Cosmid mapping of the human chorionic gonadotropin beta subunit genes by field-inversion gel electrophoresis.

A cosmid clone containing the entire hCG beta gene cluster has been isolated. The restriction map of this clone has been determined by an indirect-end-label FIGE (field inversion gel electrophoresis) method. Analysis of this cosmid clone shows that there are 6 hCG beta genes in human genomic DNA. A previously uncloned portion of the hCG beta cluster, termed the "gap" region, has been shown not to contain any sequences homologous to the hCG beta cDNA. The restriction mapping method employed in this study takes advantage of the superior resolution of FIGE for high molecular weight DNA fragments in the size range 15-50 kb. This method is broadly applicable and permits rapid and accurate restriction mapping for extended regions of genomic DNA that have been cloned into cosmid or lambda vectors.     

Genomic clone of hst with transforming activity from a patient with acute leukemia

We have previously reported the identification of a novel transforming gene, hst, in DNA samples taken from human stomach cancers and a noncancerous portion of stomach. Five clones, containing the genomic hst gene, were isolated from a human cosmid library constructed from leukocyte DNA from a patient with acute leukemia. All clones possessed transforming activity when transfected to NIH3T3 cells. From one clone, an 8.7 kb BamHI fragment was subcloned into pBR322, and this subclone was active in transforming NIH3T3 cells. This is the first isolation of transformation-competent genomic hst clones directly from a human genomic library, that is, without prior passage through NIH3T3 cells.     

Pseudomonas solanacearum genes controlling both pathogenicity on tomato and hypersensitivity on tobacco are clustered.

A pLAFR3 cosmid clone designated pVir2 containing a 25-kilobase (kb) DNA insert was isolated from a wild-type Pseudomonas solanacearum GMI1000 genomic library. This cosmid was shown to complement all but one of the nine Tn5-induced mutants which have been isolated after random mutagenesis and which have lost both pathogenicity toward tomato and ability to induce hypersensitive reaction (HR) on tobacco (hrp mutants). The insert is colinear with the genome and provides restoration of the HR-inducing ability when transferred into several Tn5-induced hrp mutants, but failed to complement deletion mutants extending on both sides of the pVir2 region. Localized mutagenesis demonstrated that the hrp genes are clustered within a 17.5-kb region of pVir2 and that this cluster probably extends on the genomic region adjacent to the pVir2 insert. A 3-kb region adjacent to the hrp cluster modulates aggressiveness toward tomato but does not control HR-inducing ability. Sequences within the hrp cluster of pVir2 have homology with the genomic DNA of Xanthomonas campestris strains representing eight different pathovars, suggesting that a set of common pathogenicity functions could be shared by P. solanacearum and X. campestris.     

Isolation and characterization of beta- and gamma-crystallin genes from rat genomic cosmid libraries

Two libraries, together containing about 10(6) colonies, have been constructed by cloning different size fractions of a partial Sau3A digest of rat genomic DNA in the cosmid vector pTM. Upon screening with two cDNA clones, one containing alpha A2-crystallin and one containing beta B1-crystallin sequences, 14 cosmid clones were isolated which were beta B1-crystallin-specific; none was found which contained alpha A2-crystallin sequences. The inserts of the beta B1 clones, which range from 35 to 45 kb in length, contain overlapping DNA segments covering more than 60 kb of rat genomic DNA. The composite BamHI restriction map of this region shows a single beta B1-crystallin gene, which is interrupted by several intronic sequences. Five recombinants hybridizing with two different rat lens gamma-crystallin cDNA clones were also isolated from these libraries. Four of these contain 31- to 41-kb inserts, whereas the fifth recombinant contains a 12.2-kb insert. Hybridization analysis with 5' and 3'-specific cDNA fragments indicates that altogether these inserts contain six gamma-crystallin genes, three of which are located on one insert of only 31 kb.     

一种用于构建红色红曲霉基因组Cosmid文库的大片段DNA制备方法

摘要：【目的】制备用于构建红色红曲霉cosmid文库的大片段基因组DNA。【方法】采用优化的酚氯仿抽提法制备DNA,并利用Sau3AI切割至平均大小为40 kb,然后使用Stratagene包装蛋白构建cosmid文库。基于PCR法使用同源探针从该文库中进行了目的基因的筛选。【结果】制备了浓度为5 μg/μL,平均片段大小大于48 kb的红色红曲霉大片段基因组DNA。利用该DNA构建的cosmid文库基因组覆盖倍数为10,并筛选到了含有目的片段的cosmid。【结论】通过该方法制备红色红曲霉大片段基因组D     

Conservation of the RD-BF-C2 organization in the pig MHC class-III region: mapping and cloning of the pig RD gene

The RD gene, named after the arginine (R) and aspartic acid (D) repeat in the central part of its protein, was initially mapped in the mouse H-2S subregion between C4 and BF. It was later mapped in the same position in the human MHC and here we show it is also conserved in the pig MHC class III region, close to the complement BF gene. A pig RD genomic clone was isolated from a γ-phage library. Hybridizations on genomic DNA separated with pulsed field gel electrophoresis identified common 220kb Nrul, 130 kb EagI and 200 kb Mlul bands for RD, BF and C2. The RD gene has also a 17 kb Kpnl and 11 kb Sad fragment in common with BFbut not with C2. The close linkage of the RD and BF genes was further established by hybridization of BF to a genomic γ-phage clone also containing the RD gene. This genomic RD clone overlaps with a γ -phage clone previously isolated and containing the complete BF gene and the 3' part of C2. The distance between RD and BF is about 6 kb. The junction between the two complement genes BF and C2 was sequenced and the BF 5' promoter region, overlapping the 3' noncoding region of C2, was compared with that of the human BF promoter. The overall homology was about 80% and all but one identified promoter elements were found in the same position in both genes. The results obtained demonstrate the RD-BF-C2 organization is strongly conserved between human, mouse and pig. No polymorphisms were detected in either the RD gene or in the BF promoter region using polymerase chain reaction and restriction fragment polymorphism analysis.     

The generation of ordered sets of cosmid DNA clones from human chromosome region 11p.

We describe progress in a continuing project aimed at the generation of an overlapping cosmid DNA clone map of the short arm of human chromosome 11. The automated procedures used to prepare DNA samples and the computerized data collection and recording systems are described. We also demonstrate the use of the clones as reagents for the rapid isolation of genomic DNAs containing smaller probed regions. We have isolated approximately 4700 human cosmid DNA clones from mouse/human hybrid cell lines that contain predominantly human chromosomal region 11p. Of the DNA in the cell lines, 60% is derived from this chromosomal region, and the remaining 40% is derived from regions of chromosomes 3, 19, and 20. A total of 4159 clones have been fingerprinted to identify potential overlaps, and we have developed 535 sets ("contigs"). Using random modeling, it is estimated that 65% of 11p must be contained in the analyzed cosmids. The database of clones has been used to identify single or overlapping clones from noncosmid DNA probes. Examples are presented. It is proposed that cosmid reference filters be distributed to requesting laboratories.     

Dispersed localization of D segments in the human immunoglobulin heavy-chain locus.

We have studied the organization of the human immunoglobulin heavy-chain genes by pulse field gel electrophoresis as well as by isolation of cosmid clones. The total length of the heavy-chain variable region locus was estimated to be approximately 3000 kb. We found that D segments including a recently isolated D5 segment were dispersed among VH segments. We identified a pseudo V segment 18 kb 3' to the D5 segment in isolated cosmid clones. A 300 kb fragment produced by MluI digestion contained VH, D, JH segments and the distance between VH and D was estimated to be approximately 240 kb. Overlapping cosmid clones containing the human D1, D2, D3, D4, JH, Cmu and C delta genes were isolated. Restriction maps of these regions indicated that the distance between D and JH is about 22 kb. A partial restriction map of the VH locus was constructed using the pulse field gel electrophoresis technique and deletion of VH segments in B cells.     

人组织型纤溶酶原激活剂(t-PA)mRNA5′端非翻译区(UTR)对其表达的调控

运用反转录-PCR技术,从黑色素瘤细胞中扩增出t—PA cDNA 5′末端460bp的片段,再经重组获得含完整5′-UTR的t—PA cDNA克隆,在兔网织红细胞裂解物中翻译和COS-7细胞中表达发现,t—PA mRNA 5′—UTR对其表达有明显的抑制作用。将t—PA mRNA 5′—UTR用苜蓿病毒RNA 5′—UTR替换,使t—PA的表达水平提高3-7倍,mRNA翻译起始区二级结构分析结果表明,翻译起始区的二级结构与t-PA的表达水平有关。     

A sequence-ready map of the human chromosome 17p telomere.

A half-YAC clone derived from human chromosome 17p was mapped at high resolution using cosmid subclone fingerprint analysis. Colinearity of the half-YAC with the telomeric human genomic DNA fragment was ascertained by RecA-assisted restriction endonuclease cleavage mapping. Previously isolated and radiation hybrid-mapped markers TEL17P37, TEL17P49, and TEL17P80 mapped 30-60 kb from the 17p terminus. This sequence-ready map permits high-resolution integration of genetic maps with the DNA sequences directly adjacent to the tip of human chromosome 17p, and will provide the cloned DNA required for ascertaining the nucleotide sequence of this subtelomeric region.     

Organization of a Chinese hamster ovary dihydrofolate reductase gene identified by phenotypic rescue.

We have constructed a genomic DNA library from a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) in the cosmid vector pHC79. By utilizing a murine dihydrofolate reductase (DHFR) cDNA clone, we have identified 66 DHFR+ clones among the 11,000 colonies screened by colony hybridization. To isolate a recombinant cosmid containing the entire DHFR gene, we have tested these colonies for their ability to rescue a DHFR- Chinese hamster ovary cell line, using the spheroplast fusion method of gene transfer developed by W. Schaffner (Proc. Natl. Acad. Sci. U.S.A. 77:2163-2167, 1980). One clone (cH1) was able to transform DHFR- cells to the DHFR+ phenotype and was shown in hybridization studies to contain all of the gene except a small portion of the 3' untranslated region. We have mapped cosmid cH1 and several overlapping cosmids with a variety of restriction enzymes and have determined the approximate positions of the five (and possibly six) exons within the DHFR gene. Differences between the sizes of homologous genes in hamster cells (24.5 kilobases [kb]) and in mouse cells (31.5 kb) are shown to reside primarily in the length of the 3' intron, which is 8 kb in the hamster gene and 16 kb in length in the mouse gene. Our studies confirm the utility of cosmid libraries for the isolation of large genes, as previously shown by R. de Saint Vincent et al. (Cell 27:267-277, 1981). In addition, a cosmid that contains a functional DHFR gene will be a useful vector for the co-amplification and subsequent overexpression of other cloned genes.     

连续3步基因抓捕构建牛αS1酪蛋白-人tPA杂合基因座

目的:构建一个利用牛αS1酪蛋白基因座完整的上下游调控序列指导人组织型纤溶酶原激活剂(tPA)基因组序列在乳腺特异性高效表达的牛αS1酪蛋白-人tPA杂合基因座。方法:采用连续3步基因抓捕的方法。首先,以pBR322载体作为骨架,插入预先无痕连接在一起的6个同源臂,构成能连续进行3次基因抓捕的抓捕载体;然后,在大肠杆菌内利用Red同源重组系统介导的缺口修复技术,第一步从含牛αS1酪蛋白基因座的细菌人工染色体(BAC)上亚克隆9 kb的牛αS1酪蛋白基因3'端侧翼序列到抓捕载体上,第二步从人tPA BAC上亚克隆17 kb的从起始密码子(ATG)到终止密码子(TGA)的人tPA基因组序列,第三步从牛αS1酪蛋白BAC上亚克隆20 kb的牛αS1酪蛋白基因5'端完整侧翼序列,并使这3个基因片段在抓捕载体上自动无痕地连接在一起,形成一个长约46kb的牛αS1酪蛋白-人tPA杂合基因座。结果:经过PCR扩增、限制性内切酶消化和序列测定验证,构建的杂合基因座中,原牛αS1酪蛋白基因组编码序列从起始密码子到终止密码子被人tPA基因组序列精确置换。结论:连续三步基因抓捕构建杂合基因座乳腺表达载体的技术,为乳腺生物反应器高效表达大载体的制备提供了探索性研究。     

Isolation and characterization of a human collagen alpha 1(I)-like gene from a cosmid library

We have isolated a human collagen alpha 1(I)-like gene from a cosmid library. The clone which contains 37kb of human DNA has been shown to contain this gene by DNA sequencing, hybrid arrest and hybrid selection assays and Northern blot hybridizations. The collagen gene sequence extends through most of the cloned DNA and must, therefore, be at least 35kb in length.