Home About us Contact
|
Home About us Contact | ||
|
|
||
Amplified Region (amplified + region)
Selected AbstractsUnscheduled DNA replication origin activation at inserted HPV 18 sequences in a HPV -18/MYC ampliconGENES, CHROMOSOMES AND CANCER, Issue 8 2007Chiara Conti Oncogene amplification is a critical step leading to tumorigenesis, but the underlying mechanisms are still poorly understood. Despite data suggesting that DNA replication is a major source of genomic instability, little is known about replication origin usage and replication fork progression in rearranged regions. Using a single DNA molecule approach, we provide here the first study of replication kinetics on a previously characterized MYC/papillomavirus (HPV18) amplicon in a cervical cancer. Using this amplicon as a model, we investigated the role DNA replication control plays in generating amplifications in human cancers. The data reveal severely perturbed DNA replication kinetics in the amplified region when compared with other regions of the same genome. It was found that DNA replication is initiated from both genomic and viral sequences, resulting in a higher median frequency of origin firings. In addition, it was found that the higher initiation frequency was associated with an equivalent increase in the number of stalled replication forks. These observations raise the intriguing possibility that unscheduled replication origin activation at inserted HPV -18 viral DNA sequences triggers DNA amplification in this cancer cell line and the subsequent overexpression of the MYC oncogene. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat. © 2007 Wiley-Liss, Inc. [source] UREASE GENE SEQUENCES FROM ALGAE AND HETEROTROPHIC BACTERIA IN AXENIC AND NONAXENIC PHYTOPLANKTON CULTURES,JOURNAL OF PHYCOLOGY, Issue 3 2009Kristopher M. Baker While urea has long been recognized as an important form of nitrogen in planktonic ecosystems, very little is known about how many or which phytoplankton and bacteria can use urea as a nitrogen source. We developed a method, targeting the gene encoding urease, for the direct detection and identification of ureolytic organisms and tested it on seven axenic phytoplankton cultures (three diatoms, two prymnesiophytes, a eustigmatophyte, and a pelagophyte) and on three nonaxenic Aureococcus anophagefferens Hargraves et Sieburth cultures (CCMP1784 and two CCMP1708 cultures from different laboratories). The urease amplicon sequences from axenic phytoplankton cultures were consistent with genomic data in the three species for which both were available. Seven of 12 phytoplankton species have one or more introns in the amplified region of their urease gene(s). The 63 urease amplicons that were cloned and sequenced from nonaxenic A. anophagefferens cultures grouped into 17 distinct sequence types. Eleven types were related to ,-Proteobacteria, including three types likely belonging to the genus Roseovarius. Four types were related to ,-Proteobacteria, including two likely belonging to the genus Marinobacter, and two types were related to ,-Proteobacteria. Terminal restriction fragment length polymorphism (TRFLP) analyses suggested that the sequenced amplicons represented approximately half of the diversity of bacterial urease genes present in the nonaxenic cultures. While many of the bacterial urease sequence types were apparently lab- or culture-specific, others were found in all three nonaxenic cultures, suggesting the possibility of specific relationships between these bacteria and A. anophagefferens. [source] Genetic Variability Analysis and Molecular Detection of Fusarium oxysporum f.sp. eustomae Isolated from Eustoma grandiflorum in Northern ItalyJOURNAL OF PHYTOPATHOLOGY, Issue 7-8 2010Yuan Li Abstract A total of 35 isolates of Fusarium oxysporum f.sp. eustomae obtained from diseased Eustoma grandiflorum plants in northern Italy, showing typical Fusarium wilt symptoms, were analysed for their genetic variability and molecular identification. Genetic diversity of the isolates was studied by using random amplified polymorphic DNA (RAPD). This analysis clustered the isolates into three groups at a genetic similarity of 69%. Sequence analysis of RAPD fragments led to the design of a pair of specific primers that amplify a 505-bp SCAR (sequence characterized amplified region) marker (SCAR505) which was used to rapidly detect F. oxysporum f.sp. eustomae on Eustoma grandiflorum plants. In a temperature-controlled chamber, detection of the pathogen by PCR was 100% successful in root and stem samples of infected but still symptomless plants. The diagnostic procedure could be completed in 1 day and allowed rapid and reliable detection of the pathogen in asymptomatic plants in the early stages of disease development. [source] Phenotypic Reaction and Genetic Analysis Using AFLP-derived SCARs for Resistance to Apple ScabJOURNAL OF PHYTOPATHOLOGY, Issue 5 2004E. M. Huaracha Abstract Six sequence-characterized amplified region (SCAR) markers linked to the apple scab resistance gene Vf were evaluated for their utility in marker-assisted selection (MAS) in apple breeding. Of the six SCARs used in this study, ACS-6 was located left of the Vf gene, ACS-7 and ACS-9 co-segregated with Vf, and ACS-8, ACS-4, ACS-5 were located right of the Vf gene. Three families derived from crosses between scab-resistant and scab-susceptible cultivars, including ,Liberty' × ,Deljub', ,Liberty' × ,Delcorf', and ,Florina' ×,Delcorf', previously screened for scab resistance following greenhouse inoculation with the fungal pathogen Venturia inaequalis, were genotyped and compared with phenotypic reactions to scab infection in the field. For each family, a subset progeny of 30 seedlings (propagated onto Malling 9 rootstock and of 7 years old) was selected based on fungal sporulation according to the following scheme. Ten seedlings with no visible scab sporulation on leaves were given phenotypic scores of 0 (deemed resistant); 10 seedlings with moderate scab sporulation were given phenotypic scores of 1.0 (deemed moderately resistant); and 10 seedlings with heavy sporulation were given phenotypic scores of 2.0 (deemed susceptible). DNA was isolated from leaf tissue collected from all 90 seedlings, parents and Malus floribunda 821, the original source of the Vf gene, and screened with all six SCARs. All six SCARs were present in the two scab-resistant parents, ,Liberty' and ,Florina', and M. floribunda 821; while, the two scab-susceptible parents, ,Deljub' and ,Delcorf', lacked all SCARs. All SCARs were either present or absent in varying numbers of seedlings in each progeny with phenotypic ratings of either 0 (resistant) or 1.0 (moderately resistant); while all seedlings with phenotypic ratings of 2.0 (susceptible) lacked all SCARs. The inconsistencies between phenotypic scab ratings and SCAR marker data are discussed. [source] Host-driven divergence in the parasitic plant Orobanche minor Sm. (Orobanchaceae)MOLECULAR ECOLOGY, Issue 19 2008C. J. THOROGOOD Abstract Many parasitic angiosperms have a broad host range and are therefore considered to be host generalists. Orobanche minor is a nonphotosynthetic root parasite that attacks a range of hosts from taxonomically disparate families. In the present study, we show that O. minor sensu lato may comprise distinct, genetically divergent races isolated by the different ecologies of their hosts. Using a three-pronged approach, we tested the hypothesis that intraspecific taxa O. minor var. minor and O. minor ssp. maritima parasitizing either clover (Trifolium pratense) or sea carrot (Daucus carota ssp. gummifer), respectively, are in allopatric isolation. Morphometric analysis revealed evidence of divergence but this was insufficient to define discrete, host-specific taxa. Intersimple sequence repeat (ISSR) marker-based data provided stronger evidence of divergence, suggesting that populations were isolated from gene flow. Phylogenetic analysis, using sequence-characterized amplified region (SCAR) markers derived from ISSR loci, provided strong evidence for divergence by clearly differentiating sea carrot-specific clades and mixed-host clades. Low levels of intrapopulation SCAR marker sequence variation and floral morphology suggest that populations on different hosts are probably selfing and inbreeding. Morphologically cryptic Orobanche taxa may therefore be isolated from gene flow by host ecology. Together, these data suggest that host specificity may be an important driver of allopatric speciation in parasitic plants. [source] Identification of a SCAR marker linked to a recessive male sterile gene (Tems) and its application in breeding of marigold (Tagetes erecta)PLANT BREEDING, Issue 1 2009Y. H. He Abstract In marigold, an F2 segregation population of 167 plants was constructed from a cross of a line (M525A) carrying the male sterility trait × an inbred line (f53f). In line M525A, the male sterility trait was controlled by the recessive gene, Tems. The intersimple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) techniques combined with bulked segregant analysis were used to develop markers linked to the trait. From a survey of the 38 ISSR primers and 170 SRAP primer combinations, only one SRAP marker that was closely linked to the target trait was identified and successfully converted into sequence characterized amplified region (SCAR) marker that was located within 2.4 cM from Tems locus. The marker was validated with five other two-type lines and in each case the male fertile plants were reliably identified. This SCAR marker therefore permits the efficient marker-assisted selection of male sterile individuals in breeding programmes of marigold and will greatly facilitate the breeding of F1 cultivars. [source] Development and characterization of SCAR markers associated with a dominant genic male sterility in rapeseedPLANT BREEDING, Issue 1 2008D. F. Hong Abstract Rs1046AB is a dominant genic male sterility (DGMS) line in rapeseed, in which the sterility has always been thought to be conditioned by the interaction of a male sterility gene (Ms) and its non-allelic restorer gene (Rf). This system provides not only a tool for assisting in recurrent selection but also a promising system for hybrid production. Based on previous studies, two amplified fragment length polymorphism markers linked with the Ms gene were converted into a dominant and a co-dominant sequence characterized amplified region (SCAR) marker, respectively. The putative linear order relationship of three dominant SCAR markers with the same genetic distance from the Rf gene, was also determined by an examination of whether the homologues of these markers are present or not in different lines carrying Rf. A bigger fragment generated by the closest marker linked to the Rf gene was observed in all lines carrying the recessive allele rf, suggesting that this marker is a co-dominant marker, which was further confirmed by nucleotide sequence comparison of these fragments. SCAR markers specific for Ms and Rf will be especially valuable in marker-assisted DGMS three-line breeding. [source] Development of SCAR markers for identification of stem rust resistance gene Sr31 in the homozygous or heterozygous condition in bread wheatPLANT BREEDING, Issue 6 2006B. K. Das Abstract The stem rust resistance gene Sr31, transferred from rye (Secale cereale) into wheat (Triticum aestivum L.) imparts resistance to all the virulent pathotypes of stem rust (Puccinia graminis f. sp. tritici) found in India. Wheat genotypes including carriers and non-carriers of the Sr31 gene were analysed using arbitrary primed polymerase chain reaction (AP-PCR). AP-PCR markers viz. SS30.2580(H) associated with the Sr31 gene and SS26.11100 associated with the allele for susceptibility were identified. Linkage between the markers and phenotypes was confirmed by analysing an F2 population obtained from a cross between a resistant and a susceptible genotype. The markers were tightly linked to the respective alleles. Both the AP-PCR markers were converted into sequence characterized amplified region (SCAR) markers, viz. SCSS30.2576 and SCSS26.11100 respectively. The markers were validated in two more segregating populations and 49 wheat genotypes. Using both markers it was possible to distinguish the homozygous from the heterozygous carriers of the Sr31 gene in the F2 generation. The markers developed in this study can be used for pyramiding of the Sr31 gene with other rust resistance genes and in marker-assisted selection. [source] Identification of a molecular marker linked to an Agropyron elongatum-derived gene Lr19 for leaf rust resistance in wheatPLANT BREEDING, Issue 3 2003D. P. Cherukuri Abstract The leaf rust resistance gene Lr19, transferred from Agropyron elongatum into wheat (Triticum aestivum L.) imparts resistance to all pathotypes of leaf rust (Puccinia recondita f.sp. tritici) in South-east Asia. A segregating F2 population from a cross between the leaf rust resistant parent ,HW 2046' carrying Lr19 and a susceptible parent ,Agra Local' was screened in the phytotron against a virulent pathotype 77-5 of leaf rust with the objective of identifying the molecular markers linked to Lr19. The gene was first tagged with a randomly amplified polymorphic DNA (RAPD) marker S73728. The RAPD marker linked to the gene Lr19 which mapped at 6.4 ± 0.035 cM distance, was converted to a sequence characterized amplified region (SCAR) marker. The SCAR marker (SCS73719) was specific to Lr19 and was not amplified in the near-isogenic lines (NILs) carrying other equally effective alien genes Lr9, Lr28 and Lr32 enabling breeders to pyramid Lr19 with these genes. [source] Geographic distribution and genetic diversity of Fusarium graminearum and F. asiaticum on wheat spikes throughout ChinaPLANT PATHOLOGY, Issue 1 2008B. Qu A large number of Fusarium graminearum and F. asiaticum isolates were collected from wheat spikes from all regions in China with a history of fusarium head blight (FHB) epidemics. Isolates were analysed to investigate their genetic diversity and geographic distribution. Sequence characterized amplified region (SCAR) analyses of 437 isolates resolved both species, with 21% being F. graminearum (SCAR type 1) and 79% being F. asiaticum (SCAR type 5). AFLP profiles clearly resolved two groups, A and B, that were completely congruent with both species. However, more diversity was detected by AFLP, revealing several subgroups within each group. In many cases, even for isolates from the same district, AFLP haplotypes differed markedly. Phylogenetic analyses of multilocus DNA sequence data indicated that all isolates of SCAR type 1, AFLP group A were F. graminearum, whilst isolates of SCAR type 5, AFLP group B were F. asiaticum, demonstrating that it is an efficient method for differentiating these two species. Both species seem to have different geographic distributions within China. Fusarium graminearum was mainly obtained from wheat growing in the cooler regions where the annual average temperature was 15°C or lower. In contrast, the vast majority of F. asiaticum isolates were collected from wheat growing in the warmer regions where the annual average temperature is above 15°C and where FHB epidemics occur most frequently. This is the first report of the distribution of, and genetic diversity within, F. graminearum and F. asiaticum on wheat spikes throughout China. [source] Variation in the response of melon genotypes to Fusarium oxysporum f.sp. melonis race 1 determined by inoculation tests and molecular markersPLANT PATHOLOGY, Issue 2 2003Y. Burger Screening of genotypes of melon (Cucumis melo) for resistance to wilt caused by Fusarium oxysporum f.sp. melonis is often characterized by wide variability in their responses to inoculation, even under carefully controlled conditions. The variability at the seedling stage of 17 genotypes susceptible to race 1 was examined in growth-chamber experiments. Disease incidence varied from 0 to 100% in a genotype-dependent manner. Using four combinations of light (60 and 90 µE m,2 s,1) and temperatures of (27 and 31°C), only light intensity showed a statistically significant effect. Marker-assisted selection for fusarium resistance breeding using cleaved amplified polymorphic sequence (CAPS) and sequence-characterized amplified region (SCAR) markers were compared using a single set of genotypes that included 24 melon accessions and breeding lines whose genotype regarding the Fom-2 gene was well characterized. The practical value of the markers for discriminating a range of genotypes and clarifying the scoring of phenotypes was also tested using a segregating breeding population which showed codominant SCAR markers to be useful in marker-assisted selection. [source] Recombination is suppressed over a large region of the rainbow trout Y chromosomeANIMAL GENETICS, Issue 6 2009R. B. Phillips Summary The previous genetic mapping data have suggested that most of the rainbow trout sex chromosome pair is pseudoautosomal, with very small X-specific and Y-specific regions. We have prepared an updated genetic and cytogenetic map of the male rainbow trout sex linkage group. Selected sex-linked markers spanning the X chromosome of the female genetic map have been mapped cytogenetically in normal males and genetically in crosses between the OSU female clonal line and four different male clonal lines as well as in outcrosses involving outbred OSU and hybrids between the OSU line and the male clonal lines. The cytogenetic maps of the X and Y chromosomes were very similar to the female genetic map for the X chromosome. Five markers on the male maps are genetically very close to the sex determination locus (SEX), but more widely spaced on the female genetic map and on the cytogenetic map, indicating a large region of suppressed recombination on the Y chromosome surrounding the SEX locus. The male map is greatly extended at the telomere. A BAC clone containing the SCAR (sequence characterized amplified region) Omy - 163 marker, which maps close to SEX, was subjected to shotgun sequencing. Two carbonyl reductase genes and a gene homologous to the vertebrate skeletal ryanodine receptor were identified. Carbonyl reductase is a key enzyme involved in production of trout ovarian maturation hormone. This brings the number of type I genes mapped to the sex chromosome to six and has allowed us to identify a region on zebrafish chromosome 10 and medaka chromosome 13 which may be homologous to the distal portion of the long arm of the rainbow trout Y chromosome. [source] Isolation of Y- and X-linked SCAR markers in yellow catfish and application in the production of all-male populationsANIMAL GENETICS, Issue 6 2009D. Wang Summary Sex controls have been performed in some farmed fish species because of significant growth differences between females and males. In yellow catfish (Pelteobagrus fulvidraco), adult males are three times larger than female adults. In this study, six Y- and X-linked amplified fragment length polymorphism fragments were screened by sex-genotype pool bulked segregant analysis and individual screening. Interestingly, sequence analysis identified two pairs of allelic genes, Pf33 and Pf62. Furthermore, the cloned flanking sequences revealed several Y- and X-specific polymorphisms, and four Y-linked or X-linked sequence characterized amplified region (SCAR) primer pairs were designed and converted into Y- and X-linked SCAR markers. Consequently, these markers were successfully used to identify genetic sex and YY super-males, and applied to all-male population production. Thus, we developed a novel and simple technique to help commercial production of YY super-males and all-male populations in the yellow catfish. [source] | ||||||||||