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PubMed2004

Bacterial artificial chromosome (BAC) library resource for positional cloning of pest and disease resistance genes in cassava (Manihot esculenta Crantz).

Pest and disease problems are important constraints of cassava production and host plant resistance is the most efficient method of combating them. Breeding for host plant resistance is considerably slowed down by the crop's biological constraints of a long growth cycle, high levels of heterozygosity and a large genetic load. More efficient methods such as gene cloning and transgenesis are required to deploy resistance genes. To facilitate the cloning of resistance genes, bacterial artificial chromosome (BAC) library resources have been developed for cassava. Two libraries were constructed from the cassava clones, TMS 30001, resistant to the cassava mosaic disease (CMD) and the cassava bacterial blight (CBB), and MECU72, resistant to cassava white fly. The TMS30001 library has 55, 296 clones with an insert size range of 40-150 kb with an average of 80 kb, while the MECU72 library consists of 92 160 clones and an insert size range of 25-250 kb average of 93 kb. Based on a genome size of 772 Mb, the TMS30001 and MECU72 libraries have a 5 and 11.3 haploid genome equivalents and a 95 and 99 chance of finding any sequence, respectively. To demonstrate the potential of the libraries, the TMS30001 library was screened by southern hybridization using a cassava analog (CBB1) of the Xa21 gene from rice that maps to a region containing a QTL for resistance to CBB as probe. Five BAC clones that hybridized to CBB1 were isolated and a Hind III fingerprint revealed 2-3 copies of the gene in individual BAC clones. A larger scale analysis of resistance gene analogs (RGAs) in cassava has also been conducted in order to understand the number and organization of RGAs. To scan for gene and repeat DNA content in the libraries, end-sequencing was performed on 2,301 clones from the MECU72 library. A total of 1705 unique sequences were obtained with an average size of 715 bp. Database homology searches using BLAST revealed that 458 sequences had significant homology with known proteins and 321 with transposable elements. The use of the library in positional cloning of pest and disease resistance genes is discussed.

Animals↗
PubMed1992

Angiotensin-II-induced expression of proto-oncogene (c-fos, jun-B and c-jun) mRNA in bovine adrenocortical fasciculata cells (BAC) is mediated by AT-1 receptors.

We have shown previously that angiotensin-II (A-II) controls proto-oncogene (c-fos, jun-B and c-jun) mRNA accumulation in bovine adrenal fasciculata cells (BAC). Since BAC contain both subtypes (AT-1 and AT-2) of the A-II receptor, we have investigated which subtype was involved in the effect of A-II on proto-oncogene mRNA by using a selective antagonist for AT-1 (DUP 753) and for AT-2 (CGP 42112A). DUP 753, but not CGP 42112A, inhibited the stimulatory effect of A-II on proto-oncogene mRNA, with ID50s of 4 x 10(-7) M, 7 x 10(-7) M and 2 x 10(-6) M for c-fos, jun-B and c-jun, respectively. Neither of the two antagonists by themselves had a direct effect on proto-oncogene mRNA. As the A-II AT-1 receptors are coupled to the phospholipase C system in BAC, we have investigated whether the A-II effects on the proto-oncogenes were mediated by protein kinase C (PKC) or by Ca2+ calmodulin. First, activation of PKC by the phorbol ester, PMA, increased the level of three proto-oncogene mRNAs, whereas calcium ionophore had no effect. Second, staurosporine, a specific inhibitor of PKC, reduced the stimulatory action of A-II on proto-oncogene mRNA by 80-90%, whereas trifluoroperazine, an inhibitor of calmodulin, had no significant effect. These results demonstrate that the effects of A-II on proto-oncogene mRNA are mediated by AT1 receptor subtypes, mainly through activation of the PKC pathway.

Angiotensin II↗
PubMed2006

Fluorescence in situ hybridization studies using BAC clones of the EVI1 locus in hematological malignancies with 3q rearrangements.

Chromosomal rearrangements involving 3q26 are a recurrent aberration in malignant myeloid disorders. Several of these rearrangements involve the EVI1 oncogene or its surrounding sequences and are associated with a poor prognosis. Fluorescence in situ hybridization (FISH) studies using bacterial artificial chromosome (BAC) clones were conducted to determine whether the EVI1 locus was rearranged in nine patients with hematological malignancies carrying 3q abnormalities. A dual-color probe was constructed with nine BACs; centromeric clones covering 1 Mb and including the EVI1 gene were labeled with a red fluorescent dye and telomeric clones covering 1 Mb were labeled with a green fluorescent dye. Two patients showed normal copies of the EVI1 locus, four patients showed one EVI1 locus rearranged (in all of them the breakpoint on 3q26 was telomeric to EVI1), one patient showed one copy of the EVI1 locus translocated to another chromosome, one patient showed one copy of the EVI1 locus rearranged and the other copy translocated, and one patient showed one extra copy of the EVI1 locus. In four cases, FISH studies using the EVI1 clones detected different 3q abnormalities not previously found by conventional cytogenetics. FISH analysis with BAC clones was a useful tool for identifying the chromosome breakpoints affecting the EVI1 locus in patients with 3q26 rearrangements.

Chromosome Aberrations↗
PubMed2004

Insight into Trichoderma reesei's genome content, organization and evolution revealed through BAC library characterization.

Trichoderma reesei is an important industrial fungus known for its ability to efficiently secrete large quantities of protein as well as its wide variety of biomass degrading enzymes. Past research on this fungus has primarily focused on extending its protein production capabilities, leaving the structure of its 33 Mb genome essentially a mystery. To begin to address these deficiencies and further our knowledge of T. reesei's secretion and cellulolytic potential, we have created a genomic framework for this fungus. We constructed a BAC library containing 9216 clones with an average insert size of 125 kb which provides a coverage of 28 genome equivalents. BAC ends were sequenced and annotated using publicly available software which identified a number of genes not seen in previously sequenced EST datasets. Little evidence was found for repetitive sequence in T. reesei with the exception of several copies of an element with similarity to the Podospora anserina transposon, PAT. Hybridization of 34 genes involved in biomass degradation revealed five groups of co-located genes in the genome. BAC clones were fingerprinted and analyzed using fingerprinted contigs (FPC) software resulting in 334 contigs covering 28 megabases of the genome. The assembly of these FPC contigs was verified by congruence with hybridization results.

Chromosome Mapping↗
PubMed2006

Construction of a contig of BAC clones spanning the region of the apple scab avirulence gene AvrVg.

The ascomycete Venturia inaequalis, causal pathogen of apple scab, underlies a gene-for-gene relationship with its host plant apple (Malus spp.). 'Golden Delicious', one of the most common cultivated apples in the world, carries the ephemeral resistance gene Vg. Avirulence gene AvrVg, matching resistance gene Vg has recently been mapped on the V. inaequalis genome. In this paper, we present the construction of a BAC library from a V. inaequalis AvrVg isolate. The library is composed of 7680 clones, with an average insert size of 80kb. By hybridization, it has been estimated that the library contains six haploid genome equivalents. Thus the V. inaequalis genome can be predicted to be approximately 100Mb in size. A chromosome walk, starting from the marker VirQ5 co-segregating with AvrVg, has been performed using the BAC library. Twelve BAC clones were identified during four steps of the chromosome walking. The size of the resulting contig is approximately 330kb.

Ascomycota↗
PubMed2004

Genetic analysis of the homeodomain transcription factor Chx10 in the retina using a novel multifunctional BAC transgenic mouse reporter.

Chx10 is a homeobox-containing transcription factor critical for progenitor cell proliferation and bipolar cell determination in the developing retina. Its expression in the retina has been reported to be restricted to these cell populations. To further understand Chx10 regulation and function, a multifunctional reporter construct consisting of GFP, alkaline phosphatase, and Cre recombinase was integrated into a BAC encoding Chx10. Stable lines of transgenic mice expressing this BAC were generated and analyzed. The reporter expression was faithful to the endogenous retinal Chx10 expression pattern and revealed a previously unappreciated locus of Chx10 expression in a subset of Müller glial cells. In addition, Chx10 reporter activity was identified in mature orJ-Chx10 mutant retinas, although these retinas lack Chx10-expressing bipolar cells. Reporter and molecular analysis showed that the reporter-expressing cells in the mutant had hallmarks of progenitor cells or partially differentiated Müller glial cells. These results strongly suggest that Chx10 promotes bipolar fate by affecting differentiation of late progenitor cells. Crosses of the Chx10 BAC reporter mice to R26R mice for fate-mapping experiments revealed that Chx10 reporter-expressing progenitor cells contribute to all mature cell types of the retina. These results demonstrate the utility of these lines for generation of mosaic or complete genetic manipulations of the retina.

Alkaline Phosphatase↗
PubMed2006

Analysis of Pax6 expression using a BAC transgene reveals the presence of a paired-less isoform of Pax6 in the eye and olfactory bulb.

Pax6, a member of the paired-family of transcription factors, exhibits restricted expression and essential functions in the developing eye, olfactory system, central nervous system, and pancreas. To understand Pax6 function, which critically depends on induction of proper expression levels during development, it is necessary to elucidate the molecular mechanisms governing Pax6 transcription. Although previous studies using classic transgenic approaches have provided a wealth of information about the distribution and types of regulatory elements involved in Pax6 regulation, genetic studies in both humans and mice indicate that these enhancers alone are not sufficient for fully regulated Pax6 expression. We report here our analysis of mice transgenic for a 160 kb mouse Pax6 BAC transgene, which was generated as a necessary first step towards testing the long-range control of Pax6 expression in vivo. We show that this BAC transgene replicates Pax6 expression in the eye. This is the first time that a reporter transgene has been expressed in a normal Pax6-like pattern in all of the tissues of the eye and defines an eye regulatory region within the Pax6 downstream regulatory region (DRR). Second, we show that this BAC transgene contains all of the cis regulatory elements required for normal Pax6 expression within the developing embryo, except for within the diencephalon and olfactory bulb. Third, we show that this transgene is subject to Pax6 autoregulation. Lastly, we identify, for the first time in mammals, an isoform of the Pax6 protein lacking the paired domain. This isoform is expressed in the developing olfactory bulb and eye. Over-expression of Pax6DeltaPD causes a microphthalmic phenotype in both Pax6(+/+) mice and Pax6(+/-) mice. These results demonstrate a role for Pax6DeltaPD in eye development, which appears to be different than that ascribed to either canonical Pax6 or Pax6(5a).

Animals↗
PubMed2004

Tissue-specific expression of a BAC transgene targeted to the Hprt locus in mouse embryonic stem cells.

The hypoxanthine phosphoribosyltransferase (Hprt) locus has been shown to have minimal influence on transgene expression when used as a surrogate site in the mouse genome. We have developed a method to transfer bacterial artificial chromosomes (BACs) as a single copy into the partially deleted Hprt locus of embryonic stem cells. BACs were modified by Cre/loxP recombination to contain the sequences necessary for homologous recombination into and complementation of the partially deleted Hprt locus. Modified BACs were shown to undergo homologous recombination into the genome intact, to be stably transmitted through the germ line of transgenic mice, and to be expressed in the proper tissue-specific manner. This technology will facilitate many studies in which correct interpretation of data depends on developmentally appropriate transgene expression in the absence of rearrangements or deletions of endogenous DNA.

Animals↗
PubMed2006

High-density comparative BAC mapping in the black muntjac (Muntiacus crinifrons): molecular cytogenetic dissection of the origin of MCR 1p+4 in the X1X2Y1Y2Y3 sex chromosome system.

The black muntjac (Muntiacus crinifrons, 2n = 8[female symbol]/9[male symbol]) is a critically endangered mammalian species that is confined to a narrow region of southeastern China. Male black muntjacs have an astonishing X1X2Y1Y2Y3 sex chromosome system, unparalleled in eutherian mammals, involving approximately half of the entire genome. A high-resolution comparative map between the black muntjac (M. crinifrons) and the Chinese muntjac (M. reevesi, 2n = 46) has been constructed based on the chromosomal localization of 304 clones from a genomic BAC (bacterial artificial chromosome) library of the Indian muntjac (M. muntjak vaginalis, 2n = 6[female symbol]/7[male symbol]). In addition to validating the chromosomal homologies between M. reevesi and M. crinifrons defined previously by chromosome painting, the comparative BAC map demonstrates that all tandem fusions that have occurred in the karyotypic evolution of M. crinifrons are centromere-telomere fusions. The map also allows for a more detailed reconstruction of the chromosomal rearrangements leading to this unique and complex sex chromosome system. Furthermore, we have identified 46 BAC clones that could be used to study the molecular evolution of the unique sex chromosomes of the male black muntjacs.

Animals↗
PubMed2001

Enhanced biodegradation of petrochemical wastewater using ozonation and BAC advanced treatment system.

The characteristics of degradation/conversion of bio-refractory and the growth of a biofilm are investigated in laboratory-scale pre-ozonation and lifted moving-bed biological activated carbon (BAC) advanced treatment processes treating phenol, benzoic acid, aminobenzoic acid and petrochemical industry wastewater which contains acrylonitrile butadiene styrene (ABS). The optimal reaction time and ozone dosage of pre-ozonation for bio-refractory conversion were determined to be 30 min and 100-200 mg O3/hr, respectively. After pre-ozonation of 30 min treatment, BOD5/COD ratio of influent and effluent increased apparently from 20 to 35%, approximately. However, the change of pH in pre-ozonation was inconspicuous. The optimal flow rate of influent and air were controlled at 1.6 l/h and 120-150 nl/min in lifted moving-bed BAC advanced treatment reactor. A COD removal efficiency of 85-95% and 70-90% may be maintained by using an organic loading of 3.2-6.3 kg COD/m3 day and 0.6-1.6 kg-COD/m3 day with an HRT of 6.0 h as secondary and advanced treatment system, respectively. The time required for the BAC bed is be regenerated by a thermal regeneration is prolonged 4-5 times more than that of GAC system. It can be estimated that the enhanced COD removal capability of the biofilm was not only due to the increase in the COD removal capability of acclimated bacteria, but also due to species succession of bacteria in bio-film ecosystem.

Adsorption↗
PubMed1997

Functional identification of the mouse circadian Clock gene by transgenic BAC rescue.

As a complementary approach to positional cloning, we used in vivo complementation with bacterial artificial chromosome (BAC) clones expressed in transgenic mice to identify the circadian Clock gene. A 140 kb BAC transgene completely rescued both the long period and the loss-of-rhythm phenotypes in Clock mutant mice. Analysis with overlapping BAC transgenes demonstrates that a large transcription unit spanning approximately 100,000 base pairs is the Clock gene and encodes a novel basic-helix-loop-helix-PAS domain protein. Overexpression of the Clock transgene can shorten period length beyond the wild-type range, which provides additional evidence that Clock is an integral component of the circadian pacemaking system. Taken together, these results provide a proof of principle that "cloning by rescue" is an efficient and definitive method in mice.

Animals↗
PubMed2001

Simple and efficient vectors for retrofitting BACs and PACs with mammalian neoR and EGFP marker genes.

Bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs) are widely used to investigate the functions of genes and genomes in mammalian cells in vitro and in vivo. We have developed a series of vectors which can simply and efficiently be retrofitted onto BACs or PACs. These vectors carry a neoR gene for selection in cells in tissue culture, including ES cells, and also an EGFP gene driven by the strong CAG promoter for quick detection of the DNA in cells. All the plasmids are retrofitted using the loxP site and Cre recombinase and some carry the gamma origin of plasmid R6K which does not function in commonly used bacteria such as DH10B. Retrofitting of PACs and BACs carrying alphoid DNA was very efficient with almost no rearrangement of the highly repetitive alphoid DNA. Following transfer into HT1080 cells and mouse oocytes in tissue culture the DNA could easily be monitored by the EGFP fluorescence.

Animals↗
PubMedDate not supplied

Influence of the Fight BAC! food safety campaign on an urban Latino population in Connecticut.

OBJECTIVE: To assess the coverage and consumer satisfaction with the Fight BAC! campaign and to evaluate the influence of the campaign on food safety knowledge, attitudes, and behaviors among a predominantly Latino population living in inner-city Hartford, Connecticut. DESIGN: A cross-sectional pre- and post-survey was administered to 500 Latino consumers in either English or Spanish. It included 30 food safety-related questions and information on the socioeconomic and demographic characteristics of participants. SETTING AND PARTICIPANTS: Respondents were selected from Latino households, with at least one child 12 years old or under, located in 5 predominantly Latino neighborhoods in inner-city Hartford. INTERVENTION: Fight BAC! media campaign. RESULTS: Seventy-three percent of respondents were exposed to at least one campaign media item and were highly satisfied with it. Recognition of the Fight BAC! logo increased from 10% to 42% between surveys (P <.001). Individuals exposed to the campaign were more likely to have a food safety knowledge score of at least 2 of a possible 4 compared with unexposed counterparts (odds ratio = 3.54; 95% CI 1.74-7.18; P <.001). They were also more likely to report defrosting meats in the refrigerator (14% vs 7%; P =.01). There was a dose-response association between the degree of campaign exposure and awareness of the term "cross-contamination." CONCLUSION AND IMPLICATIONS: Social marketing campaigns that take advantage of multiple culturally relevant media channels are likely to improve food safety awareness and bring about changes in food safety knowledge and attitudes among Latino consumers.

Adult↗
PubMed2002

Construction of a BAC contig for a 3 cM biologically significant region of mouse chromosome 1.

One QTL and genes and phenotypes have been localized in the region between 92 cM and 95cM of mouse chromosome 1. The QTL locus contributes to approximately 40% of the variation of the peak bone density between C57BL/6J (B6) and CAST/EiJ (CAST) strains. Other loci located in this chromosomal region include a neural tube defect mutant loop-tail (Lp), a lymphocyte-stimulating determinant (Lsd), and the Transgelin 2 (Tagln 2). The human chromosome region homologous to this region is 1q21-23, which also contains a QTL locus for high bone mineral density (BMD). Furthermore, it has been reported that this region may have duplicated several times in the mouse genome. Therefore, genomic sequencing of this region will provide important information for mouse genome structure, for positional cloning of mouse genes, and for the study of human homologous genes. In order to provide a suitable template for genomic sequencing by the NIH-sponsored genomic centers, we have constructed a BAC contig of this region using the RPCI-23 library. We have also identified the currently available mouse genomic sequences localized in our BAC contig. Further analysis of these sequences and BAC clones indicated a high frequency of repetitive sequences within this chromosomal area. This region also contains L1 retrotransposon sequences, providing a potential mechanism for the repetitive sequences described in the literature.

Animals↗
PubMed2003

Identification and characterisation of a melon genomic region containing a resistance gene cluster from a constructed BAC library. Microcolinearity between Cucumis melo and Arabidopsis thaliana.

A bacterial artificial chromosome (BAC) library from the dihaploid melon line 'PIT92' was constructed with a 6 times coverage of the haploid melon genome. A contig of four BACs around the MRGH63 resistance gene homologue fragment was created. The complete sequence of a 117-kb BAC clone allowed to determine two clearly defined regions, the first one containing a cluster of three resistance gene homologues. Separated by a retrotransposon, that contains large long terminal repeats, the second region presents a group of genes with a conserved distribution in two regions of the Arabidopsis genome. The detailed analysis of this region provides a description of the gene structure and the presence of repetitive sequences in a defined fragment of the genome of Cucumis melo.

Amino Acid Sequence↗
PubMed2001

A high-resolution integrated map spanning the SDHD gene at 11q23: a 1.1-Mb BAC contig, a partial transcript map and 15 new repeat polymorphisms in a tumour-suppressor region.

Chromosomal region 11q22-q23 is a frequent target for deletion during the development of many solid tumour types, including breast, ovary, cervix, stomach, bladder carcinomas and melanoma. One of the most commonly deleted subregions contains the SDHD gene, which encodes the small subunit of cytochrome b (cybS) in mitochondrial complex II (succinate-ubiquinone oxidoreductase). Germline mutations in SDHD cause hereditary paraganglioma type 1 (PGL1), and suggest a tumour suppressor role for cybS. We present a high-resolution physical map spanning SDHD, covered by 19 YACs and 20 BACs. An approximate 1.1-Mb gene-rich region around SDHD is spanned by a complete BAC contig. Twenty-six new STSs are developed from the BAC clone ends. In addition to the discovery and characterisation of 15 new simple tandem repeat polymorphisms, we provide integrated positional information for 33 ESTs and known genes, including KIAA1391, POU2AF1 (OBF1), PPP2R1B, CRYAB, HSPB2, DLAT, IL-18, PTPS, KIAA0781 and KAIA4591, which is mapped by NotI site cloning. We describe full-length transcript sequence for PPP2R1B, encoding the protein phosphatase 2A regulatory subunit A beta isoform. We also discover a processed pseudogene for USA-CYP, a cyclophilin associated with U4/U6 snRPNs, and a novel gene, DDP2, encoding a mitochondrial protein similar to the X-linked deafness-dystonia protein, which is juxtaposed 5'-to-5' to SDHD. This map will help assess this gene-rich region in PGL and in other common tumours.

3' Untranslated Regions↗
PubMed2005

Functional identification of a BAC clone from 16q24 carrying a senescence gene SEN16 for breast cancer cells.

We have identified an 85 kb BAC clone, 346J21, that carries a cell senescence gene (SEN16), previously mapped to 16q24.3. Transfer and retention of 346J21 in breast cancer cell lines leads to growth arrest after 8-10 cell doublings, accompanied by the appearance of characteristic senescent cell morphology and senescence-associated acid beta-galactosidase activity. Loss of transferred BAC results in reversion to the immortal growth phenotype of the parental cancer cell lines. BAC 346J21 restores senescence in the human breast cancer cell lines, MCF.7 and MDA-MB468, and the rat mammary tumor cell line LA7, but not in the human glioblastoma cell line T98G. We postulate that inactivation of both copies of SEN16 is required for the immortalization of breast epithelial cells at an early stage of tumorigenesis. Positional mapping of 346J21 shows that SEN16 is distinct from other candidate tumor suppressor genes reported at 16q24.

Animals↗
PubMed2000

Construction and characterization of an ovine BAC contig spanning the callipyge locus.

We describe the construction of an ovine BAC contig spanning a 4.6 centimorgan (cM) chromosome segment known to contain the callipyge (CLPG) locus. The contig comprises 21 ovine BAC clones jointly covering approximately 900 kilobases (Kb). Two gaps in the BAC contig, spanning 10 and 7.5 Kb, respectively, were bridged by long range PCR. The corresponding chromosome region was shown to be characterized by an unusually low Kb to cM ratio (164 Kb/cM) and a high density of Not1 sites (1:126 Kb) possibly reflecting a high gene density in the corresponding chromosome region. Equivalent amplification of 64 sequence tagged sites spanning the corresponding region from homozygous +/+ and CLPG/CLPG individuals disproves the hypothesis of a major deletion causing the CLPG mutation.

Animals↗