Construction and Characterization of a 10-fold Genome Equivalent Rat P1-Derived Artificial Chromosome Library

A rat PAC library was constructed in the vector pPAC4 from genomic DNA isolated from female Brown Norway rats. This library consists of 215,409 clones arrayed in 614 384-well microtiter plates. An average insert size of 143 kb was estimated from 217 randomly isolated clones, thus representing approximately 10-fold genome coverage. This coverage provides a very high probability that the library contains a unique sequence in genome screening. Tests on randomly selected clones demonstrated that they are very stable, with only 4 of 130 clones showing restriction digest fragment alterations after 80 generations of serial growth. FISH analysis using 70 randomly chosen PACs revealed no significant chimeric clones. About 7% of the clones analyzed contained repetitive sequences related to centromeric regions that hybridized to some but not all centromeres. DNA plate pools and superpools were made, and high-density filters each containing an array of 8 plates in duplicate were prepared. Library screening on these superpools and appropriate filters with 10 single-locus rat markers revealed an average of 8 positive clones, in agreement with the estimated high genomic coverage of this library and representation of the rat genome. This library provides a new resource for rat genome analysis, in particular the identification of genes involved in models of multifactorial disease. The library and high-density filters are currently available to the scientific community.     

Construction and Characterization of aMagnaporthe griseaBacterial Artificial Chromosome Library

Diaz-Perez, S. V., Crouch, V. W., and Orbach, M. J. 1996. Construction and characterization of aMagnaporthe griseabacterial artificial chromosome library.Fungal Genet. Biol.20,280–288. A bacterial artificial chromosome (BAC) library ofMagnaporthe griseacontaining 4128 clones with an average insert size of 66-kb has been constructed. This library represents seven genome equivalents ofM. griseaand has been demonstrated to be representative of the genome by screening for the presence of several single-copy genes and DNA markers. The utility of the library for use in map-based cloning projects was shown by the spanning of a nine-cosmid, 207-kb DNA contig with only 3 BAC clones. In addition, using alys1-3auxotroph, we have shown that BAC clones at least 113 kb can be transformed intoM. griseato screen for complementation of mutations. Thus, BACs isolated in chromosome walks can be rapidly screened for the presence of the sought after gene. The ease of construction of BAC libraries and of isolation and manipulation of BAC clones makes the BAC system an ideal one for physical analyses of fungal genomes.     

野生一粒小麦BAC文库的构建和鉴定

以细菌人工染色体pECBAC1为载体 ,构建了野生一粒小麦 (TriticumboeoticumBoiss)的基因组BAC文库。该文库共包含约 17万个克隆 ,平均插入片段长度为 10 4kb ,按野生一粒小麦基因组为 5 6 0 0Mb计算 ,文库覆盖了约 3倍的该物种基因组。用大麦叶绿体psbA基因和玉米线粒体atp6基因作混合探针 ,检测发现该文库中含细胞器基因组同源序列的克隆数小于 1%。该文库的建成 ,为小麦基因的克隆及基因组学研究提供了技术平台     

Wild rats (Rattus norvegicus) for mapping of disease-resistant genes

Wild rat representing a disease-resistant phenotype and genotype, was used in a crossing study with spontaneously hypertensive rat (SHR) to search for quantitative trait loci (QTL) affecting blood pressure. Therefore, one male wild rat was crossed with SHR females and F1 hybrids were transferred in a pathogen free environment by wet-hysterectomy and backcrossed onto hypertensive SHR rats resulting in first backcross hybrids (BC1). Considering that the F1 hybrids are not uniform, as are the cross hybrids of inbred rat strains, we selected 72 BC1 progeny of one F1 female, which were characterised for systolic blood pressure, measured by tail cuff method and were genetically analysed using 200 microsatellites covering the whole genome. We found suggestive linkage of blood pressure to region on chromosome 2 flanked by D2Mit8 and Fgg loci (lod score 2.3). In addition, possible interaction between genes on chromosomes 7 and 3, X and 3, 14 and 3, 13 and 11 was described, indicating that blood pressure development in the SHR might be the result of interacting genes.     

Construction and Characterization of a Magnaporthe grisea Bacterial Artificial Chromosome Library

Diaz-Perez, S. V., Crouch, V. W., and Orbach, M. J. 1996. Construction and characterization of a Magnaporthe grisea bacterial artificial chromosome library. Fungal Genet. Biol. 20, 280-288. A bacterial artificial chromosome (BAC) library of Magnaporthe grisea containing 4128 clones with an average insert size of 66-kb has been constructed. This library represents seven genome equivalents of M. grisea and has been demonstrated to be representative of the genome by screening for the presence of several single-copy genes and DNA markers. The utility of the library for use in map-based cloning projects was shown by the spanning of a nine-cosmid, 207-kb DNA contig with only 3 BAC clones. In addition, using a lys1-3 auxotroph, we have shown that BAC clones at least 113 kb can be transformed into M. grisea to screen for complementation of mutations. Thus, BACs isolated in chromosome walks can be rapidly screened for the presence of the sought after gene. The ease of construction of BAC libraries and of isolation and manipulation of BAC clones makes the BAC system an ideal one for physical analyses of fungal genomes.     

Construction and Characterization of aPlasmodium vivaxGenomic Library in Yeast Artificial Chromosomes

Here we describe the construction of a representative YAC library for the human malarial parasitePlasmodium vivax.AsP. vivaxcannot be maintained continuously under laboratory conditions, theP. vivaxDNA necessary for the library construction was isolated from a single human patient presenting himself with vivax malaria to a local hospital in the Brazilian Amazon. Thus, this YAC library is the first of its kind to be generated from patient-derived material. The YAC library consists of 560 clones with an average insert size of 180 kb. Of 9 publishedP. vivaxgenes, 8 were found to be present in the library. In addition, 12P. vivaxtelomeric YAC clones were identified.     

Construction and Some Characterization of a Yeast Artificial Chromosome Library from DNA of a Tomato Line Having Four Disease Resistance Traits

We have constructed a YAC library containing over 5000 clones of tomato (Lycopersicon esculentum Mill. cv. VFNT) DNA with an average insert size of 320 kb, which were equivalent to two haploid genomes. The tomato used here has four disease resistance traits. Therefore the library constructed should be useful for isolating genes of such traits by map-based cloning.     

Construction of a Bacterial Artificial Chromosome Library of Phytophthora infestans and Transformation of Clones into P. infestans

A bacterial artificial chromosome (BAC) library of Phytophthora infestans was constructed in a derivative of pBELOBACII that had been modified by adding a npt selectable marker gene for transforming P. infestans. A total library of 8 genome equivalents was generated and 16,128 clones with inserts averaging 75 kb (4.9 genome equivalents) were individually picked and stored as an arrayed library in microtiter plates. This coverage was confirmed by screening the library for 11 DNA loci by colony hybridization and by polymerase chain reaction of DNA pools. Transformation of P. infestans with BAC clones containing inserts of 93 to 135 kb was demonstrated. The efficiency of transformation with most BACs was noticeably higher than that with smaller plasmids. Detailed analyses of transformants obtained with a 102-kb BAC indicated that entire inserts were present in about one-quarter of the transformants.     

Effect of Moniliformis moniliformis density on distribution within the definitive host population (Rattus norvegicus)

The population dynamics of Moniliformis moniliformis was studied in ‘free-ranging’ laboratory rats, Rattus norvegicus, presented with different relative density levels of M. moniliformis in cockroaches, Periplaneta americana. Changes in selected population parameters of the negative binomial distribution were evaluated as indicators of changes in aggregation. A significant increase in the degree of aggregation of parasites occurred as a result of the increase in relative density of infective stages available to the rats. This increase in aggregation was due to the increase in over-dispersion that occurred in female rats only. The degree of aggregation in females was found to be significantly higher than that in males at both treatment levels. The best indicators of the degree of aggregation were found to be the ratio of the variance to the relative density and the ratio of the log-variance to log-relative density. Changes in k were not correlated with changes in over-dispersion or the relative density.     

可转化人工染色体文库的构建与鉴定

随着人类和植物基因组计划的实施,能容纳大片段的人工染色体载体发展迅速。而用YAC、BAC和PAC等基因组文库进行目的基因的筛选,在获得候选克隆后,通常要进行亚克隆,然后对每个亚克隆逐一进行基因功能互补试验,不仅工作量大,而且有遗漏目的基因的危险。TAC载体含P1质粒和Ri质粒的复制子,可直接转化植物,大大加速了工作进程。概括性的叙述了TAC载体的发展,TAC文库构建的程序及文库鉴定的问题。     

细菌人工染色体文库的构建及应用

细菌人工染色体（BAC）是第二代大片段DNA的克隆载体系统,具有容量大、嵌合率低、遗传特性稳定、转化效率高、插入片段易回收、操作简便等优点,因而被广泛应用于基因组较大的真核生物基因组研究中,并发挥着前所未有的重要作用。本文综述了BAC的发展,利用此载体构建基因组文库的程序和鉴定方法,及其在物理图谱构建、图位克隆、基因组测序、转基因技术等研究中的应用。     

Construction and Characterization of a Bacterial Artificial Chromosome (BAC) Library of Pacific White Shrimp, Litopenaeus vannamei

The pacific white shrimp (Litopenaeus vannamei) is one of the most economically important marine aquaculture species in the world. To facilitate gene cloning and characterization, genome analysis, physical mapping, and molecular selection breeding of marine shrimp, we have developed the techniques to isolate high-quality megabase-sized DNA from hemocyte nuclear DNA of female shrimp and constructed a bacterial artificial chromosome (BAC) genomic library for the species. The library was constructed in the Hind III site of the vector pECBAC1, consisting of 101,760 clones arrayed in 265 384-well microtiter plates, with an average insert size of 101 kb, and covering the genome approximately fivefold. To characterize the library, 92,160 clones were spotted onto high-density nylon filters for hybridization screening. A set of 18 pairs of overgo probes designed from eight cDNA sequences of L. vannamei genes were used in hybridization screening, and 35 positive clones were identified. These results suggest that the shrimp BAC libraries will provide a useful resource for screening of genomic regions of interest candidate genes, gene families, or large-sized synthetic DNA region and promote future works on comparative genomics, physical mapping, and large-scale genome sequencing in the species.     

Spatial and temporal patterns of brown rat (Rattus norvegicus) abundance variation in poultry farms

The temporal and spatial patterns of rat abundance variations were studied, and which factors might affect these patterns in poultry farms. Forty-eight poultry farms were sampled from spring 1999 to winter 2001 in Buenos Aires, Argentina. Environmental variables of farms, meteorological variables and distances among farms were registered to evaluate a possible association with rat abundance estimated by trap success. Rattus norvegicus was the dominant rat species in the study area. Rat abundance did not vary temporally and there was reproduction at all times. Trap success was more similar among neighboured farms than among farms further apart. Contrary to the abundance of other small rodents, rat abundance was not significantly related to farm characteristics. The lack of coincidence between rats and other small rodents suggests the existence of a differential response of rats to environmental factors of the poultry farms.     

Construction of a Bacterial Artificial Chromosome Library and Characterization of hrp/hrc Gene Cluster of Pseudomonas Syringae Pathovar tagetis LMG5090

Pseudomonas syringae pv. tagetis causes apical chlorosis of several plant species in the Asteraceae, including marigold. As a means to facilitate the isolation of pathogenicity genes and to characterize the genome of this bacterium, we have constructed a bacterial artificial chromosome library of P. syringae pv. tagetis strain LMG5090. The library consists of 1,536 clones with insert size ranging from 30 to 160 kb and an average size of 86 kb. Based upon colony hybridization, the BAC clone 420E23 containing the hrp/hrc gene cluster encoding the type III secretion system was identified from this library and subsequently shotgun sequenced. The hrp/hrc gene cluster of P. syringae pv. tagetis has a 23 kb sequence which contains 27 open reading frames. Comparative analysis of the hrp/hrc gene cluster of P. syringae pv. tagetis LMG5090, P. syringae pv. tomato DC3000, P. syringae pv. syringae B728a, and P. syringae pv. phaseolicola 1448A revealed that the entire hrp/hrc gene cluster of P. syringae pv. tagetis is conserved and identically arranged in all four pathovars     

Construction and Characterization of a Human Chromosome 2-Specific BAC Library

We have constructed a human chromosome 2-specific bacterial artificial chromosome (BAC) library using DNA from the somatic cell hybrid GM10826. The average size of the clones is about 63 kb. The coverage and distribution of the library were estimated by screening with known polymorphic genetic markers and fluorescence in situ hybridization (FISH). Twentyone markers tested positive when DNA pools prepared from approximately one-sixth of the library were screened with 33 known markers. This is consistent with the theoretical calculation of 63% coverage at one genomic equivalent. This suggested that the coverage of the library is approximately 5-6×. FISH analysis with 54 BACs revealed single site hybridization to chromosome 2, and the clones were distributed randomly on the chromosome. We have also performed direct sequencing of the BAC insert ends to generate sequence-tagged sites suitable for mapping and chromosome walking. This is the first reported human chromosome 2-specific BAC library and should provide a resource for physical mapping and disease searching for this chromosome.     

山羊瘤胃微生物宏基因组BAC文库的构建与分析

从山羊瘤胃液中提取混合微生物DNA,经BamHI部分酶切得到50kb～800kb的DNA片段后,将其连接到pCCIBAC载体上,转化E．coliEPI300,建立山羊瘤胃微生物BAC文库。经RFLP鉴定分析,该文库12672个克隆,平均插入片段为6lkb。该文库的构建为后续新型基因的筛选提供了材料,为进一步研究山羊瘤胃微生物奠定了基础。     

Application of Interspersed Repetitive Sequence Polymerase Chain Reaction for Construction of Yeast Artificial Chromosome Contigs

Construction of physical maps across candidate regions is one of the rate-limiting steps of positional cloning projects. To date, most physical maps have been constructed by polymerase chain reaction (PCR)-based sequence-tagged site (STS) content mapping. While effective, this technique has a number of disadvantages including problems with yeast artificial chromosome (YAC) chimerism, the time and effort required to generate new STSs from YAC ends, the cost of primer synthesis for large contiging projects, and the time, effort, and expense necessary for screening each STS in the two-tiered hierarchical YAC library screening format. An alternative strategy, interspersed repetitive sequence (IRS) PCR genomics, alleviates many of these constraints. Clonal overlap is detected by hybridization of individual IRS-PCR products to IRS-PCR product pools of the three-dimensional coordinate pools of YAC libraries in dot-blot format. Entire libraries can be screened in a single step, and multiple libraries can be screened simultaneously. Cloning YAC fragments, sequencing, and primer generation are eliminated, increasing the efficiency of contig construction and reducing the expense. In addition, the genomic location of the individual IRS-PCR products can also be simultaneously determined by screening either interspecific backcrosses or radiation hybrid panels, in dot-blot format, confirming contig extension in the region of interest.     

Construction of a Bacterial Artificial Chromosome Library of TM-1, a Standard Line for Genetics and Genomics in Upland Cotton

A bacterial artificial chromosome （BAC） library was constructed for Gossypium hirsutum acc. TM-1, a genetic and genomic standard line for Upland cotton. The library consists of 147 456 clones with an average insert size of 122.8 kb ranging from 97 to 240 kb. About 96.0% of the clones have inserts over 100 kb. Therefore, this library represents theoretically 7.4 haploid genome equivalents based on an AD genome size of 2 425 Mb. Clones were stored in 384 384- well plates and arrayed into multiplex pools for rapid and reliable library screening. BAC screening was carried out by four-round poiymerase chain reactions using 23 simple sequence repeats （SSR） markers, three sequence-related amplified polymorphism markers and one pair of primers for a gene associated with fiber development to test the quality of the library. Correspondingly, in total 92 positive BAC clones were identified with an average four positive clones per SSR marker, ranging from one to eight hits. Additionally, since these SSR markers have been localized to chromosome 12 （A12） and 26 （D12） according to the genetic map, these BAC clones are expected to serve as seeds for the physical mapping of these two homologous chromosomes, sequentially map-based cloning of quantitative trait loci or genes associated with important agronomic traits.     

基因组细菌人工染色体文库(BAC)的构建及应用

细菌人工染色体 (BAC)是一种承载DNA大片段的克隆载体系统 ,用于人、动物和植物基因组文库构建。BAC具有插入片断大、嵌合率低、遗传稳定性好、易于操作等优点。BAC文库的构建是基因组较大的真核生物基因组学研究的重要基础 ,可用于真核生物重要基因及全基因组物理作图、重要性状基因的图位克隆、基因结构及功能分析。本文主要综述了细菌人工染色体的构建与其鉴定 ,及其在物理图谱构建、图位克隆、转基因技术等研究上的应用。