Single-strand Conformation Polymorphism DNA Research Articles

Major Histocompatibility Complex gene Polymorphism in Systemic Lupus Erythematosus Patients

The major histocompatibility complex (MIC) is one of the MHC class I related genes which related withautoimmune diseases , the present study aims to investigated association between MIC gene polymorphismclass I in systematic lupus erythrymatous patients within Hilla city, the present study implemented usingwhole blood genomic DNA and single strand DNA conformation polymorphism (SSCP) for detection genepolymorphisms and haplotypes frequency, The results of present study show that 183 bp of MIC gene and ithas three haplotypes (A, B, and C) each of these haplotypes consist of two bands, non-significant differencesbetween patient and control in haplotypes frequency, B haplotype was more frequent in patinas than others(20%) while A was more frequents in control than others (22.8%). The present study concluded that MICgene may be contributed in immune response of SLE and can be used in pre diagnosis of disease. This studywas one of the serious investigators deal with several immune genes in this disease in Iraqi SLE.

Single nucleotide polymorphisms of insulin-like growth factor gene and mammographic breast density.

Aim of the studyTo analyze six single nucleotide polymorphisms (SNPs): rs1520220, rs2945834, rs5747694, rs6214, rs6219, rs5742678. An attempt was made to assess the significance of the above IGF-1 gene polymorphisms as prognostic and predictive factors in Polish women with diagnosed increased breast mammographic density.Material and methodsThe study included women diagnosed with an increased breast mammographic density (n = 98), breast cancer (n = 135) and women as a control group (n = 60). The method used to detect polymorphisms in the IGF-1 gene was the analysis of single-stranded DNA conformation polymorphism (SSCP-PCR) and Sanger’s sequencing.ResultsIn the case of rs1520220 polymorphism, the genotype CC was found to increase the risk of breast cancer (OR = 2.6 95% CI 1.01-6.5, p = 0.04). Analysis of the rs2945834 polymorphism revealed that the occurrence of the G allele reduced the risk of breast cancer, while the occurrence of the A allele increased the risk of disease almost twice (OR = 0.55 95% CI). Among women who are heterozygous in terms of rs5747694 polymorphism (TG), the risk of breast cancer is twice as high as in the control group. The SNPs in the study group did not correlate with mammographic breast density.ConclusionsThe results obtained in the course of the analysis indicate that polymorphisms rs1520220, rs2946834, rs5747694 gene IGF-1 are associated with the occurrence of breast cancer but not with increased mammographic density. Summing up, the association between the polymorphisms of IGF-1 and the risk of developing breast cancer is independent of mammographic density.

Effective screen of CRISPR/Cas9-induced mutants in rice by single-strand conformation polymorphism.

A method based on DNA single-strand conformation polymorphism is demonstrated for effective genotyping of CRISPR/Cas9-induced mutants in rice. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) has been widely adopted for genome editing in many organisms. A large proportion of mutations generated by CRISPR/Cas9 are very small insertions and deletions (indels), presumably because Cas9 generates blunt-ended double-strand breaks which are subsequently repaired without extensive end-processing. CRISPR/Cas9 is highly effective for targeted mutagenesis in the important crop, rice. For example, homozygous mutant seedlings are commonly recovered from CRISPR/Cas9-treated calli. However, many current mutation detection methods are not very suitable for screening homozygous mutants that typically carry small indels. In this study, we tested a mutation detection method based on single-strand conformational polymorphism (SSCP). We found it can effectively detect small indels in pilot experiments. By applying the SSCP method for CRISRP-Cas9-mediated targeted mutagenesis in rice, we successfully identified multiple mutants of OsROC5 and OsDEP1. In conclusion, the SSCP analysis will be a useful genotyping method for rapid identification of CRISPR/Cas9-induced mutants, including the most desirable homozygous mutants. The method also has high potential for similar applications in other plant species.

An approach to DNA polymorphism screening in SBEIIa homeologous genes of polyploid wheat (Triticum L.)

The non-transgenic manipulation of starch properties in common wheat (Triticum aestivum L.) generally implies combining mutant alleles of the particular gene copies in all three subgenomes (A, B and D). The redundancy of the hexaploid wheat chromosome set substantially complicates the identification of recessive mutations and breeding. Nevertheless, naturally occurring or induced genetic polymorphism has already been successfully exploited for the production of waxy (GBSSI-deficient) and elevated amylose (SSIIa-deficient) wheats. However, in order to achieve the amylose content above 50% of wheat endosperm starch, it may be necessary to inactivate the starch branching enzyme (SBEIIa) isoforms, as the RNAi repression results and gene expression data strongly suggest. The identification of null SBEIIa alleles and their combination in a single genotype is therefore a promising approach to the production of non-transgenic high-amylose wheat; however, wheat SBEIIa polymorphism has not been characterized as of yet. In order to develop an approach to SBEIIa mutation screening, we sequenced the SBEIIa central region (exons 9–12) from the three subgenomes of common wheat cv. Chinese Spring and the A genome of diploid einkorn T. monococcum. The genome-specific primers were developed that amplify the exons downstream from intron 11 selectively from each homeologous gene. Using a single-stranded DNA conformation polymorphism (SSCP) approach, we screened 60 wheat cultivars, landraces, and rare species for naturally occurring SNPs in exons 12, 13 and 14 of the three SBEIIa homeologs. In total, 13 SNPs were discovered in the A and B wheat genomes. Two of these SNPs affect the amino acid sequences of SBEIIa isoforms and may change the enzyme functional properties. The presence of restriction site polymorphism at SNP positions enables their easy genotyping with CAPS assays. Our results indicate that the mining for naturally occurring sequence polymorphism in starch biosynthesis genes of wheat can be successfully performed at the DNA level, providing the starting point for a search for SBEIIa mutants at a larger scale.

Genetic structure of the giant kelp Macrocystis pyrifera along the southeastern Pacific

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 420:103-112 (2010) - DOI: https://doi.org/10.3354/meps08893 Genetic structure of the giant kelp Macrocystis pyrifera along the southeastern Pacific E. C. Macaya1,2,*, G. C. Zuccarello1 1School of Biological Sciences, Victoria University of Wellington, PO Box 600, Wellington, New Zealand 2Present address: Departamento de Oceanografía, Facultad de Ciencias Naturales y Oceanográficas, Universidad de Concepción, Casilla 160-C, Concepción, Chile *Email: erasmomacaya@udec.cl ABSTRACT: We assessed the genetic structure of the giant kelp Macrocystis pyrifera across a broad latitudinal range along the southeastern Pacific coast (SEP). Specifically we analyzed the concordance of putative biogeographic breaks with genetic discontinuities and the effect of historical and contemporary events on the genetic pattern of this important seaweed. Mitochondrial DNA and single-strand DNA conformation polymorphism (SSCP) analysis for a total of 730 samples were carried out. Only 5 haplotypes were found among individuals collected along 4800 km of coastline, with very low haplotype diversity and a shallow genealogy compared with other macroalgal species. Some phylogeographic disjunctions in M. pyrifera were found to correspond roughly to established biogeographic breaks. On the southern coast we found a genetic break at 42°S (Chiloé Island) coincident with a well-known biogeographic boundary, while the genetic break found between samples in central/northern Chile (33°S) does not correspond to any known biogeographic breaks in other brown algae, but does reflect a break associated with other marine taxa. The low genetic diversity in northern Chile may be related to contemporary events (e.g. El Niño Southern Oscillation) while in southern Chile the haplotype distribution may reflect the effect of historical events (Last Glacial Maximum; LGM). Additionally, we compared the SEP data with samples from some of the subantarctic islands and New Zealand. The results showed shared haplotypes among some of the subantarctic islands and southern-central Chile, suggesting a recent colonization of the subantarctic region. The high dispersal potential of kelp rafts may also help to explain the low genetic diversity observed. We conclude that both present and historic events are responsible for the genetic structure of M. pyrifera along the SEP. KEY WORDS: Dispersal · Biogeography · ENSO · Kelp · Macrocystis pyrifera · Mitochondrial DNA Phylogeography · Southeastern Pacific · Subantarctic Full text in pdf format PreviousNextCite this article as: Macaya EC, Zuccarello GC (2010) Genetic structure of the giant kelp Macrocystis pyrifera along the southeastern Pacific. Mar Ecol Prog Ser 420:103-112. https://doi.org/10.3354/meps08893 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 420. Online publication date: December 16, 2010 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2010 Inter-Research.

Upgrading SELEX Technology by Using Lambda Exonuclease Digestion for Single-Stranded DNA Generation

The generation of single-stranded DNA (ssDNA) molecules plays a key role in the SELEX (Systematic Evolution of Ligands by EXponential enrichment) combinatorial chemistry process and numerous molecular biology techniques and applications, such as DNA sequencing, single-nucleotide polymorphism (SNP) analysis, DNA chips, DNA single-strand conformation polymorphism (SSCP) analysis and many other techniques. The purity and yield of ssDNA can affect the success of each application. This study compares the two ssDNA production methods, the strand separation by streptavidin-coated magnetic beads and alkaline denaturation and the lambda exonuclease digestion, in regard to the purity of generated ssDNA and the efficiency. Here, we demonstrate the considerable benefits of ssDNA production by lambda exonuclease digestion for in vitro selection of DNA aptamers. We believe that the generation of ssDNA aptamers using this method will greatly improve the success rate of SELEX experiments concerning the recovery of target-specific aptamers.

Identification of a Critical Novel Mutation in the Exon 1 of Androgen Receptor Gene in 2 Brothers With Complete Androgen Insensitivity Syndrome

Complete androgen insensitivity syndrome is an X-linked inherited disorder caused by mutations in the androgen receptor (AR) gene. Using polymerase chain reaction single-strand DNA conformational polymorphism and DNA sequencing, we identified a novel nonsense mutation in exon 1 of the AR gene in 2 Iranian brothers with complete androgen insensitivity syndrome. Despite a normal 46,XY karyotype, testes, and normal to elevated plasma levels of testosterone, they were born with female external genitalia and phenotype. This new mutation, a T-to-A transversion in exon 1, causes amino acid change of tyrosine (TAT) to ochre stop codon (TAA) at position 514 of the AR polypeptide. The Y514X mutation is located in a region that is normally important for the formation and function of the hormone receptor complex. We conclude that the novel Y514X mutation in the androgen receptor is the cause of complete androgen insensitivity syndrome in this family.

Multilocus Allele Polymorphism in Different Streptococcus Thermophilus Strains Isolated from Yoghurts

ABSTRACTTwenty-one Streptococcus thermophilus strains were isolated form homemade yoghourts obtained from different areas in Bulgaria. The isolated strains were determined by polyphase analysis including classical microbiological, biochemical, physiological, as well as molecular methods including PCR amplification with genus-specific primers and sequencing of the genes responsible for 16S ribosomal RNA. A recently proposed new method for intraspecies discrimination based on single strand DNA conformation polymorphism was applied for the first time on the Streptococcus genus representatives.

Loss of blood group A antigen expression in bladder cancer caused by allelic loss and/or methylation of the ABO gene

Loss of ABO blood group antigen expression has been reported in transitional cell carcinoma (TCC) of the bladder. Synthesis of the ABO blood group antigen was genetically determined by allelic variants of the ABO gene assigned on 9q34.1. We analyzed loss of heterozygosity (LOH) and promoter hypermethylation of the ABO gene in TCC and compared them with alterations of A antigen expression in TCC, dysplasia and normal urothelium. A total of 81 samples of TCC of the bladder obtained from transurethral resection (TUR) (n=44) and radical cystectomy (n=37) were examined. Expression of the A antigen was evaluated by immunohistochemical staining (IHC) using anti-A antigen monoclonal antibody. LOH of the ABO gene locus was examined by blunt-end single-strand DNA conformational polymorphism (SSCP) analysis using flouresence-based auto sequencer. Promoter hypermethylation of the ABO gene were examined by bisulfite PCR-SSCP (BiPS) analysis and/or methylation-specific PCR (MSP). Loss of A allele and/or hypermethylation were significantly associated with abnormal expression of the A antigen in cases undergoing TUR (P=0.02) and radical cystectomy (P=0.0005). For the analysis of the concomitant dysplasia in 23 cases with TCC of the bladder, the expression of the A antigen was maintained, regardless of the A allelic loss or methylation status in the tumor. In conclusion, A allelic loss and hypermethylation in the promoter region of the ABO gene showed significant correlation with reduction of A antigen expression in TCC, while the expression of the A antigen is maintained in concomitant dysplasia or normal urothelium, suggesting that loss of the ABO gene and/or its promoter hypermethylation is a specific marker for TCC.

Patients (pts) with acute myeloid leukemia (AML) and mutant RAS benefit from high-dose cytarabine (HDAC) intensification: A Cancer and Leukemia Group B (CALGB) study

6514 Background: Mutations in RAS genes occur in 15–25% of AML pts. In vitro data suggest RAS mutations enhance sensitivity to cytarabine (Ara-C). We examined whether RAS mutation status impacts response to Ara-C in vivo. Methods: 110 AML pts enrolled on CALGB 8525 and randomized to 4 cycles of 1 of 3 consolidation doses of i.v. Ara-C after achieving complete remission were analyzed for N- and K-RAS mutations by single-strand DNA conformation polymorphism and direct sequencing analyses. Results: 18 pts (16%) had mutated RAS (mutRAS). They were less often Caucasian (P=.02) and had lower % BM blasts (P=.02), but similar presenting age, sex, platelets, leukocyte count, % blood blasts, cytogenetic groups [core binding factor (CBF) vs others] and FAB subtypes to wild-type RAS (wtRAS) pts. With 11.1 yrs median follow-up, cumulative incidence of relapse (CIR) was lower with CBF AML (P=.004), consolidation with HDAC (400 mg/m2/d x 5d or 3g/m2 q12h on days 1, 3 and 5) vs low-dose Ara-C (LDAC) (100 mg/m2/d x 5d) (P=.003), but not with RAS status (P=.36). MutRAS pts assigned to HDAC had lowest likelihood of relapse (Table). Multivariable analysis to predict CIR confirmed a significant interaction of consolidation therapy and RAS status (P=.013). MutRAS pts receiving HDAC were less likely to relapse than mutRAS pts treated with LDAC and wtRAS pts treated with LDAC or HDAC. After controlling for this interaction, CBF (vs others, P=.003), HDAC (vs LDAC, P=.07), and wtRAS (vs mutRAS, P=.051), were all important predictors of lower CIR. Conclusion: AML pts carrying mutRAS benefit from higher Ara-C doses more than pts with wtRAS and, similarly to CBF AML pts, should receive consolidation with HDAC. No significant financial relationships to disclose.

Pilot study of arbitrarily primed PCR-single stranded DNA conformation polymorphism analysis for screening genetic polymorphisms related to specific phenotypes

Background To investigate relationships between phenotypes and genotypes is not simple. We propose a phenotype-to-genotype screening strategy and pooled DNA system. As a pilot study of this strategy, we used arbitrarily primed polymerase chain reaction (AP-PCR) in combination with single-stranded DNA conformation polymorphism (SSCP) to screen for genetic polymorphisms associated with longevity. Methods Study subjects were separated into 3 age groups, individuals aged >100 years, 90–99 years and 60–69 years. Genomic DNAs were prepared from each individual, pooled to represent the 5 study groups, and then the pooled genomic DNAs were subjected to AP-PCR-SSCP analysis. Results We found 1 SNP more frequently in senior citizens with longevity. The genotype frequency of the 82133G>A polymorphism of human chromosome 3 clone RP11-61K12 (AC011199) differed significantly ( P=0.0189, Fisher's exact test) between older subjects (>90 years) and younger subjects (<70 years). It is noteworthy that the strategy we describe herein was useful for identifying an SNP that showed statistically significant differences in its distribution across the subject groups. Conclusions The pooled DNA strategy and quantitative genotype discrimination can also be applied to screening for the relationship between phenotype and genotype more effectively.

Assessment of T-cell Clonality via T-cell Receptor-γ Rearrangements in Cutaneous T-cell–Dominant Infiltrates Using Polymerase Chain Reaction and Single-stranded DNA Conformational Polymorphism Assay

Discerning the pathologic significance of cutaneous T-cell infiltrates can pose a diagnostic challenge for dermatopathologists. Reactive conditions such as drug-associated lymphomatoid hypersensitivity and lymphomatoid lupus erythematosus can demonstrate lymphoid atypia and a phenotype resembling cutaneous T-cell lymphoma (CTCL). Further, lymphoid dyscrasias such as pityriasis lichenoides chronica, large plaque parapsoriasis, and atypical pigmentary purpura confuse the picture because they not only mimic CTCL but also represent prelymphomatous states with inherent malignant potential. Although the emergence of a dominant clone has been considered a clue indicative of a T-cell dyscrasia, there are reports concerning the identification of monoclonality in biopsies of reactive lymphoid infiltrates. We have conducted a modified single-stranded DNA conformational polymorphism (SSCP) assay using paraffin-embedded, formalin-fixed tissue on 92 T-cell-rich biopsies to determine the relative specificity and sensitivity of this methodology. In addition, laser capture microdissection (LCM) was performed on 22 of the 92 samples to isolate the area of interest and to compare its specificity and sensitivity with those SSCP assays performed without LCM. We found that monoclonality or oligoclonality is 86% specific for preneoplastic and neoplastic states, whereas the finding of polyclonality appears to be relatively specific for a reactive process. Some cases of reversible T-cell dyscrasia produced a molecular profile mimicking lymphoma or prelymphomatous states by virtue of monoclonality or oligoclonality. Although LCM appears to improve the sensitivity for detecting preneoplastic conditions, the relative specificity appears to be the same as that encountered with routine SSCP.

High resolution for single-strand conformation polymorphism analysis by capillary electrophoresis.

Since the successful completion of the Human Genome Project, increasing concern is being directed toward the polymorphic aspect of the genome and its clinical relevance. A form of single-strand DNA-conformation polymorphism analysis (SSCP) employing nondenaturing slab-gel electrophoresis (SGE) is applicable to the genetic diagnosis of bladder cancer from urine samples. To bring this technique into routine clinical practice, the use of capillary electrophoresis (CE) is naturally favorable in terms of speed and automation. However, the resolving power of SSCP, a prerequisite basis for reliability required in diagnostics, remains as a challenge for CE systems. We thus focused on this topic and conducted studies on CE instruments equipped with a single capillary or an array of multiple capillaries, using the resolution (Rs) as a quantitative scale for the resolving power. Polymer concentration and buffer are shown to be the decisive parameters. High Rs values of >2.5 are achieved for representative SNPs markers under the optimized conditions, without sacrificing such intrinsic advantages of CE over SGE as the 10-fold quicker migration time and operation that is reproducible, continuous, and automatic. The strategies presented broaden the limits of CE in both the current and related applications.

Characterisation of rpsL, rrs and embB mutations associated with streptomycin and ethambutol resistance in Mycobacterium tuberculosis

In order to characterise molecular mechanisms of first-line drug resistance in Mycobacterium tuberculosis and to evaluate the use of molecular markers of resistance (gene point mutations), we analysed 66 multi-drug-resistant (MDR) isolates from Latvian tuberculosis patients. They were all resistant to rifampin (RIF), isoniazid (INH) and streptomycin (SM), and 33 were resistant to ethambutol (EMB). Enzymatic digestion by MboII and nucleotide sequencing of the rpsL gene fragment detected a single nucleotide substitution K43R in 40 (61%) of the 66 SM-resistant M. tuberculosis isolates. Of the other 26 SM-resistant isolates, 16 (24%) had mutations at positions 513A→C and 516C→T of the rrs gene and 10 (15%) had the wild-type sequence. The single-stranded DNA conformation polymorphism (SSCP) method was used to detect mutations in the embB gene associated with EMB resistance. Substitutions in the embB gene were found by SSCP analysis in 15 (45%) and by sequencing in 17 (52%) of the 33 EMB-resistant isolates. Surprisingly, SSCP revealed a nucleotide mutation at codon M306 in five (15%) of 33 in vitro EMB-susceptible MDR isolates.

Molecular characterization of three novel Fanconi anemia mutations in Israeli Arabs.

In a previous study, we investigated the molecular basis of Fanconi anemia (FA) in 13 unrelated Israeli Jewish FA patients and identified four ethnicity specific mutations. In the present study we extended our study to Israeli Arab patients. We studied three consanguineous families with nine FA patients and an additional unrelated patient. DNA single-strand conformation polymorphism of each exon of the FANCA and FANCG genes was followed by sequence analysis of the aberrantly migrating fragments and by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of the splice-site mutations identified. Three unique disease-causing mutations were identified: (i) FANCA gross deletion of exons 6-31; (ii) FANCA splice-site mutation IVS 42-2A>C; (iii) FANCG splice-site mutation IVS4+3A>G. Sequence analysis of the FANCA gross deletion revealed recombination between two highly homologous Alu elements. cDNA analysis of the two splice mutations suggested intron 42 retention in FANCA IVS 42-2A>C and exon 4 skipping in FANCG IVS4+3A>G. The clinical condition of eight patients with FANCA mutations was severe. Two unique FANCA mutations and one FANCG mutation were identified in Israeli Arab FA patients. Deletion of FANCA exon 6-31 as in previously described gross deletions was within introns rich in Alu repeats. To the best of our knowledge, the FANCA IVS 42-2A>C mutation is the first in this gene to result in intron retention. Further analysis of FA mutations will enable prenatal diagnosis and a rational therapeutic approach including frequent monitoring and early bone marrow transplantation.

Estimation of the optimal electrophoretic temperature of DNA single-strand conformation polymorphism by DNA base composition.

To explore the relation between DNA base composition and the optimal single-strand conformation polymorphism (SSCP) electrophoretic temperature (T(s)), we analyzed DNA base composition and T(s) of 24 DNA fragments from different genes and found that T(s) was positively correlative with the ratio of base C/base A. T(s) could be estimated by the formula T(s) = [80 x C/(A+1)]/[2.71 + [C/(A+1)]]. T(s) could be increased dramatically by the complementary sequences in both 5'-and 3'-ends of a DNA single-strand.

Hypermethylation of an E-Cadherin ( CDH 1) Promoter Region in High Grade Transitional Cell Carcinoma of the Bladder Comprising Carcinoma In Situ

We elucidated the role of methylation in the promoter region of the 1 gene in bladder carcinogenesis, particularly in those comprising carcinoma in situ. A total of 49 cases of transitional cell carcinoma of the bladder obtained from transurethral resection were examined. Methylation status of the 1 promoter region was analyzed by methylation specific polymerase chain reaction from chemically modified DNA after Na-bisulfite treatment. Loss of heterozygosity on 16q was examined by blunt end single strand DNA conformation polymorphism using 4 tetranucleotide repeat microsatellite markers assigned on 16q13 to 22.1. E-cadherin expression was evaluated by immunostaining on formalin fixed, paraffin embedded tissue sections using anti E-cadherin murine monoclonal antibody, HECD1 and standard avidin-biotin immunoperoxidase complex technique. Analysis of the 49 bladder transitional cell carcinoma samples showed 1 promoter methylation in 23 (47%). Methylation of the 1 gene did not correlate with tumor stage (p = 0.2097) but with high grade transitional cell carcinoma (p = 0.0416). 1 promoter methylation was observed at a significantly higher frequency in the carcinoma in situ positive group than in the carcinoma in situ negative group (16 of 18 cases or 89% versus 7 of 31 or 23%, p <0.0001) and it strongly correlated with abnormal E-cadherin expression (p <0.0001). We found 16q loss of heterozygosity in 16 of 47 cases (34%), which correlated with higher histological grade (p = 0.0069) but not with the presence of the carcinoma in situ component (p = 0.1235). This study showed that 1 gene promoter methylation is strongly associated with bladder transitional cell carcinoma comprising carcinoma in situ.

Cyclin D1 Genotype, Response to Biochemoprevention, and Progression Rate to Upper Aerodigestive Tract Cancer

Altered cyclin D1 expression in advanced preinvasive lesions of the upper aerodigestive tract (UADT) is associated with an increased risk of developing cancer and histologic progression during and after combination biochemopreventive therapy (13-cis-retinoic acid, alpha-interferon, and alpha-tocopherol). Both alleles of the adenine (A)/guanine (G) cyclin D1 polymorphism located at nucleotide 870 encode two alternatively spliced transcripts, but the A allele preferentially encodes a protein with an extended half-life. We investigated whether the cyclin D1 genotype at nucleotide 870 was associated with baseline levels of cyclin D1 protein, post-treatment modulation of cyclin D1 protein levels, histologic response to treatment, and the outcome for subjects with preinvasive UADT lesions after biochemopreventive therapy. UADT tissue biopsy samples were obtained before and 6 and 12 months after biochemopreventive treatment from 31 individuals with advanced preinvasive UADT lesions. Tissues were examined for cyclin D1 genotype (by DNA single-strand conformation polymorphism analysis), for cyclin D1 protein expression (by immunohistochemistry), and for cyclin D1 gene copy number (by fluorescence in situ hybridization). Associations of cyclin D1 genotype with histologic response to therapy and time to progression to a higher degree of dysplasia or invasive cancer were investigated. All statistical tests were two-sided. The A allele was associated with increased baseline cyclin D1 expression in the parabasal epithelial layer (16 of 18 AA/AG subjects versus four of nine GG subjects; P =.02), decreased histologic response to biochemopreventive treatment (six of 21 AA/AG subjects versus four of 10 GG subjects; P =.70), decreased favorable modulation of cyclin D1 expression by the treatment (seven of 18 AA/AG subjects versus eight of nine GG subjects; P =.02), and shorter progression-free survival (P =.05). The cyclin D1 A allele was associated with a diminished modulation of normal physiologic and treatment-induced decreased expression of cyclin D1, a decreased likelihood of response to biochemopreventive intervention, and an increased rate of progression to cancer development, findings that require validation in a larger cohort.

Mutation analysis of the G4.5 gene in patients with isolated left ventricular noncompaction

Mutations in the gene G4.5, originally associated with Barth syndrome, have been reported to result in a wide spectrum of severe infantile X-linked cardiomyopathies. The purpose of this study was to investigate patients with isolated left ventricular noncompaction (LVNC) for disease-causing mutations in G4.5. In 27 patients including 10 families with isolated LVNC, mutation analysis of G4.5 was performed using single-strand DNA conformation polymorphism (SSCP) analysis and DNA sequencing. A novel splice acceptor site mutation of intron 8 of G4.5 was identified in a family with severe infantile X-linked LVNC without the usual findings of Barth syndrome. This mutation results in deletion of exon 9 from the mRNA, and is predicted to significantly disrupt the protein product. Genotype–phenotype correlation of G4.5 mutations in all 38 cases reported in the literature to date revealed that there was no correlation between location or type of mutation and either cardiac phenotype or disease severity. We suggest that males presenting with cardiomyopathy, particularly during infancy, even in the absence of the typical signs of Barth syndrome, should be evaluated for mutations in G4.5.

Microbial Community Dynamics During Composting of Organic Matter as Determined by 16S Ribosomal DNA Analysis

A culture independent survey of the microbial community dynamics during the composting of organic waste in an industrial composting process was conducted by sequence analysis of (1) universal clone coding for small-subunit rRNA-genes libraries and, in parallel, with electrophoresis and sequencing of PCR-amplified 16S rDNA fragments based on DNA single strand-conformation polymorphism (SSCP). Samples were taken from a force aerated module composting system during a two week dynamic process. In addition, to examine the beginning maturation phase, one sample was taken from force-aerated compost piles. In the initial composting stage, where a starting temperature of 41°C was recorded, a number of Enterobacteriaceae and members of the genus Lactobacillus were still detected, while different members of the low-G+C Gram-positive bacteria formed the dominant fraction of the bacterial community during the hot composting phase with temperatures fluctuating between 55 and 70°C. During the initial curing phase, where temperature had declined to 50°C, the microbial community changed accompanied by an increasing number of sequences affiliated with the Bacteroides-Cytophaga-Flexibacter group. The combined results of 16S rRNA sequence analysis demonstrated that both approaches, cloning and SSCP, have their specific advantages and limitations, but both methods were capable to detect the predominating taxa and have proved useful for the analysis of microbial community successions during composting.