Chromosome identification in the Andean common bean accession G19833 (Phaseolus vulgaris L., Fabaceae)

Characterization of all chromosomes of the Andean G19833 bean genotype was carried out by fluorescent in situ hybridization. Eleven single-copy genomic sequences, one for each chromosome, two BACs containing subtelomeric and pericentromeric repeats and the 5S and 45S ribosomal DNA (rDNA) were used as probes. Comparison to the Mesoamerican accession BAT93 showed little divergence, except for additional 45S rDNA sites in four chromosome pairs. Altogether, the results indicated a relative karyotypic stability during the evolution of the Andean and Mesoamerican gene pools of P. vulgaris.     

Molecular genetic delineation of 2q37.3 deletion in autism and osteodystrophy: report of a case and of new markers for deletion screening by PCR.

We recently studied a patient who meets criteria for autistic disorder and has a 2q37 deletion. Molecular cytogenetic studies were carried out using DNA isolated from 22 different 2q37 mapped BACs to more precisely define the extent of the chromosome deletion. We also analyzed 2q37 mapped polymorphic markers. In addition DNA sequences of BACs in the deletion region were scanned to identify microsatellite repeats. We describe four new polymorphic microsatellite repeat markers in the 2q37.3 region. These markers enabled us to determine the parental origin of the deletion in our patient. DNA from 8-13 unrelated individuals was used to determine heterozygosity estimates for these markers. We review four genes deleted in our patient - genes whose known functions and sites of expression in the brain and/or bone make them candidates for involvement in autism and/or the osteodystrophy observed in patients with 2q37.3 deletions.     

中国美利奴细毛羊BAC文库的三维PCR筛选

本研究利用中国美利奴细毛羊全基因组BAC文库,构建了可供快速筛选的两级水平的混合池,一级混合池和二级混合池(Primary pools and secondary pools).一级混合池基于每一384-well盘而构建,由盘、行,列三维混合池组成,二级混合池基于整个BAC文库而构建.设计了一种基于PCR技术的快速筛选方法,先筛选二级混合池m再根据结果筛选相应的一级混合池.利用此方法只需一步共66个PCR反应即可从BAC丈库中7.4万个克隆中筛选出1个阳性克隆,或三步100个以内的PCR反应筛选出多个阳性克隆.以绵羊基因组多态性分子标记BF94-1为引物,用一步共66个PCR反应成功筛选到1个阳性克隆373D13.     

Isolation of subtelomeric sequences of porcine chromosomes for translocation screening reveals errors in the pig genome assembly

Balanced chromosomal aberrations have been shown to affect fertility in most species studied, often leading to hypoprolificacy (reduced litter size) in domestic animals such as pigs. With an increasing emphasis in modern food production on the use of a small population of high quality males for artificial insemination, the potential economic and environmental costs of hypoprolific boars, bulls, rams etc. are considerable. There is therefore a need for novel tools to facilitate rapid, cost‐effective chromosome translocation screening. This has previously been achieved by standard karyotype analysis; however, this approach relies on a significant level of expertise and is limited in its ability to identify subtle, cryptic translocations. To address this problem, we developed a novel device and protocol for translocation screening using subtelomeric probes and fluorescence in situ hybridisation. Probes were designed using BACs (bacterial artificial chromosomes) from the subtelomeric region of the short (p‐arm) and long (q‐arm) of each porcine chromosome. They were directly labelled with FITC or Texas Red (p‐arm and q‐arm respectively) prior to application of a ‘Multiprobe’ device, thereby enabling simultaneous detection of each individual porcine chromosome on a single slide. Initial experiments designed to isolate BACs in subtelomeric regions led to the discovery of a series of incorrectly mapped regions in the porcine genome assembly (from a total of 82 BACs, only 45 BACs mapped correctly). Our work therefore highlights the importance of accurate physical mapping of newly sequenced genomes. The system herein described allows for robust and comprehensive analysis of the porcine karyotype, an adjunct to classical cytogenetics that provides a valuable tool to expedite efficient, cost effective food production.     

Localization of subtelomeric sequences of human chromosomes 1q, 11p, 13q, and 16q in the higher primates

Relative phylogenetic divergence of the members of the Pongidae family has been based on genetic evidence. The recent isolation of subtelomeric probes specific for human (HSA) chromosomes 1q, 11p, 13q, and 16q has prompted us to cross hybridize these to the chromosomes of the chimpanzee (Pan troglodytes, PTR), gorilla (Gorilla gorilla, GGO), and orangutan (Pongo pygmaeus, PPY) to search for their equivalent locations in the great apes. Hybridization signals to the 1q subtelomeric DNA sequence probe were observed at the termini of human (HSA) 1q, PTR 1q, GGO 1q, PPY 1q, while the fluorescent signals to the 11p subtelomeric DNA sequence probe were observed at the termini of HSA 11p, PTR 9p, GGO 9p, and PPY 8p. Fluorescent signals to the 13q subtelomeric DNA sequence probe were observed at the termini of HSA 13q, PTR 14q, GGO 14q, and PPY 14q, and positive signals to the 16p subtelomeric DNA sequence probe were observed at the termini of HSA 16q, PTR 18q, GGO 17q, and PPY 19q. These findings apparently suggest sequence homology of these DNA families in the ape chromosomes. Obviously, analogous subtelomeric sequences exist in apes' chromosomes that apparently have been conserved through the course of differentiation of the hominoid species. This revised version was published online in July 2006 with corrections to the Cover Date.     

An improved method for generating BAC DNA suitable for FISH

Fluorescence in situ hybridization (FISH) is commonly used to identify chromosomal aberrations such as translocations, deletions, duplications, gene fusions, and aneuploidies. It relies on the hybridization of fluorescently labeled DNA probes onto denatured metaphase chromosomes or interphase nuclei. These probes are often generated from DNA sequences cloned within bacterial artificial chromosomes (BACs). Growing these BACs in adequate amounts for FISH can be demanding. We describe FISH performed with bacteriophage Phi29 DNA polymerase amplified BAC DNA. Generating this material required significantly smaller cultures and less time than standard methods. The FISH results obtained were comparable with those obtained from standard BAC DNA. We believe this method of BAC DNA generation is useful for the entire FISH community as it improves considerably on prior methods.     

High-resolution chromosome mapping of BACs using multi-colour FISH and pooled-BAC FISH as a backbone for sequencing tomato chromosome 6

Within the framework of the International Solanaceae Genome Project, the genome of tomato (Solanum lycopersicum) is currently being sequenced. We follow a 'BAC-by-BAC' approach that aims to deliver high-quality sequences of the euchromatin part of the tomato genome. BACs are selected from various libraries of the tomato genome on the basis of markers from the F2.2000 linkage map. Prior to sequencing, we validated the precise physical location of the selected BACs on the chromosomes by five-colour high-resolution fluorescent in situ hybridization (FISH) mapping. This paper describes the strategies and results of cytogenetic mapping for chromosome 6 using 75 seed BACs for FISH on pachytene complements. The cytogenetic map obtained showed discrepancies between the actual chromosomal positions of these BACs and their markers on the linkage group. These discrepancies were most notable in the pericentromere heterochromatin, thus confirming previously described suppression of cross-over recombination in that region. In a so called pooled-BAC FISH, we hybridized all seed BACs simultaneously and found a few large gaps in the euchromatin parts of the long arm that are still devoid of seed BACs and are too large for coverage by expanding BAC contigs. Combining FISH with pooled BACs and newly recruited seed BACs will thus aid in efficient targeting of novel seed BACs into these areas. Finally, we established the occurrence of repetitive DNA in heterochromatin/euchromatin borders by combining BAC FISH with hybridization of a labelled repetitive DNA fraction (Cot-100). This strategy provides an excellent means to establish the borders between euchromatin and heterochromatin in this chromosome.     

Mapping the chromosomes of Poncirus trifoliata Raf. by BAC-FISH

In spite of the importance of Citrus in agriculture and recent progress in genetic mapping and cytogenetics of this group, chromosome mapping of Citrus species is still limited to rDNA probes. In order to obtain a better chromosome characterization of one species from this group, CMA/DAPI double staining followed by in situ hybridization using 45S rDNA and 24 BACs (BAC-FISH) were used on Poncirus trifoliata. The BACs used were obtained from a genomic library of this species and were selected by membrane hybridization using genomic DNA. Four of them were isolated from the Citrus tristeza virus (Ctv) resistance gene region. The P. trifoliata karyotype is composed of two chromosome pairs with one terminal and one proximal CMA(+) band (B type chromosomes), four chromosome pairs with a single CMA(+) band (D type) and three chromosome pairs without bands (F type). In situ hybridization with 13 of the BACs gave single copy signals on seven chromosome pairs. At least one BAC was mapped on each arm of the two B chromosome pairs. Among the four D chromosome pairs, two were identified by BACs mapped on the long arms, one has a 45S rDNA site and the other had no signal. Six BACs allowed identification of the three F chromosome pairs, with one pair hybridizing with four BACs from the Ctv resistance gene region. In summary, all nine chromosome pairs could be differentiated, seven of them by BAC-FISH, while the other two chromosomes could be recognized by the CMA(+) band pattern and 45S rDNA sites. This first BAC-FISH map gives a general framework for comparative genome structure and evolutionary studies in Citrus and Poncirus, allowing the integration of genetic and physical maps when these BACs are included.     

Red-Mediated Transposition and Final Release of the Mini-F Vector of a Cloned Infectious Herpesvirus Genome

Bacterial artificial chromosomes (BACs) are well-established cloning vehicles for functional genomics and for constructing targeting vectors and infectious viral DNA clones. Red-recombination-based mutagenesis techniques have enabled the manipulation of BACs in Escherichia coli without any remaining operational sequences. Here, we describe that the F-factor-derived vector sequences can be inserted into a novel position and seamlessly removed from the present location of the BAC-cloned DNA via synchronous Red-recombination in E. coli in an en passant mutagenesis-based procedure. Using this technique, the mini-F elements of a cloned infectious varicella zoster virus (VZV) genome were specifically transposed into novel positions distributed over the viral DNA to generate six different BAC variants. In comparison to the other constructs, a BAC variant with mini-F sequences directly inserted into the junction of the genomic termini resulted in highly efficient viral DNA replication-mediated spontaneous vector excision upon virus reconstitution in transfected VZV-permissive eukaryotic cells. Moreover, the derived vector-free recombinant progeny exhibited virtually indistinguishable genome properties and replication kinetics to the wild-type virus. Thus, a sequence-independent, efficient, and easy-to-apply mini-F vector transposition procedure eliminates the last hurdle to perform virtually any kind of imaginable targeted BAC modifications in E. coli. The herpesviral terminal genomic junction was identified as an optimal mini-F vector integration site for the construction of an infectious BAC, which allows the rapid generation of mutant virus without any unwanted secondary genome alterations. The novel mini-F transposition technique can be a valuable tool to optimize, repair or restructure other established BACs as well and may facilitate the development of gene therapy or vaccine vectors.     

Using comparative genomics to reorder the human genome sequence into a virtual sheep genome

Background

Is it possible to construct an accurate and detailed subgene-level map of a genome using bacterial artificial chromosome (BAC) end sequences, a sparse marker map, and the sequences of other genomes?

Results

A sheep BAC library, CHORI-243, was constructed and the BAC end sequences were determined and mapped with high sensitivity and low specificity onto the frameworks of the human, dog, and cow genomes. To maximize genome coverage, the coordinates of all BAC end sequence hits to the cow and dog genomes were also converted to the equivalent human genome coordinates. The 84,624 sheep BACs (about 5.4-fold genome coverage) with paired ends in the correct orientation (tail-to-tail) and spacing, combined with information from sheep BAC comparative genome contigs (CGCs) built separately on the dog and cow genomes, were used to construct 1,172 sheep BAC-CGCs, covering 91.2% of the human genome. Clustered non-tail-to-tail and outsize BACs located close to the ends of many BAC-CGCs linked BAC-CGCs covering about 70% of the genome to at least one other BAC-CGC on the same chromosome. Using the BAC-CGCs, the intrachromosomal and interchromosomal BAC-CGC linkage information, human/cow and vertebrate synteny, and the sheep marker map, a virtual sheep genome was constructed. To identify BACs potentially located in gaps between BAC-CGCs, an additional set of 55,668 sheep BACs were positioned on the sheep genome with lower confidence. A coordinate conversion process allowed us to transfer human genes and other genome features to the virtual sheep genome to display on a sheep genome browser.

Conclusion

We demonstrate that limited sequencing of BACs combined with positioning on a well assembled genome and integrating locations from other less well assembled genomes can yield extensive, detailed subgene-level maps of mammalian genomes, for which genomic resources are currently limited.     

Comparative cytogenetic mapping between the lima bean (Phaseolus lunatus L.) and the common bean (P. vulgaris L.)

The common bean (Phaseolus vulgaris) and lima bean (P. lunatus) are among the most important legumes in terms of direct human consumption. The present work establishes a comparative cytogenetic map of P. lunatus, using previously mapped markers from P. vulgaris, in association with analyses of heterochromatin distribution using the fluorochromes chromomycin A3 (CMA) and 4′,6-diamidino-2-phenylindole (DAPI) and localization of the 5S and 45S ribosomal DNA (rDNA) probes. Seven BACs selected from different common bean chromosomes demonstrated a repetitive pericentromeric pattern corresponding to the heterochromatic regions revealed by CMA/DAPI and could not be mapped. The subtelomeric repetitive pattern observed for BAC 63H6 in most of the chromosome ends of common bean was not detected in lima bean, indicating lack of conservation of this subtelomeric repeat. All chromosomes could be identified and 16 single-copy clones were mapped. These results showed a significant conservation of synteny between species, although change in centromere position suggested the occurrence of pericentric inversions on chromosomes 2, 9 and 10. The low number of structural rearrangements reflects the karyotypic stability of the genus.     

Contig assembly of bacterial artificial chromosome clones through multiplexed fluorescence-labeled fingerprinting

A rapid multiplexed fingerprinting method has been developed for bacterial artificial chromosome (BAC) contig assembly. Defined subsets of BAC DNA fragments that result from digestion by three paired restriction endonucleases are labeled with unique fluorescent F-ddATP for each subset. Lists of the labeled fragment size are generated by an ABI 377 DNA sequencer and the GeneScan analysis software and then processed by an assembly program, FPC (Fingerprinted Contigs), to produce contig maps. Data obtained from the multiplexed labeling permit detection of smaller overlaps than is observed when data from a single double-digest are analyzed. The method has been tested on 98 BACs from chromosome 22 regions where large-scale sequencing is under way and also through simulation, using randomly generated BAC clones derived from existing DNA sequence data. In each case, contig assembly results demonstrated the advantages of multiplexed fingerprinting.     

Analysis of the subtelomeric regions of macronuclear gene-sized DNA molecules of the hypotrichous ciliate Stylonychia lemnae: Implications for the DNA fragmentation process during macronuclear development?

The subtelomeric regions of macronuclear gene-sized DNA molecules from Stylonychia lemnae were analyzed. The results obtained indicate that these regions show a highly ordered and common sequence organization: Immediately adjacent to the telomeric sequence a short inverted repeat sequence is found, followed by another 7–9 bp inverted repeat sequence at approximately position 40. A 10 bp consensus sequence found in the subtelomeric regions of all gene-sized DNA molecules is found at approximately position 60 and in addition at about the same position palindromic sequences showing no homology to each other are localized. The biological significance of this sequence organization is discussed. © 1993Wiley-Liss, Inc.     

The canine sarcoglycan delta gene: BAC clone contig assembly, chromosome assignment and interrogation as a candidate gene for dilated cardiomyopathy in Dobermann dogs

Dilated cardiomyopathy (DCM) is a common disease of the myocardium recognized in human, dog and experimental animals. Genetic factors are responsible for a large proportion of cases in humans, and 17 genes with DCM causing mutations have been identified. The genetic origin of DCM in the Dobermann dogs has been suggested, but no disease genes have been identified to date. In this paper, we describe the characterization and evaluation of the canine sarcoglycan delta (SGCD), a gene implicated in DCM in human and hamster. Bacterial artificial chromosomes (BACs) containing the canine SGCD gene were isolated with probes for exon 3 and exons 4-8 and were characterized by Southern blot analysis. BAC end sequences were obtained for four BACs. Three of the BACs overlapped and could be ordered relative to each other and the end sequences of all four BACs could be anchored on the preliminary assembly of the dog genome sequence (www. ensembl.org). One of the BACs of the partial contig was localized by fluorescent in situ hybridization to canine chromosome 4q22, in agreement with the dog genome sequence. Two highly informative polymorphic microsatellite markers in intron 7 of the SGCD gene were identified. In 25 DCM-affected and 13 non DCM-affected dogs seven different haplotypes could be distinguished. However, no association between any of the SGCD variants and the disease locus was apparent.     

BAC representation of two low‐copy regions of the genome of Arabidopsis thaliana

Two regions of Arabidopsis chromosome 4, totalling 4.7 Mb, were assayed for representation in the TAMU and IGF BAC libraries. A directed approach to BAC identification was developed. Gel-purified DNA samples of YACs selected from the YAC-based physical map of chromosome 4 were used to probe high-density colony arrays of the BAC libraries. Strategies were developed that allowed the efficient construction of restriction maps and BAC contigs. Four hundred and sixty-four BACs were mapped, assembled into two complete contigs and used to analyse genomic representation. These BACs provided a mean of 9.4-fold redundant coverage, with a range of 2- to 22-fold. The representation provided by the two libraries showed almost coincident peaks and troughs, with a periodicity of approximately 200 kb. These results demonstrate that, provided both TAMU and IGF libraries are used in their entirety, BACs should provide an excellent resource for both physical mapping and sequencing of the Arabidopsis genome.     

Chromosome landing at the bacterial blight resistance gene<Emphasis Type="Italic"> Xa4</Emphasis> locus using a deep coverage rice BAC library

Xa4 is a dominantly inherited rice gene that confers resistance to Philippine race 1 of the bacterial blight pathogen Xanthomonas oryzae pv. oryzae in rice. In order to isolate the gene by positional cloning, a bacterial artificial chromosome (BAC) library was constructed from genomic DNA isolated from an Xa4-harboring accession, IRBB56. The library contains 55,296 clones with an average insert size of 132 kb, providing 14 rice genome equivalents. Three DNA markers closely linked to Xa4 were used to screen the library. The marker RS13, a resistance gene analogue that co-segregates with Xa4, identified 18 clones, of which four and six, respectively, were simultaneously detected by the other two markers, G181 and L1044. Fingerprinting and Southern analysis indicated that these clones overlapped and define an interval spanning 420 kb. In an F2 population derived from an indica variety, IR24, and its Xa4-containing near isogenic line (NIL), IRBB4, the susceptible plants were screened in order to map the Xa4 gene genetically and physically. Out of 24 insert ends isolated from the BACs in the contig, three revealed polymorphisms between IR24 and IRBB4. Two insert ends, 56M22F and 26D24R, flanked Xa4 on each side. Based on the overlap of the BACs, six overlapping clones were considered to include the Xa4 allele, one of which, 106P13, was chosen for further investigation.     

Delivery of bacterial artificial chromosomes into mammalian cells with psoralen-inactivated adenovirus carrier.

Molecular biology has many applications where the introduction of large (>100 kb) DNA molecules is required. The current methods of large DNA transfection are very inefficient. We reasoned that two limits to improving transfection methods with these large DNA molecules were the difficulty of preparing workable quantities of clean DNA and the lack of rapid assays to determine transfection success. We have used bacterial artificial chromosomes (BACs) based on the Escherichia coli F factor plasmid system, which are simple to manipulate and purify in microgram quantities. Because BAC plasmids are kept at one to two copies per cell, the problems of rearrangement observed with YACs are eliminated. We have generated two series of BAC vectors bearing marker genes for luciferase and green fluorescent protein (GFP). Using these reagents, we have developed methods of delivering BACs of up to 170 kb into mammalian cells with transfection efficiency comparable to 5-10 kb DNA. Psoralen-inactivated adenovirus is used as the carrier, thus eliminating the problems associated with viral gene expression. The delivered DNA is linked to the carrier virus with a condensing polycation. Further improvements in gene delivery were obtained by replacing polylysine with low molecular weight polyethylenimine (PEI) as the DNA condensing agent.     

Exo1 and Rad24 differentially regulate generation of ssDNA at telomeres of Saccharomyces cerevisiae cdc13-1 mutants

Cell cycle arrest in response to DNA damage depends upon coordinated interactions between DNA repair and checkpoint pathways. Here we examine the role of DNA repair and checkpoint genes in responding to unprotected telomeres in budding yeast cdc13-1 mutants. We show that Exo1 is unique among the repair genes tested because like Rad9 and Rad24 checkpoint proteins, Exo1 inhibits the growth of cdc13-1 mutants at the semipermissive temperatures. In contrast Mre11, Rad50, Xrs2, and Rad27 contribute to the vitality of cdc13-1 strains grown at permissive temperatures, while Din7, Msh2, Nuc1, Rad2, Rad52, and Yen1 show no effect. Exo1 is not required for cell cycle arrest of cdc13-1 mutants at 36 degrees but is required to maintain arrest. Exo1 affects but is not essential for the production of ssDNA in subtelomeric Y' repeats of cdc13-1 mutants. However, Exo1 is critical for generating ssDNA in subtelomeric X repeats and internal single-copy sequences. Surprisingly, and in contrast to Rad24, Exo1 is not essential to generate ssDNA in X or single-copy sequences in cdc13-1 rad9Delta mutants. We conclude that Rad24 and Exo1 regulate nucleases with different properties at uncapped telomeres and propose a model to explain our findings.     

Comparative cytogenetics of human chromosome 3q21.3 reveals a hot spot for ectopic recombination in hominoid evolution

Fluorescence in situ hybridization mapping of fully integrated human BAC clones to primate chromosomes, combined with precise breakpoint localization by PCR analysis of flow-sorted chromosomes, was used to analyze the evolutionary rearrangements of the human 3q21.3-syntenic region in orangutan, siamang gibbon, and silvered-leaf monkey. Three independent evolutionary breakpoints were localized within a 230-kb segment contained in BACs RP11-93K22 and RP11-77P16. Approximately 200 kb of the human 3q21.3 sequence was not present on the homologous orangutan, siamang, and Old World monkey chromosomes, suggesting a genomic DNA insertion into the breakpoint region in the lineage leading to humans and African great apes. The breakpoints in the orangutan and siamang genomes were narrowed down to 12- and 20-kb DNA segments, respectively, which are enriched with endogenous retrovirus long terminal repeats and other repetitive elements. The inserted DNA segment represents part of an ancestral duplication. Paralogous sequence blocks were found at human 3q21, approximately 4 Mb proximal to the evolutionary breakpoint cluster region; at human 3p12.3, which contains an independent orangutan-specific breakpoint; and at the subtelomeric and pericentromeric regions of multiple human and orangutan chromosomes. The evolutionary breakpoint regions between human chromosome 3 and orangutan 2 as well their paralogous segments in the human genome coincide with breaks of chromosomal synteny in the mouse, rat, and/or chicken genomes. Collectively our data reveal reuse of the same short recombinogenic DNA segments in primate and vertebrate evolution, supporting a nonrandom breakage model of genome evolution.     

Interchromosomal exchange of a large subtelomeric segment in a Plasmodium falciparum cross.

Duplications and interchromosomal transpositions of chromosome segments are implicated in the genetic variability of Plasmodium falciparum malaria parasites. One parasite clone, HB3, was shown to lack a subtelomeric region of chromosome 13 that normally carries a PfHRPIII gene. We show here that the chromosome 13 segment carrying PfHRPIII was replaced in HB3 by a duplicated terminal segment from chromosome 11. Mapping results indicate that the segment includes at least 100-200 kb of subtelomeric DNA and contains duplicated copies of the Pf332 and RESA-2 genes. We followed inheritance of this duplication in a genetic cross between the HB3 and another P.falciparum clone, Dd2, that is euploid for the Pf332, RESA-2 and PfHRPIII genes. Three types of progeny from the cross showed expected inheritance forms: a Dd2 euploid parent type, an HB3 aneuploid parent type, and a recombinant euploid type that carried PfHRPIII from Dd2 chromosome 13 and Pf332 from HB3 chromosome 11. However, a fourth euploid progeny type was also observed, in which the chromosome 13 segment from HB3 was transposed back to replace the terminus of chromosome 11. Three of 14 individual progeny were of this type. These findings suggest a mechanism of recombination from subtelomeric pairing and exchange between non-homologous chromosomes in meiosis.