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Artificial Chromosome (artificial + chromosome)
Kinds of Artificial Chromosome Selected AbstractsComparison of PERV genomic locations between Asian and European pigsANIMAL GENETICS, Issue 1 2010W. Y. Jung Summary Xenotransplantation from pigs provides a possible solution to the shortage of human organs for allotransplantation. Porcine endogenous retroviruses (PERVs) are a possible obstacle to using porcine organs in addition to the immunological barriers. Three main types of PERVs (A, B and C) have been previously investigated in diverse pig breeds. To examine the copy numbers of PERVs and their genomic locations in the Korean native pig genome, we screened a BAC (Bacterial Artificial Chromosome) library with PERV-specific protease primers for initial recognition of PERV-positive clones and three sets of envelope-specific primers for the identification of PERV types. A total of 45 PERV-positive clones, nine PERV-A and 36 PERV-B, have been identified from the library screening and the BAC contigs were constructed using the primers designed from BAC end sequences (BESs). These primers were also used for SCH (Somatic Cell Hybrid) and RH (Radiation Hybrid) mapping of the PERV-positive clones. The results indicate that 45 PERV-positive BAC clones belong to nine contigs and a singleton. SCH and IMpRH (INRA-Minnesota Porcine Radiation Hybrid) mapping results indicated that there are at least eight separate PERV genomic locations, consisting of three PERV-A and five PERV-B. One contig could not be mapped, and two contigs are closely located on SSC7. Southern blotting indicates there may be up to 15 additional sites. Further investigation of these clones will contribute to a general strategy to generate PERV-free lines of pigs suitable for xenotransplantation. [source] A case of intersexuality in pigs associated with a de novo paracentric inversion 9 (p1.2; p2.2)ANIMAL GENETICS, Issue 1 2002A. Pinton In several mammalian species, genetic defects can be responsible for the interruption of and/or the deviation from the sequential steps of normal gonadal differentiation, leading to a sex-reversal syndrome. In pigs, female-to-male sex-reversal conditions are particularly frequent, but their aetiologies remain unclear. Chromosomal abnormalities that co-occur with sex-reversal disorders can be useful in the identification of loci containing responsible or susceptibility genes. This report describes a female-to-male SRY -negative intersex pig with a de novo paracentric inversion of the short arm of one chromosome 9 (p1.2; p2.2). We have fine mapped the proximal chromosomal breakpoint of this rearrangement because it corresponded to a region potentially involved in the pig intersexuality. Fluorescent in situ hybridization (FISH) experiments carried out with Bacterial Artificial Chromosome (BAC) clones located within the critical region defined by genetic linkage analysis and ordered on the porcine RH map allowed us to locate the proximal breakpoint between markers SW2571 and SW539. Further investigations are currently in progress to find new markers inside this interval, in order to determine the BAC in which the break occurred. [source] Further delineation of 9q22 deletion syndrome associated with basal cell nevus (Gorlin) syndrome: Report of two cases and review of the literatureCONGENITAL ANOMALIES, Issue 1 2009Kayono Yamamoto ABSTRACT Basal cell nevus syndrome (BCNS; Gorlin syndrome) is an autosomal dominant disorder, characterized by a predisposition to neoplasms and developmental abnormalities. BCNS is caused by mutations in the human homolog of the Drosophila patched gene-1, PTCH1, which is mapped on chromosome 9q22.3. Nonsense, frameshift, in-frame deletions, splice-site, and missense mutations have been found in the syndrome. Haploinsufficiency of PTCH1, which is caused by interstitial deletion of 9q22.3, is also responsible for the syndrome. To date, 19 cases with interstitial deletion of long arm of chromosome 9 involving the region of q22 have been reported. We describe two unrelated patients with some typical features of BCNS associated with deletion of 9q21.33-q31.1 and determined the boundary of the deletion by fluorescence in situ hybridization (FISH) with bacterial artificial chromosome (BAC) clones. The results showed that the size of deletions is between 15.33 and 16.04 Mb in patient 1 and between 18.08 and 18.54 Mb in patient 2. Although the size and breakpoints were different from those of previously reported cases, the clinical features are common to patients with 9q22 deletion associated with BCNS. Delineation of the 9q22 deletions and further consideration of the genes responsible for the characteristic manifestations may provide insight into this newly recognized deletion syndrome. [source] Characterization of HLA DR3/DQ2 transgenic mice: a potential humanized animal model for autoimmune disease studiesEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 1 2003Dan Chen Abstract Linkage studies indicate close associations of certain HLA alleles with autoimmune diseases. To better understand how specific HLA alleles are related to disease pathogenesis, we have generated an HLA DR3/DQ2 transgenic mouse utilizing a 550-kb yeast artificial chromosome (YAC) construct containing the complete DR,, DR,1, DR,3, DQ,, and DQ, regions. The transgenic mouse (4D1/C2D) in an I-A,o background appears healthy with no signs of autoimmune diseases. Lymphoid tissues as well as CD4+ T cells develop normally. Characterization of the transgene expression demonstrates that ,90% of B cells express high levels of DR3 and 50,70% of B cells express DQ2. CD11c+ dendritic cells express high levels of DR and DQ. Approximately12,18% of resting T cells are positive for DR expression, and further up-regulation to 40,50% expression is seen upon activation with anti-CD3/anti-CD28 mAb. These results suggest that the transgenic construct confers a high fidelity to the normal human temporal and spatial expression profile. Analysis of T cell receptor repertoire in transgenic mice confirms that DR3/DQ2 are able to mediate thymic selection. Furthermore, transgenic mice respond to a DR3-restricted antigen, demonstrating antigen processing and presentation by antigen-presenting cells (APC). Purified T cells from ovalbumin (OVA)-immunized 4D1 mice respond to human APC co-cultured with OVA, suggesting appropriate antigen/DR3 or DQ2 recognition by murine T cells. Immunoglobulin isotype switching is also observed, indicating functional T-B cognate interactions. Thus, the DR3/DQ2 transgenic mouse has normal lymphoid development and functionality that are mediated by HLA transgenes and can be used to investigate HLA-associated immunological questions. [source] Identification of brain neurons expressing the dopamine D4 receptor gene using BAC transgenic miceEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2006Daniela Noaín Abstract The dopamine D4 receptor (D4R) has received considerable interest because of its higher affinity for atypical antipsychotics, the extremely polymorphic nature of the human gene and the genetic association with attention deficit and hyperactivity disorder (ADHD). Several efforts have been undertaken to determine the D4R expression pattern in the brain using immunohistochemistry, binding autoradiography and in situ hybridization, but the overall published results present large discrepancies. Here, we have explored an alternative genetic approach by studying bacterial artificial chromosome (BAC) transgenic mice that express enhanced green fluorescent protein (EGFP) under the transcriptional control of the mouse dopamine D4 receptor gene (Drd4). Immunohistochemical analysis performed in brain sections of Drd4 -EGFP transgenic mice using an anti-EGFP polyclonal antibody showed that transgenic expression was predominant in deep layer neurons of the prefrontal cortex, particularly in the orbital, prelimbic, cingulate and rostral agranular portions. In addition, discrete groups of Drd4 -EGFP labelled neurons were observed in the anterior olfactory nucleus, ventral pallidum, and lateral parabrachial nucleus. EGFP was not detected in the striatum, hippocampus or midbrain as described using other techniques. Given the fine specificity of EGFP expression in BAC transgenic mice and the high sensitivity of the EGFP antibody used in this study, our results indicate that Drd4 expression in the adult mouse brain is limited to a more restricted number of areas than previously reported. Its leading expression in the prefrontal cortex supports the importance of the D4R in complex behaviours depending on cortical dopamine (DA) transmission and its possible role in the etiopathophysiology of ADHD. [source] Developmental elimination of ectopic projection sites for the transgenic OR gene that has lost zone specificity in the olfactory epitheliumEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2003Hiroko Nakatani Abstract In rodents, olfactory receptor (OR) genes are expressed in one of four zones in the olfactory epithelium (OE), and olfactory sensory neurons (OSNs) expressing the same OR project their axons to a specific set of glomeruli on the olfactory bulb (OB). Using the yeast artificial chromosome (YAC) transgenic system, we have analysed the expression of the murine OR gene MOR29A of the MOR28 cluster located on chromosome 14. Although expression of the endogenous MOR29A was restricted to the most dorsomedial zone, the transgenic MOR29A (Tg MOR29A) was expressed in all four zones of the OE. When the OB of the transgenic mouse was analysed, the axons of the OSNs expressing Tg MOR29A were found to project not only to the dorsal side but also to the ventral side of the OB as well. The ectopic projection sites on the ventral side gradually disappear during postnatal development. Naris occlusion prevents this elimination, suggesting that odorant stimulation is involved in eliminating the ectopic projection sites. [source] Artificial chromosome libraries of Streptomyces coelicolor A3(2) and Planobispora rosea,FEMS MICROBIOLOGY LETTERS, Issue 1 2003Rosa Alduina Abstract Using an Escherichia coli,Streptomyces shuttle vector derived from a bacterial artificial chromosome (BAC), we developed methodologies for the construction of BAC libraries of filamentous actinomycetes. Libraries of Streptomyces coelicolor, the model actinomycete, and Planobispora rosea, a genetically intractable strain, were constructed. Both libraries have an average insert size of 60 kb, with maximal insert larger than 150 kb. The S. coelicolor library was evaluated by selected hybridisations to Dra I fragments and by end sequencing of a few clones. Hybridisation of the P. rosea library to selected probes indicates a good representation of the P. rosea genome and that the library can be used to facilitate the genomic analysis of this actinomycete. [source] Rearrangement of upstream sequences of the hTERT gene during cellular immortalizationGENES, CHROMOSOMES AND CANCER, Issue 11 2009Yuanjun Zhao Telomerase expression, resulting from transcriptional activation of the hTERT gene, allows cells to acquire indefinite proliferative potential during cellular immortalization and tumorigenesis. However, mechanisms of hTERT gene activation in many immortal cell lines and cancer cells are poorly understood. Here, we report our studies on hTERT activation using genetically related pairs of telomerase-negative (Tel,) and -positive (Tel+) fibroblast lines. First, whereas transiently transfected plasmid reporters did not recapitulate the endogenous hTERT promoter, the promoter in chromosomally integrated bacterial artificial chromosome (BAC) reporters was activated in a subset of Tel+ cells, indicating that activation of the hTERT promoter required native chromatin context and/or distal regulatory elements. Second, the hTERT gene, located near the telomere of chromosome 5p, was translocated in all three Tel+ cell lines but not in their parental precrisis cells and Tel, immortal siblings. The breakage points were mapped to regions upstream of the hTERT promoter, indicating that the hTERT gene was the target of these chromosomal rearrangements. In two Tel+ cell lines, translocation of the endogenous hTERT gene appeared to be the major mechanism of its activation as the activity of hTERT promoter in many chromosomally integrated BAC reporters, with intact upstream and downstream neighboring loci, remained relatively low. Therefore, our results suggest that rearrangement of upstream sequences is an important new mechanism of hTERT promoter activation during cellular immortalization. The chromosomal rearrangements likely occurred during cellular crisis and facilitated by telomere dysfunction. Such translocations allowed the hTERT promoter to escape from the native condensed chromatin environment. © 2009 Wiley-Liss, Inc. [source] TNFAIP3 is the target gene of chromosome band 6q23.3-q24.1 loss in ocular adnexal marginal zone B cell lymphomaGENES, CHROMOSOMES AND CANCER, Issue 1 2008Keiichiro Honma The genomic aberrations in extra nodal marginal zone B cell lymphoma vary according to their anatomical origin. This polarization is a reflection of the participation of different genes in the lymphomagenesis of marginal zone B cell lymphoma. We previously demonstrated by means of genome-wide array comparative genomic hybridization (CGH) that the genomic profile of ocular adnexal marginal zone B cell lymphoma is distinct from that of pulmonary or nodal marginal zone B cell lymphoma. The novel finding was a recurrent deletion of a 2.9-Mb region at chromosome band 6q23.3-q24.1, including homozygous loss, in ocular adnexal marginal zone B cell lymphoma. For a more detailed examination of the deletions of 6q23.3-24.1, we used contig bacterial artificial chromosome (BAC) array CGH, containing 24 BAC clones covering the 2.9-Mb region, to analyze nine cases with 6q23.3-q24.1 loss. We narrowed the minimal common region down to a length of 586 kb with two genes and four expressed sequence tags (ESTs). All of these genes and ESTs were subjected to RT-PCR and real-time quantitative RT-PCR. Correlation between genomic loss and expression level was found only for TNFAIP3, demonstrating that TNFAIP3 is a target gene of 6q deletion in ocular adnexal marginal zone B cell lymphoma. TNFAIP3 is an inhibitor of NF-kB signaling so that loss of this gene may play an important role in lymphomagenesis and suggests that TNFAIP3 may act as a tumor suppressor gene in ocular adnexal marginal zone B cell lymphoma. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat. © 2007 Wiley-Liss, Inc. [source] Chromosome 8 BAC array comparative genomic hybridization and expression analysis identify amplification and overexpression of TRMT12 in breast cancer,GENES, CHROMOSOMES AND CANCER, Issue 7 2007Virginia Rodriguez Genomic changes in chromosome 8 are commonly observed in breast cancer cell lines and tumors. To fine map such genomic changes by comparative genomic hybridization (CGH), a high resolution (100 kb) chromosome 8 array that can detect single copy changes was developed using Phi29 DNA polymerase amplified BAC (bacterial artificial chromosome) DNA. The BAC array CGH resolved the two known amplified regions (8q21 and 8q24) of a breast cancer cell line (SKBR3) into nine separate regions including six amplicons and three deleted regions, all of which were verified by Fluorescence in situ hybridization. The extent of the gain/loss for each region was validated by qPCR. CGH was performed with a total of 8 breast cancer cell lines, and common regions of genomic amplification/deletion were identified by segmentation analysis. A 1.2-Mb region (125.3,126.5 Mb) and a 1.0-Mb region (128.1,129.1 Mb) in 8q24 were amplified in 7/8 cell lines. A global expression analysis was performed to evaluate expression changes associated with genomic amplification/deletion: a novel gene, TRMT12 (at 125.5 Mb), amplified in 7/8 cell lines, showed highest expression in these cell lines. Further analysis by RT-qPCR using RNA from 30 breast tumors showed that TRMT12 was overexpressed >2 fold in 87% (26/30) of the tumors. TRMT12 is a homologue of a yeast gene encoding a tRNA methyltransferase involved in the posttranscriptional modification of tRNAPhe, and exploring the biological consequence of its altered expression, may reveal novel pathways in tumorigenesis. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat. Published 2007 Wiley-Liss, Inc. [source] A novel gene, MDS2, is fused to ETV6/TEL in a t(1;12)(p36.1;p13) in a patient with myelodysplastic syndromeGENES, CHROMOSOMES AND CANCER, Issue 1 2002María D. Odero ETV6/TEL is the first transcription factor identified that is specifically required for hematopoiesis within the bone marrow. This gene has been found to have multiple fusion partners of which 16 have been cloned. Fluorescence in situ hybridization (FISH) analysis in a patient with myelodysplastic syndrome (MDS) revealed a t(1;12)(p36;p13) involving ETV6, with the breakpoint in this gene between exon 2 and exon 3. We report here the cloning of a novel ETV6 partner located on 1p36.1, involved in the t(1;12). 3, RACE-PCR from RNA identified a novel sequence fused to exon 2 of ETV6. Database searches localized this sequence in a bacterial artificial chromosome (BAC) mapped to 1p36 by fingerprint analysis. This result was confirmed by FISH using this BAC as probe. 5, and 3, RACE experiments with primers from this novel sequence were carried out on RNA from a healthy donor and identified a novel full-length mRNA, which we named MDS2 (myelodysplastic syndrome 2). RT-PCR experiments were performed on a panel of human cDNAs to analyze the expression pattern of this gene and they revealed four splicing variants. RT-PCR analysis showed that ETV6-MDS2, but not the reciprocal MDS2-ETV6 fusion transcript, was expressed in the bone marrow of the patient. The product of the ETV6-MDS2 fusion transcript predicts a short ETV6 protein containing the first 54 amino acids of ETV6 plus four novel amino acids, lacking both the PTN and the DNA-binding domains. Possible mechanisms to account for the development of MDS in this patient are discussed. © 2002 Wiley-Liss, Inc. [source] Identification of evolutionarily conserved regulatory elements in the mouse Fgf8 locusGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 1 2006Friedrich Beermann Abstract The secreted signaling molecule fibroblast growth factor 8 (Fgf8) is an essential component of certain embryonic signaling centers including the mid-hindbrain (isthmic) organizer, the first branchial arch (BA1), and the apical ectodermal ridge (AER). In these signaling centers Fgf8 transcripts are expressed in a dynamic and transient fashion, but the mechanism by which this highly specific expression pattern is established remains largely unknown. We used DNA sequence comparisons coupled to transgenic approaches to obtain insight into the structure and function of regulatory elements in the Fgf8 locus. First, a bacterial artificial chromosome (BAC) containing the mouse Fgf8 gene partially rescues the embryonic lethality of Fgf8- deficient mice and controls Fgf8 -specific gene expression of a coinjected lacZ reporter transgene. Second, sequence comparison of vertebrate Fgf8 loci revealed evolutionarily highly conserved noncoding sequences that were unexpectedly located mainly 3, of the Fgf8 coding region. Third, in transgenic mice some of these elements were sufficient to target expression to the AER, tail bud, and brain, including the isthmic organizer, indicating that they may represent Fgf8 cis-acting elements. Collectively, these data identify novel regulatory elements of the Fgf8 gene sufficient to drive expression to regions of known Fgf8 activity. genesis 44:1,6, 2006. © 2006 Wiley-Liss, Inc. [source] Zebrafish Cx35: Cloning and characterization of a gap junction gene highly expressed in the retinaJOURNAL OF NEUROSCIENCE RESEARCH, Issue 6 2003Elizabeth McLachlan Abstract The vertebrate connexin gene family encodes protein subunits of gap junction channels, which provide a route for direct intercellular communication. Consequently, gap junctions play a vital role in many developmental and homeostatic processes. Aberrant functioning of gap junctions is implicated in many human diseases. Zebrafish are an ideal vertebrate model to study development of the visual system as they produce transparent embryos that develop rapidly, thereby facilitating morphological and behavioral testing. In this study, zebrafish connexin35 has been cloned from a P1 artificial chromosome (PAC) library. Sequence analysis shows a high degree of similarity to the Cx35/36 orthologous group, which are expressed primarily in nervous tissue, including the retina. The gene encodes a 304-amino acid protein with a predicted molecular weight of approximately 35 kDa. Injection of zebrafish Cx35 RNA into paired Xenopus oocytes elicited intercellular electrical coupling with weak voltage sensitivity. In development, Cx35 is first detectable by Northern analysis and RT-PCR, at 2 days post-fertilization (2 dpf), and in the adult it is expressed in the brain and retina. Immunohistochemical analysis revealed that the Cx35 protein is expressed in two sublaminae of the inner plexiform layer of the adult retina. A similar pattern was seen in the 4 and 5 dpf retina, but no labeling was detected in the retina of earlier embryos. © 2003 Wiley-Liss, Inc. [source] Induction of early murine cytomegalovirus infection by different reporter gene-associated recombinant virusesJOURNAL OF VIRAL HEPATITIS, Issue 6 2006U. Drebber Summary., Murine cytomegalovirus (MCMV) has provided useful models for acute, chronic and latent CMV infection because of its similarities in structure and biology with human CMV. We report the induction of acute MCMV hepatitis with different bacterial artificial chromosome (BAC)-cloned virus constructs [MCMV-SEAP which includes the gene for secreted alkaline phosphatase (SEAP) under Rous sarcoma virus (RSV)-promoter control, MCMV-GFP which includes the gene for enhanced green fluorescent protein (eGFP) under HCMV-ie promoter control, MCMV-HBs includes the gene for hepatitis B surface antigen (HBsAg) under simian virus (SV)40-promoter control and the DeltaMC95.21 virus in which the m152 gene was deleted and substituted by the reporter gene lacZ] in order to elucidate the histopathological changes together with different reporter-gene products in the liver tissue and the effect of the deletion of a certain gene. All the virus constructs induced a similar mild acute hepatitis which had its climax from days 3 to 5 post-infection in immunocompetent mice. In situ, the reporter-gene products beta-galactosidase and secreted alkaline phosphatase could be visualized in relation to the inflammatory changes. The composition of the invading cell populations did not change even in the absence of the m152 gene. Additionally discrete inflammatory changes were seen in kidney and serosa while the other organs were not involved. This model helps us understand the immunological and histopathological mechanisms of the CMV-induced hepatitis, which plays an important role especially in the immunocompromised patient. The morphological changes can be analysed while the respective reporter gene product is expressed by the virus construct. [source] SNPs in ecological and conservation studies: a test in the Scandinavian wolf populationMOLECULAR ECOLOGY, Issue 2 2005J. M. SEDDON Abstract Single nucleotide polymorphisms (SNPs) have the potential to become the genetic marker of choice in studies of the ecology and conservation of natural populations because of their capacity to access variability across the genome. In this study, we provide one of the first demonstrations of SNP discovery in a wild population in order to address typical issues of importance in ecology and conservation in the recolonized Scandinavian and neighbouring Finnish wolf Canis lupus populations. Using end sequence from BAC (bacterial artificial chromosome) clones specific for dogs, we designed assays for 24 SNP loci, 20 sites of which had previously been shown to be polymorphic in domestic dogs and four sites were newly identified as polymorphic in wolves. Of the 24 assayed loci, 22 SNPs were found to be variable within the Scandinavian population and, importantly, these were able to distinguish individual wolves from one another (unbiased probability of identity of 4.33 × 10,8), providing equivalent results to that derived from 12 variable microsatellites genotyped in the same population. An assignment test shows differentiation between the Scandinavian and neighbouring Finnish wolf populations, although not all known immigrants are accurately identified. An exploration of the misclassification rates in the identification of relationships shows that neither 22 SNP nor 20 microsatellite loci are able to discriminate across single order relationships. Despite the remaining obstacle of SNP discovery in nonmodel organisms, the use of SNPs in ecological and conservation studies is encouraged by the advent of large scale screening methods. Furthermore, the ability to amplify extremely small fragments makes SNPs of particular use for population monitoring, where faecal and other noninvasive samples are routinely used. [source] Y chromosome microsatellite isolation from BAC clones in the greater white-toothed shrew (Crocidura russula)MOLECULAR ECOLOGY RESOURCES, Issue 1 2006LORI J. LAWSON HANDLEY Abstract We constructed a microsatellite library from four Crocidura russula Y chromosome-specific bacterial artificial chromosome (BAC) clones. Only one of eight microsatellites was male-specific, despite genome walking to obtain more flanking sequence and testing of 93 primer combinations. Potential reasons for this low success are discussed. The male-specific locus, CRY3, was genotyped in 90 males, including C. russula from across the species range and two related species. The large difference in CRY3 allele size between eastern and western lineages supports earlier reports of high divergence between them. Despite polymorphism of CRY3 in Morocco, only one allele was found throughout the whole of Europe, consistent with previous studies that suggest recent colonization of Europe from a small number of Moroccan founders. [source] Five hundred and fifty microsatellite markers for the study of the honeybee (Apis mellifera L.) genomeMOLECULAR ECOLOGY RESOURCES, Issue 2 2003Michel Solignac Summary Microsatellites are currently considered the most useful genetic markers with wide applications in genomics, quantitative and population genetics. We present here the structure of the core sequence of 552 microsatellites, together with the sequences of the primers and the length of the sequenced allele. These microsatellites were isolated from several libraries constructed from either fractions of total genomic DNA or from clones of a bacterial artificial chromosome (BAC) library. All 552 loci are polymorphic in the honeybee. Many of them were also successfully amplified in three other species of Apis: A. cerana (58%), A. dorsata (59%) and A. florea (38%). A summary of the variability of 36 loci in the three main evolutionary lineages of A. mellifera is given. [source] Fluorescence in situ hybridization analysis with a tissue microarray: ,FISH and chips' analysis of pathology archivesPATHOLOGY INTERNATIONAL, Issue 8 2010Haruhiko Sugimura Practicing pathologists expect major somatic genetic changes in cancers, because the morphological deviations in the cancers they diagnose are so great that the somatic genetic changes to direct these phenotypes of tumors are supposed to be correspondingly tremendous. Several lines of evidence, especially lines generated by high-throughput genomic sequencing and genome-wide analyses of cancer DNAs are verifying their preoccupations. This article reviews a comprehensive morphological approach to pathology archives that consists of fluorescence in situ hybridization with bacterial artificial chromosome (BAC) probes and screening with tissue microarrays to detect structural changes in chromosomes (copy number alterations and rearrangements) in specimens of human solid tumors. The potential of this approach in the attempt to provide individually tailored medical practice, especially in terms of cancer therapy, is discussed. [source] Repeated fluorescence in situ hybridization by a microwave-enhanced protocolPATHOLOGY INTERNATIONAL, Issue 9 2006Yasuhiko Kitayama A novel re-hybridization protocol for pathology archive sections that uses microwave-assisted fluorescence in situ hybridization (FISH) is described. Stripping the probe from the pathology archive sections with HCl and re-hybridizing with the next probe by intermittent microwave irradiation generated clear signals without background noise. Repeated stripping and hybridization with numerous bacterial artificial chromosome (BAC)-derived probes would identify the profile of genome-wide changes in small lesions on sections. [source] Efficient cloning of plant genomes into bacterial artificial chromosome (BAC) libraries with larger and more uniform insert sizePLANT BIOTECHNOLOGY JOURNAL, Issue 3 2004Boulos Chalhoub Summary The construction of bacterial artificial chromosome (BAC) libraries remains relatively complex and laborious, such that any technological improvement is considered to be highly advantageous. In this study, we addressed several aspects that improved the quality and efficiency of cloning of plant genomes into BACs. We set the ,single tube vector' preparation method with no precipitation or gel electrophoresis steps, which resulted in less vector DNA damage and a remarkable two- to threefold higher transformation efficiency compared with other known vector preparation methods. We used a reduced amount of DNA for partial digestion (up to 5 µg), which resulted in less BAC clones with small inserts. We performed electrophoresis in 0.25 × TBE (Tris, boric acid, ethylenediaminetetraacetic acid) buffer instead of 0.5 × TBE, which resulted in larger and more uniformly sized BAC inserts and, surprisingly, a two- to threefold higher transformation efficiency, probably due to less contamination with borate ions. We adopted a triple size selection that resulted in an increased mean insert size of up to 70 kb and a transformation efficiency comparable with that of double size selection. Overall, the improved protocol presented in this study resulted in a five- to sixfold higher cloning efficiency and larger and more uniformly sized BAC inserts. BAC libraries with the desired mean insert size (up to 200 kb) were constructed from several plant species, including hexaploid wheat. The improved protocol will render the construction of BAC libraries more available in plants and will greatly enhance genome analysis, gene mapping and cloning. [source] First-generation SNP/InDel markers tagging loci for pathogen resistance in the potato genomePLANT BIOTECHNOLOGY JOURNAL, Issue 6 2003Andreas M. Rickert Summary A panel of 17 tetraploid and 11 diploid potato genotypes was screened by comparative sequence analysis of polymerase chain reaction (PCR) products for single nucleotide polymorphisms (SNPs) and insertion-deletion polymorphisms (InDels), in regions of the potato genome where genes for qualitative and/or quantitative resistance to different pathogens have been localized. Most SNP and InDel markers were derived from bacterial artificial chromosome (BAC) insertions that contain sequences similar to the family of plant genes for pathogen resistance having nucleotide-binding-site and leucine-rich-repeat domains (NBS-LRR-type genes). Forty-four such NBS-LRR-type genes containing BAC-insertions were mapped to 14 loci, which tag most known resistance quantitative trait loci (QTL) in potato. Resistance QTL not linked to known resistance-gene-like (RGL) sequences were tagged with other markers. In total, 78 genomic DNA fragments with an overall length of 31 kb were comparatively sequenced in the panel of 28 genotypes. 1498 SNPs and 127 InDels were identified, which corresponded, on average, to one SNP every 21 base pairs and one InDel every 243 base pairs. The nucleotide diversity of the tetraploid genotypes (, = 0.72 × 10,3) was lower when compared with diploid genotypes (, = 2.31 × 10,3). RGL sequences showed higher nucleotide diversity when compared with other sequences, suggesting evolution by divergent selection. Information on sequences, sequence similarities, SNPs and InDels is provided in a database that can be queried via the Internet. [source] Isolation of two microsatellite markers from BAC clones of the Vf scab resistance region and molecular characterization of scab-resistant accessions in Malus germplasm,PLANT BREEDING, Issue 4 2004B. A. Vinatzer Abstract A polymerase chain reaction (PCR)-based method was developed to isolate microsatellite markers from large-insert genomic DNA clones of bacterial artificial chromosome (BAC) libraries. The method is fast and economic since it does not require subcloning. It was applied to isolate a microsatellite marker from a BAC clone of the chromosomal region containing the apple scab resistance gene Vf. The Vf gene of Malus floribunda 821 is the most widely used source of scab resistance in apple breeding. A second microsatellite was found on the extremity of a BAC clone flanking the Vf locus. The two microsatellites allowed the identification of the presence of the Vf gene in the scab-resistant accessions M. micromalus SA573-3, ,Golden Gem', M. prunifolia 19651 and MA 16 not previously known to carry this gene. They were also used to verify the correctness of the published genealogical tree of the Vf cultivar ,Florina', in which a probable mistake was identified. This analysis shows the importance of genotyping the Vf locus when choosing scab-resistant germplasm as parents in breeding programmes. [source] Novel microsatellite markers for the analysis of Phytophthora infestans populationsPLANT PATHOLOGY, Issue 3 2006A. K. Lees Co-dominant microsatellite molecular markers for Phytophthora infestans were developed and their potential for monitoring the genetic variation in populations was demonstrated in the UK, across Europe and worldwide. Markers were developed according to two strategies. First, several thousand P. infestans expressed sequence tag (EST) and bacterial artificial chromosome (BAC) sequences were screened for the presence of simple sequence repeat (SSR) motifs, and, of these, 100 candidate loci were selected for further investigation. Primer pairs developed to these loci were tested against a panel of 10 P. infestans isolates and approximately 10% were shown to be polymorphic and therefore appropriate for further testing. Secondly, the construction and screening of a partial genomic library resulted in the development of one additional polymorphic marker. The resulting 12 SSR markers were converted to higher-throughput fluorescence-based assays and used in combination with two previously published markers to characterize a wider collection of 90 P. infestans isolates from the UK and six other countries. Several isolates from the closely related species P. mirabilis, P. ipomoea and P. phaseoli collected from around the world were also genotyped using these markers. Amongst the 90 isolates of P. infestans examined, considerable SSR diversity was observed, with 68 different genotypes and an average of 3·9 (range 2,9) alleles per locus. When other Phytophthora species were genotyped, all loci were successfully amplified and the majority were polymorphic, indicating their transferability for the potential study of other closely related taxa. [source] Haplotype divergence in Beta vulgaris and microsynteny with sequenced plant genomesTHE PLANT JOURNAL, Issue 1 2009Juliane C. Dohm Summary We characterized two overlapping sugar beet (Beta vulgaris) bacterial artificial chromosome (BAC) clones representing different haplotypes. A total of 254 kbp of the genomic sequence was determined, of which the two BACs share 92 kbp. Eleven of 15 genes discovered in the sequenced interval locate to the overlap region. The haplotypes differ in exons by 1% (nucleotide level) and in non-coding regions by 9% (6% mismatches, 3% gaps; alignable regions only). Large indels or high sequence divergence comprised 11% of either sequence. Of such indels, 68 and 45%, respectively, could be attributed to haplotype-specific integration of transposable elements. We identified novel repeat candidates by comparing the two BAC sequences to a set of genomic sugar beet sequences. Synteny was found with Arabidopsis chromosome 1 (At1), At2 and At4, Medicago chromosome 7, Vitis chromosome 15 and paralogous regions on poplar chromosomes II and XIV. [source] BAC-based upgrading and physical integration of a genetic SNP map in Atlantic salmonANIMAL GENETICS, Issue 1 2010S. Lorenz Summary A better understanding of the genotype,phenotype correlation of Atlantic salmon is of key importance for a whole range of production, life history and conservation biology issues attached to this species. High-density linkage maps integrated with physical maps and covering the complete genome are needed to identify economically important genes and to study the genome architecture. Linkage maps of moderate density and a physical bacterial artificial chromosome (BAC) fingerprint map for the Atlantic salmon have already been generated. Here, we describe a strategy to combine the linkage mapping with the physical integration of newly identified single nucleotide polymorphisms (SNPs). We resequenced 284 BAC-ends by PCR in 14 individuals and detected 180 putative SNPs. After successful validation of 152 sequence variations, genotyping and genetic mapping were performed in eight salmon families comprising 376 individuals. Among these, 110 SNPs were positioned on a previously constructed linkage map containing SNPs derived from expressed sequence tag (EST) sequences. Tracing the SNP markers back to the BACs enabled the integration of the genetic and physical maps by assigning 73 BAC contigs to Atlantic salmon linkage groups. [source] Assignment of 128 genes localized on human chromosome 14q to the IMpRH mapANIMAL GENETICS, Issue 4 2009T. Shimogiri Summary To provide a gene-based comparative map and to examine a porcine genome assembly using bacterial artificial chromosome-based sequence, we have attempted to assign 128 genes localized on human chromosome 14q (HSA14q) to a porcine 7000-rad radiation hybrid (IMpRH) map. This study, together with earlier studies, has demonstrated the following. (i) 126 genes were incorporated into two SSC7 RH linkage groups by CarthaGene analysis. (ii) In the remaining two genes, TOX4 linked to TCRA located in SSC7 by two-point analysis, whereas SIP1 showed no significant linkage with any gene/marker registered in the IMpRH Web Server. (iii) In the two groups, the gene clusters located from 19.9 to 36.5 Mb on HSA14q11.2-q13.3 and from 64.0 to 104.3 Mb on HSA14q23-q32.33 respectively were assigned to SSC7q21-q26. (iv) Comparison of the gene order between the present RH map and the latest porcine sequence assembly revealed some inconsistencies, and a redundant arrangement of 16 genes in the sequence assembly. [source] Genome-wide genetic diversity of ,Nici', the DNA source for the CHORI-260 turkey BAC library and candidate for whole genome sequencingANIMAL GENETICS, Issue 3 2009L. D. Chaves Summary Vertebrate whole genome sequence assembly can benefit from a priori knowledge of variability in the target genome, with researchers often selecting highly inbred individuals for sequencing. However, for most species highly inbred research lines are lacking, requiring the use of an outbred individual(s). Here we examined the source DNA [Nicholas inbred (Nici)] of the CHORI-260 turkey bacterial artificial chromosome (BAC) library through analysis of microsatellites and BAC sequences. Heterozygosity of Nici was compared with that of individuals from several breeder lines. Seventy-eight microsatellites were screened for polymorphism in a total of 43 birds, identifying an average individual heterozygosity of 0.39, with Nici at 0.35. Additional loci (total of 147) were examined on a subset of individuals to obtain better genome coverage. The mean heterozygosity for this subset was 0.33 with Nici at 0.31. Examination of approximately 200 kb of genome sequence identified SNPs in the order of one per 200 bp in Nici. These data suggest that the heterozygosity of Nici is comparable to other birds of selected breeder lines and that whole genome sequencing would result in an abundant resource of genome-wide polymorphisms. [source] Genomic structure and gene order of swine chromosome 7q1.1,q1.2ANIMAL GENETICS, Issue 1 2006M. Tanaka Summary To clarify the structure of the porcine genomic region that contains quantitative trait loci (QTL) related to fat, we constructed a bacterial artificial chromosome (BAC) contig of the region from DST to SRPK1 on porcine chromosome 7 and performed low-redundancy ,skim' shotgun sequencing of the clones that composed a minimum tiling path of the contig. This analysis revealed that the gene order from VPS52 to SRPK1 is conserved between human and swine and that comparison with the human sequence identified a rearrangement in the swine genome at the proximal end of VPS52. Analysis of the nucleotide sequences of three BAC clones that included the rearrangement point demonstrated that COL21A1 and DST, which were not present in the corresponding human region, were located adjacent to the rearrangement point. These results provide useful information about the genomic region containing QTL for fat in pigs and help to clarify the structure of the so-called ,extended-class II' region distal to the porcine major histocompatibility complex class II region. [source] Characterization of the porcine melanocortin 2 receptor gene (MC2R,)ANIMAL GENETICS, Issue 6 2002K. Jacobs A porcine bacterial artificial chromosome (BAC) clone, containing the melanocortin 2 receptor gene (MC2R) was isolated. The complete coding sequence of the MC2R gene, contained in 1 exon, was determined. Polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) was performed on a 241-bp coding fragment. An AluI polymorphism, detecting a silent mutation, was found and typed on unrelated animals of five different pig breeds. The Meishan, Piétrain and Large White breeds differ significantly in allele frequencies from the Landrace and Czech Meat Pig breeds. The melanocortin 5 receptor gene (MC5R) was detected by PCR in the same BAC clone, as could be expected from the human and porcine mapping data. PCR-SSCP was performed on a 200-bp coding of MC5R, but no polymorphisms were detected. The BAC clone was mapped to Sscr6q27 by fluorescent in situ hybridization (FISH). A (CA)n microsatellite (SGU0002), isolated from the BAC, was localized on chromosome 6 by RH mapping near marker SW1473 and by linkage mapping on the MARC reference family at the same position as the marker SW2173 (97 cM). Allele frequencies, heterozygosity and polymorphism information contents (PIC) values were calculated for the five different pig breeds examined. The transcription of both genes in porcine liver, heart, kidney, fat, brain, pancreas, stomach, bladder, ovaries, lung, spleen, skin, adrenal gland and muscle tissues was examined by reverse transcriptase-polymerase chain reaction. Transcription was detected in skin and adrenal gland tissues for MC2R, while a positive signal was detected for MC5R in kidney, fat, pancreas, skin, adrenal gland and spleen tissues. [source] Construction and evaluation of a porcine bacterial artificial chromosome libraryANIMAL GENETICS, Issue 1 2000K Suzuki Summary A porcine bacterial artificial chromosome (BAC) library consisting of 103 488 clones has been constructed. The average insert size in the BAC vector was calculated to be 133 kb based on the examination of 189 randomly selected clones, indicating that the library contained 4.4 genome equivalents. The library can be screened by two-step PCR. The first screening step is performed on 22 superpools, each containing 4704 clones (49×96 well plates). In the second screening step, 49 plates comprising a superpool are arrayed in a 7×7 matrix and 4D-PCR is performed. Screening of the library superpools by PCR for 125 marker sequences selected from different regions of swine genome revealed 123 sequences, indicating that the library is not biased. Subsequent screenings (4D-PCR) were successfully applied for identification of clones containing each marker sequence. This porcine BAC library and the PCR screening system are useful for isolation of genomic DNA fragments containing desired sequences. [source] | |||||||||||||||||||||||||||||||