Human immunodeficiency virus type 1 gag-pol frameshifting is dependent on downstream mRNA secondary structure: demonstration by expression in vivo.

The human immunodeficiency virus type 1 (HIV-1) Gag-Pol fusion polyprotein is produced via ribosomal frameshifting. Previous studies in vitro and in Saccharomyces cerevisiae have argued against a significant role for RNA secondary structure 3' of the shift site, in contrast with other systems, in which such structure has been shown to be required. Here we show, by expressing the HIV-1 gag-pol domain in cultured vertebrate cells, that a stem-loop structure 3' of the HIV-1 shift site is indeed important for wild-type levels of frameshifting in vivo.     

Immobilization and characterization of D-amino acid oxidase.

Optimal conditions with respect to pH, concentration of glutaraldehyde and enzyme, and order of addition of enzyme and crosslinking reagent were established for the immobilization of hog kidney D-amino acid oxidase to an attapulgite support. Yields of 40 to 70% were generally attained although when low concentrations of enzyme were used yields were consistently greater than 100%. It is suggested that this is due to a dimer leads to monomer shift at low protein concentrations. The stability of soluble D-amino acid oxidase was dependent on the buffer in which it was stored (pyrophosphate-phosphate greater than borate greater than Tris). Stability of immobilized enzyme was less than soluble in pyrophosphate-phosphate buffer, but storage in the presence of FAD improved stability. In addition, treatment of stored, immobilized enzyme with FAD before assay restored some of its activity. The immobilized D-amino acid oxidase was less stable to heat (50 degrees C) than the soluble enzyme from pH 6 to 8 but was more stable above and below these values. Apparent Km values for D-alanine, D-valine, and D-tryptophan decreased for the immobilized enzyme compared to the soluble.     

Inhibition of bacterial perchlorate reduction by zero-valent iron

Perchlorate was reduced by a mixed bacterial culture over a pH range of 7.0–8.9. Similar rates of perchlorate reduction were observed between pH 7.0 and 8.5, whereas significantly slower reduction occurred at pH 8.9. Addition of iron metal, Fe(0), to the mixed bacterial culture resulted in slower rates of perchlorate reduction. Negligible perchlorate reduction was observed under abiotic conditions with Fe(0) alone in a reduced anaerobic medium. The inhibition of perchlorate reduction observed in the presence of Fe(0) is in contrast to previous studies that have shown faster rates of contaminant reduction when bacteria and Fe(0) were combined compared to bacteria alone. The addition of Fe(0) resulted in a rise in pH, as well as precipitation of Fe minerals that appeared to encapsulate the bacterial cells. In experiments where pH was kept constant, the addition of Fe(0) still resulted in slower rates of perchlorate reduction suggesting that encapsulation of bacteria by Fe precipitates contributed to the inhibition of the bacterial activity independent of the effect of pH on bacteria. These results provide the first evidence linking accumulation of iron precipitates at the cell surface to inhibition of environmental contaminant degradation. Fe(0) was not a suitable amendment to stimulate perchlorate-degrading bacteria and the bacterial inhibition caused by precipitation of reduced Fe species may be important in other combined anaerobic bacterial–Fe(0) systems. Furthermore, the inhibition of bacterial activity by iron precipitation may have significant implications for the design of in situ bioremediation technologies for treatment of perchlorate plumes.     

Within- and between-individual sequence variation among ITS1 copies in the meadow grasshopper Chorthippus parallelus indicates frequent intrachromosomal gene conversion

Sequencing multiple copies of the ITS1 region revealed the coexistence of two or more haplotypes within the genome of Chorthippus parallelus. Using a PCR-RFLP approach, the ITS1 numbers and frequencies of haplotypes present in each of 40 individuals were investigated, revealing a consistent lack of homogeneity. For each individual, the level of intra-individual variation was estimated from a sample of 20 ITS1 copies. The level of differentiation in haplotype frequency among individuals was then estimated by maximum likelihood using models based on the Dirichlet distribution. This confirmed the existence of significant levels of variation among individuals within each population studied. The most likely turnover mechanism that could generate this pattern of variation is gene conversion, operating at the intrachromosomal level. Furthermore, the discovery of linkage disequilibrium among the ITS1 haplotypes of C. parallelus suggests that intrachromosomal gene conversion occurs more frequently than interchromosomal recombination. Subspecies of C. parallelus showed significantly different haplotype distributions following about 0.5 Myr of divergence. With respect to the process of concerted evolution, we show that homogenization of repeats is slow relative to speciation, and the standing variation among individuals is sufficient for selection to operate.     

Homoeologous exchange is a major cause of gene presence/absence variation in the amphidiploid Brassica napus

Homoeologous exchanges (HEs) have been shown to generate novel gene combinations and phenotypes in a range of polyploid species. Gene presence/absence variation (PAV) is also a major contributor to genetic diversity. In this study, we show that there is an association between these two events, particularly in recent Brassica napus synthetic accessions, and that these represent a novel source of genetic diversity, which can be captured for the improvement of this important crop species. By assembling the pangenome of B. napus, we show that 38% of the genes display PAV behaviour, with some of these variable genes predicted to be involved in important agronomic traits including flowering time, disease resistance, acyl lipid metabolism and glucosinolate metabolism. This study is a first and provides a detailed characterization of the association between HEs and PAVs in B. napus at the pangenome level.     

The Amyloid Precursor Protein Is Not Enriched in Caveolae-Like, Detergent-Insoluble Membrane Microdomains

Abstract: The amyloid precursor protein may be processed by several different pathways, one of which produces the amyloid β-peptide βA4 present in the amyloid plaques characteristic of Alzheimer's disease. A recent report suggested that axonal-amyloid precursor protein is present in a membrane fraction "with caveolae-like properties." In the present study we have isolated detergent-insoluble, caveolae-like membranes from both mouse cerebellum and the human neuroblastoma cell line SH-SY5Y. Detergent-insoluble membranes from mouse cerebellum retained nearly all of the glycosylphosphatidylinositol-anchored proteins—alkaline phosphatase, 5'-nucleotidase, and the F3 protein—while excluding the majority of the plasmalemmal marker protein alkaline phosphodiesterase I. Although the inositol trisphosphate receptor was highly enriched in this detergent-insoluble fraction, neither amyloid precursor protein nor clathrin immunoreactivity could be detected. Similar results were obtained with SH-SY5Y cells, where 5'-nucleotidase activity was enriched at least 30-fold in the detergent-insoluble membranes, but no amyloid precursor protein or clathrin immunoreactivity could be detected. Caveolin could not be detected in microsomal membranes from either mouse cerebellum or SH-SY5Y cells. These observations suggest that amyloid precursor protein is not normally present in detergent-insoluble, caveolae-like membrane microdomains.