Roentgenologic characteristics of small bronchoalveolar cancer of the lung under conditions of image processing using linear filtration (roentgenomorphologic comparison)

X-ray and morphological signs were compared in 50 patients with histologically verified bronchioloalveolar cancer (BAC). Particular attention was drawn to a study of an x-ray picture after computer processing of a tomographic image using a special technique. The most characteristic x-ray signs of BAC were subpleural localization, the presence of a fragmented scar in a tumor, and ill defined outlines. The peculiarities of an x-ray picture of small-size BAC were determined by the morphological structure of a tumor, a degree of its differentiation that could be detected in most cases only after image processing by the linear filtration technique.     

Use of array CGH in the evaluation of dysmorphology, malformations, developmental delay, and idiopathic mental retardation

The clinical implementation of array comparative genomic hybridization has revolutionized the diagnosis of patients with syndromic or nonsyndromic mental retardation. Multiple studies of hundreds of patients with idiopathic mental retardation, and normal karyotype and/or subtelomeric testing using genome-wide microarray platforms with approximately 2000 to >30,000 (tiling-path) interrogating BAC/PAC probes have detected chromosome abnormalities in up to 17% of cases. Surprisingly, some of the pathogenic changes are mosaic and not detectable in conventional karyotyping. Commercially available genome-wide microarrays with >300,000 synthesized oligonucleotide probes enable higher resolution and sensitivity and will probably replace the BAC/PAC arrays in clinical laboratories.     

Pentanol and Benzyl Alcohol Attack Bacterial Surface Structures Differently

The genus Methylobacterium tolerates hygiene agents like benzalkonium chloride (BAC), and infection with this organism is an important public health issue. Here, we found that the combination of BAC with particular alcohols at nonlethal concentrations in terms of their solitary uses significantly reduced bacterial viability after only 5 min of exposure. Among the alcohols, Raman spectroscopic analyses showed that pentanol (pentyl alcohol [PeA]) and benzyl alcohol (BzA) accelerated the cellular accumulation of BAC. Fluorescence spectroscopic assays and morphological assays with giant vesicles indicated that PeA rarely attacked membrane structures, while BzA increased the membrane fluidity and destabilized the structures. Other fluorescent spectroscopic assays indicated that PeA and BzA inactivate bacterial membrane proteins, including an efflux pump for BAC transportation. These findings suggested that the inactivation of membrane proteins by PeA and BzA led to the cellular accumulation but that only BzA also enhanced BAC penetration by membrane fluidization at nonlethal concentrations.     

Construction, characterization and FISH mapping of a bacterial artificial chromosome library of Chinese pangolin (Manis pentadactyla)

Chinese pangolins as a representative species in the order Pholidota have highly specified morphological characters and occupy an important place in the mammalian phylogenetic tree. To obtain genomic data for this species, we have constructed a bacterial artificial chromosome (BAC) library of Chinese pangolin. The library contains 208,272 clones with an average insert size of 122.1 kb and represents approximately eight times the Chinese pangolin haploid genome (if we assume that the Chinese pangolins have a genome size similar to human). One hundred and twenty randomly-selected BAC clones were mapped onto Chinese pangolin chromosomes by fluorescence in situ hybridization (FISH), showing a largely unbiased chromosomal distribution. Several clones containing repetitive DNA and ribosomal DNA genes were also found. The BAC library and FISH mapped BAC clones are useful resources for comparative genomics and cytogenetics of mammals and in particular, the ongoing genome sequencing project of Chinese pangolins.     

细菌人工染色体基因组文库构建方法的改进

目的:建立一种改进的更简便、易操作的细菌人工染色体(BAC)文库构建方法。方法:在构建猪霍乱沙门氏菌基因组大片段DNA的BAC文库时,对改进的基因组BAC文库构建方法和常规的BAC文库构建方法进行比较。结果:利用改进的方法可简便快速地构建猪霍乱沙门氏菌基因组BAC文库。结论:使用2种方法构建BAC文库,其转化效率,以及在BAC克隆中插入的DNA片段的大小和BAC克隆的稳定性等都相同,从而表明改进的方法更简单、更方便,它能使BAC文库的构建更为高效。     

Morphologic and immunocytochemical studies of bronchioloalveolar carcinoma at Duke University Medical Center, 1968-1986

Between 1968 and 1986, the tumor registries at Duke University Medical Center and Durham VA Medical Center accumulated a total of 193 patients with a diagnosis of bronchioloalveolar carcinoma (BAC). All available histologic sections of primary lung tumors and all available respiratory and pleural cytologic material were reviewed for 135 cases having an initial histologic diagnosis of BAC and no history of a primary nonpulmonary adenocarcinoma. Thirty-nine cases showed a pure BAC pattern in histologic sections; 76 showed a dominant BAC pattern with focal areas of fibrosis and acinar differentiation; 16 were carcinomas with a focal BAC pattern; and 4 were adenocarcinomas lacking a BAC pattern. Of the 115 cases with at least a dominant BAC pattern, 51 showed predominantly mucinous differentiation while 64 showed predominantly nonmucinous differentiation. Adequate cytologic material was available for review from 111 patients. For cases having at least a dominant BAC pattern, tumor cells were present in 77 of 172 adequate sputums (44.8%), 36 of 133 bronchoscopy specimens (27.1%), 15 of 18 needle aspirates (83.3%) and 14 of 15 pleural fluids (93.3%). Of all patients in this group, 60.2% had at least one specimen positive for malignancy. No cytologic features clearly distinguished adenocarcinomas having only focal bronchioloalveolar differentiation from those with a pure or dominant BAC pattern. A significant degree of overlap was observed in the cytologic features of mucinous and nonmucinous tumors. Histologic sections from 19 mucinous and 16 nonmucinous tumors were stained with monoclonal antibody B72.3: all showed some staining, with no significant difference in degree of staining between the two groups. This suggests that expression of the tumor-associated glycoprotein TAG-72 is independent of mucinous differentiation.     

Non-gridded library: a new approach for BAC (bacterial artificial chromosome) exploitation in hexaploid wheat (Triticum aestivum)

The feasibility of exploiting non-gridded bacterial artificial chromosome (BAC) libraries and some major factors affecting the efficiency of handling such libraries were studied in hexaploid wheat. Even for a bacterial culture containing only 55% recombinants, some 2000 BAC clones with inserts ranging from 45 to 245 kb could be pooled. The pooled BAC clones could be amplified by culturing for up to 6 h without losing any target clones. These results imply that even for hexaploid wheat, which has an extremely large genome, some 250 pools are sufficient for a BAC library that should satisfy many research objectives. This non-gridded strategy would dramatically reduce the cost and make robotic equipment non-essential in exploiting BAC technology. To construct a representative library and to minimise clone competition, thawing and re-freezing ligation mixtures and bacterial cultures should be avoided in BAC library construction and application.     

Effect of temperature and benzalkonium chloride on nitrate reduction

The effect of temperature and benzalkonium chloride (BAC) on nitrate reduction was investigated in batch assays using a mixed nitrate reducing culture. Nitrate was transformed completely, mainly through denitrification, to dinitrogen at 5, 10, 15 and 22 °C. In the absence of BAC, reduction of individual nitrogen oxides had different susceptibility to temperature and transient nitrite accumulation was observed at low temperatures. When the effect of BAC was tested up to 100 mg/L from 5 to 22 °C, denitrification was inhibited at and above 50 mg BAC/L with transient nitrite accumulation at all temperatures. The effect of BAC was described by a competitive inhibition model. Nitrite reduction was the denitrification step most susceptible to BAC, especially at low temperatures. BAC was not degraded during the batch incubation and was mostly biomass-adsorbed. Overall, this study shows that low temperatures exacerbate the BAC inhibitory effect, which in turn is controlled by adsorption to biomass.     

Efficient cloning of plant genomes into bacterial artificial chromosome (BAC) libraries with larger and more uniform insert size

The construction of bacterial artificial chromosome (BAC) libraries remains relatively complex and laborious, such that any technological improvement is considered to be highly advantageous. In this study, we addressed several aspects that improved the quality and efficiency of cloning of plant genomes into BACs. We set the 'single tube vector' preparation method with no precipitation or gel electrophoresis steps, which resulted in less vector DNA damage and a remarkable two- to threefold higher transformation efficiency compared with other known vector preparation methods. We used a reduced amount of DNA for partial digestion (up to 5 microg), which resulted in less BAC clones with small inserts. We performed electrophoresis in 0.25 x TBE (Tris, boric acid, ethylenediaminetetraacetic acid) buffer instead of 0.5 x TBE, which resulted in larger and more uniformly sized BAC inserts and, surprisingly, a two- to threefold higher transformation efficiency, probably due to less contamination with borate ions. We adopted a triple size selection that resulted in an increased mean insert size of up to 70 kb and a transformation efficiency comparable with that of double size selection. Overall, the improved protocol presented in this study resulted in a five- to sixfold higher cloning efficiency and larger and more uniformly sized BAC inserts. BAC libraries with the desired mean insert size (up to 200 kb) were constructed from several plant species, including hexaploid wheat. The improved protocol will render the construction of BAC libraries more available in plants and will greatly enhance genome analysis, gene mapping and cloning.     

Purification of BAC DNA for high-efficiency transgenesis

An unresolved bottleneck in bacterial artificial chromosome (BAC) transgenesis is low efficiency generation of founder mice because of suboptimal quality of the manipulated BAC DNA. Using mini-gel electrophoresis and electro-elution that circumvents CsCl(2) centrifugation, column chromatography, and resin purifications, we have used RECOCHIP, a commercially available dialysis cassette for the purification of BAC DNA that generates transgenic founders with up to 80% efficiency.     

Construction of a full bacterial artificial chromosome （BAC） library of Oryza sativa genome

We have constructed a full BAC library for the superior early indica variety of Oryza sativa,Guang Lu Ai 4.The MAX Efficiency DH10B with increased stability of inserts was used as BAC host cells.The potent pBelo BACII with double selection markers was used as cloning vector.The cloning efficiency we have reached was as high as 98%,and the transformation efficiency was raised up to 10^6 transformants/μg of large fragment DNA.The BAC recombinant transformants were picked at random and analyzed for the size of inserts,which turned out to be of 120 kb in length on average.We have obtained more than 20,000 such BAC clones.According to conventional probability equation,they covered the entire rice genome of 420,000 kb in length.The entire length of inserts of the library obtained has the 5-to 6-fold coverage of the genome.To our knowledge,this is the first reported full BAC library for a complex genome.     

Construction and characterization of a bacterial artificial chromosome library of thermo-sensitive genie male-sterile rice 5460S

In order to develop a detailed physical map of the thermo-sensitive genie male-sterile (TGMS) gene-encompassing region and finally clone the TGMS gene, a high-quality rice bacterial artificial chromosome (BAC) library from TGMS rice 5460S was constructed. The method of constructing BAC library was examined and optimized. The 5460S library consists of 19 584 BAC clones with an average insert size of 110 kb, which represents about 5 times rice haploid genome equivalents. Rice inserts of up to 140 kb and 250 kb were isolated and appeared stable after 100 generations of serial growth. Hybridization of BAC clones with mitochondrial and chloroplastic genes as probes demonstrated that this library has no organellar contamination. The 5460S library was screened with 3 molecular markers linked to tmsl gene as probes and at least 1 BAC clone was identified with each probe. The insert ends of positive clones were successfully isolated using thermal asymmetric interlaced PCR (TAIL-PCR) technique.     

Construction of a BAC library for Haplochromis chilotes,a cichlid fish from Lake Victoria

Cichlid fishes in Lake Victoria are model organisms for studying rapid radiation and speciation. On the way to examine the molecular basis of how these cichlid fishes achieved such a remarkable morphological diversification, we constructed a bacterial artificial chromosome (BAC) library derived from a cichlid species, Haplochromis chilotes, from Lake Victoria. The library includes 157,056 clones with the average insert size of 128 kb, corresponding to a 10-fold coverage of the H. chilotes genome. Given that the cichlid fishes endemic to Lake Victoria are closely related to one another phylogenetically and their genomes are nearly identical, this BAC library can be utilized to isolate genes from the more than 200 Haplochromine cichlid species in Lake Victoria.     

BAC clones generated from sheared DNA

BAC libraries generated from restriction-digested genomic DNA display representational bias and lack some sequences. To facilitate completion of genome projects, procedures have been developed to create BACs from DNA physically sheared to create fragments extending up to 200 kb. The DNA fragments were repaired to create blunt ends and ligated to a new BAC vector. This approach has been tested by generating BAC libraries from Drosophila DNA with insert lengths between 50 and 150 kb. The libraries lack chimeric clone problems as determined by mapping paired BAC-end sequences to the assembled fly genome sequence. The utility of "sheared" libraries was demonstrated by closure of a previous clone gap and by isolation of clones from telomeric regions, which were notably absent from previous Drosophila BAC libraries.     

A novel method for increasing the transformation efficiency of Escherichia coli-application forbacterial artificial chromosome library construction.

Bacterial artificial chromosome (BAC) libraries play a pivotal role in genomics studies. A crucial step in BAC library construction is the transformation of Escherichia coli by electroporation. Absolute efficiency (cfu/microgram DNA) is affected by a number of factors including the topological form and treatment of DNA samples. Here we report a simple new protocol using tRNA assisted precipitation that increased transformation efficiency by 70-fold for BAC ligations and up to 400-fold for plasmid ligations. The mechanism may involve altering or stabilizing the topographical form of the DNA molecules.     

适用于链霉菌大片段基因组DNA 克隆和异源表达的细菌人工染色体(BAC)载体的构建及应用

【目的】很多链霉菌来源的天然产物的生物合成基因簇往往很大,用传统的cosmid载体很难完整的克隆和异源表达。本研究通过载体改造,成功构建出一个新的细菌人工染色体(BAC)载体,用于链霉菌来源的天然产物生物合成基因簇的克隆及异源表达实验。【方法】从复合型载体pCUGIBAC1出发,通过λRED介导的PCR-targeting方法,用链霉素抗性基因替换掉原有的氯霉素抗性基因标记,同时插入链霉菌中常用的安普拉霉素抗性标记、转移起始位点oriT、φC31整合酶基因int、整合位点attP等元件。【结果】成功构建出可装载链霉菌大片段DNA的BAC载体pMSBBACs。使用pMSBBACs构建出链霉菌U27的基因组BAC文库,平均插入片段大小为100 kb。选取其中一个大小为140 kb的BAC质粒进行功能验证,实验证明通过接合转移和原生质体转化的方法都能够将这个大型BAC质粒导入链霉菌模式菌株,并通过位点特异性重组整合到染色体中进行异源表达。【结论】BAC载体pMSBBACs可成功用于放线菌大片段基因组DNA的克隆和异源表达实验。     

BAC trimming: minimizing clone overlaps

Bacterial vectors containing large inserts of genomic DNA are now the standard substrates for large-scale genomic sequencing. Long overlaps between some clones lead to considerable redundant effort. A method for deleting defined regions from bacterial artificial chromosome (BAC) inserts, using homologous recombination, was applied to minimize the overlap between successive BAC clones. This procedure, called trimming, was carried out in the recA(-) BAC host. We have precisely deleted up to 70 kb of DNA from BACs that were to be sequenced. This method requires minimal prior characterization of the clones: collections of BAC end sequences or STS-based maps will accelerate the process. BAC trimming will be useful in both small and large genome sequencing projects and will be of particular utility for gap closure in finishing phases.     

Construction and characterization of a bacterial artificial chromosome library of Sorghum bicolor.

The construction of representative large insert DNA libraries is critical for the analysis of complex genomes. The predominant vector system for such work is the yeast artificial chromosome (YAC) system. Despite the success of YACs, many problems have been described including: chimerism, tedious steps in library construction and low yields of YAC insert DNA. Recently a new E.coli based system has been developed, the bacterial artificial chromosome (BAC) system, which offers many potential advantages over YACs. We tested the BAC system in plants by constructing an ordered 13,440 clone sorghum BAC library. The library has a combined average insert size, from single and double size selections, of 157 kb. Sorghum inserts of up to 315 kb were isolated and shown to be stable when grown for over 100 generations in liquid media. No chimeric clones were detected as determined by fluorescence in situ hybridization of ten BAC clones to metaphase and interphase S.bicolor nuclei. The library was screened with six sorghum probes and three maize probes and all but one sorghum probe hybridized to at least one BAC clone in the library. To facilitate chromosome walking with the BAC system, methods were developed to isolate the proximal ends of restriction fragments inserted into the BAC vector and used to isolate both the left and right ends of six randomly selected BAC clones. These results demonstrate that the S. bicolor BAC library will be useful for several physical mapping and map-based cloning applications not only in sorghum but other related cereal genomes, such as maize. Furthermore, we conclude that the BAC system is suitable for most large genome applications, is more 'user friendly' than the YAC system, and will likely lead to rapid progress in cloning biologically significant genes from plants.     

利用染色体区段混合BAC探针鉴定食管癌细胞中的染色体畸变

染色体畸变是恶性肿瘤细胞的重要遗传学特征, 文章旨在应用BAC DNA克隆鉴定食管癌细胞中的染色体臂和染色体区段的畸变。针对染色体各区段选取5~10个1 Mb BAC DNA, 分别混合制备成特定染色体区段的BAC DNA混合克隆, 然后将染色体臂上覆盖所有区段的上述混合克隆进一步混合制备成特定染色体臂BAC DNA混合克隆。利用简并寡核苷酸引物聚合酶链反应(Degenerate oligonucleotide primed PCR, DOP-PCR)标记染色体臂探针, 利用切口平移法(Nick translation)标记染色体区段探针, 并对食管癌细胞中期染色体进行荧光原位杂交(Fluorescence in situ hybridization, FISH)分析。正常人外周血淋巴细胞中期染色体FISH结果显示, 上述方法标记的探针具有较高的特异性。进一步利用染色体臂混合探针, 确定了多个食管癌细胞中的染色体重排所涉及的特定染色体臂; 利用染色体区段混合探针, 鉴定出KYSE140的t(1q;7q)衍生染色体中1q上的断点范围位于1q32-q41。文章成功建立了1 Mb BAC DNA混合克隆探针标记技术, 并鉴定出多个食管癌细胞中的染色体臂和染色体区段畸变, 不仅为利用M-FISH技术鉴定肿瘤细胞中的染色体畸变提供了更为准确的方法, 而且还可能进一步将该法推广应用于恶性血液病的核型分析以及产前诊断。     

Targeted analysis of orthologous phytochrome A regions of the sorghum,maize, and rice genomes using comparative gene-island sequencing

A "gene-island" sequencing strategy has been developed that expedites the targeted acquisition of orthologous gene sequences from related species for comparative genome analysis. A 152-kb bacterial artificial chromosome (BAC) clone from sorghum (Sorghum bicolor) encoding phytochrome A (PHYA) was fully sequenced, revealing 16 open reading frames with a gene density similar to many regions of the rice (Oryza sativa) genome. The sequences of genes in the orthologous region of the maize (Zea mays) and rice genomes were obtained using the gene-island sequencing method. BAC clones containing the orthologous maize and rice PHYA genes were identified, sheared, subcloned, and probed with the sorghum PHYA-containing BAC DNA. Sequence analysis revealed that approximately 75% of the cross-hybridizing subclones contained sequences orthologous to those within the sorghum PHYA BAC and less than 25% contained repetitive and/or BAC vector DNA sequences. The complete sequence of four genes, including up to 1 kb of their promoter regions, was identified in the maize PHYA BAC. Nine orthologous gene sequences were identified in the rice PHYA BAC. Sequence comparison of the orthologous sorghum and maize genes aided in the identification of exons and conserved regulatory sequences flanking each open reading frame. Within genomic regions where micro-colinearity of genes is absolutely conserved, gene-island sequencing is a particularly useful tool for comparative analysis of genomes between related species.