Detection of Tannerella forsythia and/or Prevotella intermedia might be useful for microbial predictive markers for the outcome of initial periodontal treatment in Koreans

A proportion of diseased sites in periodontal disease do not respond to the initial treatment, which might be due in part to the presence of specific microbial pathogens. The aim of this study was to clarify the value of microbial screening for predicting the outcome of periodontal treatment in Koreans using a polymerase chain reaction (PCR). This study enrolled 32 adults with periodontal disease. Microbial and clinical examinations were performed at the baseline and after the initial treatment (professional toothbrushing, scaling, and root planing). Subgingival plaque samples were taken from four sites in each subject (total 128 samples). PCR was used to detect the four putative pathogenic bacteria. There was an improvement in the average of each clinical measurement after the initial treatment. However, approximately half of the sites exhibiting bleeding upon probing (BOP) at the baseline still exhibited bleeding after treatment. There was a close association between the presence of BOP and the presence of Tannerella forsythia (formerly Bacteroides forsythus) and/or Prevotella intermedia. Furthermore, the sites harboring both T. forsythia and P. intermedia at the baseline had a poorer response to treatment than the sites where these two species were not detected. Therefore, microbial screening for T. forsythia and P. intermedia might be useful for predicting the treatment outcome in Koreans.     

Inducible Excision of Selectable Marker Gene from Transgenic Plants by the Cre/<Emphasis Type="Italic">lox</Emphasis> Site-specific Recombination System

In a plant transformation process, it is necessary to use marker genes that allow the selection of regenerated transgenic plants. However, selectable marker genes are generally superfluous once an intact transgenic plant has been established. Furthermore, they may cause regulatory difficulties for approving transgenic crop release and commercialization. We constructed a binary expression vector with the Cre/lox system with a view to eliminating a marker gene from transgenic plants conveniently. In the vector, recombinase gene cre under the control of heat shock promoter and selectable marker gene nptII under the control of CaMV35S promoter were placed between two lox P sites in direct orientation, while the gene of interest was inserted outside of the lox P sites. By using this vector, both cre and nptII genes were eliminated from most of the regenerated plants of primary transformed tobacco through heat shock treatment, while the gene of interest was retained and stably inherited. This autoexcision strategy, mediated by the Cre/lox system and subjected to heat shock treatment to eliminate a selectable marker gene, is easy to adopt and provides a promising approach to generate marker-free transgenic plants.     

Urinary marker of oral pregnenolone administration

Pregnenolone (PREG) can potentially be abused by athletes to maintain an equilibration of the steroidal environment after sex steroids administrations. Five men volunteers orally ingested 50 mg PREG to determine optimal urinary markers for detection of this steroid. Our findings show that ingestion of PREG has no significant effects on the testosterone/epitestosterone (T/E) and testosterone/luteinizing hormone (T/LH) ratios, whereas variable changes on the carbon isotopic values of three T metabolites: androsterone, etiocholanolone, 5beta-androstane-3alpha,17beta-diol (5beta-androstanediol) together with 16(5alpha)-androsten-3alpha-ol (androstenol) and 5beta-pregnane-3alpha,20alpha-diol (pregnanediol) have been observed. The difference between the carbon isotopic values (delta13C-values) of androstenol and pregnanediol is potentially the most reliable marker of exogenous PREG administration in males. For all subjects, the differences differ by 3.0 per thousand or more over a period of about 10 h and for both of them the detection window for positivity is extended over 40 h.     

Intron-flanking EST–PCR markers: from genetic marker development to gene structure analysis in Rhododendron

With a long-term goal of constructing a linkage map of Rhododendron enriched with gene-specific markers, we utilized Rhododendron catawbiense ESTs for the development of high-efficiency (in terms of generating polymorphism frequency) PCR-based markers. Using the gene-sequence alignment between Rhododendron ESTs and the genomic sequences of Arabidopsis homologs, we developed ‘intron-flanking‘ EST–PCR-based primers that would anneal in conserved exon regions and amplify across the more highly diverged introns. These primers resulted in increased efficiency (61% vs. 13%; 4.7-fold) of polymorphism-detection compared with conventional EST–PCR methods, supporting the assumption that intron regions are more diverged than exons. Significantly, this study demonstrates that Arabidopsis genome database can be useful in developing gene-specific PCR-based markers for other non-model plant species for which the EST data are available but genomic sequences are not. The comparative analysis of intron sizes between Rhododendron and Arabidopsis (made possible in this study by aligning of Rhododendron ESTs with Arabidopsis genomic sequences and the sequencing of Rhododendron genomic PCR products) provides the first insight into the gene structure of Rhododendron. Electronic Supplementary Material Supplementary material is available for this article at     

Construction of an integrated map of rose with AFLP, SSR, PK, RGA, RFLP, SCAR and morphological markers

A high-density genetic map with a number of anchor markers has been created to be used as a tool to dissect genetic variation in rose. Linkage maps for the diploid 94/1 population consisting of 88 individuals were constructed using a total of 520 molecular markers including AFLP, SSR, PK, RGA, RFLP, SCAR and morphological markers. Seven linkage groups, putatively corresponding to the seven haploid rose chromosomes, were identified for each parent, spanning 487 cM and 490 cM, respectively. The average length of 70 cM may cover more than 90% of the rose genome. An integrated map was constructed by incorporating the homologous parental linkage groups, resulting in seven linkage groups with a total length of 545 cM. The present linkage map is currently the most advanced map in rose with regard to marker density, genome coverage and with robust markers, giving good perspectives for QTL mapping and marker-assisted breeding in rose. The SSR markers, together with RFLP markers, provide good anchor points for future map alignment studies in rose and related species. Codominantly scored AFLP markers were helpful in the integration of the parental maps.     

Review: intellectual property aspects of plant transformation

One of the recurring themes of the debates concerning the application of genetic transformation technology has been the role of Intellectual Property Rights (IPR). This term covers both the content of patents and the confidential expertise usually related to methodology and referred to as 'Trade Secrets'. This review explains the concepts behind patent protection, and discusses the wide-ranging scope of existing patents that cover all aspects of transgenic technology, from selectable markers and novel promoters to methods of gene introduction. Although few of the patents in this area have any real commercial value, there are a small number of key patents that restrict the 'freedom to operate' of new companies seeking to exploit the methods. Over the last 20 years, these restrictions have forced extensive cross-licensing between ag-biotech companies and have been one of the driving forces behind the consolidation of these companies. Although such issues are often considered of little interest to the academic scientist working in the public sector, they are of great importance in any discussion of the role of 'public-good breeding' and of the relationship between the public and private sectors.     

Marker assisted selection for the improvement of two antagonistic traits under mixed inheritance

A Monte Carlo simulation was used to investigate the potential of Marker Assisted Selection (MAS) in a multiple-trait situation. Only additive effects were considered. The base population was assumed to be in linkage equilibrium and, next, the population was managed over 15 discrete generations, 10 males and 50 females were chosen out of the 100 candidates of each sex. Performance for two traits was simulated with an overall heritability of a given trait equal to 0.25 or 0.10 and the overall genetic correlation between traits was generally equal to -0.4 except in one case where it was equal to 0. The model involved one biallelic QTL, accounting for 10 or 20% of the genetic variance of a given trait, plus polygenes. Initial allelic frequencies at the QTL were generally equal to 0.5 but in one case were equal to 0.1 and 0.9. A marker with 120 different alleles in the 60 founder parents was simulated in the vicinity of the QTL. Two values of the recombination rate between these two loci were considered, 0.10 and 0.02. The genetic evaluation was based on a multiple-trait BLUP animal model, accounting (MAS) or not (conventional BLUP) for marker information. Two sets of simulations were run: (1) a "missing data"case, with males having no record for one of the traits, and (2) a "secondary trait"case, with one trait having a weight in the aggregate genotype 4 times less than the other trait and the QTL acting only on this secondary trait. In the first set, evaluation methods were found to mainly affect the accuracy of overall genetic values prediction for the trait with missing data. In comparison with BLUP, MAS led to an extra overall genetic response for the trait with missing data, which was strongly penalised under the conventional BLUP, and to a deficit in response for the other trait. This more balanced evolution of the two traits was obtained, however, at the expense of the long-term overall cumulated response for the aggregate genotype, which was 1 to 2.5% lower than the one obtained under the conventional BLUP. In the second set of simulation, in the case of low initial frequency (0.1) of the QTL allele favourable to the secondary trait, MAS was found to be substantially more efficient to avoid losing this allele than BLUP only when the QTL had a large effect and the marker was close. More benefits should be expected from MAS with more specific applications, such as early selection of animals, or by applying dynamic procedures i.e. letting the respective weights to QTL and polygenic values in the selection criterion vary across generation.     

Genetic characterization of non-spoilage variant isolated from beer-spoilage Lactobacillus brevis ABBC45

AIMS: To characterize the non-spoilage variant obtained from beer-spoilage Lactobacillus brevis ABBC45C and to identify a potential genetic marker capable of discriminating beer-spoilage L. brevis strains from non-spoilers. METHODS AND RESULTS: A non-spoilage variant was obtained from beer-spoilage L. brevis ABBC45C by repeatedly subculturing the strain at 37 degrees C. Genetic characterization of the variant revealed that 12,605 bp portion of one plasmid, designated pRH45II, was lost in the variant. The sequence analysis indicates the presence of 12 ORFs in the deleted region of pRH45II. The PCR and Southern hybridization study revealed that the homologues of ORF5 found in the deleted region were present in all of the beer-spoilage L. brevis strains examined in this study. In contrast, the homlogues appeared to be absent in non-spoilage L. brevis strains. CONCLUSIONS: The presence or absence of ORF5 homologues was found to be highly correlated with the beer-spoilage ability of L. brevis strains, indicating this ORF is potentially a useful genetic marker capable of differentiating beer-spoilage strains among L. brevis. SIGNIFICANCE AND IMPACT OF THE STUDY: A non-spoilage variant was successfully isolated from beer-spoilage L. brevis ABBC45C. This study could facilitate the understanding of mechanisms underlying beer-spoilage ability of L. brevis.     

Spontaneous ectopic recombination in cell-type-specific Cre mice removes loxP-flanked marker cassettes in vivo

Conditional gene targeting using the Cre/loxP technology generally includes integration of a selection marker cassette flanked by loxP recognition sites (floxed) in the target gene locus. Subsequent marker removal avoids possible impairment of gene expression or mosaicism due to partial and total deletions after Cre-mediated recombination in vivo. The use of deleter Cre mice for in vivo marker removal in floxed connexin43 mice revealed considerable mosaicism, but no selective marker removal. In addition, we noted that several Cre transgenic lines displayed spontaneous ectopic activity, reminiscent of deleter Cre mice, and required the confirmation of cell type-specific deletion in every individual mouse. When we used myosin heavy chain promoter Cre (alphaMyHC-Cre) mice for cardiomyocyte specific deletion, we observed, in addition to cardiomyocyte-restricted or complete excision, selective marker removal in a subgroup of mice as well. Thus, selective marker removal can be achieved as a byproduct of cell-type restricted deletion.