Single Bacterial Artificial Chromosome Clone Research Articles

Transgene Recombineering in Bacterial Artificial Chromosomes.

Bacterial Artificial Chromosome (BAC) libraries are a valuable research resource. Any oneof the clones in these libraries can carry hundreds of thousands of base pairs of genetic information. Often the entire coding sequence and significant upstream and downstream regions, including regulatory elements, can be found in a single BAC clone. BACs can be put to many uses, such as to study the function of human genes in knockout mice, to drive reporter gene expression in transgenic animals, and for gene discovery. In order to use BACs for experimental purposes it is often desirable to genetically modify them by introducing reporter elements or heterologous cDNA sequences. It is not feasible to use conventional DNA cloning approaches to modify BACs due to their size and complexity, thus a specialized field "recombineering" has developed to modify BAC clones through the use of homologous recombination in bacteria with short homology regions. Genetically engineered BACs can then be used in cell culture, mouse, or rat models to study cancer, neurology, and genetics.

The Rvi15 (Vr2) Apple Scab Resistance Locus Contains Three TIR-NBS-LRR Genes

Scab caused by the pathogen Venturia inaequalis is considered the most important fungal disease of cultivated apple (Malus x domestica Borkh.). In all, 16 monogenic resistances against scab have been found in different Malus spp. and some of them are currently used in apple breeding for scab-resistant cultivars. However, the self incompatibility and the long generation time of Malus spp. together with the high standards of fruit quality demanded from the fresh market render the breeding of high-quality cultivars in apple a long and expensive task. Therefore, the cloning of disease resistance genes and the use of the cloned genes for the transformation of high-quality apple cultivars could be an approach to solve these drawbacks. We report the construction of a bacterial artificial chromosome (BAC) contig spanning the Rvi15 (Vr2) apple scab resistance locus using two GMAL 2473 BAC libraries. A single BAC clone of the contig was sufficient to span the resistance locus. The BAC clone was completely sequenced, allowing identification of a sequence of 48.6 kb going from the two closest markers (ARGH17 and 77G20RP) bracketing Rvi15 (Vr2). Analysis of the 48.6-kb sequence revealed the presence of three putative genes characterized by a Toll and mammalian interleukin-1 receptor protein nucleotide-binding site leucine-rich repeat structure. All three genes were found to be transcribed.

Molecular cytogenetic characterization of eight small supernumerary marker chromosomes originating from chromosomes 2, 4, 8,18, and 21 in three patients

Small supernumerary marker chromosomes (sSMCs) are a morphologically heterogeneous group of additional structurally abnormal chromosomes that cannot be identified unambiguously by conventional banding techniques alone. Molecular cytogenetic methods enable detailed characterization of sSMCs; however, in many cases interpretation of their clinical significance is problematic. The aim of our study was to characterize precisely sSMCs identified in three patients with dysmorphic features, psychomotor retardation and multiple congenital anomalies. We also attempted to correlate the patients' genotypes with phenotypes by inclusion of data from the literature. The sSMCs were initially detected by G-banding analysis in peripheral blood lymphocytes in these patients and were subsequently characterized using multicolor fluorescence in situ hybridization (M-FISH), (sub)centromere-specific multicolor FISH (cenM-FISH, subcenM-FISH), and multicolor banding (MCB) techniques. Additionally, the sSMCs in two patients were also studied by hybridization to whole-genome bacterial artificial chromosome (BAC) arrays (array-CGH) to map the breakpoints on a single BAC clone level. In all three patients, the chromosome origin, structure, and euchromatin content of the sSMCs were determined. In patient RS, only a neocentric r(2)(q35q36) was identified. It is a second neocentric sSMC(2) in the literature and the first marker chromosome derived from the terminal part of 2q. In the other two patients, two sSMCs were found, as M-FISH detected additional sSMCs that could not be characterized in G-banding analysis. In patient MK, each of four cell lines contained der(4)(:p11.1-->q12:) accompanied by a sSMC(18): r(18)(:p11.2-->q11.1::p11.2-->q11.1:), inv dup(18)(:p11.1-->q11.1::q11.1-->p11.1:), or der(18) (:p11.2-->q11.1::q11.1-->p11.1:). In patient NP, with clinical features of trisomy 8p, three sSMCs were characterized: r(8)(:p12-->q11.1::q11.1-->p21:) der(8) (:p11.22-->q11.1::q11.1-->p21::p21-->p11.22:) and der(21)(:p11.1-->q21.3:). The BAC array results confirmed the molecular cytogenetic results and refined the breakpoints to the single BAC clone resolution. However, the complex mosaic structure of the marker chromosomes derived from chromosomes 8 and 18 could only be identified by molecular cytogenetic methods. This study confirms the usefulness of multicolor FISH combined with whole-genome arrays for comprehensive analyses of marker chromosomes.

An eIF4E allele confers resistance to an uncapped and non‐polyadenylated RNA virus in melon

The characterization of natural recessive resistance genes and virus-resistant mutants of Arabidopsis have implicated translation initiation factors of the 4E family [eIF4E and eIF(iso)4E] as susceptibility factors required for virus multiplication and resistance expression. To date, viruses controlled by these genes mainly belong to the family Potyviridae. Melon necrotic spot virus (MNSV) belongs to the family Tombusviridae (genus Carmovirus) and is an uncapped and non-polyadenylated RNA virus. In melon, nsv-mediated resistance is a natural source of recessive resistance against all strains of MNSV except MNSV-264. Analyses of chimeras between non-resistance-breaking and resistance-breaking strains have shown that the avirulence determinant maps to the 3'-untranslated region (3'-UTR) of the viral genome. Using a combination of positional cloning and microsynteny analysis between Arabidopsis thaliana and melon, we genetically and physically delimited the nsv locus to a single bacterial artificial chromosome clone and identified the melon eukaryotic translation initiation factor 4E (Cm-eIF4E) as a candidate gene. Complementation analysis using a biolistic transient expression assay, confirmed Cm-eIF4E as the product of nsv. A single amino acid change at position 228 of the protein led to the resistance to MNSV. Protein expression and cap-binding analysis showed that Cm-eIF4E encoded by a resistant plant was not affected in it's cap-binding activity. The Agrobacterium-mediated transient expression of the susceptibility allele of Cm-eIF4E in Nicotiana benthamiana enhanced MNSV-264 accumulation. Based on these results, a model to explain melon resistance to MNSV is proposed. These data, and data from other authors, suggest that translation initiation factors of the eIF4E family are universal determinants of plant susceptibility to RNA viruses.

Construction and Identification of Bacterial Artificial Chromosome Library for 0‐613‐2R in Upland Cotton

AbstractA bacterial artificial chromosome (BAC) library containing a large genomic DNA insert is an important tool for genome physical mapping, map‐based cloning, and genome sequencing. To isolate genes via a map‐based cloning strategy and to perform physical mapping of the cotton genome, a high‐quality BAC library containing large cotton DNA inserts is needed. We have developed a BAC library of the restoring line 0‐613‐2R for isolating the fertility restorer (Rf1) gene and genomic research in cotton (Gossypium hirsutum L.). The BAC library contains 97 825 clones stored in 255 pieces of a 384‐well microtiter plate. Random samples of BACs digested with the NotI enzyme indicated that the average insert size is approximately 130 kb, with a range of 80–275 kb, and 95.7% of the BAC clones in the library have an average insert size larger than 100 kb. Based on a cotton genome size of 2 250 Mb, library coverage is 5.7 × haploid genome equivalents. Four clones were selected randomly from the library to determine the stability of the BAC clones. There were no different fingerprints for 0 and 100 generations of each clone digested with NotI and HindIII enzymes. Thus, the stability of a single BAC clone can be sustained at least for 100 generations. Eight simple sequence repeat (SSR) markers flanking the Rf1 gene were chosen to screen the BAC library by pool using PCR method and 25 positive clones were identified with 3.1 positive clones per SSR marker.(Managing editor: Li‐Hui Zhao)

Two highly representative rice BAC libraries of japonica cv Tainung 67 suitable for rice structural and functional genomic research

Two deep-coverage bacterial artificial chromosome (BAC) libraries of Oryza sativa japonica cv. Tainung 67 (TNG 67), a popular genetic stock in breeding programs and scientific research in Taiwan, have been constructed to facilitate positional cloning of rice genes and to analyze variety-specific genome composition, toward rice structural and functional genomic studies. Good high-molecular-weight DNA was produced well by nuclei preparation from two- to three-week-old seedlings, partially digested by either HindIII or EcoRI, and two cycles of size selection by pulsed-field gel electrophoresis (PFGE). The HindIII library consists of 45,312 clones, near lack of false positive clones, with an estimated average insert size of 138.4 kb and coverage of 15.1× haploid genome equivalents. The EcoRI library consists of 9984 clones, 2% of false positive clones, with an estimated average insert size of 137.8 kb and coverage of 3.2× haploid genome equivalents. Nine single-copy sequence tagged site (STS) markers, located on different chromosomes, were used to screen the two libraries, which a single BAC clone double-spotted on three filters, and hybridized 16–29, with an average of 21.2 BACs. As a result, these two libraries can complement each other and cover nearly 100% of the rice genome, consequently providing efficient tools to isolate any genes of interest. Resources, high-density filters, individual clones, and whole libraries, are available for public distribution and may be accessed at the Institute of Plant and Microbial Biology, Academia Sinica, or Arizona Genomic Institute (AGI).

Genomic organization and differential expression of two polygalacturonase-inhibiting protein genes fromMedicago truncatula

Two adjacent polygalacturonase-inhibiting protein (PGIP) genes were characterized from the model legumeMedicago truncatula. MtPGIP1 andMtPGIP2 were isolated from a single bacterial artificial chromosome clone identified from library-screening with cDNA probes. Ten and nine characteristic stretches of leucine-rich repeats, respectively, were identified from the predicted MtPCIP1 and MtPGIP2, showing 58% sequence identity at the amino acid level. TheseMtPGIP genes are likely present as a small gene family. Transcripts encoding MtPGIP1 were expressed highly in the flowers and at low levels in the roots and stems, whereas those encoding MtPGIP2 were not detected in any untreated organs. Inoculation of theM. truncatula cultivar “Jemalong” with the pathogenic fungusColletotrichum trifolii induced a hypersensitive response and the expression of both genes. The two genes were also expressed in response to the application of jasmonic acid, although mechanical wounding induced onlyMtPGIP1 and salicylic acid induced neither. Abiotic stresses, such as high-salt, cold, or drought, induced the expression ofMtPGIP1, whereas low-temperature stress inducedMtPGIP1 only. Consistent with these observations, sequence elements specific to plant defense and stress responses were identified, in varying numbers, from the putative promoter regions of the two genes. Furthermore, supportive of their putative functional roles, bacterially expressed recombinant MtPGIP1 and MtPGIP2 inhibited fungal polygalacturonase activity. Therefore, these results suggest thatMtPGIP1 andMtPGIP2, copies that presumably arose from duplication, are regulated by separate signaling pathways and likely play roles in response to pathogenic and environmental stresses.

OCGR array: an oral cancer genomic regional array for comparative genomic hybridization analysis

Genetic alterations have been recognized as important events in the carcinogenesis of oral squamous cell carcinoma (OSCC) and have been used as predictors of progression risk. In this study, we have designed an oral cancer-specific human bacterial artificial chromosome (BAC) array, called the oral cancer genomic regional array (OCGR), to detect and fine map copy number alterations in OSCC. This array contains a total of ∼45 Mbp coverage of nine chromosomal regions reported to be involved in the progression of oral cancer. We demonstrate the detection of copy number alterations in 14 microdissected clinical specimens in each of the nine regions. These include both copy number increases and decreases. Although the number of regions selected for this first generation array is small, we observed multiple segmental changes. In some cases, we observed single BAC clone alterations at 7p11 and 11q13 which contain EGFR and cyclin D1 respectively highlighting the need for high resolution detection techniques. Array comparative genomic hybridization (CGH) complements traditional methods for detecting genetic alterations in OSCC (such as microsatellite and CGH analysis) by improving the detection of segmental copy number alterations to single BAC clone resolution. This work represents the first attempt at the construction of an oral cancer-specific CGH array.

Amplification of a 280-Kilobase Core Region at the ERBB2 Locus Leads to Activation of Two Hypothetical Proteins in Breast Cancer

Amplification of the ERBB2 oncogene at 17q12 is clinically the most relevant genetic aberration in breast cancer and several studies have linked ERBB2 activation to poor clinical outcome. The development of targeted antibody-based therapy for ERBB2-overexpressing tumors and the possible role of ERBB2 as a predictor of chemotherapy treatment response have further emphasized the essential role of ERBB2 in breast cancer. Here, we performed a detailed characterization of the molecular events occurring at the ERBB2 amplicon in primary breast tumors. Analysis of the amplicon structure in 330 breast tumors by fluorescence in situ hybridization to a tissue microarray revealed a 280-kb common region of amplification that contains 10 transcribed sequences, including eight known genes. The expression levels of these 10 transcripts were determined in 36 frozen samples of grade-matched ERBB2-amplified and -nonamplified (as determined by fluorescence in situ hybridization) primary breast tumors by using quantitativereal-time reverse transcriptase-polymerase chain reaction. A highly significant association between amplification and expression levels was observed for six of these genes, including ERBB2 and two uncharacterized hypothetical proteins, MGC9753 and MGC14832. These results support the recent findings on the influence of copy number on gene expression levels and highlight novel genes that might contribute to the clinical behavior of ERBB2-amplified breast tumors.

High-resolution genetic and physical mapping of the cauliflower high-beta-carotene gene Or ( Orange).

Mutation in the cauliflower gene Or causes high levels of beta-carotene to accumulate in various tissues of the plant that are normally devoid of carotenoids. To decipher the molecular basis by which Or regulates carotenoid accumulation, we have undertaken the isolation of Or by a map-based cloning strategy. Two previously isolated, locus-specific, sequence-characterized amplified region (SCAR) markers that flank Or were employed for the analysis of a large segregating population consisting of 1632 F(2) individuals, and a high-resolution genetic linkage map of the Or locus region was developed. To facilitate positional cloning, we constructed a cauliflower genomic library in a bacterial artificial chromosome (BAC) vector, using high molecular weight DNA from Or homozygotes. The BAC library comprises 60,288 clones with an average insert size of 110 kb, and represents an estimated 10-fold coverage of the genome. A BAC contig encompassing the Or locus was established by screening the library with a marker that is closely linked to Or and by identifying overlapping BAC clones by chromosome walking. Physical mapping delimited the Or locus to a 50-kb DNA fragment within a single BAC clone, which corresponds to a genetic interval of 0.3 cM.

Complete sequence of the 45-kb mouse ribosomal DNA repeat: analysis of the intergenic spacer☆

DNA from a single bacterial artificial chromosome clone was used to sequence the mouse ribosomal DNA intergenic spacer from the 3′ end of the 45S pre-RNA to the spacer promoter (Accession No. AF441733). This made possible the assembly of a complete mouse ribosomal DNA repeat unit (45,309 bp long, TPA Accession No. BK000964). Analysis of the intergenic spacer (IGS) showed a high density of simple sequence repeats and transposable elements. The IGS contains two long sequence blocks, which are repeated tandemly. Some of the sequences in these blocks are also present in other parts of the IGS. A difference in the mutation rate along the mouse IGS was observed. The significance of sequence motifs in the IGS for transcription enhancement, transcription termination, origin of replication, and nucleolar organization is discussed.

Tandemly duplicated Safener-induced glutathione S-transferase genes from Triticum tauschii contribute to genome- and organ-specific expression in hexaploid wheat.

Glutathione S-transferase (GST) gene expression was examined in several Triticum species, differing in genome constitution and ploidy level, to determine genome contribution to GST expression in cultivated, hexaploid bread wheat (Triticum aestivum). Two tandemly duplicated tau class GST genes (TtGSTU1 and TtGSTU2) were isolated from a single bacterial artificial chromosome clone in a library constructed from the diploid wheat and D genome progenitor to cultivated wheat, Triticum tauschii. The genes are very similar in genomic structure and their encoded proteins are 95% identical. Gene-specific reverse transcriptase-polymerase chain reaction analysis revealed differential transcript accumulation of TtGSTU1 and TtGSTU2 in roots and shoots. Expression of both genes was induced by herbicide safeners, 2,4-dichlorophenoxyacetic acid and abscisic acid, in the shoots of T. tauschii; however, expression of TtGSTU1 was always higher than TtGSTU2. In untreated seedlings, TtGSTU1 was expressed in both shoots and roots, whereas TtGSTU2 expression was only detected in roots. RNA gel-blot analysis of ditelosomic, aneuploid lines that are deficient for 6AS, 6BS, or 6DS chromosome arms of cultivated, hexaploid bread wheat showed differential genome contribution to safener-induced GST expression in shoots compared with roots. The GST genes from the D genome of hexaploid wheat contribute most to safener-induced expression in the shoots, whereas GSTs from the B and D genomes contribute to safener-induced expression in the roots.

Physical and genetic mapping of barley (Hordeum vulgare) germin-like cDNAs.

Germin with oxalate oxidase and superoxide dismutase activity is a homohexamer of six manganese-containing interlocked beta-jellyroll monomers with extreme resistance to heat and proteolytic degradation [Woo, E.-J., Dunwell, J. M., Goodenough, P. W., Marvier, A. C. & Pickersill, R. W. (2000) Nat. Struct. Biol. 7, 1036-1038]. This structure is conserved in germin-like proteins (GLPs) with other enzymatic functions and characteristic for proteins deposited in plant cell walls in response to pathogen attack and abiotic stress. Comparative nucleotide and amino acid sequence analyses of 49,610 barley expressed sequence tags identified 124 germin and germin-like cDNAs, which distributed into five subfamilies designated HvGER-I to HvGER-V. Representative cDNAs for these subfamilies hybridized to 67 bacterial artificial chromosome (BAC) clones from a library containing 6.3 genomic equivalents. Twenty-six BAC clones hybridized to the subfamily IV probe and identified a gene-rich region including clone 418E1 of 96 kb encoding eight GLPs (i.e., 1 gene per 12 kb). This BAC clone lacked highly repeated sequences and mapped to the subtelomeric region of the long arm of chromosome 4(4H). Among the six genes of the contig expressed in leaves, one specifies a protein known to be associated with papilla formation in the epidermis upon powdery mildew infection. Three structural genes for oxalate oxidase are present in subfamily I and eight GLPs of various functions in the other subfamilies. These genes map at loci in chromosomes 1(7H), 2 (2H), 3(3H), 4(4H), and 7(5H). Some are present on a single BAC clone. The results are discussed in relation to cereal genome organization.

Physical mapping, BAC-end sequence analysis, and marker tagging of the soilborne nematicidal bacterium, Pseudomonas synxantha BG33R.

A bacterial artificial chromosome (BAC) library was constructed for the genome of the rhizosphere-inhabiting fluorescent pseudomonad Pseudomonas synxantha BG33R. Three thousand BAC clones with an average insert size of 140 kbp and representing a 70-fold genomic coverage were generated and arrayed onto nylon membranes. EcoRI fingerprint analysis of 986 BAC clones generated 23 contigs and 75 singletons. Hybridization analysis allowed us to order the 23 contigs and condense them into a single contig, yielding an estimated genome size of 5.1 Mb for P. synxantha BG33R. A minimum-tile path of 47 BACs was generated and end-sequenced. The genetic loci involved in ring nematode egg-kill factor production in BG33R Tn5 mutants, 246 (vgrG homolog), 1122 (sensor kinase homolog), 1233 (UDP-galactose epimerase homolog), 1397 (ferrisiderophore receptor homolog), and 1917 (ribosomal subunit protein homolog), have been mapped onto the minimum-tile BAC library. Two of the genetic regions that flank Tn5 insertions in BG33R egg-kill-negative mutants 1233 and 1397 are separated by a single BAC clone. Fragments isolated by ligation-mediated PCR of the Tn5 mutagenized regions of 29 randomly selected, non-egg-kill-related, insertion mutants have been anchored onto the ordered physical map of P. synxantha.

Differential regulation of a family of apyrase genes from Medicago truncatula.

Four putative apyrase genes were identified from the model legume Medicago truncatula. Two of the genes identified from M. truncatula (Mtapy1 and Mtapy4) are expressed in roots and are inducible within 3 h after inoculation with Sinorhizobium meliloti. The level of mRNA expression of the other two putative apyrases, Mtapy2 and Mtapy3, was unaffected by rhizobial inoculation. Screening of a bacterial artificial chromosome library of M. truncatula genomic DNA showed that Mtapy1, Mtapy3, and Mtapy4 are present on a single bacterial artificial chromosome clone. This apyrase cluster was mapped to linkage group seven. A syntenic region on soybean linkage group J was found to contain at least two apyrase genes. Screening of nodulation deficient mutants of M. truncatula revealed that two such mutants do not express apyrases to any detectable level. The data suggest a role for apyrases early in the nodulation response before the involvement of root cortical cell division leading to the nodule structure.

Characterization of the Human Talin (TLN) Gene: Genomic Structure, Chromosomal Localization, and Expression Pattern

Talin is a high-molecular-weight cytoskeletal protein, localized at cell–extracellular matrix associations known as focal contacts. In these regions, talin is thought to link integrin receptors to the actin cytoskeleton. Talin plays a key role in the assembly of actin filaments and in spreading and migration of various cell types. Talin proteins are found in a wide variety of organisms, from slime molds to humans. The human Talin (HGMW-approved symbol TLN) gene was previously mapped to chromosome 9p, but little was known of its sequence and genomic structure. To characterize human TLN further, we have isolated a single bacterial artificial chromosome clone, harboring the entire gene. The gene extends over more than 23 kb and consists of 57 exons. We have localized TLN to human chromosome band 9p13 by both fluorescence in situ hybridization and radiation hybrid mapping. Northern blot analysis detected TLN expression in various human tissues, including leukocytes, lung, placenta, liver, kidney, spleen, thymus, colon, skeletal muscle, and heart. Based on its chromosomal location, expression pattern, and protein function, we considered TLN as a candidate gene for cartilage-hair hypoplasia (CHH), an autosomal recessive metaphyseal chondrodysplasia, previously mapped to 9p13. We sequenced the entire TLN coding sequence in several CHH patients, but no functional mutations were detected.