Abstract. Striated muscle contraction is elicited by the release of stored calcium ions through ryanodine receptor channels in the sarcoplasmic reticulum, ryr-1 is a C. elegans ryanodine receptor homologue that is expressed in body-wall muscle cells used for locomotion.Using genetic methods, we show that ryr-1 is the previously identified locus uric-68. First, transposon-induced deletions within ryr-1 are alleles of unc-68. Second, transformation of unc-68 mutants with ryr-1 genomic DNA results in rescue of the Unc phenotype, unc-68 mutants move poorly, exhibiting an incomplete flaccid paralysis, yet have normal muscle ultrastructure. The mutants are insensitive to the paralytic effects of ryanodine, and lack detectable ryanodine-binding activity. The Unc-68 phenotype suggests that ryanodine receptors are not essential for excitation-contraction coupling in nematodes, but act to amplify a (calcium) signal that is sufficient for contraction.

Maryon EB, Molloy SA, Kaplan JH. Cellular glutathione plays a key role in copper uptake mediated by human copper transporter 1.

Linear DNA injected into Xenopus laevis oocyte nuclei recombines with high efficiency if homologous sequences are present at overlapping molecular ends. We found that injected linear DNA was degraded by a 5'-*3' strand-specific exonuclease activity during incubation in the oocyte nucleus to leave a heterogeneous population of 3'-tailed molecules. Decreasing the concentration of DNA injected increased the heterogeneity and the average rate of degradation. The 3' tails created were relatively stable; among molecules persisting after overnight incubation, many had 3' tails intact to within 10 bases of the original ends. DNA molecules that were efficient substrates for homologous recombination in oocytes were also partially degraded, leaving 3' tails. We found no evidence for other potent nuclease activities. If molecules with recessed 3'-OH ends were injected, endogenous polymerase efficiently resynthesized complementary strands before degradation of the 5' tails occurred. 3'-tailed molecules are plausible intermediates in the initiation of homologous recombination events in Xenopus oocyte nuclei.Studies on a variety of organisms have shown a correlation between double-strand breaks in DNA and increased levels of homologous recombination (16,33,35,39). When introduced into cells, linear DNA is generally more efficient at recombination than circular DNA, and in most situations recombination events are focused near the ends of linear molecules. Double-strand breaks made in vivo can also stimulate recombination events at the site of the break (16, 39).Several models have been proposed for the initiation of eucaryotic homologous recombination events by DNA duplex ends. Typically, these start with degradation at the ends by hypothetical strand-specific exonucleases, leaving molecules with single-stranded tails (22,30,38). There is genetic and biochemical evidence in procaryotes for exonuclease activity in end-initiated recombination events. Genes known to encode strand-specific exonucleases are required for certain bacteriophage and bacterial recombination pathways in which recombination events are elevated near ends (32,39). Furthermore, the bacterial RecA protein can interact in vitro with DNA molecules having single-stranded tails and catalyze the invasion of homologous duplex DNA, creating crossed-strand recombination intermediates (41). Proteins from a variety of organisms have been shown to have analogous strand transfer activities (13,(17)(18)(19)25). It has not been proven, however, that DNA molecules which have undergone exonucleolytic degradation initiate procaryotic recombination events in vivo, nor have such molecules been physically isolated.Little genetic or biochemical evidence exists for the involvement of specific exonucleases in eucaryotic homologous recombination, but two laboratories have recently identified probable physical intermediates of recombination events initiated by double-strand breaks in Saccharomyces cerevisiae (16,36 cules had undergone strand-specific exonucleolytic degradation before ...

Cu is an essential micronutrient, and its role in an array of critical physiological processes is receiving increasing attention. Among these are wound healing, angiogenesis, protection against reactive oxygen species, neurotransmitter synthesis, modulation of normal cell and tumor growth, and many others. Free Cu is absent inside cells, and a network of proteins has evolved to deliver this essential, but potentially toxic, metal ion to its intracellular target sites following uptake. Although the total body content is low (∼100 mg), dysfunction of proteins involved in Cu homeostasis results in several well-characterized human disease states. The initial step in cellular Cu handling is its transport across the plasma membrane, a subject of study for only about the last 25 years. This review focuses on the initial step in Cu homeostasis, the properties of the major protein, hCTR1, that mediates Cu uptake, and the status of our understanding of this highly specialized transport system. Although a high-resolution structure of the protein is still lacking, an array of biochemical and biophysical studies have provided a picture of how hCTR1 mediates Cu(I) transport and how Cu is delivered to the proteins in the intracellular milieu. Recent studies provide evidence that the transporter also plays a key protective role in the regulation of cellular Cu via regulatory endocytosis, lowering its surface expression, in response to elevated Cu loads.

The major human copper uptake protein, hCTR1, has 190 amino acids and a predicted mass of 21 kDa. hCTR1 antibodies recognize multiple bands in SDS-PAGE centered at 35 kDa. Part of this increased mass is due to N-linked glycosylation at Asn-15. We show that in mammalian cells the N15Q mutant protein trafficked to the plasma membrane and mediated copper uptake at 75% of the rate of wild-type hCTR1. We demonstrate that the extracellular amino terminus of hCTR1 also contains O-linked polysaccharides. Glycosidase treatment that removed O-linked sugars reduced the apparent mass of hCTR1 or N15Q mutant protein by 1-2 kDa. Expression of amino-terminal truncations and alanine substitution mutants of hCTR1 in HEK293 and MDCK cells localized the site of O-linked glycosylation to Thr-27. Expression of alanine substitutions at Thr-27 resulted in proteolytic cleavage of hCTR1 on the carboxyl side of the T27A mutations. This cleavage produced a 17-kDa polypeptide missing approximately the first 30 amino acids of hCTR1. Expression of wild-type hCTR1 in mutant Chinese hamster ovary cells that were unable to initiate O-glycosylation also resulted in hCTR1 cleavage to produce the 17-kDa polypeptide. The 17-kDa hCTR1 polypeptide was located in the plasma membrane and mediated copper uptake at about 50% that of the rate of wildtype hCTR1. Thus, O-linked glycosylation at Thr-27 is necessary to prevent proteolytic cleavage that removes half of the extracellular amino terminus of hCTR1 and significantly impairs transport activity of the remaining polypeptide.

No abstract

When DNA molecules are injected into Xenopus oocyte nuclei, they can recombine with each other. With bacteriophage lambda DNAs, it was shown that this recombination is stimulated greatly by introduction of double-strand breaks into the substrates and is dependent on homologous overlaps in the recombination interval. With plasmid DNAs it was shown that little or no recombination occurs between circular molecules but both intra-and intermolecular events take place very efficiently with linear molecules. As with the lambda substrates, homology was required to support recombination; no simple joining of ends was observed. Blockage of DNA ends with nonhomologous sequences interfered with recombination, indicating that ends are used directly to initiate homologous interactions. These observations are combined to evaluate possible models of recombination in the oocytes. Because each oocyte is capable of recombining nanogram quantities of linear DNA, this system offers exceptional opportunities for detailed molecular analysis of the recombination process in a higher organism.

Background:Copper enters human cells through pores formed by trimeric hCTR1 transporters that require intramembrane methionines near the extracellular side. Results: The copper transport rate is increased by mutations on the intracellular side of hCTR1. Conclusion: hCTR1 elements on the intracellular side affect the copper transport rate and response to high copper. Significance: The mutations provide unexpected insight into the hCTR1 transport mechanism.