Cleavage map of BK virus DNA with restriction endonucleases MboI and HaeIII.

Specific cleavage of BK virus (MM) DNA with restriction endonuclease MboI gives rise to 10 fragments. Two techniques were used to determine the location of these fragments on the viral genome with respect to the three known sites for HindIII cleavage. In the first method, reciprocal digestion, individual MboI fragments were digested with HindIII and individual HindIII fragments were digested with MboI. In the second method, single-end 32P-labeled HindIII subfragments were partially digested with MboI, and then the sizes of the radioactive partial products were used to deduce the nearest neighboring fragment. Information from these two methods is more than adequate to map all the MboI enzyme sites. Cleavage of BK virus (MM) DNA with restriction enzyme HaeIII produces 21 fragments. With the aid of the same two methods, these fragments have also been ordered with respect to the known map locations of the HindIII and MboI sites.     

Physical maps of bovine papillomavirus type 1 and type 2 genomes.

Physical maps of bovine papillomavirus type 1 and type 2 (BPV-1 and BPV-2) DNA were constructed from analysis of the electrophoretic mobilities of restriction endonuclease cleavage fragments from dual digests. BPV-1 DNA was sensitive to Hind III, HindIII, EcoRI, HpaI, AND BamHI, with all but HindII yielding single scissions. BPV-2 DNA was resistant to EcoRI, and HindIII had one cleavage site whereas HpaI, BamHI, and HindII yielded multiple fragments. Of four BPV-1 isolates examined, DNA from one isolate was resistant to HindIII, and another DNA isolate was resistant to BamHI. The three BPV-2 isolates examined were uniformly sensitive to the restriction endonucleases employed.     

Cloning and cleavage site mapping of DNA from bovine herpesvirus 1 (Cooper strain).

Sequences representative of most of the bovine herpesvirus 1 (Cooper strain) DNa were cloned in the plasmid vector pBR322 at the HindIII site. EcoRI, HpaI, and BamHI restriction endonuclease sites were mapped in each of the cloned fragments, and this information was used to construct a restriction endonuclease cleavage site map of the entire viral genome for the four enzymes.     

Restriction endonuclease DNA analysis of Leptospira interrogans serovars Icterohaemorrhagiae and Copenhageni

Leptospira interrogans serovar icterohaemorrhagiae strains Ictero No. I and RGA and serovar copenhageni strains M20, Shiromizu and Shibaura were examined by restriction endonuclease DNA analysis. Fifteen endonucleases (AluI, BamHI, BglII, EcoRI, HaeIII, HhaI, HindIII, KpnI, PstI, SacI, SalI, SmaI, StyI, XbaI and XhoI) were used as the digesting enzymes. Strain Ictero No. I showed endonuclease cleavage patterns which differed from those of the other four strains only when it was digested with enzymes KpnI and HindIII. When digested with KpnI, an extra band of about 5.4 kb was clearly produced, and when digested with HindIII, an extra band of about 25 kb was produced. When the other 13 enzymes were used, no differences were found between the endonuclease cleavage patterns among the five strains. Moreover, strains RGA, M20, Shiromizu and Shibaura could not be distinguished by the restriction endonuclease DNA analysis using all 15 endonucleases. In addition, six newly isolated leptospires from patients with leptospirosis and from Rattus norvegicus were compared with the Ictero No. I and M20 strains, by restriction endonuclease DNA analysis using enzymes KpnI and HindIII. Three leptospires belonging to serovar icterohaemorrhagiae showed the same endonuclease cleavage patterns as the M20 strain. The other three strains, which belong to serovar copenhageni, showed almost the same endonuclease cleavage patterns as the M20 strain; only the Kai ima 702 strain produced an extra band which was not identical to the Ictero No. I-specific extra band when digested with HindIII. The leptospiral restriction endonuclease DNA analysis has revealed taxonomic structures that are unrecognized by serology alone.     

Identification of the avian myeloblastosis virus genome. I. Identification of restriction endonuclease fragments associated with acute myeloblastic leukemia.

The proviral DNA of chicken peripheral blood leukemic myeloblasts was analyzed by restriction endonuclease digestion and Southern blotting. Two restriction endonuclease-generated fragments, an EcoRI 2.2-megadalton (Md) and a HindIII 2.6-Md fragment, were present upon enzyme cleavage of all leukemic myeloblast DNA preparations in addition to endogenous or helper-specific fragments. Neither of these fragments was derived from viral DNA of the two known myeloblastosis-associated viruses (MAV-1 and MAV-2). In contrast, DNA similarly treated from the erythrocytes of leukemic chickens showed only small amounts of the two avian myeloblastosis virus-specific fragments, whereas the helper virus-specific fragments were present in the amount seen in MAV-producing chicken embryo fibroblasts. The appearance of the EcoRI 2.2-Md and HindIII 2.6-Md specific fragments in all leukemic myeloblast DNA preparations indicates they are presumably part of the leukemogenic genome that must be present to induce acute myeloblastic leukemia.     

应用CRISPR/Cas13b编辑技术治疗TNNT2R141W转基因小鼠的扩张型心肌病

本研究旨在应用CRISPR/Cas13b系统对TNNT2R141W转基因扩张型心肌病（dilated cardiomyopathy,DCM）小鼠（DCM小鼠）进行探索性治疗,尝试发现治疗扩张型心肌病的一种新方式,为CRISPR/Cas13b系统在体内应用提供实验基础。随机设计11种Cas13b-TNNT2 gRNA并成功构建表达质粒,把它和人源TNNT2过表达质粒共同转染到293T细胞中,通过实时定量PCR（quantitative real-time PCR,Q-PCR）检测人源TNNT2 mRNA的表达水平。结果显示,gRNA 2引导Cas13b敲低目标基因的效率最高,达到80%（P<0.0001）。把gRNA2表达质粒包装到慢病毒载体中转导出生后1天的DCM小鼠原代心肌细胞,Q-PCR检测结果表明CRISPR/Cas13b系统对人源TNNT2 mRNA的敲低效率达到55%（P<0.01）。把PspCas13b和gRNA2的表达载体分别包装到AAV9病毒载体中,然后将200 μL 约1×1012 AAV9病毒颗粒通过尾静脉注射到4月龄DCM小鼠体内,待注射小鼠发育至5月龄时,Q-PCR检测结果显示,AAV9+DCM组TNNT2R141W表达水平较未注射组对照明显下降至40%（P<0.01）。对5月龄野生型（WT）、DCM（未注射病毒组）和AAV9+DCM（基因组编辑工具注射组）三组小鼠的心脏形态、心功能、心肌纤维化和心力衰竭等表型的观察结合显示：DCM小鼠的心脏形态异常,而AAV9+DCM小鼠心脏形态趋于正常;对三组小鼠的心脏进行超声心动图并对心功能指标进行统计发现,DCM组较WT组小鼠的左心室射血分数（left ventricular percent ejection fraction,LV EF%）、左心室短轴缩短率（left ventricular percent fractional shortening,LV FS%）分别下降了50.4%（P<0.0001）,55.1%（P<0.0001）,而AAV9+DCM组较DCM组小鼠的LV EF%、LV FS%分别上升了66.5%（P<0.01）,77.0%（P<0.01）;通过Q-PCR和天狼星红染色检测三组小鼠的心脏纤维化程度,结果显示DCM组较WT组小鼠的Col3a1和Postn两种纤维化基因,分别高表达5.2倍（P<0.001）、4.5倍（P<0.01）,而AAV9+DCM组较DCM组小鼠两种基因表达分别下降了2.0倍（P<0.05）、1.4倍（NS）,天狼星红染色结果显示纤维化区域明显下降;通过Q-PCR和蛋白质免疫印迹分别检测三组小鼠的心脏心力衰竭基因Nppb mRNA和Nppa蛋白质的表达水平,结果表明DCM组较WT组小鼠Nppb mRNA表达上升14.2倍（P<0.01）,而AAV9+DCM组较DCM组小鼠Nppb mRNA表达明显下降下降2.8倍（P<0.05）,Nppa蛋白质表达趋势与Nppb相同。把gRNA 5和含有R141W突变（gRNA 5T）和正常的TNNT2 mRNA（gRNA 5V）序列分别组合转染到293T细胞中,通过Q-PCR检测两种序列mRNA的表达水平。结果显示,gRNA 5T序列表达效率为30%（P<0.0001）,而并未检测到gRNA 5V mRNA的敲低。本研究通过设计靶向TNNT2R141W mRNA的gRNA,特异性敲低TNNT2R141W转基因小鼠体内突变的mRNA,有效改善了转基因小鼠的心功能,为临床进一步探索扩张型心肌病的治疗奠定了实验室基础。     

Molecular cloning and physical mapping of murine cytomegalovirus DNA.

Murine cytomegalovirus (MCMV) Smith strain DNA is cleaved by restriction endonuclease HindIII into 16 fragments, ranging in size from 0.64 to 22.25 megadaltons. Of the 16 HindIII fragments, 15 were cloned in plasmid pACYC177 in Escherichia coli HB101 (recA). The recombinant plasmid clones were characterized by cleavage with the enzymes XbaI and EcoRI. In addition, fragments generated by double digestion of cloned fragments with HindIII and XbaI were inserted into the plasmid vector pACYC184. The results obtained after hybridization of 32P-labeled cloned fragments to Southern blots of MCMV DNA cleaved with HindIII, XbaI, EcoRI, BamHI, ApaI, ClaI, EcoRV, or KpnI allowed us to construct complete physical maps of the viral DNA for the restriction endonucleases HindIII, XbaI, and EcoRI. On the basis of the cloning and mapping experiments, it was calculated that the MCMV genome spans about 235 kilobase pairs, corresponding to a molecular weight of 155,000,000. All fragments were found to be present in equimolar concentrations, and no cross-hybridization between any of the fragments was seen. We conclude that the MCMV DNA molecule consists of a long unique sequence without large terminal or internal repeat regions. Thus, the structural organization of the MCMV genome is fundamentally different from that of the human cytomegalovirus or herpes simplex virus genome.     

Restriction enzyme analysis of the Plasmid ColIb DNA

Summary Plasmid ColIb (61.5 Mdal) was digested with restriction enzymes EcoRI and HindIII. The DNA digestion products were separated by electrophoresis on 1.2% agarose gels. There were identified 22 fragments of ColIb DNA generated by the endonuclease EcoRI and 21 fragments produced by HindIII. Molecular weights of the fragments were estimated. The total molecular weight of the fragments generated by EcoRI was 61.42 Mdal and for HindIII fragments 62.79 Mdal.     

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.     

Restriction endonuclease from Haemophilus gallinarum (HgaI) cleaves polyoma DNA at four locations.

A restriction endonuclease obtained from Haemophilus gallinarum (hgaI) cleaves polyoma DNA at four specific sites. Using the EcoRI, HindIII, and HpaII endonuclease restriction sites as reference, the four HgaI cleavage sites were mapped at 0.02, 0.14, 0.27, and 0.48 fractional lengths, clockwise, from the single EcoRI cleavage site.     

Biochemical studies on bovine adenovirus type 3. III. Cleavage maps of viral DNA by restriction endoncleases EcoRI, BamHI, and HindIII.

Cleavage of bovine adenovirus type 3 (BAV3) DNA by restriction endonucleases EcoRI, BamHI, and HindIII yielded 7 (A to G), 5 (A to E), and 12 (A to L) fragments, respectively. The order of these fragments has been determined to be GDACBFE for EcoRI fragments, AEBDC for BamHI fragments, and JEBKACDHFGIL for HindIII fragments, and cleavage sites of these enzymes have been mapped on the genome of BAV3. BAV3 preparation contains incomplete virus whose genome has a deletion of about 13% of complete virus genome. Restriction endonuclease digestion of the incomplete virus DNA revealed that EcoRI E and F, BamHI C and HindIII G, I, and L fragments were deleted. Therefore, the deleted region of incomplete virus DNA is located near the right-hand end of the BAV3 DNA molecule, a result consistent with our previous electron-microscopic observations on heteroduplex molecules formed between complete and incomplete BAV3 DNA.     

Physical map of the bacteriophage T5 genome based on the cleavage products of the restriction endonucleases SalI, SmaI, BamI, and HpaI.

A physical map of the bacteriophage T5 genome was constructed by ordering the fragments produced by cleavage of T5 DNA with the restriction endonucleases SalI (4 fragments), SmaI (4 fragments), BamI (5 fragments), and HpaI (28 fragments). The following techniques were used to order the fragments. (i) Digestion of DNA from T5 heat-stable deletion mutants was used to identify fragments located in the deletable region. (ii) Fragments near the ends of the T5 DNA molecule were located by treating T5 DNA with lambda exonuclease before restriction endonuclease cleavage. (iii) Fragments spanning other restriction endonuclease cleavage sites were identified by combined digestion of T5 DNA with two restriction endonucleases. (iv) The general location of some fragments was determined by isolating individual restriction fragments from agarose gels and redigesting the isolated fragments with a second restriction enzyme. (v) Treatment of restriction digests with lambda exonuclease before digestion with a second restriction enzyme was used to identify fragments near, but not spanning, restriction cleavage sites. (vi) Exonucleases III treatment of T5 DNA before restriction endonuclease cleavage was used to locate fragments spanning or near the natural T5 single-chain interruptions. (vii) Analysis of the products of incomplete restriction endonuclease cleavage was used to identify adjacent fragments.     

Aerobactin iron uptake sequences in plasmid ColV-K30 are flanked by inverted IS1-like elements and replication regions.

By using Southern blot hybridization procedures, we found that a specific sequence within a 16.3-kilobase HindIII restriction fragment of pColV-K30 was also present in at least three other pColV-K30 HindIII fragments. Restriction endonuclease mapping of these HindIII fragments indicated that two of these repeated sequences, identified as IS1-like, occur in reverse orientation adjacent to the ends of the aerobactin iron uptake region, also included in the 16.3-kilobase HindIII fragment. There are two distinct replication regions enclosing the IS1-flanked aerobactin genes. A pColV-K30 BamHI restriction endonuclease fragment, carrying one of these replication regions, showed homology with the F plasmid EcoRI fragment f5, which carries the F replication sequences.     

Organization of the yeast ribosomal RNA gene cluster via cloning and restriction analysis.

The restriction endonuclease EcoR1 cleaves Saccharomyces cerevisiae DNA, which codes for ribosomal RNA (rRNA), into seven fragments, A second restriction endonuclease, HindIII, cleaves the same yeast ribosomal DNA into two fragments. These two restriction enzymes each yield DNA segments that total about 5.9 megadaltons. The "repeat unit" of the yeast genes coding for rRNA is thus about 5.9 megadaltons or about 9000 base pairs long. The two HindIII-cleaved DNA fragments as well as one of the EcoR1-cleaved DNA fragments were purified and amplified by cloning in Escherichia coli. Three of the seven EcoR1-generated DNA fragments could then be ordered by treating the two cloned HindIII DNA fragments with EcoR1. This led the assignment of the two HindIII restriction sites. The various restriction DNA fragments were hybridized directly from the gel utilizing 32P-labeled 5 S, 5.8 S, 18 S, and 25 S rRNA. Identification of the various DNA restriction segments then led to the final ordering of the DNA fragments. The gene coding for the 5 S RNA is adjacent to the gene coding for the 35 S precursor rRNA. These two groups of genes thus occur as a cluster in the following sequence: [5 S-spacer]-[spacer-18 S-5.8 S-25 S-spacer]-[spacer-5 S]. The actual map of the DNA restriction fragments is presented.     

A general method for inserting specific DNA sequences into cloning vehicles

A general method has been developed to introduce any double-stranded DNA molecule into cloning vehicles at different restriction endonuclease sites. In this method a chemically synthesized decadeoxyribonucleotide duplex, containing a specific restriction endonuclease sequence, is joinlex DNA is cut by the same restriction endonuclease to generate the cohesive ends. It is then inserted into the restriction endonuclease cleavage site of the cloning vehicle. To demonstrate the feasibility of this new method, we have inserted separately the synthetic lac operator DNA at the Bam I and HindIII cleavage sites of the plasmid pMB9 DNA.     

Cloning, physical mapping and cross-hybridization of the canine adenovirus types 1 and 2 genomes

Cloned HindIII fragments of the type 1 and type 2 canine adenovirus (canAd) genomes were mapped with respect to the BamHI, EcoRI, HincII, PaeR7 and PstI restriction endonuclease cleavage sites. Considering the large differences found between the canAd-1 and canAd-2 DNA molecules, in terms of number and location of restriction sites, cross-hybridization experiments were performed. Homologous DNA sequences were located on the canAd-1 and canAd-2 physical maps. Both viruses are genetically related to the extent of 57%. Our results confirm the existence of two distinct Ad species in the dog.     

Restriction endonuclease map of Euglena gracilis chloroplast DNA.

A physical map of the Euglena gracilis chloroplast genome has been constructed, based on cleavage sites of Euglena gracilis chloroplast DNA treated with bacterial restriction endonucleases. Covalently close, circular chloroplast DNA is cleaved by restriction endonuclease SalI into three fragments and by restriction endonuclease BamHI into six fragments. These nine cleavage sites have been ordered by fragment molecular weight analysis, double digestions, partial digestions, and by digestion studies of isolated DNA fragments. A fragment pattern of the products of EcoRI restriction endonuclease digestion of Euglena chloroplast DNA is also described. One of these fragments has been located on the cleavage site map.     

DNA cleavage of lambda and phi 80 transducing phages carrying the argA, argECBH, argF and argI operons of Escherichia coli K-12 with the restriction endonucleases EcoRI, SmaI and HindIII.

DNA isolated from each of the seven arginine transducing phages lambdaargA2cI857susS7, phi80ppc argECBH, phi80argF, phi80argF ilambdacI857, lambdaargF2, lambdaargF23 and lambdaargI valScI857susS7 has been specifically cleaved by the restriction endonucleases EcoRI, SmaI and HindIII. The DNA fragments resulting from single, and in some cases, double endonuclease digests were separated by electrophoresis in agarose and also in polyacrylamide gel. The electrophoretic patterns thus obtained were compared with those produced by digestion of DNA isolated from the corresponding lambda and phi80 parental phages. The majority of cleavage sites produced by the action of these restriction enzymes on arginine transducing DNA have been physically mapped.