Construction and utility of a human chromosome 22-specific Fosmid library

We have previously demonstrated the capability of the Fosmid vector based on Escherichia coli F-factor replicon to stably propagate cosmid-sized human genomic DNA fragments. Using the Fosmid vector, we have constructed and arrayed a 10 × human chromosome 22-specific library, partly by picking human positive clones from a total Fosmid library constructed using DNA from human-hamster hybrid cell line containing human chromosome 22, and partly by using flow-sorted chromosomal DNA. The clones and physical contig maps of the clones in the library will serve as a valuable resource for detailed analysis of the chromosome by providing reliable materials for high resolution mapping and sequencing. In order to efficiently built physical maps for the chromosomal regions of interest spanning several hundred kilobases to a megabase, it is necessary to rapidly identify subsets of the Fosmid clones from the library that cover such regions. In this report, we describe a method of using random amplification products derived from YAC clones to rapidly identify a subset of Fosmid clones that cover a specific genomic subregion.     

Construction and characterization of a NotI linking library of human chromosome 21

Effective procedures have been developed for constructing NotI linking libraries starting from chromosome-specific genomic libraries. Fifteen different single copy and two rDNA NotI linking clones from human chromosome 21 were identified in two libraries. Their chromosomal origin was confirmed, and regional location established using hybrid cell panels. Hybridization experiments with these probes revealed pairs of genomic NotI fragments, each ranging in size from less than 0.05 to 4.0 Mb. Many fragments displayed cell type variation. The total size of the NotI fragments detected in a human fibroblast cell line (GM6167) and mouse hybrid cell containing chromosome 21 as its only human component (WAV17) were approximately 32 and 34 Mb, respectively. If these fragments were all non-overlapping, this would correspond to about 70% of the 50-Mb content estimated for the whole chromosome. The linking clones will be enormously useful in the subsequent construction of a NotI restriction map of this chromosome. Characterization of these clones indicates the presence of numerous additional sites for other enzymes that recognize sequences containing CpG. Thus most NotI linking clones appear to derive from CpG islands and probably identify the 5' end of genes.     

Isolation and regional localisation of DNA sequences from a human chromosome 11-specific cosmid library

Summary A cosmid library has been prepared in the lorist-B vector from a mouse/human somatic cell hybrid containing region 11q23-11pter as the only human component. This chromosome region is stably maintained in the hybrid as a result of translocation onto one copy of mouse chromosome 13. Individual cosmids containing human DNA were isolated by their ability to hybridise with total human DNA, digested with either HindIII or EcoRI, and 33 individual unique sequences were identified. These fragments were then isolated and subcloned into the bluescribe plasmid vector. Regional localisation of these unique sequences was achieved using a panel of somatic cell hybrids containing different overlapping deletions of chromosome 11. The majority of the 33 mapped sequences derived from the long arm of chromosome 11. Two clones were located within the 11p13–p14 region, which is associated with a predisposition to Wilms' tumour. These probes supplement those already mapped to this chromosome and will assist in the generation of a detailed chromosome 11 linkage map.     

Microdissection of the Prader-Willi syndrome chromosome region and identification of potential gene sequences

The Prader-Willi syndrome chromosome region on the long arm of human chromosome 15 was microdissected and microcloned from 20 GTG-banded metaphase chromosomes, and 5000 recombinant clones were obtained. Of these clones, 39% identify single-copy human DNA sequences, most of which map to the dissected chromosome region and are evolutionarily conserved in other species. Three of eleven clones studied in detail are deleted in several patients with Prader-Willi syndrome. The microclones will be useful for the physical characterization of the Prader-Willi syndrome chromosome region and the identification of the affected genes in this disease.     

Construction and characterization of a NotI-BsuE linking library from the human X chromosome.

We describe the construction and characterization of methylation-resistant sequence-tagged NotI linking clones specific for the X chromosome, referred to as NotI-BsuE linking clones. The approach consists of methylating the X-chromosome-specific cloned DNA with BsuE methylase (M. BsuE), an enzyme that methylates the first C residue in the CGCG sequence, followed by selection of the methylation-resistant NotI sites by insertion of a kanamycin-resistance gene in the clones cleavable by NotI. The frequent occurrence of NotI sites in CpG islands is expected to cause methylation of a large number of NotI sites with BsuE methylase, thereby rendering them resistant to NotI cleavage. Thus, the combination of M. BsuE and NotI yields less frequent cutting than the NotI alone. We have isolated, partially sequenced, and characterized 113 NotI-BsuE linking clones, and mapped 50 clones to various regions along the chromosome.     

Construction of a bacterial artificial chromosome (BAC) library and identification of overlapping BAC clones with chromosome 4-specific RFLP markers in rice

 To facilitate construction of physical map of the rice genome, a bacterial artificial chromosome (BAC) library of IR64 genomic DNA was constructed. It consists of 18 432 clones and contains 3.28 rice genomic equivalents. The insert size ranged from 37 to 364 kb with an average of 107 kb. We used 31 RFLP markers on chromosome 4 to screen the library by colony hybridization. Sixty eight positive clones were identified with 2.2 positive clones per RFLP marker. The positive clones were analyzed to generate 29 contigs whose sizes ranged from 50 to 384 kb with an average of 145.6 kb. Chromosome walking was initiated for ten contigs linked to resistance genes. Thirty eight BAC clones were obtained and two contigs were integrated. Altogether, they covered 5.65 Mb (15.1%) of chromosome 4. These contigs may be used as landmarks for physical mapping of chromosome 4, and as starting points for chromosome walking towards the map-based cloning of disease resistance genes which were located nearby. Received: 15 November 1996 / Accepted: 24 January 1997     

染色体特异DNA文库的构建与鉴定

为构建人类21号染色体特异DNA文库, 以应用于人类遗传疾病的鉴定和研究, 文章采用循环温度梯度法溶解释放微分离的人外周血细胞21号染色体DNA, 将其进行简并寡核苷酸引物PCR(Degenerate oligo nucleotide primer-PCR, DOP-PCR)扩增后, 利用100~500 bp和500~2 000 bp分段回收纯化的两种不同片段大小的DOP-PCR产物构建染色体特异DNA文库, 并分别采用荧光原位杂交(Florescence in situ hybridization, FISH)和斑点杂交对DOP-PCR产物的来源和随机取样的文库克隆进行检测以评估所构建DNA文库的特异性。结果表明: 循环温度梯度法能有效溶解释放微分离的21号染色体DNA; 通过对DOP-PCR产物的分段回收纯化和克隆, 增加了大片段DNA的连接效率; 利用FISH技术和斑点杂交双重鉴定实验证明了文库的特异性, 从而构建了21号染色体特异的DNA文库, 并建立了构建染色体特异DNA文库及检测其特异性的方法, 为21号染色相关遗传疾病的鉴定和研究奠定了基础。     

An improved pulsed-field polyacrylamide gel electrophoresis system for physical selection of linking clones: isolation of SfiI linking clones from a chromosome 21-specific library

We had previously developed an efficient procedure for selective cloning of rare-cutter linking fragments that is based on physical separation of linking clone DNAs by pulsed-field polyacrylamide gel electrophoresis (PF-PAGE). An advantage of the physical selection procedure over the conventional cloning-based ones utilizing the insertion of selection marker or vector sequences into the rare-cutter sites is that it can be readily applied to the selection of linking fragments for rare-cutters, generating ambiguous cohesive end sequences such as SfiI (GGCCNNNN/NGGCC). In the present work, the physical separation procedure was improved by introducing a discontinuous buffer system into PF-PAGE, and its feasibility was exemplified by the selective isolation of SfiI linking clones from a human chromosome 21-specific library. This simple and efficient procedure will provide a useful tool for genome analysis.     

11p15.5-specific libraries for identification of potential gene sequences involved in Beckwith-Wiedemann syndrome and tumorigenesis.

Constitutional and somatic chromosomal abnormalities of the chromosome 11p15 region are involved in an overgrowth malformation syndrome, the Beckwith-Wiedemann syndrome (BWS), and in several types of associated tumors. The bias in parental origin for the different etiologic forms of this syndrome and for loss of heterozygosity in the tumors suggests that a gene (or genes) mapping to this region undergoes genomic imprinting. However, the precise localization of the locus (or loci) for the BWS and associated tumors is still unknown and more markers are required. We therefore isolated 11p15 markers from two libraries: the first one obtained by microdissection of the chromosome 11p15.5 region and the second one, a phage library, constructed from a hybrid cell line containing this region as its sole human DNA. Of 19 microclones isolated from the microdissection library, 11 were evolutionarily conserved. Four phage clones were isolated; one (D11S774) detected a highly informative variable number of tandem repeats (VNTR) and another (D11S773) a biallelic polymorphism. These clones were sublocalized using a panel of somatic cell hybrids that defines eight physical intervals in 11p15.5. Twenty-one clones map to the distal interval that harbors the BWS locus.     

Construction of a human chromosome 3 specific NotI linking library using a novel cloning procedure.

Two new diphasmid vectors (lambda SK17 and SK22) and a novel procedure to construct linking libraries are described. A partial filling-in reaction provides counter-selection against false linking clones in the library, and obviates the need for supF selection. The diphasmid vectors, in combination with the novel selection procedure, have been used to construct a chromosome 3 specific NotI linking library from a human chromosome 3/mouse microcell hybrid cell line. The application of the new vectors and the strong biochemical and biological selections resulted in a library of 60,000 NotI linking clones. As practically all of them are real NotI linking clones (no false recombinants) the library represents approximately 3,000 human recombinants (equal to 10-15 genomic equivalents of chromosome 3). Previously published methods for construction of linking libraries are compared with the procedure described in the present paper. The advantages of the new vectors and the novel protocol are discussed.     

Construction and regional localization of clones from a NotI linking library from human chromosome 17q

A NotI linking library was constructed from a somatic cell hybrid containing chromosome 17q as its only human material. A total of 112 human clones were assigned to nine regions of 17q using a somatic cell hybrid mapping panel. The library includes clones that detect the acute promyelocytic leukemia and von Recklinghausen neurofibromatosis translocation breakpoints at 17q11.2-12 and 17q11.2, respectively, on pulsed-field gel electrophoresis. The mapped clones represent over 50% of the estimated number of NotI sites on 17q, and therefore constitute an important resource for long-distance mapping.     

An improved pulsed-field polyacrylamide gel electrophoresis system for physical selection of linking clones: Isolation of SfiI linking clones from a chromosome 21-specific library

We had previously developed an efficient procedure for selective cloning of rare-cutter linking fragments that is based on physical separation of linking clone DNAs by pulsed-field polyacrylamide gel electrophoresis (PF-PAGE). An advantage of the physical selection procedure over the conventional cloning-based ones utilizing the insertion of selection marker or vector sequences into the rare-cutter sites is that it can be readily applied to the selection of linking gragments for rare-cutters, generating ambiguous cohesive end sequences such as SfiI (GGCCNNNN/NGGCC). In the present work, the physical separation procedure was improved by introducing a discontinuous buffer system into PF-PAGE, and its feasibility was exemplified by the selective isolation of SfiI linking clones from a human chromosome 21-specific library. This simple and efficient procedure will provide a useful tool for genome analysis.     

Construction of poplar (Populus tremula) chromosome 1-specific DNA library by using a microdissection technique

A method for single-chromosome microdissection and microcloning was established in forest plants using poplar (Populus tremula) as a model. By use of meristematic cell division in root tip and the wall degradation hypotonic method, well-spread poplar metaphase chromosome spreads showing low contamination were quickly prepared and fitted for chromosome microdissection. An individual chromosome 1 was microdissected from the metaphase spreads of poplar root-tip cells with a fine glass needle controlled by a micromanipulator. The dissected chromosome was amplified in vitro by theSau3A linker adaptor-mediated PCR technique, by which 200- to 3000-bp smear DNA fragments were obtained. Southern hybridization results showed that the PCR products from the single poplar chromosome were homogeneous with poplar genomic DNA, indicating that DNA from the single chromosome has been successfully amplified. Next, the second-round PCR products from the single chromosome 1 were cloned into T-easy vectors to generate a DNA library of the chromosome 1. About 3×10⁵ recombinant clones were obtained. Evaluation based on 160 randomly selected clones showed that the sizes of the cloned inserts varied from 230–2200 bp, with an average of 800 bp. Therefore, this research suggests that microdissection and microcloning of single small chromosomes in forest plants is feasible.     

Construction and analysis of an EMBL-3 phage library containing partially digested human chromosome 21-specific DNA inserts (15-20 kb)

In the mouse-human hybrid cell line SCC 16-5, chromosome 21 is the only human chromosome present. Fractions highly enriched for this chromosome were obtained by applying the chromosome velocity sedimentation technique to this cell line. DNA prepared from these chromosomal fractions was partially digested with Mbo I, size fractionated on an NaCl gradient, and cloned in the EMBL-3 phage vector. The phage library thus prepared was highly enriched for human chromosome 21-specific recombinant DNA sequences 15-20 kb long. Of the approximately 21,000 phage clones obtained, at least 99% were recombinant. Following phage plaque filter hybridization and Southern blotting, it was found that half of the recombinants were positive for human repetitive DNA. Almost all phages harbored highly or middle repetitive human or mouse DNA sequences owing to the large size of the recombinant inserts. In this library, the human chromosome 21 is represented approximately four times. All human recombinants studied thus far contained DNA inserts originating from chromosome 21 only. The employed cloning strategy is discussed with regard to utility, purity, quality, and completeness of chromosome-specific recombinant DNA libraries.     

Construction of a bacterial artificial chromosome (BAC) library for citrus and identification of BAC contigs containing resistance gene candidates

A BAC library was constructed from the genomic DNA of an intergeneric Citrus and Poncirus hybrid. The library consists of 24,576 clones with an average insert size of 115 kb, representing approximately seven haploid genome equivalents and is able to give a greater than 99% probability of isolating single-copy citrus DNA sequences from this library. High-density colony hybridization-based library screening was performed using DNA markers linked to the citrus tristeza virus (CTV) resistance gene and citrus disease resistance gene candidate (RGC) sequences. Between four and eight clones were isolated with each of the CTV resistance gene-linked markers, which agrees with the library’s predicted genome coverage. Three hundred and twenty-two clones were identified using 13 previously cloned citrus RGC sequences as probes in library screening. One to four fragments in each BAC were shown to hybridize with RGC sequences. One hundred and nine of the RGC BAC clones were fingerprinted using a sequencing gel-based procedure. From the fingerprints, 25 contigs were assembled, each having a size of 120–250 kb and consisting of 2–11 clones. These results indicate that the library is a useful resource for BAC contig construction and molecular isolation of disease resistance genes. Received: 22 May 2000 / Accepted: 25 September 2000     

Construction of a NotI linking library and isolation of new markers close to the Huntington''s disease gene.

Linking clones contain sequences flanking recognition sites for enzymes cutting rarely in mammalian DNA. They can be used to obtain and correlate both physical and genetic mapping information over subregions of mammalian chromosomes. We have constructed and used a NotI linking clone library representing unmethylated NotI sites from HHW693 DNA, a hamster hybrid cell line containing 4p15-4pter and a fragment of 5p as its only human chromosome contribution. Human clones were identified by hybridisation with a cloned human repeat sequence, and localised further to subregions of human chromosome 4p15-4pter using a panel of additional hybrids. Clones from the region distal to the DNA probes (D4S10, D4S43, D4S95) linked to the Huntington's disease mutation, were further analysed. Four markers close to the HD gene: D4S111, D4S113, D4S114 and clone 417 are described here. In addition to serving as markers in physical and genetic mapping experiments, these linking clones provide probes next to cleavable NotI sites, and can therefore be used to screen NotI based chromosome jumping libraries. They also provide indications for potential gene sequences, identifiable as evolutionarily conserved sequences.     

Construction of a bacterial artificial chromosome library of Medicago truncatula and identification of clones containing ethylene-response genes

 To facilitate genome analysis and map-based cloning of symbiotic genes in the model legume Medicago truncatula, a bacterial artificial chromosome (BAC) library was constructed. The library consists of 30 720 clones with an average insert size of approximately 100 kb, representing approximately five haploid-genome equivalents. The frequency of BAC clones carrying inserts of chloroplast DNA was estimated to be 1.4%. Screening of the library with single- or low-copy genes as hybridization probes resulted in the detection of 1–12 clones per gene. Hybridization of the library with repeated sequences such as rDNA genes and transposon-like elements of M. truncatula revealed the presence of 60 and 374 BAC clones containing the two sequences, respectively. The BAC library was pooled for screening by polymerase chain reaction (PCR)-amplification. To demonstrate the utility of this system, we used primers designed from a conserved region of the ein3-like loci of Arabidopsis thaliana and isolated six unique BAC clones from the library. DNA gel-blot and sequence analyses showed that these ein3-like clones could be grouped into three classes, an observation consistent with the presence of multiple ein3-like loci in M. truncatula. These results indicate that the BAC library represents a central resource for the map-based cloning and physical mapping in M. truncatula and other legumes. Received: 27 July 1998 / Accepted: 5 August 1998     

Construction and evaluation of a porcine bacterial artificial chromosome library

A porcine bacterial artificial chromosome (BAC) library consisting of 103,488 clones has been constructed. The average insert size in the BAC vector was calculated to be 133 kb based on the examination of 189 randomly selected clones, indicating that the library contained 4.4 genome equivalents. The library can be screened by two-step PCR. The first screening step is performed on 22 superpools, each containing 4704 clones (49 x 96 well plates). In the second screening step, 49 plates comprising a superpool are arrayed in a 7 x 7 matrix and 4D-PCR is performed. Screening of the library superpools by PCR for 125 marker sequences selected from different regions of swine genome revealed 123 sequences, indicating that the library is not biased. Subsequent screenings (4D-PCR) were successfully applied for identification of clones containing each marker sequence. This porcine BAC library and the PCR screening system are useful for isolation of genomic DNA fragments containing desired sequences.     

A bacterial artificial chromosome library for soybean and identification of clones near a major cyst nematode resistance gene

 We constructed a bacterial artificial chromosome (BAC) library for soybean (Glycine max) consisting of approximately 30 000 clones with an average insert size of 120 kilobase pairs. The library was successfully screened with restriction fragment length polymorphism (RFLP) and microsatellite markers tightly linked to a major resistance gene for the cyst nematode, Heterodera glycines. Since many soybean RFLPs hybridize to duplicate loci, BACs homologous to duplicate RFLP loci were distinguished by digestion with the restriction enzyme originally used to map the RFLP, followed by a comparison of the hybridizing fragments. Linkage mapping of BAC clones identified with markers linked to the cyst nematode resistance gene demonstrated that these clones were located at the expected chromosomal positions and that there were no indications of chimeras within the genomic inserts. Received: 3 July 1997/Accepted: 26 August 1997