胡子鲇mtDNA多态性及限制性酶切图谱

用８种限制性内切酶对胡子鲇（ＣｌａｒｉａｓｆｕｓｃｕｓＬａｃｅｐｅｄｅ）肝脏线粒体ＤＮＡ（ＭｉｔｏｃｈｏｎｄｒｉａｌＤＮＡ,ｍｔＤＮＡ）进行了分析。ＸｈｏⅠ、ＥｃｏＲⅠ、ＰｓｔⅠ、ＢａｍＨⅠ、ＸｂａⅠ、ＨｉｎｄⅢ在ｍｔＤＮＡ分子上分别有２、３、１、１、３和５个切点。胡子鲇种内存在ｍｔＤＮＡ酶切片段长度多态性（Ｒｅｓｔｒｉｃｔｉｏｎｆｒａｇｍｅｎｔｌｅｎｇｔｈｐｏｌｙｍｏｒｐｈｉｓｍｓ,ＲＦＬＰ）。经ＢｇｌⅠ、ＢｇｌⅡ酶解,ｍｔＤＮＡ都出现两种酶切类型,Ⅰ型各具２个片段,Ⅱ型各具１个片段。ｍｔＤＮＡ分子量为１０．２４２×１０６ｕ,长度约为１６．６８ｋｂ。用双酶解法建立了胡子鲇ｍｔＤＮＡ的限制性酶切图谱,并对ＲＦＬＰ现象进行了分析。     

团头鲂线粒体DNA的限制性内切酶图谱

用ＢａｍＨⅠ,ＢｇｌⅠ,ＢｇｌⅡ,ＥｃｏＲⅠ,ＨｐａⅠ,ＫｐｎⅠ,ＰｓｔⅠ,ＳａｃⅠＳａｌⅠ,ＸｂａⅠ,和ＸｈｏⅠ１１种限制性内切酶对团头鲂（ＭｅｇａｌｏｂｒａｍａａｍｂｌｙｃｅｐｈａｌａＹｉｈ）的线粒体ＤＮＡ（ｍｔＤＮＡ）进行了单酶切,其切点数依次为２,３,２,３,３,１、０,１,０,０和０;经琼脂糖凝胶电泳测算出各酶切片断大小,得出团头鲂ｍｔＤＮＡ分子长约１６０２０±３５６碱基对（ｂｐ）,分子量６．３７×１０１８ｕ（原子质量）,构建了团头鲂ｍｔＤＮＡ的限制酶图。     

大鳞副泥鳅线粒体DNA九种限制性内切酶酶切图谱

大鳞副泥鳅ｍｔＤＮＡ经１１种限制性内切酶（ＢａｍＨＩ,ＢｇｌＩ,ＢｇｌⅡ,ＥｃｏＲＩ,ＨｐａＩ,ＫｐｎＩ,ＰｓｔＩ,ＳａｃＩ,ＳａｌＩ,ＸｂａＩ和ＸｈｏＩ）单酶完全酶解获得２３个酶切位点,这些酶酶切位点数依次分别是：２、７、０、３、３、０、３、１、１、２和１。通过琼脂糖凝胶电泳测定大鳞副泥鳅ｍｔＤＮＡ平均分子量为１０．３３±０．２２×１０６ｕ（原子质量）;其分子长约１６７２０±３５０碱基对（ｂｐ）。采用双酶完全酶解法构建了大鳞副泥鳅ｍｔＤＮＡ限制性内切酶酶切图谱。     

海南黄牛和徐闻黄牛线粒体DNA的多态性及其品种分化关系

本文以ＡｐａⅠ、ＡｖａⅠ、ＢａｍＨⅠ、ＢｇｌⅠ、ＢｇｌⅡ、ＤｒａⅠ、ＥｃｏＲⅠ、ＥｃｏＲⅤ、ＨｉｎｄⅢ、ＨｐａⅠ、ＰｓｔⅠ、ＳａｌⅠ、ＳｃａⅠ和ＸｈｏⅠ等１４种限制性内切酶分析来自海南岛的海南黄牛和雷州半岛的徐闻黄牛的线粒体ＤＮＡ限制性片段长度多态性（ｍｔＤＮＡＡＲＦＬＰ）。结果只有一种限制性内切酶（ＳａｌⅠ）在海南黄牛中检测到多态性,并且其中的Ｃ型（１５．０,１．３）尚未见报道。我们的结果还显示,两个品种６个个体的ｍｔＤＮＡ基因单倍型全部表现为Ａ型,即瘤牛的血统。徐闻黄牛和海南黄牛ｍｔＤＮＡ极低的遗传变异度表明两个品种的亲缘关系很近,从而在分子生物学水平为其合称为雷琼黄牛提供了佐证。     

隼形目鹰科四种鸟类线粒体DNA研究

本实验采用ＡｐａＩ,ｆＢａｍＨＩ,ＢｇｉＩＩ,ＥｃｏＲＩ,ＥｃｏＲＶ,ＨｉｎｄⅢＨｐａＩ,ＫｐｎＩ,ＰｓｔＩ,ＰｒｕＩＩ,ＳａｌＩ,ＳｃａＩ,ＸｂａＩ和ＸｈｏＩ１４种限制性内切酶,对鹰科４种鸟类即雀鹰（Ａｃｃｉｐｉｔｅｒｎｉｓｕｓ）,松雀鹰（Ａ．ｖｉｒｇａｔｕｓ）苍鹰（Ａ．ｇｅｎｔｉｌｉｓ）和灰险鹰（Ｂｕｆｔａｔｕｒｉｎｄｉｃｕｓ）ｍｔＤＮＡ限制性片段长度多态分析。结果表明,４种鸟类中雀鹰和松１主     

树鼠mtDNA限制性内切酶图谱及其与小家鼠褐家鼠的亲缘关系

本实验用ＡｐｅⅠ,ＡｖａⅠ,ＢａｍＨⅠ,ＢｃｌⅠ,ＢｇｌⅠ,ＣｌａⅠ,ＥｃｏＲⅠ,ＥｃｏＲＶ,ＨｐａⅠ,ＰｓｔⅠ,ＰｖｕⅡ,ＳｃａⅠ,ＸｂａⅠ等１３种限制性内切酶分析树鼠（Ｃｈｉｒｏｍｙｓｃｕｓｃｈｉｒｏｐｅｓ）的ｍｔＤＮＡ限制性片段长度多态性,并用双酶解法构建了其中８种酶的限制性内切酶图谱。根据限制性片段差异法和分子钟,计算并讨论树鼠和小家鼠（Ｍｕｓｍｕｓｃｕｌｕｓ）、褐家鼠（Ｒａｔｔｕｓｎｏｒｖｅｇｉｃｕｓ）的ｍｔＤＮＡ遗传距离和亲缘关系。结果表明树鼠与褐家鼠的关系较接近,两者的分歧时间在距今１５００─２０００万年前,即处于中新世早中期。     

板齿鼠线粒体DNA的研究

本文应用ＡｐａＩ、ＢａｍＨＩ、ＢｃｌＩ、ＢｇｌＩ、ＣｌａＩ、ＥｃｏＲＩ、ＥｃｏＲＶ、ＨｉｎｄＩＩ、ＰｓｔＩ、ＰｖｕＩＩ、ＳａｃＩ、ＳｃａＩ和ＸｂａＩ等１３种限制性内切酶对板齿鼠线粒体ＤＮＡ（ｍｔＤＮＡ）进行限制性片段长度多态（ＲＦＬＰ）分析,并用双酶解法构建其限制性内切酶图谱。结果表明板齿鼠存在３种ｍｔＤＮＡ单倍型,可通过限制酶ＰｖｕＩＩ、ＨｉｎｄＩＩ和ＡｐａＩ区分,呈现ＤＮＡ多态性和种内遗传变异。与小家鼠、褐家鼠ｍｔＤＮＡ限制性片段的数据相比较,板齿鼠和这两种鼠ｍｔＤＮＡ存在明显差异。板齿鼠ｍｔＤＮＡ限制性内切酶图谱的建立,为进一步系统研究鼠科动物的遗传分化提供了依据。     

褐家鼠线粒体DNA遗传多态性的研究

通过碱变性法提取线粒体ＤＮＡ,用地高辛标记的探针Ｓｏｕｔｈｅｒｎ杂交限制性酶切多态性（ＲＦＬＰ）分析,研究中国家鼠Ｒａｔｔｕｓｎｏｒｖｅｇｉｃｕｓ遗传多态性。采用ＡｐａⅠ、ＡｖａⅠ、ＢａｎＨＩ、ＢｃｌⅠ、ＢｇｌⅠ、ＣｌａⅠ、ＥｃｏＲⅠ、ＥｃｏＲⅤ、ｈｉｎｄⅢ、ＰｖａⅡ、ＳｃａⅠ和ＸｂａⅠ等１２种限制性内切酶分析来自我国８个地区２６只褐家鼠的线粒体ＤＮＡ,共检出２０种限制性态型和１１种ｍｔＤＮＡ单倍     

用RFLP和PCR—RFLP技术研究东北虎和华南虎线粒体DNA多态性

采用ｍｔＤＮＡＲＦＬＰ和ＰＣＲＲＦＬＰ技术研究了东北虎和华南虎的ｍｔＤＮＡ的多态性。在ｍｔＤＮＡＲＦＬＰ研究中,分离纯化了东北虎和华南虎肝、肾和心脏组织的ｍｔＤＮＡ,用２０种识别６碱基对的限制性内切酶消化,结果只有１种限制性内切酶（ＸｂａⅠ）检测到多态性片段,其余１９种限制性内切酶消化产生的限制性格局在东北虎和华南虎完全一致。在ＰＣＲＲＦＬＰ研究中,用ＰＣＲ技术分别扩增了东北虎和华南虎ｍｔＤＮＡ的控制区（ｃｏｎｔｒｏｌｒｅｇｉｏｎ）,用８种识别４碱基对的限制性内切酶分别对扩增产物进行消化,结果只有１种限制性内切酶（ＲｓａⅠ）检测到多态性片段。ｍｔＤＮＡＲＦＬＰ及ＰＣＲＲＦＬＰ的结果均提示东北虎和华南虎之间的遗传距离极小。这可能与下列因素有关：两者分布区间无天然隔离屏障;具有强扩散能力;近几百年才被相互隔离。     

中国大陆若干群体的黑果蝇的线粒体DNA多态性研究

本文研究了果蝇Ｄ．ｖｉｒｉｌｉｓ种群Ｄ．ｖｉｒｉｌｉｓ线粒体ＤＮＡ（ｍｉｔｏｃｈｏｎｄｒｉａｌＤＮＡ,ｍｔＤＮＡ）的多态性。用９种限制性内切酶ＸｂａⅠ,ＥｃｏＲⅠ,ＰｓｔⅠ,ＨｉｎｄⅢ,ＢｇｌⅡ,ＳａｃⅠ,ＳｃａⅠ,ＥｃｏＲＶ和ＰｕｖⅡ,对青岛、南京、上海、宁波与泉州５个Ｄ．ｖｉｒｉｌｉｓ群体的ｍｔＤＮＡ进行了限制性片段长度多态性（ｒｅｓｔｒｉｃｔｉｏｎｆｒａｇｍｅｎｔｓｌｅｎｇｔｈｐｏｌｙｍｏｒｐｈｉｓｍ,ＲＦＬＰ）的研究。在５群体中,发现５种不同的酶切图谱,它们彼此之间的遗传差异π为０．４６％－１．７６％,群体内遗传差异πｉｊ为０．００％－０．３３％,群体间的差异ｄｘｙ,为０．００％－０．８２％。分布于中国大陆的Ｄ．ｖｉｒｉｌｉｓ的群体间遗传差异在总遗传差异中所占比例γｓｔ值为２４．６２％。我们发现,Ｄ．ｖｉｒｉｌｉｓ的栖息环境对ｍｔＤＮＡ的遗传变异有十分明显的影响,而不同地理纬度的群体之间其遗传距离并无倾群（ｃｌｉｎｅ）表现。     

滑鼠蛇肝线粒体DNA的限制酶图谱

用六种限制性内切酶BamHⅠ、EcoRⅠ、PstⅠ、BglⅠ、BglⅡ和SalⅠ对滑鼠蛇肝脏线粒体DNA(mtDNA)进行酶解。发现BglⅡ、PstⅠ、BamHⅠ、BglⅠ和EcoRⅠ在滑鼠蛇肝mtDNA上分别有1、2、3、3和4个切点。SalⅠ不能切割滑鼠蛇肝mtDNA。根据滑鼠蛇肝mtDNA的单酶、双酶完全酶解及部分酶解片段的数目和分子量,建立了滑鼠蛇肝mtDNA的限制酶图谱。     

Restriction cleavage map of mitochonrial DNA from the yeast Saccharomyces cerevisiae.

Mitochondrial DNA (mtDNA) from the yeast Saccharomyces cerevisiae was cleaved by restriction endonucleases Eco RI, Hpa I, Bam HI, Hind III, Pst I, and Sal I, yielding 10, 7, 5, 6, 1, and 1 fragments, respectively. A physical ordering of the restriction sites on yeast mtDNA has been derived. Yeast mtDNA cannot be isolated as intact molecules, and it contains nicks and gaps which complicate the use of conventional fragment mapping procedures. Nevertheless, the position of each of the restriction sites was obtained primarily by reciprocal redigestion of isolated restriction fragments. This procedure was supplemented by co-digestion of mtDNA with a multisite enzyme and a single-site enzyme (i.e., Sal I or Pst I) which provided a unique orientation for overlapping fragments cleaved by Sal I or Pst I. The data obtained from these approaches were confirmed by analysis of double and triple enzyme digests. Analysis of partial digest fragments was used for positioning of the smallest Eco RI fragment. A comparison of mtDNA from four grande strains (MH41-7B, 19d, TR3-15A, and MH32-12D) revealed similar, but slightly varying restriction patterns, with an identical genome size for each of approximately 5 X 10(-7) d or 75 kb. A fifth grande strain, D273-10B from S. cerevisiae, revealed restriction patterns different from those of the above strains, with a smaller genome size of 70 kb.     

The cDNA Cloning and Nucleotide Sequence of the Gene Encoding Nonstrucure Protein of Rice Dwarf Virus Genome Segment 10

The results of cloning and sequencing the gene encoding nonstructure protein of the rice dwarf virus (RDV) gtnome segment 10 with polymerase chain reaction(PCR) technique were reported. The amplified PGR product was cloned into Hind Ⅱ site of plasmid pGEM3zf(-) and analysed with restriction enzymes. The physical map of the cloned fragment has been constructed, the insert is 1150 bp in length with restriction enzyme sites of Sac Ⅰ, Hind Ⅲ, NdeⅠ, BamH Ⅰ, etc. Besides, two restriction enzyme sites Bgl Ⅱ and EcoR Ⅰ have been separetely added in the 5 and 3 end of the segment in order to be cloned into plant intermediate vector in a convenient way. The fragments cleaved by the above-mentioned restriction enzymes were subcloned and the DNA sequence of full length of segment 10 was determined. In comparison with the RDV epidemic in Japan, the nucleotide sequence and deduced amino acid sequence of cloned segment 10 are 96.03% and 97.17% in homology respectively.     

Assembly of the mitochondrial membrane system. Physical map of the Oxi3 locus of yeast mitochondrial DNA

The oxi3 locus of yeast mitochondrial DNA is currently thought to code for Subunit 1 of cytochrome oxidase (Tzagoloff, A., Macino, G., and Sebald, W. (1979) Annu. Rev. Biochem. 48, 419-441). The respiratory competent strain of Saccharomyces cerevisiae D273-10B/A48 was used to obtain cytoplasmic "petite" clones enriched for genetic markers in the oci3 locus. The most complex clone studied (DS6) was ascertained to have a mitochondrial genome with a tandemly repeated segment of mtDNA 16.5 kilobases in length. The oxi3 locus was dissected by mutagenesis of DS6 with ethidium bromide and selection of new clones having less complex genotypes. Six derivative clones with genome sizes ranging from 2.3 to 6.1 kilobases have been extensively analyzed. Most of the restriction sites present in the segments of mtDNA retained by the clones have been mapped, thereby providing a detailed restriction map of the oxi3 gene. Based on the physical locations of the most distal oxi3 mutations, the gene spans approximately 10,000 nucleotides and occupies the region of wild type mtDNA from 44 to 58 map units.     

人乙型肝炎病毒(HBV)adr亚型基因组的多态性

由乙型肝炎adr亚型病毒(HBVadr)携带者26人的混合血清,得到了HBVadr基因组克隆株(PADR)158株,对这些克隆株进行四种限制性内切酶(BglⅡ,HindⅢ,PstⅠ,XhoⅠ)切点测定,并对其中S株的13种限制性内切酶图谱进行比较研究,发现同为adr亚型病毒,其基因组的限制性酶切图谱存在差异。另外,通过HindⅢ)切点得到的12个克隆株(PADR-H),也进行了酶切图谱分析。在这170个克隆株中,已经发现了5种类型的HBVadr基因组限制性酶切图谱,其中有6种酶(AvaⅠ,EglⅠ,BglⅡ,HincⅡ,HindⅢ,HpaⅠ)的7个变异点。本文报道了HBVadr基因组的多态性现象。     

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。     

Physical and functional mapping of rat-liver mitochondrial DNA

Summary Rat-liver mitochondrial DNA (mtDNA) contains 2 cleavage sites of the restriction endonuclease XbaI. The molecular sizes of restriction fragments are 6.6×10⁶ and 3.7×10⁶ D. The results of partial cleavage of mtDNA with EcoRI allow the fragment F (0.32×10⁶ D) to be localized in the sequence ABCEGFHDA. The functional map of mtDNA is constructed for two genes of the ATP-ase mRNAs from rat-liver mitochondria. Molecular hybridization shows that the ATPase genes are located in fragment B and in the GEHD area of mtDNA EcoRI cleavage.     

Polymorphism of the Noncoding Region of the Mitochondrial Genome in Three Kazakh Populations Inhabiting Different Areas of Kazakhstan and in DNA Samples from Ancient Populations of the Kazakhstani Altai

The polymorphism of the major noncoding region of mitochondrial DNA (mtDNA D loop, 528 bp) has been studied in samples from three modern Kazakh populations (from Almaty, the Semipalatinsk Region, and the Altai Mountains) and in DNA samples of ancient human populations of the Kazakhstani Altai. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis for 13 restriction sites, including BamHI, EcoRV, Sau3AI (one site each), KpnI (two sites), HaeIII (three sites), and RsaI (five sites) were used. The frequency distributions of all sites have been determined. The gene diversity (h) and the genetic distances between different Kazakh populations and other populations of the world have been calculated. The RFLP analysis of the mtDNA control region of fossil samples has been performed similarly to the analysis of modern mtDNA samples. Two fossil mtDNA samples from burial mound 11 are monomorphic with respect to all restriction sites analyzed.     

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.     

用富集文库克隆人胰岛素基因组基因

通过构建可富集人胰岛素基因的λ噬菌体文库,克隆了人胰岛素基因组基因．首先从中国人血液白细胞中提取到人基因组ＤＮＡ,用ＥｃｏＲⅠ和ＢｇｌⅡ对基因组ＤＮＡ进行全酶切,经０．４％琼脂糖凝胶电泳,特异回收９．５ｋｂ左右的ＤＮＡ片段．将该片段与λＥＭＢＬ３／ＢａｍＨⅠ臂连接,构建成一个特殊的人基因组λ噬菌体文库（富集文库）,效价为２×１０４．同时采用ＰＣＲ方法及用引物Ⅰ：５′ＧＧＡＣＡＧＧＣＴＡＣＡＴＣＡＧＧＡＡＧＡＧＧ３′,引物Ⅱ：５′ＣＴＧＣＧＴＣＴＡＡＴＴＧＣＡＧＴＡＧＴＴＣ３′,从人基因组ＤＮＡ中扩增出一段含胰岛素基因的１．３６ｋｂＤＮＡ片段,做为放射性标记探针,对文库进行了噬菌斑原位杂交筛选,从１×１０４个噬菌斑中筛选到一个含人胰岛素基因组基因的阳性克隆,并进一步完成了亚克隆和该基因１７３２ｂｐＤＮＡ序列的测定．结果该基因的１７３２ｂｐＤＮＡ序列包括部分５′端和３′端与国外发表的人胰岛素α型等位基因的序列相同