell line. S2 cells express four bestrophins (dbest1-4) and have an endogenous CaC current. The CaC current is abolished by several RNAi constructs to dbest1 and dbest2, but not dbest3 or dbest4. The endogenous CaC current was mimicked by expression of dbest1 in HEK cells, and the rectification and relative permeability of the current were altered by replacing F81 with cysteine. Single channel analysis of the S2 bestrophin currents revealed an approximately 2-pS single channel with fast gating kinetics and linear current-voltage relationship. A similar channel was observed in CHO cells transfected with dbest1, but no such channel was seen in S2 cells treated with RNAi to dbest1. This provides definitive evidence that bestrophins are components of native CaC channels at the plasma membrane.     

An exploration of the sequence of a 2.9-Mb region of the genome of Drosophila melanogaster: the Adh region.

A contiguous sequence of nearly 3 Mb from the genome of Drosophila melanogaster has been sequenced from a series of overlapping P1 and BAC clones. This region covers 69 chromosome polytene bands on chromosome arm 2L, including the genetically well-characterized "Adh region." A computational analysis of the sequence predicts 218 protein-coding genes, 11 tRNAs, and 17 transposable element sequences. At least 38 of the protein-coding genes are arranged in clusters of from 2 to 6 closely related genes, suggesting extensive tandem duplication. The gene density is one protein-coding gene every 13 kb; the transposable element density is one element every 171 kb. Of 73 genes in this region identified by genetic analysis, 49 have been located on the sequence; P-element insertions have been mapped to 43 genes. Ninety-five (44%) of the known and predicted genes match a Drosophila EST, and 144 (66%) have clear similarities to proteins in other organisms. Genes known to have mutant phenotypes are more likely to be represented in cDNA libraries, and far more likely to have products similar to proteins of other organisms, than are genes with no known mutant phenotype. Over 650 chromosome aberration breakpoints map to this chromosome region, and their nonrandom distribution on the genetic map reflects variation in gene spacing on the DNA. This is the first large-scale analysis of the genome of D. melanogaster at the sequence level. In addition to the direct results obtained, this analysis has allowed us to develop and test methods that will be needed to interpret the complete sequence of the genome of this species.Before beginning a Hunt, it is wise to ask someone what you are looking for before you begin looking for it. Milne 1926     

Characterization and regulation of T-type Ca2+ channels in embryonic stem cell-derived cardiomyocytes

T-type Ca2+ channels may play a role in cardiac development. We studied the developmental regulation of the T-type currents (ICa,T) in cardiomyocytes (CMs) derived from mouse embryonic stem cells (ESCs). ICa,T was studied in isolated CMs by whole cell patch clamp. Subsequently, CMs were identified by the myosin light chain 2v-driven green fluorescent protein expression, and laser capture microdissection was used to isolate total RNA from groups of cells at various developmental time points. ICa,T showed characteristics of Cav3.1, such as resistance to Ni2+ block, and a transient increase during development, correlating with measures of spontaneous electrical activity. Real-time RT-PCR showed that Cav3.1 mRNA abundance correlated (r2 = 0.81) with ICa,T. The mRNA copy number was low at 7+4 days (2 copies/cell), increased significantly by 7+10 days (27/cell; P < 0.01), peaked at 7+16 days (174/cell), and declined significantly at 7+27 days (25/cell). These data suggest that ICa,T is developmentally regulated at the level of mRNA abundance and that this regulation parallels measures of pacemaker activity, suggesting that ICa,T might play a role in the spontaneous contractions during CM development.     

Oxido-reductive titrations of cytochrome c oxidase followed by EPR spectroscopy

Experiments are described on oxido-reductive titrations of cytochrome c oxidase as followed by low-temperature EPR and reflectance spectroscopy. The reductants were cytochrome c or NADH and the oxidant ferricyanide. Experiments were conducted in the presence and absence of either cytochrome c or carbon monoxide, or both. An attempt is made to provide a complete quantitative balance of the changes observed in the major EPR signals. During reduction, the maximal quantity of heme represented in the high-spin ferric heme signals (g ～ 6; 2) is 25% of the total heme present, and during reoxidation 30%. With NADH reduction there is little difference between the pattern of disappearance of the low-spin ferric heme signals in the absence or presence of cytochrome c. The copper and high-spin heme signals, however, disappear at higher titrant concentrations in the presence of cytochrome c than in its absence. In these titrations, as well as in those with ferrocytochrome c, the quantitative balance indicates that, in addition to EPR-detectable components, EPR-undetectable components are also reduced, increasingly so at higher titrant concentrations. The quantity of EPR-undetectable components reduced appears to be inversely related to pH. A similar inverse relationship exists between pH and appearance of high-spin signals during the titration. At pH 9.3 the quantity of heme represented in the high-spin signals is < 5%, whereas it approximately doubles from pH 7.4 to pH 6.1. In the presence of CO less of the low-spin heme and copper signals disappears for the same quantity of titrant consumed, again implying reduction of EPR undetectable components. At least one of these components is represented in a broad absorption band centered at 655 nm. The stoichiometry observed on reoxidation, particularly in the presence of CO, is not compatible with the notion that the copper signal represents 100% of the active copper of the enzyme as a pair of interacting copper atoms.     

Effect of the effluent released from the canine internal mammary artery after intraluminal and extraluminal perfusion of acetylcholine and adenosine diphosphate

Segments of the canine internal mammary artery (35 mm in length) were suspended in vitro in an organ chamber containing physiological salt solution (95% O₂/5% CO₂, pH = 7.4, 37°C). Segments were individually cannulated and perfused at 5 ml/minute using a roller pump. Vasorelaxant activity of the effluent from the perfused internal mammary arteries was bioassayed by measuring the decrease in tension induced by the effluent of the coronary artery endothelium-free ring which had been contracted with prostaglandin F_2α (2 × 10^-6 M). Intraluminal perfusion of adenosine diphosphate (10^-5 M) induced significant increase in relaxant activity in the effluent from the perfused blood vessel. However, when adenosine diphosphate (10^-5 M) was added extraluminally to the internal mammary artery, no change in relaxant activity in the effluent was noted. In contrast, acetylcholine produced significant increase in the relaxant activity on the effluent of the perfused internal mammary artery with both intraluminal and extraluminal perfusion. The intraluminal and extraluminal release of endothelium-derived relaxing factor (EDRF) by acetylcholine (10^-5 M) can be inhibited by site-specific administration of atropine (10^-5 M). These experiments indicate that certain agonists can induce the release of EDRF only by binding to intravascular receptors while other agonists can induce endothelium-dependent vasodilatation by acting on neural side receptors.