Buoyant density and hybridization analysis of human DNA sequences, including three satellite DNAs.

Total human DNA was fractionated from the three types of Cs2SO4 gradient used to prepare satellites I, II and III. Three satellite DNAs were found: satellite I with a mean buoyant density of 1.6888 g/ml comprising about 1.3% of the total, satellite II with a mean buoyant of 1.696 g/ml, comprising about 1% of the total an satellite III with a mean buoyant density of 1.699 g/ml comprising about 2.2% of the total. The buoyant densities of these satellites after purification were 1.686, 1.694 and 1.697 m/gl, respectively. A preparation with the attributes of satellite IV was isolated from the shoulder region of a satellite III preparative gradient. In situ hybridization using complementary RNA showed that the three satellites were located predominantly on chromosomes 9, Y, 15 and 1. Satellite II also showed marked hybridization to chromosome 16. Satellites I and II and III cross-hybridized to each other but satellites I and II did not. On the basis of our hybridization data, we suggest that some of the same sequences which comprise satellite III also comprise satellite I an II.

Elution, detection, and quantification of polio I, bacteriophages, Salmonella montevideo, and Escherichia coli O157:H7 from seeded strawberries and tomatoes.

This study compared the effect of different physical and chemical treatments of strawberries and tomatoes to determine their ability to recover seeded viral and bacterial pathogens from produce surfaces. Solutions of salts, amino acids, complex media, and detergents were compared as eluants. Phosphate-buffered saline (PBS) containing 0.1% Tween 80 eluted the highest number of seeded microorganisms. Elution with this defined solution was then compared under different conditions of physical agitation. Rotary shaking for 20 min at 36 degrees C eluted higher numbers of viruses and bacteria than did low- or high-speed stomaching. Commercially available and laboratory prepared bacteriological differential media were compared for their ability to recover and distinguish eluted Salmonella Montevideo and Escherichia coli O157:H7 strains from seeded produce. The recovery of seeded bacterial pathogens was low when differential media containing selective ingredients were used (MacConkey sorbitol agar, XLD agar, MacConkey agar). Highest recoveries were obtained on a medium consisting of tryptic soy agar supplemented with sodium thiosulfate and ferric ammonium citrate compared with selective media that inhibited up to 50% of the growth of the eluted microorganisms.

Photoaffinity labeling of creatine kinase with 2-azido- and 8-azidoadenosine triphosphate: identification of two peptides from the ATP-binding domain.

Two different analogs of ATP, [gamma-32P]2N3ATP and and [gamma-32P]8N3ATP, were used to photoaffinity label the MM and BB isoforms of rabbit cytosolic creatine kinase. Evidence that photoinsertion was within the ATP-binding domain was as follows: (1) Assays for creatine phosphate production demonstrated that [gamma-32]2N3ATP and [gamma-32P]8N3ATP are substrates for creatine kinase. (2) Enzymatic activity was inhibited by photolabeling with either analog. (3) Saturation of photoinsertion was observed for both analogs. Half-maximal saturation was observed at 5 microM [gamma-32P]2N3ATP or 12 microM (gamma-32P]8N3ATP. (4) Photoinsertion of both probes could be decreased by micromolar levels of ATP. Immobilized Al3+ affinity chromatography and HPLC were used to isolate the peptides modified by these probes. Overlapping sequence analysis of the isolated peptides from the tryptic and chymotryptic digests of the photolabeled MM isoform revealed that [gamma-32P]8N3ATP photoinserted into the peptide region corresponding to Val279-Arg291, whereas [gamma-32P]2N3-ATP photoinserted into Val236-Lys241. The corresponding peptide (Ile279-Arg291 and Val236-Lys241) from the BB isoform were shown to be selectively modified. We conclude that amino acid residues within the peptide regions 236-241 and 279-291 of rabbit cytosolic creatine kinase are localized within the binding domain for the adenine moiety of ATP. The results also demonstrate the effectiveness and selectivity of Al3+ as the chelating agent in immobilized metal affinity chromatography for the isolation of photolabeled peptides as well as its potential to enhance retention of radiolabel during HPLC.

PEN2 is not a genetic risk factor for Alzheimer's disease in a large family sample.

PEN2 is a reasonable Alzheimer's disease (AD) candidate gene because it is a necessary component of the gamma-secretase complex that generates beta-amyloid peptide. Moreover, its gene (PEN2) maps to a highly significant linkage region on chromosome 19q13. Four common polymorphisms in PEN2 were tested for genetic association with AD in a large and carefully ascertained AD family sample (789 subjects from 202 nuclear families) using single-locus and haplotype-based analyses. These results do not suggest PEN2 to be a major AD risk factor.