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A study was conducted to analyze the development and partial characterization of turkey genomic libraries enriched for CCT, TG and GAT simple sequence repeats. The primary library was created using standard techniques. It was then improved for each of the three simple sequence repeats by single-primer polymerase chain reaction. Results indicated that simple sequence repeats are abundant in the genomes of turkeys.

In a recent study, we showed how novel stem-loop DNA probes on dendron-modified aldehyde substrates could be used to detect synthetic nucleic acid targets without amplification. In this article, we demonstrate the application of stem-loop DNA probes as arrays for the detection of specific families and genera of methane-producing bacteria from sludge samples harvested from an anaerobic digester using 16S ribosomal RNA (rRNA) signatures. Specific 16S rRNA could be detected in samples that had 0.2ng/μl total sludge RNA without any target amplification., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Specificity and sensitivity are important factors affecting DNA microarrays. Stem-loop DNA probes (SLPs) can be more specific in their recognition of target sequences than linear DNA probes, but unless they are carefully designed, surface interactions can disrupt the native stem-loop structure. In this study, we show how dendron-modified surfaces with well-defined, uniform spacing of aldehyde chemical functionalities offer an ideal substrate to immobilize SLPs and use them to detect nucleic acid targets. The mesospacing provided by the dendron-modified surfaces produces a solution-like environment that allows the SLPs to detect target nucleic acids at concentrations as low as 1pM in concentration., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The gene expression profile in rat brain was examined using microarrays in rats fed lithium chloride for 7 days (subacute) or 42 days (chronic). Brain lithium concentrations were 0.39 mM and 0.79 mM (therapeutically relevant), at 7 and 42 days, respectively. Of the 4132 genes represented in the microarrays, 25 genes were downregulated by at least twofold and none was upregulated after 7 days of treatment. Expression of 50 genes was downregulated by at least two-fold at 42 days, without any being upregulated. Lithium treatment for 7 days did not affect at a measurable extent expression of 37 of the 50 genes that were downregulated at 42 days. Genes whose expression was changed at 42 days coded for a number of receptors, protein kinases, transcription and translation factors, markers of energy metabolism, and signal transduction. Thus, chronic lithium at a therapeutically relevant concentration reduced expression of a large number of genes involved in multiple signaling and other pathways, without increasing expression at a comparable extent.

Background: A method that provides standardized data and is relatively inexpensive and capable of high throughput is a prerequisite to the development of a meaningful gene expression database suitable for conducting multi-institutional clinical studies based on expression measurement. Standardized RT (StaRT)-PCR has all these characteristics. In addition, the method must be reproducible. StaRT-PCR has high intralaboratory reproducibility. The purpose of this study is to determine whether StaRT-PCR provides similar interlaboratory reproducibility., Methods and Results: In a blinded interlaboratory study, expression of ten genes was measured by StaRT-PCR in a complementary DNA sample provided to each of four laboratories. The average coefficient of variation for interlaboratory comparison of the nine quantifiable genes was 0.48. In all laboratories, expression of one of the genes was too low to be measured., Conclusion: Because StaRT-PCR data are standardized and numerical and the method is reproducible among multiple laboratories, it will allow development of a meaningful gene expression database.

A physical map internal to the markers DXS1368 and DXS228 was developed for the p11.4 region of the human X chromosome. Twenty-four BACs and 10 PACs with an average insert size of 149 kb were aligned to form a contig across an estimated 1.4 Mb of DNA. This contig, which has on average fourfold clone coverage, was assembled by STS and EST content analysis using 46 markers, including 8 ESTs, two retinally expressed genes, and 22 new STSs developed from BAC- and PAC-derived DNA sequence. The average intermarker distance was 30 kb. This physical map provides resources for high-resolution mapping as well as suitable clones for large-scale sequencing efforts in Xp11.4, a region known to contain the gene for complete X-linked congenital stationary night blindness., (Copyright 2000 Academic Press.)

Background: The pigmentary purpuras (PPs) are a heterogeneous group of dermatoses defined by specific clinicopathologic features but sharing, at the light microscopic level, superficially disposed dermal lymphocytic infiltrates and hemorrhage. The term atypical pigmentary purpura (APP) is used by the authors in reference to cases of PP in which individual lesions, although clinically presenting as PP, show morphological features typically associated with mycosis fungoides (MF) including Sezary cells and epidermotropism. The integrated concept of lymphocyte atypia and PP is a confusing and enigmatic one to which reference in the literature has been previously made. Specifically, there are reports of PP presaging fully evolved MF, lymphoid atypia has been identified in lesions of routine PP and MF with purpuric features has been described. The clinical, light microscopic, and genomic features of biopsied lesions showing pathological features of APP and which clinically were consistent with PP is explored., Design: The light microscopy of skin biopsy specimens from 34 patients with a pathological diagnosis of APP was correlated to medical and drug histories. In 14 cases, adequate tissue was present in the paraffin blocks to allow DNA extraction. The polymerase chain reaction (PCR) was used in these 14 cases to explore for rearrangement of the T-cell receptor. Fisher's exact test and pair wise exact tests were used to assess the significance of histological differences between cases determined by dinical features to be of MF- or drug-related origin, or to be idiopathic in nature., Results: Of 34 patients, 7 were held to have MF related PP; specifically these patients had violaceous, infiltrative, variably purpuric plaques on trunk, buttocks, and thighs accompanied by typical PP lesions which occurred either concomitant to or preceded the MF lesions. In 10 cases, a diagnosis of idiopathic PP was made whereby the clinical presentation was characteristic of PP; there were no concomitant lesions suspicious for MF and a drug-based origin was excluded. A drug-based origin was established in 17 patients based on lesional onset related to initiation (5 patients) and/or resolution after discontinuation (12 patients) of drugs including calcium channel blockers, lipid-lowering agents, beta-blockers, angiotensin-converting enzyme (ACE) inhibitors, antihistamines, antidepressants, or analgesics. There was considerable overlap histologically between all 3 groups including the degree of lymphoid atypia in the dermis, the presence of dermal-based Sezary cells, the degree and pattern of epidermotropism, the paucity of other inflammatory cell elements, and the presence of laminated dermal sclerosis. Morphological features predictive of MF related APP over the other 2 groups were intraepidermal lymphocytes which were more atypical than the dermal-based infiltrate. Intraepidermal Sezary cells were less frequent in biopsies of drug-related APP relative to idiopathic PP (IPP) and MF related PP. PCR studies conducted in 14 cases (2 cases of MF, 6 cases of drug-related APP, and 6 cases of IPP) revealed clonality in 2 cases of drug-related APP and 2 cases of IPP; the 2 studied MF-related cases did nor show clonal restriction., Conclusion: APP should not be equated with purpuric MF; it is not necessarily a precursor lesion of MF, and may be of drug-based origin. Clinical features are critical to the final assessment because there is overlap pathologically in the 3 clinical subtypes of APP.

To generate sequence-ready templates for the gene-rich Xp11.23 region, we have constructed a 1.5-Mb bacterial artificial chromosome (BAC) contig spanning the interval between the DNA markers OATL1 and DXS255. The contig includes 28 BACs, ranging in size from 58 to 258 kb with an average size of 135 kb, which provide 2.5-fold coverage of the region. The BAC contig was constructed based entirely on the content of 40 DNA markers from a previously established YAC contig and 11 new markers developed from BAC-end DNA sequences, 4 of which were required to close gaps in the map. There was no evidence of rearrangement, instability, or chimerism in any of the BAC clones. The BAC cloning system appears to provide robust and total physical coverage of this gene-rich region with clones that are suitable for DNA sequencing.

Cloned cDNAs for the human mitochondrial DNA polymerase gamma (POLG) were identified by homology with the yeast mitochondrial DNA polymerase catalytic subunit (MIP). Fluorescence in situ hybridization (FISH) of human and mouse bacterial artificial chromosomes (BACs), hybridized by radioactively labeled POLG cDNAs, mapped to human chromosome band 15q24-->q26, as well as to mouse chromosome band 7E. Direct sequencing of the BAC DNA without subcloning confirmed the presence of both human POLG and mouse mitochondrial DNA polymerase gamma (Polg) in the respective BACs.

Pseudomonas cepacia colonization of the lung is associated with increased morbidity and mortality for cystic fibrosis (CF) patients. The lack of a sensitive detection method for Pseudomonas cepacia in CF sputum has resulted in controversy regarding its epidemiology. We designed a PCR method to detect P. cepacia using P. cepacia 16 S rRNA sequences as the amplification target region. The PCR amplification with purified DNA as template yielded the expected 209-bp products from P. cepacia, but not from related Pseudomonas species of medical importance or other bacteria which have been reported to colonize CF patients. In serial dilution experiments as few as 10(2) P. cepacia CFU were detectable. When sputum samples from three CF patients chronically colonized with P. cepacia and P. aeruginosa were analyzed, P. cepacia was detected in all three specimens by PCR, but only in two when selective culture was performed. Our data support the potential role of PCR technology in the rapid, sensitive, and definitive detection of P. cepacia in CF sputum samples, even in the context of concomitant P. aeruginosa colonization.

Lcr-plasmids of yersiniae are known to mediate a unique low calcium response characterised by restriction of growth in vitro with induction of putative virulence factors including yersiniae outer membrane-peptides (YOPs) and V antigen (Lcr+). A medium was developed that permitted expression of high yields of V by Yersinia pestis KIM in large fermenter vessels. Immunoblots of specific precipitates prepared by prior molecular sieving showed that native unaggregated V exists as a monomeric 37,000 dalton peptide. Fractionation by precipitation with (NH4)2SO4 and chromatography on phenyl-Sepharose, DEAE cellulose, Sephacryl S200, calcium hydroxyapatite, and Sephadex G200 yielded highly purified antigen as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of parallel preparations from Lcr+ and Lcr- yersiniae. However, yields of V obtained by this process were unexpectedly low. As determined from immunoblots with monospecific polyclonal and monoclonal anti-V, this loss of activity occurred as a function of evident degradation at every step of purification yielding antigenic fragments of about 36,000, 34,000, 31,000, 30,000, and 28,000 daltons. Neutral or acidic pH favored hydrolysis; insignificant cleavage occurred in viable Lcr+ cells of Y. pestis or in culture supernatant fluids. V in neutral cytoplasm from Yersinia pseudotuberculosis or Yersinia enterocolitica did not undergo comparable degradation.

Age and do-not-resuscitate (DNR) orders were experimentally manipulated with a 2 X 2 factorial design using four vignettes which were randomly assigned to 95 staff nurses from four sites in a mid-Atlantic metropolitan area. Attitudes toward aggressiveness of nursing care were measured using the same 13-item 6-point Likert scale with all vignettes. Two replications with 183 nursing students and 86 intensive care nurses from six sites followed. Both increased age and DNR orders significantly, p less than .05 and .01, reduced aggressiveness of nursing care attitudes in all three studies. However, attitudes toward care still remained in the moderately aggressive range, which is more aggressive than current patient classification systems describe for DNR patient care.