Restriction enzyme analysis of Bacillus subtilis bacteriophage phi 105 DNA

The recognition sites on phi 105 DNA for the restriction endonucleases EcoRI, Bg/II, SmaI, KpnI, SstI, SalI, XhoI, NcoI, PstI, HindIII, ClaI, EcoRV and MluI have been mapped. The sites for EcoRI are shown to be different from those published earlier. The DNA from phi 105 contains no recognition sites for the endonucleases BamHI and XbaI.     

Effect of site-specific endonuclease digestion on the thyP3 gene of bacteriophage phi 3T and the thyP11 gene of bacteriophage rho11.

phi 3T and rho11 are closely related bacteriophages of Bacillus subtilis which can "convent" thymine auxotrophs to thymine prototrophs upon infection or transfection. The effect of endonuclease digestion on the ability of both bacteriophage and prophage DNA from phi eT and rho11 to transform for thymine prototrophy was determined. All of the endonucleases tested: BamHI, Bg/II, BsuRI, EcoRI, HindII+ III, and HpaII reduced the efficiency of thyP transformation to an equal extent in prophage and bacteriophage DNA. Only HpaII completely abolished thyP transformation. The reduction in transformation with BamHI, Bg/II, BsuRI, EcoRI, and HpaII fragments is size related. The thyP transforming fragments generated by these endonucleases are potentially clonable.     

'Red pigment' from ADE-2 mutants of S. cerevisiae prevents DNA cleavage by restriction endonucleases

Protection of DNA from cleavage by restriction endonucleases EcoRI, HindIII, BamHI, and Bg/II with red pigment, produced by ADE-2 mutants of Saccharomyces cerevisiae is demonstrated. Purification of yeast DNA from pigment can be achieved by chromatography on hydroxyapatite columns.     

Restriction map of permuted DNA of Erwinia carotovora temperate bacteriophage 59

The cyclic permutation and terminal redundancy were found in the genomes of Erwinia carotovora temperate bacteriophage 59 by electron microscopic studies. The headful mechanism for bacteriophage DNA cleavage and packaging during the phage morphogenesis was confirmed by the restriction analysis technique. Restriction map of the bacteriophage 59 DNA was constructed for restriction endonucleases BamHI, Bg1II, Eco31, Sa1I, SmaI, EcoRI.     

Structure and organization of the highly repeated and interspersed 1.3 kb EcoRI-Bg1II sequence family in mice.

EcoRI digestion of total mouse DNA yields a prominant 1.3 kb fragment amounting to between 1 and 2% of the mouse genome. The majority of the 1.3 kb EcoRI fragments have a single Bg1II site 800 bp from one end. This EcoRI-Bg1II sequence family shows HindIII and HaeIII sequence heterogeneity. We have cloned representatives of the EcoRI-Bg1II gene family in Charon 16A and studied their structure and organization within the genome. The cloned 1.3 kb fragments show the expected restriction enzyme patterns as well as additional heterogeneity. Representatives of the EcoRI-Bg1II sequence family were found to be interspersed throughout the mouse genome as judged by CsCl density gradient centrifugation experiments. Family members were also found to be organized in higher order repeating units. Homologous sequences were also found in other rodent species including rat and Chinese hamster. Cross hybridization between a cloned 1.3 kb mouse fragment and a cloned CHO repeated sequence is of special interest since the latter has been shown to contain sequences homologous to the Human A1uI family by nucleotide sequencing.     

Restriction endonuclease analysis of mitochondrial DNA of three farm animal species: Cattle, sheep and goat

Five restriction endonucleases (HindIII, BgIII, EcoRI, EcoRV and BamHI) were employed to analyse mitochondrial DNA of cattle, sheep and goat. The results showed completely different restriction patterns of mtDNA among the three bovid species. A total of 11, 16, and 17 restriction fragments in cattle, sheep and goat respectively, were detected by the five restriction endonucleases. Average total sizes of mtDNA of cattle, sheep and goat were found to be 16.49 ± 0.18, 16.30 ± 0.25 and 16.44 ± 0.08 kb, respectively. The mtDNA cleavage patterns were identical for all seven individuals belonging to two cattle breeds and for 10 individuals from one sheep breed.     

Isolation and characterization of a Pseudomonas aeruginosa bacteriophage with a very limited host range

A Pseudomonas aeruginosa bacteriophage, phi PLS743, with extremely limited host range has been isolated. It belongs to the virus family Podoviridae, morphological type C1, and possesses a head diameter of 45 nm. The phage has a buoyant density in CsCl of 1.516 g/cm3, and its mass is 45 x 10(6) daltons. The phage particles are composed of double-stranded DNA (49.9 mol% G + C; 42.4 kilobase pairs) and 11 structural proteins (66% by weight). The major head protein, P5, has a Mr of 34,500. The DNA is not cut by SalI or XhoI restriction endonucleases, but is cut by PvuII (1 site), KpnI and BglII (2 sites), PvuI (4 sites), BamHI (7 sites), EcoRI (9 sites), and HindIII (12 sites). A restriction endonuclease map is presented.     

Convenient vectors for cloning and sequencing EcoRI and HindIII fragments

The polylinker regions of plasmid pUC and bacteriophage M13mp vectors have been specifically modified to provide alternative positions for cloning and reexcising EcoRI and HindIII fragments; the EcoRI and HindIII sites have been moved internal to BamHI and Bg/II sites. The location of EcoRI and HindIII sites in these HinEco vectors allows either selective linearization or excision of the cloned fragments at unique flanking sites.     

Isolation and organization of calf ribosomal DNA.

Ribosomal DNA (rDNA) from calf was isolated by three density gradient centrifugations. The first centrifugation in Cs2S04/BAMD was used to obtain partially resolved dG+dC-rich fractions from total DNA. The second and third centrifugations, in Cs2S04/Ag+, led to the isolation of an rDNA fraction characterized by a symmetrical band in CsCl, p = 1.724 g/cm3. This new procedure appears to be generally suitable for the isolation of rDNA and other dG+dC-rich repeated genes. The organization of isolated calf rDNA has been studied by restriction enzyme digestion and by hybridization with cloned rDNA from Xenopus laevis. The repeat unit of calf rDNA has a molecular weight of 21x10(6) and is split by EcoR1 into two fragments, 16x10(6) and 5.0x10(6), and by BamHI into seven fragments. EcoRI and BamHI sites have been mapped. Most of the 18S and 28S RNA genes and the transcribed spacer are contained in the small EcoRI fragment, while the non-transcribed spacer is localized in the large EcoRI fragment. This spacer showed length heterogeneity within a single individual; such heterogeneity is limited to two regions of the spacer.     

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。     

Comparative restriction analysis of HindIII-F-fragments of DNA from 4 strains of vaccinia virus

The localization of KpnI, SacI, XhoI, AvaI, PstI, BglI, BamHI, EcoRI, PmiI, SalI, BglII, restriction endonuclease cleavage sites in HindIII-F-fragments of DNA from vaccinia strains WR, Copenhagen, LIVP and neurovaccine has been detected. The fragments have been shown to differ in the number of AvaI, EcoRI and BamHI sites. The fragments also differ from the analogue of Tian Tan vaccinia strain in the pattern of restriction by AvaI, XhoI, PstI, EcoRI and BamHI endonucleases.     

A restriction map of cauliflower mosaic virus DNA (strain PV 147). Mapping of the cleavage sites of HhaI, SacI, AvaI, PvuII, PstI, XbaI, EcoRI, Bg/II, HincII, HpaII and HindII + III.

The virion-extracted DNA (Mr5 x 10(6)) of cauliflower mosaic virus (CaMV) has three single-stranded interruptions. The mapping of this DNA using eleven restriction endonucleases (HhaI, SacI, AvaI, PvuII, PstI, XbaI, EcoRI, Bg/II, HincII, HpaII and HindII + III) is reported here. The existence of the three single-stranded breaks complicates the identification and the molecular weight determination of fragments produced by HpaII, HindIII and HindII + III. Indeed the electrophoretic mobility of some fragments in which a single-stranded discontinuity is located is modified, and the fluorescence of ethidium bromide complexed with these fragments is reduced as compared to that observed for the other fragments existing in a molar ratio. These drawbacks were overcome by performing experiments of nick-translation of CaMV DNA with Escherichia coli DNA polymerase I. FRom the data it follows that the CaMV DNA molecule bears bears 1 site for HhaI and SacI, 2 for AvaI and PvuII, 3 for PstI, 4 for XbaI, 5 for EcoRI, 6 for Bg/II and HincII, 11 for HpaII and 15 for HindII + III. The corresponding fragments have all been ordered and precisely located providing a suitable map for further investigations connected with the study of the fine structure and the function of the CaMV genome.     

Physical mapping of the ribosomal DNA region of Euglena gracilis chloroplast DNA.

1. The relative positions of endo R . EcoRI and endo R . Bg/II cleavage sites are mapped within the linked DNA fragments Bam-E-E-D of the Euglena gracilis chloroplast DNA. 2. The DNA segment Bam-E-E-D contains three contiguous repeated segments of approximately 5600 base pairs. 3. Each repeated segment can code for an rRNA gene (16-S and 23-S).     

Comparative structure and evolution of goat and sheep satellite I DNAs.

The satellite I DNAs of domestic goat (Capra hircus) and domestic sheep (Ovis aries) have been studied using molecular hybridisation and restriction enzyme analysis. Both satellite DNAs are composed of repeat units of 820 base pairs in length, but their restriction maps, although similar, differ in certain respects. Thus the majority of sheep satellite I repeat units have two EcoRI sites and one AluI site, whereas the majority of goat satellite I repeat units have one EcoRI site and two AluI sites. The sheep satellite I repeat units with the two EcoRI sites are much more homogeneous than the repeats forming the remainder of the satellite, as judged by the difference in the melting temperatures of native and reassociated molecules. DNAs from species of wild sheep and goats were screened for the presence of these repeat units, and they appear to have been amplified during the radiation of the Ovis genus. Goat satellite I is composed of a single sequence type which has changed through base substitution until the sequence now shows considerable heterogeneity. It is proposed that the major sequence types of these two satellite DNAs were amplified by different saltatory replication events at different times in the evolution of the group.     

Isolation, cloning and characterization of argF gene DNA from Escherichia coli K-12.

A 1650 base pair (BP) fragment carrying the entire argF structural gene with its associated control regions was isolated from an EcoRI/BamHI digest of phi80argFilambda cI857 DNA. This segment was cloned using the EcoRI and BamHI cleavage sites in the plasmid pBR322. A preliminary restriction map of the argF region was prepared. RNA polymerase binding studies indicated that the argF promoter is located approx. 30 base pairs from the EcoRI terminus of the cloned DNA segment.     

Physical map of the Rhodobacter sphaeroides bacteriophage phi RsG1 genome and location of the prophage on the host chromosome.

We constructed a physical map of the 50-kilobase-pair (kb) DNA of the temperate Rhodobacter sphaeroides bacteriophage phi RsG1, with the relative positions of the cleavage sites for the nine restriction endonucleases KpnI, HindIII, XbaI, ClaI, BclI, EcoRV, EcoRI, BglII, and BamHI indicated. Using biotinylated phi RsG1 DNA as a probe in hybridization studies, we detected homologies with virus DNA and fragments of restriction endonuclease-digested host chromosomal DNA but not with plasmid DNA. This indicates that the prophage is integrated into the host chromosome. In addition, the use of specific probes such as the 10.4-kb BglII A fragment and the 2.65-kb BamHI H fragment allowed the determination of the position of phage attachment site (attP).     

Simple method of cloning eukaryote DNA: production of certain new ribosomal genes of Drosophila

We propose a simple method which allows to receive a collection of clones containing recombinant plasmids. It is based on the ligation of the longer fragment of pBR332 formed by EcoRI and BamH1 with eukaryotic DNA (from Drosophila melanogaster embryo in this case) partially cleaved with EcoRI and BamHI. This approach gave us 10(4) colonies from 1 microgram of Drosophila DNA and 0.1 microgram of the BamHI--EcoRI "vector". About 0.5% of all clones carried the fragments of ribosomal genes with insertions in the 26S gene. Ribosomal genes lacking insertions did not enter the collection due to some peculiarities in their restriction map. The sites of cleavage are mapped in eight recombinant plasmide for HindIII, BamHI and EcoRI. These maps show that some insertions within 26S gene have not been cloned earlier. The mean length of cloned fragments is 11.8 kilobases, the mean number of EcoRI and BamHI restriction sites are 1.2 and 1.0, respectively. The electrophoretical screening of plasmids using cetyl trimethyl ammonium bromide was developed.     

Physical mapping of a large-plaque mutation of adenovirus type 2.

We have developed a simple method based on cotransfection of overlapping DNA restriction fragments for construction of recombinants of adenovirus type 2 (Ad2) and Ad5. When Ad2 DNA digested with restriction endonuclease EcoRI was cotransfected with Ad5 DNA digested with SalI, recombination occurred between Ad2 EcoRI-A (map position 0 to 59) and Ad5 SalI-A (map position 45 to 100). Analysis of the recombinant DNAs by digestion with EcoRI or BamHI restriction endonucleases indicated that, as expected, recombination had occurred in overlapping sequences (map position 45 to 59) between the Ad2 EcoRI-A fragment and the Ad5 SalI-A fragment. By using this method, several recombinants were constructed between a large-plaque (lp) mutant of Ad2 and wild-type Ad5. Cleavage of the recombinant genomes with restriction endonucleases BamHI, EcoRI, and HindIII revealed that the lp mutation is located within the left 41% of Ad2 genome.     

Characterization of restriction endonuclease maps of hepatitis B viral DNAs

The HBV DNA isolated from Dane particles of 9 patients' plasma was cloned into the EcoRI or BamHI site of the pUC8 plasmids. Two plasmids with full length HBV DNA and four plasmids containing the HBV surface antigen gene were obtained. Based on our cloned HBV DNA and a comparison with 7 complete sequences and 5 restriction endonuclease patterns of HBV DNA published by others, we can recognize common restriction sites shared by different subtypes (adw, adr, ayw, and adyw): (1) a HincII site in the S gene, (2) a BamHI site in the X region, and (3) two BglII sites in the C gene. In addition adw has specific sites for HincII, BamHI, and PstI in the pre-S region. A unique XhoI site is present in the pre-S region in all subtypes except for adw.     

Variation in the number of alpha-globin loci in sheep

Southern blot analysis was used to compare sheep and goat restriction- endonuclease maps of the DNA region containing the alpha-globin genes. The identical digestion patterns observed in both species with three endonucleases (BamHI, BstEII, and PstI) show that in sheep a single chromosome normally bears two nonallelic alpha-globin genes positioned at the same distance as in goat. Variant digestion patterns with enzymes that cleave outside (BamHI and HindIII) and within (EcoRI) the alpha-globin loci allowed us to infer that chromosomes with different numbers of alpha-globin loci are also present in sheep. In particular, in the 60 sheep considered, four individuals were heterozygous (alpha alpha/alpha alpha alpha) and one was homozygous (alpha alpha alpha/alpha alpha alpha) for chromosomes with three loci and one individual was heterozygous for a chromosome with four loci (alpha alpha/alpha alpha alpha alpha). This variation in the number of copies of alpha-globin loci can be explained by means of unequal crossovers.