Human repeat element-mediated PCR: cloning and mapping of chromosome 10 DNA markers.

Repeat element-mediated PCR can facilitate rapid cloning and mapping of human chromosomal region-specific DNA markers from somatic cell hybrid DNA. PCR primers directed to human repeat elements result in human-specific DNA synthesis; template DNA derived from a somatic cell hybrid containing the human chromosomal region of interest provides region specificity. We have generated a series of repeat element-mediated PCR clones from a reduced complexity somatic cell hybrid containing a portion of human chromosome 10. The cloning source retains the centromere and tightly linked flanking markers, plus additional chromosome 10 sequences. Twelve new inter-Alu, two inter-L1, and four inter-Alu/L1 repeat element-mediated PCR clones were mapped by hybridization to Southern blots of repeat element-mediated PCR products amplified from somatic cell hybrid DNA templates. Two inter-Alu clones mapped to the pericentromeric region. We propose that a scarcity of Alu elements in the pericentromeric region of chromosome 10 contributed to the low number of clones obtained from this region. One inter-Alu clone, pC11/A1S-6-c23, defines the D10S94 locus, which is tightly linked to MEN2A and D10Z1.     

应用ＣＡＴＳ法分离和鉴定猪ＧＦＡＰ基因的研究

根据比较锚定序列宗踪（ＣＡＴＳ）法,选择人和小鼠胶质细胞原纤维酸性蛋白（ＧＦＡＰ）基因的同源区域设计引,用ＰＣＲ方法从二花脸猪基因组中分离到４１２bp的基因片段,经与基因资料库中已训功能基因的同源性比较,该片段可鉴定为猪的ＧＦＡＰ基因,利用猪－啮齿类体细胞杂克隆板将ＧＦＡＰ基因定位于猪１２号染色体１２p11-(2/3)P13区域。     

Assignment of 60 human ESTs in cattle

As part of the human genome study, large-scale cDNA sequencing has produced thousands of Expressed Sequence Tags (ESTs). Généthon has mapped in human 10,000 of these ESTs and has shown that the primers of about 1000 ESTs could amplify bovine DNA. In this work, we have analyzed 233 primer pairs provided by Genethon, to assign type I sequences to the bovine genome by using a hamster-bovine somatic cell hybrid panel. Among these 233 primer pairs, 109 gave a specific PCR product with bovine genomic DNA, but for 50% the size of the PCR product was the same in cattle and hamster, requiring SSCP analysis. Finally, 60 ESTs were assigned to the bovine genome, and among them 46 were found on the bovine chromosome expected from heterologous painting data between cattle and human. Received: 16 December 1999 / Accepted: 6 May 2000     

猪肝细胞和培养上清液中猪内源性逆转录病毒的检测

建立了猪肝细胞及其培养上清液中猪内源性逆转录病毒(PERV)的检测方法,探讨了其在猪肝细胞生物人工肝应用中的意义。以PERV gag基因为靶序列,选用特定的引物,PCR检测中国实验用小型猪肝细胞PERV前病毒DNA;RT-PCR检测猪、犬、大鼠以及HBV阳性病人血清和猪肝细胞培养6h、24h时的上清液PERV RNA,同时检测猪肝细胞猪线粒体DNA(mtDNA)。研究结果表明：检测5份中国实验用小型猪血清、肝细胞及培养猪肝细胞24h时的上清液PERV均为阳性,而5份培养猪肝细胞6h时的上清液、5份犬血清、5份大鼠血清和5份HBV阳性病人血清PERV检测结果均为阴性,猪肝细胞中均可检测到猪mtDNA。因此,中国实验用小型猪肝细胞携带PERV;PERV可释放到血清中;猪肝细胞培养24h后该病毒颗粒已释放到培养液中;PCR和RT-PCR方法检测PERV具有特异性强、简便的特点。     

Mapping and expression studies of the mir17-92 cluster on pig Chromosome 11

We have identified the first porcine microRNA (miRNA) cluster (the mir17-92 cluster) and localized it to the q-arm of pig Chromosome 11. The miRNA cluster was found by sequence similarity search with human miRNA sequences against the pig genomic data generated within the Sino-Danish pig genome project. The resulting data contained three complete and two incomplete miRNA precursors of seven miRNAs from the human mir17-92 cluster. Because there is a 100% sequence identity between the four pig miRNAs and the corresponding human miRNAs, the sequences of three unavailable pig miRNAs were derived from the human data. The expression profiles of seven studied miRNAs were analyzed by hybridization to Northern blots containing five porcine tissues: cerebellum, cortex, hippocampus, kidney, and liver. In order to determine the localization of the mir17-92 cluster in the pig genome, we mapped it by PCR in the porcine somatic cell hybrid (SCH) panel and in the INRA-University of Minnesota (INRA-UMN) porcine radiation hybrid (IMpRH) panel. The PCR results enabled us to localize this cluster to the q-arm of pig Chromosome 11 and map it in relation to two microsatellites. Our study presents the first expression analyses of miRNAs in pig and adds information for further functional studies in this species.     

Mapping of EST- and STS-markers in the human genome using a panel fo radiation hybrids

Ten DNA markers were localized in the human genome by a screening procedure against the radiation hybrid somatic cell panel (GeneBridge 4 RH Panel) using polymerase chain reaction (RH mapping method). DNA markers were developed to nucleotide sequences adjacent to NotI sites of human chromosome 3 (NotI-STS markers) and also to nucleotide sequences of human cDNA (EST markers). Three EST markers mapped (B10164, S16R and 18F5R) were localized in the human genome for the first time. Marker B10164 was found to be homologous to the nucleotide sequence of the BASP1 gene coding a major receptor protein. Markers S16R and 18F5R presumably tagged new genes, because no homologies were revealed among the nucleotide sequences presented in the databases. For four NotI-STS, more precise localization on human chromosome 3 was determined. On the basis of the data obtained, the NotI map may be integrated with other types of physical maps of human chromosome 3. RH mapping with a standard commercial panel of radiation hybrid somatic cells provided a chance to integrate the data obtained into international databases and existing integrated human chromosomal maps.     

Establishment of a Partially Informative Porcine Somatic Cell Hybrid Panel and Assignment of the Loci for Transition Protein 2 (TNP2) and Protamine 1 (PRM1) to Chromosome 3 and Polyubiquitin (UBC) to Chromosome 14

Porcine lymphocytes and fibroblasts were fused with 3 different permanent rodent cell lines, and 21 stable somatic cell hybrid lines were established. These hybrid cell lines were characterized cytogenetically by sequential QFQ banding and chromosome painting using fluorescence in situ hybridization with porcine DNA. The lines were further characterized by PCR analysis with primer pairs derived from genes with confirmed mapping information. Using this panel, we assigned the locus encoding polyubiquitin (UBC) to chromosome 14, and the transition protein 2 locus (TNP2) and protamine loci (PRM1 and PRM2) to chromosome 3. Two chromosomal localizations have been further refined by radioactive in situ hybridization. UBC maps to chromosome 14q12-q15 and TNP2 to 3p11-p12.     

Chromosomal mapping and zygosity check of transgenes based on flanking genome sequences determined by genomic walking.

Transgenes can affect transgenic mice via transgene expression or via the so-called positional effect. DNA sequences can be localized in chromosomes using recently established mouse genomic databases. In this study, we describe a chromosomal mapping method that uses the genomic walking technique to analyze genomic sequences that flank transgenes, in combination with mouse genome database searches. Genomic DNA was collected from two transgenic mouse lines harboring pCAGGS-based transgenes, and adaptor-ligated, enzyme restricted genomic libraries for each mouse line were constructed. Flanking sequences were determined by sequencing amplicons obtained by PCR amplification of genomic libraries with transgene-specific and adaptor primers. The insertion positions of the transgenes were located by BLAST searches of the Ensembl genome database using the flanking sequences of the transgenes, and the transgenes of the two transgenic mouse lines were mapped onto chromosomes 11 and 3. In addition, flanking sequence information was used to construct flanking primers for a zygosity check. The zygosity (homozygous transgenic, hemizygous transgenic and non-transgenic) of animals could be identified by differential band formation in PCR analyses with the flanking primers. These methods should prove useful for genetic quality control of transgenic animals, even though the mode of transgene integration and the specificity of flanking sequences needs to be taken into account.     

Comparative mapping between humans and pigs: localization of 58 anchorage markers (TOASTs) by use of porcine somatic cell and radiation hybrid panels

To increase the number of Type I markers that are directly informative for comparative mapping, 58 anchorage markers, TOASTs (Traced Orthologous Amplified Sequence Tags), were mapped in pig. With specific consensus primers, 76 TOASTs were tested in pig: 50 were regionally localized in pig on a somatic cell hybrid panel (SCHP), and 51 were mapped on the whole genome, INRA/University of Minnesota porcine Radiation Hybrid panel (IMpRH). Comparison of marker positions on RH and cytogenetic maps indicated general concordance except for two chromosomal regions. For RH mapping, all markers, apart from one, were significantly linked (LOD > 4.8) to a marker of the first-generation radiation hybrid map. Localization of new markers on the initial map is necessary for drawing a framework map as shown for Chromosome Sscr 14. The addition of four TOASTs has enabled us to propose an improved map, using a threshold likelihood ratio of 1000/1. At the whole-genome level, this work significantly increased (by 50%) the number of precisely mapped genes on the porcine RH map and confirmed that the IMpRH panel is a valuable tool for high-resolution gene mapping in pig. Porcine PCR products were sequenced and compared with human sequences to verify their identity. Most of the localizations made it possible to either confirm or refine the previous comparative data between humans and pigs obtained through heterologous chromosomal painting or gene mapping. Moreover, the use of TOASTs in mapping studies appears to be a complement to other strategies using CATS, human ESTs, or heterologous FISH with BACs which had already been applied to improve the gene density of comparative genomic maps for mammals. Received: 15 March 2000 / Accepted: 27 July 2000     

应用RNA干扰沉默猪内源性反转录病毒

目的:尝试应用RNA干扰(RNAi)沉默猪源PK-15细胞中的猪内源性反转录病毒(PERV),并通过反转录酶活性及pol基因相对荧光定量PCR检测沉默效果。方法:依据GenBank公布的PERV pol基因序列,采用Invitro-gen公司的BLOCK-iT RNAi Designer软件设计Stealth小干扰RNA(siRNA)序列;将合成的siRNA转染PK-15细胞,72 h后检测细胞上清PERV反转录酶活性及细胞内pol基因拷贝数并评价沉默效果。结果:反转录酶活性及pol基因拷贝数检测结果表明,设计的3条Stealth siRNA序列中,位于pol基因3272～3296 bp的序列能有效沉默PERV。结论:RNAi方法可有效使猪源PK-15细胞中的PERV沉默,为进一步研究天然抗病毒分子与PERV的相互作用提供了实验基础,同时也为猪源异种移植研究中去除PERV提供了一种可供尝试的方法。     

"PCR-karyotype" of human chromosomes in somatic cell hybrids

Amplification of human DNA sequences in 16 monochromosomal somatic cell hybrids containing different human chromosomes were performed by the polymerase chain reaction (PCR) using primer directed at human-specific regions of Alu or L1, the two major classes of interspersed repetitive sequences (IRS-PCR). A chromosome-specific pattern of amplification products was observed on agarose gels run with ethidium bromide, producing a "PCR-karyotype." This simple gel analysis provides a rapid method for identifying and monitoring the human chromosomal content of monochromosomal somatic cell hybrids without conventional cytogenetic analysis. Hybrids containing multiple human chromosome produce complex gel patterns, but identification of chromosome content can be achieved by hybridization of PCR products against a reference panel of monochromosomal or highly reduced hybrids representing each human chromosome. This dot-blot method also enables identification of human marker chromosomes or translocated pieces in hybrids that are not identifiable by cytogenetic methods. These IRS-PCR methods should greatly reduce the need for more laborious cytogenetic, isozyme, and Southern blot characterizations of human-rodent cell hybrids.     

Isolation and analysis of retroviral integration targets by solo long terminal repeat inverse PCR

Upon retroviral infection, the genomic RNA is reverse transcribed to make proviral DNA, which is then integrated into the host chromosome. Although the viral elements required for successful integration have been extensively characterized, little is known about the host DNA structure constituting preferred targets for proviral integration. In order to elucidate the mechanism for the target selection, comparison of host DNA sequences at proviral integration sites may be useful. To achieve simultaneous analysis of the upstream and downstream host DNA sequences flanking each proviral integration site, a Moloney murine leukemia virus-based retroviral vector was designed so that its integrated provirus could be removed by Cre-loxP homologous recombination, leaving a solo long terminal repeat (LTR). Taking advantage of the solo LTR, inverse PCR was carried out to amplify both the upstream and downstream cellular flanking DNA. The method called solo LTR inverse PCR, or SLIP, proved useful for simultaneously cloning the upstream and downstream flanking sequences of individual proviral integration sites from the polyclonal population of cells harboring provirus at different chromosomal sites. By the SLIP method, nucleotide sequences corresponding to 38 independent proviral integration targets were determined and, interestingly, atypical virus-host DNA junction structures were found in more than 20% of the cases. Characterization of retroviral integration sites using the SLIP method may provide useful insights into the mechanism for proviral integration and its target selection.     

Discrimination between alpha-satellite DNA sequences from chromosomes 21 and 13 by using polymerase chain reaction.

alpha-Satellite subfamilies from chromosomes 21 and 13 are almost identical in sequence and cannot be distinguished from each other by hybridization techniques. A general method based on membrane-bound PCR is described here, allowing the discrimination of alpha-satellite DNA sequences from each of these two chromosomes, after detection by Southern blot hybridization. The PCR conditions were developed using somatic hybrid DNAs. The method was tested in membrane-bound PCR by using the alpha-satellite bands from a Southern blot of a CEPH family. The chromosomal origin of these bands, previously determined by linkage analysis, was confirmed by this method.     

Comparative and physical mapping of 111 previously reported and 105 new porcine microsatellites

Here we report radiation hybrid mapping of 105 new porcine microsatellite markers on the IMpRH₇₀₀₀ radiation hybrid panel. In addition, we searched flanking sequences of these markers, as well as 673 previously reported RH-mapped microsatellite markers, for orthology to human sequences. Eighty-seven new and 111 previously mapped sequences exhibited orthology to human sequences. Using a stringent sequence alignment, 25 microsatellite-flanking sequences were found to be highly similar to genic sequences, whereas 173 were similar to non-genic sequences in the human genome. Five markers were located near known breakpoints of synteny between human and swine.     

Toward a long-range map of human chromosomal band 22q11

Human chromosome band 22q11 is involved in numerous chromosomal rearrangements. A long-range molecular map of this region would allow the more precise localization of the various breakpoints of these rearrangements. Toward this goal we have constructed a genomic DNA library that allows the isolation of DNA clones that are directly adjacent to NotI sites. NotI was chosen because it is a restriction enzyme that digests infrequently in the human genome. The genomic DNA used in this library was from a human/hamster hybrid cell line that has a chromosome 22 as the only visible human chromosome. Two clones were isolated and mapped to different regions of 22q11 using a somatic cell hybrid mapping panel. A long-range restriction map flanking the NotI site of each of these two clones was produced using NotI and other infrequently cutting enzymes. Both NotI sites analyzed were located in HTF islands, regions often associated with the 5' end of genes. Thus, the NotI map of 22q11 may also aid in the cloning of undiscovered genes, giving a starting point for the study of duplication/deficiency syndromes of the region.     

Construction of a swine BAC library: application to the characterization and mapping of porcine type C endoviral elements.

A porcine bacterial artificial chromosome (BAC) library was constructed using the pBeloBAC11 vector. It comprised 107,520 clones with an average insert size of 135 kb, representing an almost fivefold coverage of the swine haploid genome. Screening of the library allowed recovery of one to eight clones for 142 unique markers located all over the genome, while it failed for only one marker. About 4% chimeric clones were found. The library was also screened for the protease gene of type C porcine endoviral sequences (PERVs), and 62 clones were recovered, all but two of which contained one protease gene. We found 20 protease sequences (PERV-1 to PERV-20) which, despite differing by point mutations, were all coding sequences. The most frequent sequence, PERV-2, was 100% similar to a protease sequence expressed in the porcine PK-15 cell line. Most of the clones harbored envelope genes. Thirty-three BAC clones were mapped by fluorescence in situ hybridization to 22 distinct locations on 14 chromosomes, including the X and Y chromosomes. These overall results indicate that there is generally one PERV copy per integration site. Although PERV sequences were not tandemly arranged, clusters of integration sites were observed at positions 3p1.5 and 7p1.1. Southern blot experiments revealed 20-30 PERV copies in the Large White pig genome studied here, and variations in PERV content among pigs of different breeds were observed. In conclusion, this BAC collection represents a significant contribution to the swine large genomic DNA cloned insert resources and provides the first detailed map of PERV sequences in the swine genome. This work is the first step toward identification of potential active sites of PERV elements.