It is clear that the construction of large insert DNA libraries is important for map-based gene cloning, the assembly of physical maps, and simple screening for specific genomic sequences. The bacterial artificial chromosome (BAC) system is likely to be an important tool for map-based cloning of genes since BAC libraries can be constructed simply and analyzed more efficiently than yeast artificial chromosome (YAC) libraries. BACs have significantly expanded the size of fragments from eukaryotic genomes that can be cloned in Escherichia coli as plasmid molecules. To facilitate the isolation of molecular-biologically important genes in Ashbya gossypii, we constructed Ashbya chromosome-specific BAC libraries using pBeloBAC11 and pBACwich vectors with an average insert size of 100 kb, which is equivalent to 19.8X genomic coverage. pBACwich was developed to streamline map-based cloning by providing a tool to integrate large DNA fragments into specific sites in chromosomes. These chromosome-specific libraries have provided a useful tool for the further characterization of the Ashbya genome including positional cloning and genome sequencing.
Ascomycota* ; Chromosomes, Artificial, Bacterial ; Chromosomes, Artificial, Yeast ; Clone Cells ; Cloning, Organism ; DNA ; Escherichia coli ; Gene Library ; Genome ; Mass Screening ; Plasmids

OBJECTIVETo annotate the human genome 3p24-p25 478 kb complete sequence.

METHODSThe protein-coding genes in the genomic sequence were identified by using ab initio gene finding, homology-based similarity database searching and all or partial mRNA aligning with genomic sequence, and the content feature of the genomic sequence were analyzed by using EMBOSS package.

RESULTSTwo known genes SLC6A1 and SLC6A11 were identified; as well as the GC content of this genomic sequence was 47% and 3 putative CpG islands were predicted in the genomic sequence, located in 130,685-131,516 bp, 307,090-307,870 bp and 415,585-416,308 bp, respectively.

CONCLUSIONSThe methods, as mentioned above, might be used for annotating the biological information in the genomic sequence, such as gene structure, GC content, CpG island.

Base Sequence ; Chromosome Mapping ; Chromosomes, Artificial, Bacterial ; Chromosomes, Human, Pair 3 ; Genome, Human ; Human Genome Project ; Humans ; Molecular Sequence Data

In order to optimize the conditions of construction BAC library, the transformation efficiency of E. coli DH10B was studied in this paper. Our data prove much higher competence of electroporation (reaches 2.19 x 10(10) cfu/microg pUC19 DNA) when harvesting the cells between an OD550 of 0.7 - 0.8. Five different electric field strength (from 9 kV/cm to 25 kV/cm) and three different sized plasmid vector DNAs including pUC19 DNA, pECBAC1 DNA and pCLD04541 DNA, as well as three bacterial artificial chromosomes (BACs) ranging from 40 to 190 kb and their mixture were used to discover the transformation efficiency changes under various conditions. Our data show maximum transformation efficiency and optimal electric field strength of plasmid DNAs drop dramatically with increasing size of the DNA. Molecules of 190 kb transform more than 50-fold less well, on a molar basis, than molecules of 40 kb. And the optimal voltage gradient is strongly dependent on the different sized molecules, for instance, pUC19 reaches the highest transformation efficiency at 21 kV/cm, while the 180 kb BAC DNA gets its best efficiency at 13 kV/cm. This paper demonstrates that conditions may be selected which increase the average size of BAC clones generated by electroporation and could be widely applied in large-insert genome library construction.
Chromosomes, Artificial, Bacterial ; genetics ; DNA, Bacterial ; chemistry ; genetics ; Electroporation ; methods ; Escherichia coli ; genetics ; Molecular Weight ; Plasmids ; genetics ; Transformation, Genetic

EphA/ephrin-A mediated signaling has emerged as a key mechanism regulating axon guidance and topographic mapping, particularly in the well-characterized visual system from the retina to the superior colliculus (SC). In this study, EphA8 bacterial artificial chromosome (BAC) was manipulated to contain a floxed eGFP and human ephrin-A5 expression cassette using homologous recombination method. In the mice containing the recombinant BAC, it was shown that GFP is expressed in an anterior>posterior gradient in the SC. Furthermore, when these mice were crossed with the transgenic mice expressing Cre under the EphA8 promoter, it was evident that a GFP expression cassette was eliminated, and that human ephrin-A5 was ectopically expressed in the anterior region of the SC. This transgenic model would be useful to analyze the role of ephrin-A5 in the SC during the retinocollicular topography formation.
Animals ; Axons ; Chromosomes, Artificial, Bacterial ; Ephrin-A5 ; Homologous Recombination ; Humans ; Mice ; Mice, Transgenic ; Retina ; Superior Colliculi

For rapid screening, we constructed two levels pools (primary and secondary pools) of the bacterial artificial chromosome (BAC) library of Chinese fine wool merino sheep. The primary pools were based on the individual 384-well microtiter plate and were prepared with a three-dimensional pooling scheme. Three dimension (plate, row and column) pools were made for each. The secondary pools were based on the entire BAC library. We developed a PCR based strategy to identify positive BACs from sheep BAC library. First, we analyzed secondary pools DNAs, according to the result, we analyzed correlative primary pools. It was one-step screening (66 PCR reactions) that we could screen a single positive clone from 74 000 BACs by our method, or three-step screening (less than 100 PCR reactions) could screen more clones. By one-step screening (66 PCR reactions), we screened successfully a positive clone 373D13 with polymorphism marker BF94-1.
Animals ; Chromosomes, Artificial, Bacterial ; genetics ; Gene Library ; Polymerase Chain Reaction ; methods ; Polymorphism, Genetic ; Sheep ; genetics

To rapidly and accurately manipulate genome such as gene deletion, insertion and site mutation, the whole genome of a very virulent strain Md5 of Marek's disease virus (MDV) was inserted into bacterial artificial chromosome (BAC) through homogeneous recombination. The recombinant DNA was electroporated into DH10B competent cells and identified by PCR and restriction fragment length polymorphism analysis. An infectious clone of Md5BAC was obtained following transfection into chicken embryo fibroblast (CEF) cells. Furthermore, a lorf10 deletion mutant was constructed by two step Red-mediated homologous recombination. To confirm the specific role of gene deletion, the lorf10 was reinserted into the original site of MDV genome to make a revertant strain. All the constructs were rescued by transfection into CEF cells, respectively. The successful packaging of recombinant viruses was confirmed by indirect immunofluorescence assay. The results of growth kinetics assay and plaques area measurement showed that the lorf10 is dispensable for MDV propagation in vitro. Overall, this study successfully constructed an infectious BAC clone of MDV and demonstrated its application in genome manipulation; the knowledge gained from our study could be further applied to other hepesviruses.
Animals ; Chick Embryo ; Chickens ; Chromosomes, Artificial, Bacterial ; DNA, Recombinant ; Herpesvirus 2, Gallid/genetics* ; Marek Disease

BACKGROUND: Williams syndrome is characterized by supravalvular aortic stenosis, mental retardation and peculiar facial appearance. Its genetic etiology is considered to be hemizygotic deletion in Chromosome 7q11.23 which includes the elastin gene. We examined the deletion in Korean Williams syndrome patients and their parents. MATERIALS AND METHOD: Sixteen patients were selected through careful clinical examination including echocardiography and cardiac angiography. Hemizygotic deletion of elastin gene was determined in patients and 21 parents with fluorescent in situ hybridization (FISH) technique using the bacterial artificial chromosome clone 244H3 probe or commercial WSCR probe. RESULTS: FISH showed hemizygotic deletion of chromosome 7 in all sixteen patients but none of their parents showed deletion. CONCLUSION: Hemizygotic deletion of elastin gene can be determined by FISH with new probe 244H3 in clinically suspected Williams syndrome patients.
Angiography ; Aortic Stenosis, Supravalvular ; Chromosomes, Artificial, Bacterial ; Chromosomes, Human, Pair 7 ; Clone Cells ; Echocardiography ; Elastin* ; Humans ; In Situ Hybridization, Fluorescence* ; Intellectual Disability ; Parents ; Williams Syndrome*

PURPOSE: Williams syndrome is characterized by supravalvular aortic stenosis, mental retardation and peculiar facial appearance. Its genetic etiology is considered to be a hemizygotic deletion in Chromosome 7q11.23, which includes the elastin gene. We examined the hemizygotic deletion of Chromosome 7q11.23 in 12 Korean Williams syndrome patients and 8 patients with isolated supravalvular aortic stenosis and performed deletion mapping in the Williams syndrome patients. METHODS: Hemizygotic deletion was determined with fluorescence in situ hybridization(FISH) using the bacterial artificial chromosome clone 244H3, which has the genomic DNA sequence of elastin gene, as a probe. For the deletion mapping, polymorphism analysis of 10 Williams syndrome patients and their parents was done with 9 dinucleotide repeat sequence polymorphic markers(D7S499, D7S672, D7S653, ELN, D7S2472, D7S1870, D7S2518, D7S675 and D7S669). RESULTS: In the Williams syndrome patients, FISH showed deletion in all. In patients with isolated supravalvular aortic stenosis, FISH showed deletion in one, partial deletion in another and no deletion in the other six patients. Polymorphism analysis showed that alleles at three loci(ELN, D7S2472 and D7S1870) were commonly deleted in the Williams syndrome patients. Paternal alleles were deleted in six patients and maternal alleles were deleted in four. CONCLUSION: Hemizygotic deletion could be detected in Williams syndrome patients with FISH and the commonly deleted loci were ELN, D7S2472 and D7S1870. Most patients with isolated supravalvular aortic stenosis showed no deletion with FISH and the genetic defect should be much smaller than what FISH could detect.
Alleles ; Aortic Stenosis, Supravalvular* ; Base Sequence ; Chromosomes, Artificial, Bacterial ; Chromosomes, Human, Pair 7* ; Clone Cells ; Dinucleotide Repeats ; Elastin ; Fluorescence ; Genes, vif ; Humans ; Intellectual Disability ; Parents ; Williams Syndrome*

PURPOSE: Williams syndrome is characterized by supravalvular aortic stenosis, mental retardation and peculiar facial appearance. Its genetic etiology is considered to be a hemizygotic deletion in Chromosome 7q11.23, which includes the elastin gene. We examined the hemizygotic deletion of Chromosome 7q11.23 in 12 Korean Williams syndrome patients and 8 patients with isolated supravalvular aortic stenosis and performed deletion mapping in the Williams syndrome patients. METHODS: Hemizygotic deletion was determined with fluorescence in situ hybridization(FISH) using the bacterial artificial chromosome clone 244H3, which has the genomic DNA sequence of elastin gene, as a probe. For the deletion mapping, polymorphism analysis of 10 Williams syndrome patients and their parents was done with 9 dinucleotide repeat sequence polymorphic markers(D7S499, D7S672, D7S653, ELN, D7S2472, D7S1870, D7S2518, D7S675 and D7S669). RESULTS: In the Williams syndrome patients, FISH showed deletion in all. In patients with isolated supravalvular aortic stenosis, FISH showed deletion in one, partial deletion in another and no deletion in the other six patients. Polymorphism analysis showed that alleles at three loci(ELN, D7S2472 and D7S1870) were commonly deleted in the Williams syndrome patients. Paternal alleles were deleted in six patients and maternal alleles were deleted in four. CONCLUSION: Hemizygotic deletion could be detected in Williams syndrome patients with FISH and the commonly deleted loci were ELN, D7S2472 and D7S1870. Most patients with isolated supravalvular aortic stenosis showed no deletion with FISH and the genetic defect should be much smaller than what FISH could detect.
Alleles ; Aortic Stenosis, Supravalvular* ; Base Sequence ; Chromosomes, Artificial, Bacterial ; Chromosomes, Human, Pair 7* ; Clone Cells ; Dinucleotide Repeats ; Elastin ; Fluorescence ; Genes, vif ; Humans ; Intellectual Disability ; Parents ; Williams Syndrome*

OBJECTIVETo determine the origin of a supernumerary small marker chromosome found in a fetus using prenatal BACs-on-Beads (BoBs) and single nucleotide polymorphism array (SNP-array) assays.

METHODSThe fetal sample was subjected to chromosomal karyotyping and BoBs analysis, and the results were validated with genome-wide scanning using a SNP microarray.

RESULTSThe fetus was found to have a 47,XX,+mar karyotype. BoBs analysis indicated that there was an amplification between 18p11.32 and 18p11.21, which was verified by the SNP-array assay as a 18.3 Mb duplication occurring at 18p11.32q11.1.

CONCLUSIONThe karyotype of the fetus was determined as 47,XX,+der18(18p11.32?18q11.1::18q11.1?18p11.32). The duplication has involved important genes including SMCHD1, LPIN2 and TGIF1, which may result in severe malformations in the fetus.

Adult ; Aneuploidy ; Chromosomes, Artificial, Bacterial ; genetics ; Chromosomes, Human, Pair 18 ; genetics ; Female ; Humans ; Karyotyping ; Microarray Analysis ; methods ; Polymorphism, Single Nucleotide ; Pregnancy ; Prenatal Diagnosis ; methods