The fully-active nature of synthetic and hydrolytic activities of glucose-6-phosphatase of intact nuclear membrane

Carbamyl-P: glucose phosphotransferase, mannose-6-P: glucose phosphotransferase, and mannose-6-P and glucose-6-P phosphohydrolase activities of D-glucose-6-P phosphohydrolase (EC 3.1.3.9) have been demonstrated in avian and mammalian liver (and kidney) nuclear membrane. In marked contrast with activities of this enzyme of fragmented endoplasmic reticulum (“microsomes”), those of the intact membrane of isolated nuclei are totally, or nearly-totally, manifest without the need for preliminary activation by detergents or similar treatments. Disruption of nuclei and isolation of nuclear membranes results in the acquisition of detergent-sensitivity of such activities. Physiological implications of these observations are discussed.     

Ethylene Directly Inhibits Foliar Gas Exchange in Glycine max

Gas exchange of individual attached leaves of soybean, Glycine max (L.) Merr cv Davis, was monitored during exposure to exogenous ethylene (C₂H₄) to test the hypothesis that the effects of C₂H₄ on net photosynthesis (P_N) and stomatal conductance to H₂O vapor (g_s) are direct and not mediated by changes in leaf orientation to light. Leaflets were held perpendicular to incident light in a temperature-controlled cuvette throughout a 5.5 hour exposure to 10 microliters per liter C₂H₄. Declines in both P_N and g_s were evident within 2 hours and became more pronounced throughout the exposure period. In C₂H₄ treated plants, P_N and g_s decreased to 80 and 62%, respectively, of the rates in control plants. Because epinastic movement of the leaflets was prohibited by the cuvette, the observed declines in P_N and g_s were a direct effect of C₂H₄ rather than the result of reduced light interception caused by changing leaf angle.     

Synthesis in vitro of a seven amino acid peptide encoded in the leader RNA of Rous sarcoma virus

Sequences of avian retroviral RNAs suggest that short open reading frames in the putatively untranslated leader sequences might direct the synthesis of small peptides. Previous analyses indicate that translation of Rous sarcoma virus (RSV) RNA in vitro faithfully reflects translation of the viral RNA in the chick cell. Accordingly, we sought to determine if the heptapeptide LP1, encoded in the open reading frame closest to the 5' end of RSV RNA, could be synthesized in vitro since this would strongly suggest that it might also be synthesized in vivo. Here we confirm that RSV RNA directs the synthesis of LP1 in rabbit reticulocyte lysates. LP1 is rapidly degraded in the lysate by an aminopeptidase activity. On the basis of the following observations, we propose that the open reading frame encoding LP1 plays a role in the life cycle of avian retroviruses. The LP1 open reading frame is ubiquitous with respect to position and length in 12 strains of avian retrovirus. In the amino acid sequences of the 12 strains, only three of the seven residues are invariant. On the basis of the conservation of the -3 and +4 nucleotides flanking the AUG codon, the strengths of initiation for translation of LP1 are approximately the same in the different viruses. The LP1 open reading frame is positioned in front of sites on retrovirus RNA that are required for initiation of cDNA synthesis and for packaging of the RNA into mature virus.     

Immunochemical analysis of the structure of the signature domains of thrombospondin-1 and thrombospondin-2 in low calcium concentrations

Thrombospondins (TSPs) undergo conformational changes upon removal of calcium. The eight C-type and five N-type calcium-binding repeats of TSP-2 form a circuitous wire that, in 2 mm calcium, interacts at its ends with more N-terminal epidermal growth factor (EGF)-like modules, EGF2 and EGF3, and the C-terminal lectin-like module. These components, along with the other EGF-like module(s), form the signature domain of TSPs. Characterization of conformation-sensitive epitopes of monoclonal antibodies to human TSP-2 and its TSP-1 homolog have given insights into the structure of the signature domain in the absence of calcium. The epitope for 4B6.13 anti-TSP-2 was localized to His-722 and Leu-703 in repeat 1C of the wire; recognition only occurred in constructs that included EGF3, the rest of the wire, and the lectin-like module and in the presence of calcium. The epitope for C6.7 anti-TSP-1 was localized to Glu-609 in the EGF2 module. The C6.7 epitope was preferentially recognized when EGF2 was expressed in the context of EGF1, EGF3, the wire, and the lectin-like module. Preferential recognition of the C6.7 epitope did not require calcium. Rotary shadowing electron microscopy of TSP-1 has shown elongation of the stalk and diminution of the C-terminal globule. We propose a model whereby at low calcium concentrations the lectin-like module drops away from EGF3 concomitant with changes in conformation of the wire and loss of the 4B6.13 epitope. A critical feature of the model is interaction of repeat 12N of the wire with EGF2 in both the presence and absence of calcium.     

Power to detect risk alleles using genome-wide tag SNP panels

Advances in high-throughput genotyping and the International HapMap Project have enabled association studies at the whole-genome level. We have constructed whole-genome genotyping panels of over 550,000 (HumanHap550) and 650,000 (HumanHap650Y) SNP loci by choosing tag SNPs from all populations genotyped by the International HapMap Project. These panels also contain additional SNP content in regions that have historically been overrepresented in diseases, such as nonsynonymous sites, the MHC region, copy number variant regions and mitochondrial DNA. We estimate that the tag SNP loci in these panels cover the majority of all common variation in the genome as measured by coverage of both all common HapMap SNPs and an independent set of SNPs derived from complete resequencing of genes obtained from SeattleSNPs. We also estimate that, given a sample size of 1,000 cases and 1,000 controls, these panels have the power to detect single disease loci of moderate risk (λ ~ 1.8–2.0). Relative risks as low as λ ~ 1.1–1.3 can be detected using 10,000 cases and 10,000 controls depending on the sample population and disease model. If multiple loci are involved, the power increases significantly to detect at least one locus such that relative risks 20%–35% lower can be detected with 80% power if between two and four independent loci are involved. Although our SNP selection was based on HapMap data, which is a subset of all common SNPs, these panels effectively capture the majority of all common variation and provide high power to detect risk alleles that are not represented in the HapMap data.     

U1A inhibits cleavage at the immunoglobulin M heavy-chain secretory poly(A) site by binding between the two downstream GU-rich regions

The immunoglobulin M heavy-chain locus contains two poly(A) sites which are alternatively expressed during B-cell differentiation. Despite its promoter proximal location, the secretory poly(A) site is not expressed in undifferentiated cells. Crucial to the activation of the secretory poly(A) site during B-cell differentiation are changes in the binding of cleavage stimulatory factor 64K to GU-rich elements downstream of the poly(A) site. What regulates this change is not understood. The secretory poly(A) site contains two downstream GU-rich regions separated by a 29-nucleotide sequence. Both GU-rich regions are necessary for binding of the specific cleavage-polyadenylation complex. We demonstrate here that U1A binds two (AUGCN(1-3)C) motifs within the 29-nucleotide sequence and inhibits the binding of cleavage stimulatory factor 64K and cleavage at the secretory poly(A) site.