Distribution of endogenous murine leukemia virus DNA sequences among mouse chromosomes.

We used mouse-Chinese hamster somatic cell hybrids which lose mouse chromosomes to examine the distribution of murine leukemia virus DNA sequences in the genome of A/HeJ mice. We analyzed total cellular DNA from various hybrid clones for the presence of viral sequences by molecular hybridization and used the Southern blot hybridization procedure to identify viral DNA in cellular restriction endonuclease fragments. Our results show that murine leukemia virus DNA sequences are distributed among many mouse chromosomes in this strain. Chromosome 4 was shown to contain murine leukemia virus DNA sequences.     

Eucaryotic chromosome transfer: production of a murine-specific cosmid library from a neor-linked fragment of murine chromosome 17.

We recently developed a procedure for the molecular analysis of specific mammalian chromosomal fragments. This procedure allows for the transfer of contiguous chromosomal fragments, varying in size from a fraction to several centimorgans in length, from the donor cell of one species into a recipient cell of a different species. Specifically, we inserted a neor gene, encoded by a recombinant retrovirus, into the murine major histocompatibility complex (MHC). Metaphase chromosome transfers with this neor-tagged chromosome into recipient hamster, primate, and canine fibroblasts produced a panel of primary neor transferents, each containing a portion of, or all of, the murine MHC. A cosmid library was made from one such transferent, CHMD(D)B1. Cosmid clones were divided, using species-specific repeat probes, into those containing murine (donor) DNA sequences and those containing sequences derived from the recipient cell. The murine-specific cosmids were clustered into overlapping DNA segments by restriction enzyme digest analysis of the cosmid DNAs coupled with Southern blot analysis with, as probes, murine-specific repeat sequences and nick-translated murine genomic DNA. These cosmid clusters were analyzed for their position within or outside of the MHC, using recombinant mouse strains, and for the presence within them of known murine MHC genes.     

Structure of products of the Moloney murine leukemia virus endogenous DNA polymerase reaction.

We have investigated the process by which the single-stranded RNA genome of Moloney murine leukemia virus is copied into DNA in vitro. DNA synthesis if initiated near the 5' end of the genome, and the elongation of the growing chain occurs by a jumping mechanism whereby the DNA synthesized at the 5' end of the genome is elongated along the 3' end. Unique DNA fragments synthesized beyond the 5' end of the genome in vitro have, at their 5' and 3' ends, copies of unique sequences from the 5' and 3' ends of the genome. These flank a copy of the 49- to 60-nucleotide terminally redundant sequence. These results indicate that the terminal redundancy serves as a "bridge" to allow a DNA molecule synthesized at the 5' end of the genome to serve as a primer for synthesis from the 3' end.     

Cloning of the Escherichia coli gene for the stringent starvation protein

Summary In order to clone the Escherichia coli gene for the stringent starvation protein (SSP), we determined its N-terminal sequence as well as the sequence of two peptide fragments obtained by cyanogen bromide cleavage of the protein. We then chemically synthesized four sets of oligodeoxyribonucleotide mixtures that represented possible codon combinations for parts of these amino acid sequences. The synthetic oligonucleotides were labelled with ³²P at their 5-termini and used as hybridization probes to detect DNA fragments containing the complementary sequences. Genomic Southern hybridization of E. coli chromosomal DNA gave up to ten DNA fragments hybridizing with each probe but only a few hybridized with two or more of the probes. The latter fragments were coloned in pBR322. By determining partial base sequences with a rapid method and examining proteins encoded by the DNA fragments, we were able to show that we had isolated a clone containing the complete SSP structural gene.Abbreviations SSP stringent starvation protein - PTH phenylthiohydantoin     

Evidence for the Asiatic origin of endogenous AKR-type murine leukemia proviruses.

A restriction endonuclease cleavage map of the genome of AKV, the endogenous, ecotropic leukemia virus of AKR mice, has been derived. By using this map and analyzing DNA from congenic mice, we have defined four DNA fragments diagnostic for AKV proviruses. Analysis of DNAs from 10 strains of American laboratory mice revealed that all strains carrying inducible, ecotropic murine leukemia viruses yielded DNAs which contained the four DNA fragments diagnostic for AKV. Virus-negative strains lacked these fragments in their DNA. Screening DNA from 23 additional mice revealed that, among these mice, only mice from Asia gave rise to the DNA fragments diagnostic of an AKV provirus. We conclude that all of the endogenous ecotropic murine leukemia proviruses in American laboratory mice are closely related since they share a common set of restriction endonuclease cleavage sites. These proviruses appear to derive from the East Asian ancestors of these mouse strains. Analysis of DNA from six selected mice with an additional restriction endonuclease showed that greater than 97% of the nucleotide sequences in each provirus are contigous and that these endogenous proviruses are indistinguishable from proviruses introduced by exogenous infection.     

Molecular cloning and physical mapping of the tupaia herpesvirus genome.

Purified virion DNA of about 200 kilobase pairs of tupaia herpesvirus strain 2 was cleaved with EcoRI or HindIII restriction endonuclease. Restriction fragments representing the complete viral genome including both termini were inserted into the EcoRI, HindIII, and EcoRI-HindIII sites of the bacterial plasmid pAT153. Restriction maps for the restriction endonucleases EcoRI and HindIII were constructed with data derived from Southern blot hybridizations of individual viral DNA fragments or cloned DNA fragments which were hybridized to either viral genome fragments or recombinant plasmids. The analysis revealed that the tupaia herpesvirus genome consists of a long unique sequence of 200 kilobase pairs and that inverted repeat DNA sequences of greater than 40 base pairs do not occur, in agreement with previous electron microscopic data. No DNA sequence homology was detectable between the tupaia herpesvirus DNA and the genome of murine cytomegalovirus, which was reported to have a similar structure. In addition, seven individual isolates of tupaia herpesvirus were characterized. The isolates can be grouped into five strains by their DNA cleavage patterns.     

Isolation and characterization of a 15-kilobase genomic sequence coding for part of the Pro alpha 2 chain of sheep type I collagen

DNA fragments, prepared by partial Eco RI digestion of fetal sheep liver genomic DNA, were used to prepare a "library" of amplified genomic sequences with the lambda vector Charon 4A. Several recombinant plaques were identified by their ability to hybridize to 32P-labeled cDNA prepared from fetal sheep tendon type I procollagen mRNA. Two of these recombinant DNA bacteriophages (SpC3 and SpC7) were identified as containing procollagen pro alpha 2 gene sequences by their ability to specifically anneal to procollagen pro alpha 2 mRNA. Restriction endonuclease and hybridization to a cloned pro alpha 2 cDNA demonstrated that approximately half (2.5 kilobases) of the pro alpha 2 mRNA sequence is distributed over 15 kilobases of genomic DNA. Restriction maps of SpC3 and SpC7 demonstrated that these two DNA fragments contain overlapping sequences of the pro alpha 2 gene. Electron microscopy and R-loop analysis of SpC3 revealed that at least 12 to 16 intervening sequences are distributed throughout the length of this gene fragment.     

Kindred DNA amplification from two distinct populations of cDNA fragments

Kindred DNA amplification is a novel and cost-effective method developed to isolate common cDNA fragments between two distinct cDNA populations. Unlike subtractive hybridization, which discards common sequences, kindred DNA amplification isolates and amplifies these sequences within a single hybridization procedure. The utility of this method is demonstrated by cloning the genes in common between two different but metabolically homologous muscles, murine ventricular myocardium and soleus. The reliability of kindred DNA amplification was confirmed by Southern hybridization.     

PCR-based gene targeting of the inducible nitric oxide synthase (NOS2) locus in murine ES cells,a new and more cost-effective approach

Gene targeting by double homologous recombination in murine embryonic stem (ES) cells is a powerful tool used to study the cellular consequences of specific genetic mutations. A typical targeting construct consists of a neomycin phosphotransferase (neo) gene flanked by genomic DNA fragments that are homologous to sequences in the target chromosomal locus. Homologous DNA fragments are typically cloned from a murine genomic DNA library. Here we describe an alternative approach whereby the inducible nitric oxide synthase (NOS2) gene locus is partially mapped and homologous DNA sequences obtained using a long-range PCR method. A 7 kb NOS2 amplicon is used to construct a targeting vector where theneo gene is flanked by PCR-derived homologous DNA sequences. The vector also includes a thymidine kinase (tk) negative-selectable marker gene. Following transfection into ES cells, the PCR-based targeting vector undergoes efficient homologous recombination into the NOS2 locus. Thus, PCR-based gene targeting can be a valuable alternative to the conventional cloning approach. It expedites the acquisition of homologous genomic DNA sequences and simplifies the construction of targeting plasmids by making use of defined cloning sites. This approach should result in substantial time and cost savings for appropriate homologous recombination projects.     

Integrated Moloney murine leukemia virus DNA studied by using complementary DNA which does not recognize endogenous related sequences

By preannealing a radioactive, representative Moloney murine leukemia virus (M-MuLV) cDNA with large excesses of AKR 70S viral RNA, an M-MuLV-specific cDNA has been prepared. When hybridized to restriction enzyme fragments of M-MuLV-infected mouse cell DNA, the preannealed probe recognizes integrated M-MuLV DNA and does not recognize endogenous related DNA sequences found in uninfected mouse cells. The viral DNA sequences recognized by the preannealed probe are spread throughout the viral genome, although some sequences are recognized less efficiently. By using this preannealed probe, multiple integrations of M-MuLV DNA have been detected in infected fibroblasts and in an M-MuLV-induced tumor. Integrated viral DNA fragments smaller than the complete viral genome have also been detected. By using this preannealed probe to examine a mass-infected culture of mouse fibroblasts, no evidence for a strongly preferred site for M-MuLV integration could be found.     

Rapid identification of hybridization probes for chromosomal walking

The presence of repeated elements in restriction fragments used as hybridization probes for chromosomal walking poses a major obstacle to the success of this gene-cloning strategy. This report describes a simple and rapid means of identifying restriction fragments devoid of repeated sequences and therefore useful as hybridization probes for chromosomal walking. Restriction fragments derived from a genomic DNA clone are Southern blotted and hybridized to nick-translated total genomic [32P]DNA. Fragments of the genomic clone that contain high abundance sequences (i.e., repeated elements) hybridize strongly to their nick-translated counterparts, which, due to their high copy number, comprise a significant proportion of the total genomic DNA probe. Conversely, fragments containing single-copy or low-abundance sequences do not hybridize, as their nick-translated counterparts are poorly represented in the total genomic DNA probe. These latter fragments, by virtue of their low-abundance sequences, are well suited as probes for chromosomal walking. Ensuring the absence of repeated elements in restriction fragments prior to their purification and utilization as chromosomal walking probes results in marked savings of time, effort and materials.     

Methods for rapid cloning and detection for sequencing of cloned inverse PCR-generated DNA fragments adjacent to known sequences in bacterial chromosomes.

Since the invention of PCR, many adaptation techniques have been developed for sequencing DNA fragments flanking known sequences. Of them, inverse PCR is a matter of interest because of the simplicity of its principle. However, the protocols for inverse PCR introduced so far consist of some time-consuming procedures, and with them, we cannot "walk" chromosomes too far since the number of suitable restriction enzymes is limited. Our experiments led to confirming simpler technical approaches applicable to the case of bacterial chromosomes, that is, designing two end-specific "contextual" sequences with which we can quickly detect the desired clones of targeted DNA fragments by simply analyzing PCR products, employing "the minimum value of the desired fragments" as a "discriminating minimum" value to decrease contaminant DNA fragments, and creating a new tandem of two cleaved end fragments of a known sequence ("reordering") for PCR amplification in combination with cloning of the inverse PCR-generated DNA. With the improvements, we could both simplify the procedures and broaden the capacity of the inverse PCR in "walking" chromosomes.     

AKR thymic lymphomas involving mink cell focus-inducing murine leukemia viruses have a common region of provirus integration

Newly acquired proviruses related to a mink cell focus-inducing murine leukemia virus were detected in low copy number in restriction endonuclease-digested DNAs from thymic lymphomas of AKR/J mice. These extra proviruses were not present in DNAs of either normal thymus or leukemic brain tissues. Extra tumor-specific DNA fragments generated by restriction endonucleases either were identical in size or fell into similar size classes, suggesting a common site(s) of provirus integration. Characterization of extra EcoRI DNA fragments for mink cell focus-inducing viral sequences revealed that all of them contained large terminal repeat sequences and that a significant number represented proviruses with deletions.     

利用CRISPR/Cas9对基因组中高度同源DNA片段编辑多样性的遗传学研究

在基因组中,编码区存在许多高度相似的基因簇或基因群(多拷贝基因),非编码区也存在大量的重复序列。这些重复序列能通过改变染色体的三维结构调控基因的转录,对于生物体的遗传与进化起到了重要的作用。其高度同源的特征使得利用CRISPR/Cas9技术进行基因组编辑时面临更加复杂的状况。如果编辑的片段是二倍体或多倍体,还会产生各条染色单体上的编辑情况不相同的现象。为此本文选择了2个位于同一染色体相距11 kb的高度同源300 bp片段(L1和L2)进行CRISPR介导的DNA片段编辑。采用一对sgRNA(分别共同靶向两片段的上、下游位点)引导Cas9对HepG2细胞两个高度相似的DNA片段进行切割。片段编辑的细胞进一步单克隆化后,对获得的22个L1/L2编辑的CRISPR单克隆细胞株进行详细的基因型鉴定。结果发现除了这两个DNA片段本身被删除外,它们之间的大片段也存在被删除的现象,三个片段的各种反转组合也很频繁。该研究结果对于采用CRISPR/Cas9系统编辑多拷贝基因或重复序列,尤其是对二倍体或多倍体生物进行基因组编辑时具有重要的借鉴和参考价值。     

Mouse DNA contamination in human tissue tested for XMRV

Background

We used a PCR-based approach to study the prevalence of genetic sequences related to a gammaretrovirus, xenotropic murine leukemia virus-related virus, XMRV, in human prostate cancer. This virus has been identified in the US in prostate cancer patients and in those with chronic fatigue syndrome. However, with the exception of two patients in Germany, XMRV has not been identified in prostate cancer tissue in Europe. Most putative associations of new or old human retroviruses with diseases have turned out to be due to contamination. We have looked for XMRV sequences in DNA extracted from formalin-fixed paraffin- embedded prostate tissues. To control for contamination, PCR assays to detect either mouse mitochondrial DNA (mtDNA) or intracisternal A particle (IAP) long terminal repeat DNA were run on all samples, owing to their very high copy number in mouse cells.

Results

In general agreement with the US prevalence, XMRV-like sequences were found in 4.8% of prostate cancers. However, these were also positive, as were 21.5% of XMRV-negative cases, for IAP sequences, and many, but not all were positive for mtDNA sequences.

Conclusions

These results show that contamination with mouse DNA is widespread and detectable by the highly sensitive IAP assay, but not always with less sensitive assays, such as murine mtDNA PCR. This study highlights the ubiquitous presence of mouse DNA in laboratory specimens and offers a means of rigorous validation for future studies of murine retroviruses in human disease.     

DNA sequences homologous to the T DNA region of Agrobacterium tumefaciens are present in diverse Rhizobium species

Summary DNA sequences homologous to the T DNA region of the octopine-type Ti plasmid from Agrobacterium tumefaciens are present in different Rhizobium species. Plasmid DNA from each of two R. leguminosarum, two R. meliloti, and four slow-growing Rhizobium strains examined contain restriction endonuclease fragments that hybridize with the T DNA region, or with DNA sequences at or near the adjacent Ti plasmid transfer (ra) region. Four different BamHI fragments that contain homology to the T DNA region were cloned from R. leguminosarum 300 plasmid DNA. Cloned fragments of 5.9 kb and 10.3 kb hybridize to each other and are homologous to sequences which map at the right boundary region (EcoRI fragment 24) of the core T DNA. Ti plasmid sequences homologous to those present in cloned fragments of 10.9 kb and 2.0 kb map in adjacent fragments near the tra genes, approximately 10 kb to the right of the core T DNA.