Two arginines in the cytoplasmic C-terminal domain are essential for voltage-dependent regulation of A-type K+ current in the Kv4 channel subfamily

Contributions of the C-terminal domain of Kv4.3 to the voltage-dependent gating of A-type K+ current (IA) were examined by (i) making mutations in this region, (ii) heterologous expression in HEK293 cells, and (iii) detailed voltage clamp analyses. Progressive deletions of the C terminus of rat Kv4.3M (to amino acid 429 from the N terminus) did not markedly change the inactivation time course of IA but shifted the voltage dependence of steady state inactivation in the negative direction to a maximum of -17 mV. Further deletions (to amino acid 420) shifted this parameter in the positive direction, suggesting a critical role for the domain 429-420 in the voltage-dependent regulation of IA. There are four positively charged amino acids in this domain: Lys423, Lys424, Arg426, and Arg429. The replacement of the two arginines with alanines (R2A) resulted in -23 and -13 mV shifts of inactivation and activation, respectively. Additional replacement of the two lysines with alanines did not result in further shifts. Single replacements of R426A or R429A induced -15 and -10 mV shifts of inactivation, respectively. R2A did not significantly change the inactivation rate but did markedly change the voltage dependence of recovery from inactivation. These two arginines are conserved in Kv4 subfamily, and alanine replacement of Arg429 and Arg432 in Kv4.2 gave essentially the same results. These effects of R2A were not modulated by co-expression of the K+ channel beta subunit, KChIPs. In conclusion, the two arginines in the cytosolic C-terminal domain of alpha-subunits of Kv4 subfamily strongly regulate the voltage dependence of channel activation, inactivation, and recovery.     

A double staining method for differentiating between two classes of mycobacterial catalase in polyacrylamide electrophoresis gels

Mycobacteria produce two classes of catalase, designated T and M. Only the T-catalase also has a peroxidase-like function. When a 3,3'-diaminobenzidine (DAB) peroxidase stain was applied to polyacrylamide gel electrophoresis gels, followed by a ferricyanide negative stain for catalase, isoenzymes of T-catalase appeared as dark bands within a zone of clearing in the green background; the M-catalase appeared only as a clear zone. Heated and unheated preparations could be used to demonstrate the presence of comigrating bands of M and T. The application of the ferricyanide stain after the DAB stain of T-catalase resulted in marked intensification of the positive bands of T-catalase due to nonenzymatic, peroxide-independent reduction of the ferricyanide by the DAB product.     

The New York Consortium on Membrane Protein Structure (NYCOMPS): a high-throughput platform for structural genomics of integral membrane proteins

The New York Consortium on Membrane Protein Structure (NYCOMPS) was formed to accelerate the acquisition of structural information on membrane proteins by applying a structural genomics approach. NYCOMPS comprises a bioinformatics group, a centralized facility operating a high-throughput cloning and screening pipeline, a set of associated wet labs that perform high-level protein production and structure determination by x-ray crystallography and NMR, and a set of investigators focused on methods development. In the first three years of operation, the NYCOMPS pipeline has so far produced and screened 7,250 expression constructs for 8,045 target proteins. Approximately 600 of these verified targets were scaled up to levels required for structural studies, so far yielding 24 membrane protein crystals. Here we describe the overall structure of NYCOMPS and provide details on the high-throughput pipeline.     

Molecular mechanisms for environmentally induced and evolutionarily rapid redistribution (plasticity) of meiotic recombination

It has long been known (circa 1917) that environmental conditions, as well as speciation, can affect dramatically the frequency distribution of Spo11/Rec12-dependent meiotic recombination. Here, by analyzing DNA sequence-dependent meiotic recombination hotspots in the fission yeast Schizosaccharomyces pombe, we reveal a molecular basis for these phenomena. The impacts of changing environmental conditions (temperature, nutrients, and osmolarity) on local rates of recombination are mediated directly by DNA site-dependent hotspots (M26, CCAAT, and Oligo-C). This control is exerted through environmental condition-responsive signal transduction networks (involving Atf1, Pcr1, Php2, Php3, Php5, and Rst2). Strikingly, individual hotspots modulate rates of recombination over a very broad dynamic range in response to changing conditions. They can range from being quiescent to being highly proficient at promoting activity of the basal recombination machinery (Spo11/Rec12 complex). Moreover, each different class of hotspot functions as an independently controlled rheostat; a condition that increases the activity of one class can decrease the activity of another class. Together, the independent modulation of recombination rates by each different class of DNA site-dependent hotspots (of which there are many) provides a molecular mechanism for highly dynamic, large-scale changes in the global frequency distribution of meiotic recombination. Because hotspot-activating DNA sites discovered in fission yeast are conserved functionally in other species, this process can also explain the previously enigmatic, Prdm9-independent, evolutionarily rapid changes in hotspot usage between closely related species, subspecies, and isolated populations of the same species.     

Characterization of Clostridia by Gas Chromatography: I. Differentiation of Species by Cellular Fatty Acids

Fatty acids of 41 strains representing 13 species of Clostridium were extracted directly from whole cells and examined as methyl esters by gas-liquid chromatography. Both visual and quantitative comparisons of the resulting chromatograms for the presence and relative amounts of large major peaks allowed rapid differentiation of C. perfringens, C. sporogenes, and C. bifermentans from each other and from 10 other species. Each of the three former species possessed a different characteristic fatty acid methyl ester profile that was exhibited by all strains tested within the respective species. Culture age and growth media influenced the relative proportions of certain of the acids, but such differences did not limit species differentiation.     

Production of Glutaric Acid: a Useful Criterion for Differentiating Pseudomonas diminuta from Pseudomonas vesiculare

A gas-liquid chromatographic procedure was used to determine short-chain acids produced by Pseudomonas diminuta and P. vesiculare after growth on Trypticase soy agar. Each of nine strains of P. diminuta produced glutaric acid, whereas none of the strains of P. vesiculare produced this acid.     

NOTE—THE STA-1 MUTATION PREVENTS ASSEMBLY OF STARCH GRANULES IN NITROGEN-STARVED CELLS AND SERVES AS A USEFUL MORPHOLOGICAL MARKER DURING SEXUAL REPRODUCTION IN CHLAMYDOMONAS MONOICA (CHLOROPHYCEAE)

Iodine staining of clones of nitrogen-starved Chlamydomonas cells was used to screen for mutants with altered levels or altered composition of storage starch. Mutations leading to defects in quantity or morphology of starch granules not only can provide information on storage starch biosynthesis and granule assembly but can also be used as morphological markers in genetic and cell biological studies. A mutant of Chlamydomonas monoica Strehlow devoid of starch granules was obtained following ultraviolet mutagenesis. Nitrogen-starved cells of the sta-1 strain lacked pyrenoidal starch granules and granules normally associated with thylakoid membranes. The mutant phenotype was the consequence of a single Mendelian mutation that appeared to affect granule assembly rather than starch biosynthesis per se and that had no effect on vegetative growth, sexual reproduction, or zygospore viability.