No abstract

Reduction and alkylation of protein disulfides prior to IEF, when performed directly in a centrifugal ultrafiltration device, provides an effective means of terminating the alkylation reaction, concentrating the proteins for analysis, and removing ionic impurities that interfere with IEF. When cells were lysed in "buffers" that support the activity of enzymes such as lysozyme and benzonase, the conductivity of the resulting lysate was an order of magnitude higher than when lysis was induced by chaotropic urea detergent solutions. Following reduction and alkylation, the conductivity of both lysates was lowered by ultrafiltration to the 0.1-0.2 mS/cm range in preparation for IEF. The detergent 3-(4-heptyl)phenyl 3-hydroxypropyl dimethylammonio propanesulfonate (C7BzO), which favors the solubilization of proteins, but which interferes with SDS equilibration and second dimension PAGE, was effectively removed by ultrafiltration and exchanged with CHAPS without measurable loss of protein. Disparate protein patterns of Rhodopseudomonas palustris lysates were revealed by two-dimensional gel electrophoresis depending on which reagent was used to induce cell lysis.

A common problem in the analysis of forensic human DNA evidence, or for that matter any nucleic acid analysis, is the presence of contaminants or inhibitors. Contaminants may copurify with the DNA, inhibiting downstream PCR or they may present samples effectively as containing fewer templates than exist in the PCR, even when the actual amount of DNA is adequate. Typically, these challenged samples exhibit allele imbalance, allele dropout, and sequence-specific inhibition, leading to interpretational difficulties. Lessening the effects of inhibitors may increase the effective yield of challenged low template copy samples. High pressure may alter some inhibitors and render them less effective at reducing the yield of PCR products. In an attempt to enhance the amplicon yield of inhibited DNA samples, pressure cycling technology was applied to DNA exposed to various concentrations of hematin (0, 1.25, 2.5, 5, and 7 μM) and humic acid (0, 1.25, 2.5, 5, and 7 ng/μL). The effect of high pressure on the inhibitors, and subsequently the PCR process, was assessed by measuring DNA quantity by quantitative PCR and evaluating short tandem repeat typing results. The results support that pressure cycling technology reduces inhibitory effects and thus, in effect, enhances yield of contaminated amplified products of both hematin and humic acid contaminate samples. Based on the results obtained in this study, this method can improve the ability to type challenged or inhibited DNA samples.

No abstract

No abstract

No abstract

The gene for the catalytic RNA subunit of RNase P has been isolated from several Enterobacteriaceae by complementation of an Escherichia coli strain that is temperature-sensitive for RNase P activity. The selection procedure relies on the ability of the heterologous gene products to function enzymatically in E. coli. This procedure obviates the need for positive results in DNA blot hybridization experiments or for the purification of holoenzyme to identify the RNA component of RNase P and its corresponding gene from organisms other than E. coli. Comparisons of the variations in sequences provide the basis for a refined two-dimensional model of the secondary structure of Ml RNA.RNase P is an enzyme essential for the formation of the 5' termini of tRNA molecules in Escherichia coli. It consists of a catalytic RNA subunit and a protein cofactor (1). RNase P-like activities have also been found in extracts of many organisms (2). Hybrid RNase P holoenzymes consisting of the catalytic RNA subunit from one organism and the protein cofactor from a different, distantly related organism can be formed in vitro (2, 3). This capability to form hybrid ribonucleoprotein enzyme can be the basis of a selection technique in a suitable host for genes from different organisms. Here we report a rapid method for the selection ofrnpB genes from the bacterial family Enterobacteriaceae, the family to which E. coli belongs. The method of selection depends on the ability of the product of a subcloned rnpB gene to complement (presumably by forming functional enzyme in vivo) E. coli FS101, a strain temperature-sensitive in RNase P activity. This method is generally applicable to the selection of any rnpB gene that can function in E. coli, irrespective of whether the appropriate DNA is capable of yielding a positive signal in a blot hybridization assay.A model has been proposed for the secondary structure of Ml RNA from E. coli that is based on a base-pairing scheme upon which additional constraints have been imposed by data from limited digestion with ribonucleases (4). This model has been refined to incorporate results from the sequence analysis of the rnpB gene from Salmonella typhimurium (5). To verify the structural features inferred from these data, it is advantageous to compare the sequences of genes (rnpB genes) encoding the analog to Ml RNA. For example, a model of the secondary structure for P RNA from Bacillus subtilis (the analog of Ml RNA) can be drawn with some features that are similar to the structural features proposed for Ml RNA (6). Comparisons of nucleotide sequences from homologous genes from diverse species have indeed been used to test the validity of models for the secondary structure of stable RNAs (7). Data from this study have revealed aspects of the transcriptional control regions of the genes coding for the analog to Ml RNA from various Enterobacteriaceae and have also been used to suggest alternative conformations for parts of the model of secondary structure of Ml RNA that was proposed by Guerrier-Takad...

No abstract