Change in the ultraviolet spectrum of solubilized Ca2+-dependent ATPase from sarcoplasmic reticulum due to binding with Ca2+ ions.

Solubilized sarcoplasmic reticulum (SSR) was prepared by solubilizing fragmented sarcoplasmic reticulum (FSR) with a nonionic detergent (C12E8) then displacing the detergent with Tween 80, using a DEAE-cellulose column. The UV absorption of SSR decreased reversibly at about 286 and 292 nm on removal of free Ca2+ ions, while no change in the fluorescence spectrum was detectable. On the other hand, the fluorescence intensity of FSR decreased 3-4% on removal of free Ca2+ ions, as previously reported by Dupont [(1976) Biochem. Biophys. Res. Commun. 71, 544-550]. The UV absorption of FSR increased reversibly at about 270-280 nm on removal of free Ca2+ ions, but the rate of the change was very slow (k = about 0.1 min-1).     

Phylogenetics of Tribe Collabieae (Orchidaceae,Epidendroideae) Based on Four Chloroplast Genes with Morphological Appraisal

Collabieae (Orchidaceae) is a long neglected tribe with confusing tribal and generic delimitation and little-understood phylogenetic relationships. Using plastid matK, psaB, rbcL, and trnH-psbA DNA sequences and morphological evidence, the phylogenetic relationships within the tribe Collabieae were assessed as a basis for revising their tribal and generic delimitation. Collabieae (including the previously misplaced mycoheterotrophic Risleya) is supported as monophyletic and nested within a superclade that also includes Epidendreae, Podochileae, Cymbidieae and Vandeae. Risleya is nested in Collabiinae and sister to Chrysoglossum, a relationship which, despite their great vegetative differences, is supported by floral characters. Ania is a distinct genus supported by both morphological and molecular evidence, while redefined Tainia includes Nephelaphyllum and Mischobulbum. Calanthe is paraphyletic and consists four clades; the genera Gastrorchis, Phaius and Cephalantheropsis should be subsumed within Calanthe. Calanthe sect. Ghiesbreghtia is nested within sect. Calanthe, to which the disputed Calanthe delavayi belongs as well. Our results indicate that, in Collabieae, habit evolved from being epiphytic to terrestrial.     

A DNA Damage Checkpoint Pathway Coordinates the Division of Dikaryotic Cells in the Ink Cap Mushroom Coprinopsis cinerea

The fungal fruiting body or mushroom is a multicellular structure essential for sexual reproduction. It is composed of dikaryotic cells that contain one haploid nucleus from each mating partner sharing the same cytoplasm without undergoing nuclear fusion. In the mushroom, the pileus bears the hymenium, a layer of cells that includes the specialized basidia in which nuclear fusion, meiosis, and sporulation occur. Coprinopsis cinerea is a well-known model fungus used to study developmental processes associated with the formation of the fruiting body. Here we describe that knocking down the expression of Atr1 and Chk1, two kinases shown to be involved in the response to DNA damage in a number of eukaryotic organisms, dramatically impairs the ability to develop fruiting bodies in C. cinerea, as well as other developmental decisions such as sclerotia formation. These developmental defects correlated with the impairment in silenced strains to sustain an appropriated dikaryotic cell cycle. Dikaryotic cells in which chk1 or atr1 genes were silenced displayed a higher level of asynchronous mitosis and as a consequence aberrant cells carrying an unbalanced dose of nuclei. Since fruiting body initiation is dependent on the balanced mating-type regulator doses present in the dikaryon, we believe that the observed developmental defects were a consequence of the impaired cell cycle in the dikaryon. Our results suggest a connection between the DNA damage response cascade, cell cycle regulation, and developmental processes in this fungus.     

Carcinogens can induce homologous recombination between duplicated chromosomal sequences in mouse L cells.

The ability of a series of DNA-damaging agents to induce homologous intrachromosomal recombination between duplicated genes in the chromosome of mouse cells was investigated. The target cells were the thymidine kinase-deficient mouse L-cell strain 333M, which contains a single integrated copy of a plasmid with two herpes simplex virus thymidine kinase (Htk) genes, each containing an 8-base-pair XhoI linker inserted at a unique site. Expression of a functional Htk enzyme requires a productive recombinational event between the two nonfunctional genes. The spontaneous rate of recombination in this strain is 3 per 10(6) cells per generation. The agents tested represent physical carcinogens (UV and ionizing radiation), a simple alkylating agent (N-methyl-N'-nitro-N-nitrosoguanidine), an alkylating cross-linking agent (mitomycin C), and a reactive metabolite of a polycyclic aromatic hydrocarbon ((+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene [BPDE] ). The background frequency of tk+ recombinants in the untreated population averaged 18 X 10(-6) +/- 5 X 10(-6). Ionizing radiation had little or no effect on recombination; exposure to mitomycin C, N-methyl-N'-nitro-N-nitrosoguanidine, BPDE, or UV, at doses that lowered the survival to between 90 and 10% of the control, caused a dose-dependent increase in frequency of recombinants, reaching 50 X 10(-6) to 100 X 10(-6). No tk+ cells could be generated with a control cell line that contained only one mutant copy of the Htk gene. Molecular hybridization analysis showed that 85 to 90% of the tk+ recombinants retained the Htk gene duplication, consistent with nonreciprocal transfer of wild-type genetic information, gene conversion. In the rest, only a single copy of the Htk gene remained, reflecting a single reciprocal exchange within a chromatid or a single unequal exchange between sister chromatids. Each recombinant tested contained an XhoI-resistant (wild-type) Htk gene.     

On the toroidal condensed state of closed circular DNA

The influence of double helix torsional elasticity on the compaction and structure of circular DNA compact form is studied theoretically in the case when the compact (globular) form has torus shape. For closed circular DNA the topological invariant, the linking number, yields a strict connection between conformation of the double helix considered as unifilar homopolymer and elastic energy of torsional twisting. The contribution of torsional elasticity to the free energy of the toruslike globule is calculated. This contribution is shown to be proportional to the square of superhelical density. Allowance of the torsional elasticity decreases the equilibrium radius of the toruslike globule formed by circular DNA. Closure of linear DNA into a ring widens the stability range of the relatively short DNA compact form and tightens it for long DNA.     

Import and processing of the precursor of cytochrome P-450(SCC) by bovine adrenal cortex mitochondria

Isolated bovine adrenal cortex mitochondria imported in vitro synthesized pre-P-450(SCC) and processed it to the mature form. Partial radio-sequencing of the processed P-450(SCC) gave a result identical with that for authentic P-450(SCC). Rat liver mitochondria also imported pre-P-450(SCC) and processed it to the mature form, whereas bovine heart mitochondria were unable to import and process pre-P-450(SCC) although both mitochondrial preparations imported and processed pre-adrenodoxin. The pre-P-450(SCC) processing activity of bovine adrenal cortex mitochondria was associated with the matrix side surface of the inner membrane. The processing protease could be solubilized by sodium cholate and partially purified by ammonium sulfate fractionation. The partially purified processing protease cleaved pre-P-450(SCC) at the correct position. It was also active in processing pre-P-450(11 beta) but inactive toward pre-adrenodoxin. Bovine heart mitochondria lacked the processing activity to pre-P-450(SCC). The localization of pre-P-450(SCC) and mature P-450(SCC) in bovine adrenal cortex mitochondria was examined. Mature P-450(SCC) processed by the mitochondria was found associated with the matrix-side surface of the inner membrane, which is the correct location of P-450(SCC) in the cell. In the presence of o-phenanthroline, pre-P-450(SCC) was imported into the organelles without being processed and remained soluble in the matrix. The incorporation of newly processed mature P-450(SCC) into the inner membrane was also observed when pre-P-450(SCC) was incubated with inner membrane vesicles. Mature P-450(SCC) generated in vitro from pre-P-450(SCC) by the partially purified processing protease was incorporated not only into the inner membrane vesicles but also into bovine adrenal cortex microsomes. These findings suggested that the processing of pre-P-450(SCC) occurred prior to the incorporation of mature-P-450(SCC) into the inner membrane.