Analysis of production brewing strains of yeast by DNA fingerprinting

P. WIGHTMAN, D.E. QUAIN AND P. G. MEADEN. 1996. Production brewing strains of the yeast Saccharomyces cerevisiae were analysed by DNA fingerprinting, using a Southern blotting and hybridization procedure and employing the Tyl-15 transposon as a probe. The ability to differentiate readily between strains was very dependent on the restriction enzyme used to digest the DNA prior to Southern blotting and hybridization; the enzymes Eco RI, Pst I and Sal I were found to be particularly useful in this respect. The method was applicable to the differentiation of both ale and lager yeasts, and was sufficiently sensitive to distinguish between very closely related strains. DNA fingerprinting by this approach confirmed, for example, that a flocculent strain isolated during a production-scale fermentation with a lager yeast was genotypically different from the parent.     

Translation in Escherichia coli mini-cells containing hamster mitochondrial DNA-Co1E1 - Ampr recombinant plasmids.

Hamster mitochondrial DNA is cleaved into two fragments (4.2 and 11.4 kilobase pairs of DNA (kb)) by the restriction enzyme, Eco RI. Recombinant DNA molecules formed in vitro between an Escherichia coli plasmid, Co1E1 - Ampr, and Eco RI-digested hamster mitochondrial DNA were transformed into E. coli K12. The translation products of the parent plasmid, Co1E1 - Ampr, and recombinant plasmid DNAs containing (i) the 4.2 kb mitochondrial DNA fragment and (ii) the 11.4 kb fragment were characterized on sodium dodecyl sulfate-polyacrylamide gels using bacterial mini-cell lysates. The Co1E1 - Ampr plasmid specifies at least six polypeptides whose structural genes comprise 56% of the plasmid DNA. Insertion of hamster mitochondrial DNA at the Eco RI site of the plasmid alters the relative rate of synthesis of these six polypeptides and induces the occurrence of a new band on sodium dodecyl sulfate-polyacrylamide gels which is probably not specified by the inserted mitochondrial DNA sequences.     

大熊猫线粒体DNA的九种限制酶图谱

本文用9种限制性内切酶（BamHⅠ,BglⅠ,BglⅡ,EcoRⅠ,EcoRⅤ,PstⅠ,PvuⅡ,SalⅠ,XhoⅠ）分析大熊猫的线粒体DNA（mtDNA）。构建其中5种酶（BamHⅠ,EcoRⅠ,EcoRⅤ,PstⅠ,PvuⅡ）的mtDNA物理图谱。大熊猫mtDNA的分子大小约为16.4 Kb,酶切位点是随机分布。我们的结果为进一步研究大熊猫mtDNA进化提供了基础资料。     

人工雌核发育草鱼近交F1代线粒体DNA限制性内切酶分析

结合差速离心法、饱和酚／氯仿／异戊醇抽提等技术．提取并纯化了雌核发育草鱼近交F．代的线粒体DNA(mtDNA)．用8种限制性内切酶对mtDNA酶切分析．结果表明：雌核发育草鱼近交Fl代mtDNA分子大小为16、77kb,除无sail酶切位点外,其余7种酶EcoRI、BalI、。YbaI、BarnHI、PstI、XhoI各产生4、4、3、3、3、2、2个切点。与已报道的普通草鱼一致．雌核发育草鱼近交F1代与普通草鱼间未检测出限制性片断长度多态性差异、这表明,雌核发育草鱼F。代与普通草鱼mtDNA遗传结构具有同一性、     

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     

大麦DNA单限制性酶切选择性扩增多态性技术及其优化

建立和优化了大麦DNA单限制性酶切选择性扩增多态性技术体系(SADF).分别用限制酶PstI、EcoRI和MseI酶切大麦基因组DNA,再与各自相应的人工接头连接,使用带三个选择碱基的引物进行选择性扩增.结果表明:采用分别优化建立的标准PCR扩增检测体系,六个识别碱基的PstI和EcoRI的SADF均能得到丰富稳定的带形,四个识别碱基的MseI不能得到明显谱带.SADF扩增产物片段大小范围为:PstI一般在200～2000bp,而EcoRI在200～1000bp;两者在不同大麦品种中均能检测到多态性,可用于不同大麦品种的检测,但PstI得到的带型明显优于EcoRI,在大麦中得到的片段具有范围宽、分布均匀、易于观察、多态性高等特点。 Abstract:A method named selective amplification DNA fragments (SADF) using single restrictive enzyme was established and optimized in barley.The barley genome DNA was first restricted into fragments of varied length with PstI,EcoRI and MseI respectively,then ligated with synthetic adapters.The ligated sets of fragments were used as templates for PCR amplification.Ideal amplification results could be obtained with different amplification procedure for PstI and EcoRI.Except MseI,both PstI and EcoRI could obtain abundant and reproducible bands,but SADF of PstI was more suitable for barley studies because of wider,more recognizable and polymorphic distribution of bands.     

Physical map of the bacteriophage L (Salmonella typhimurium)

Abstract A circular restriction map of the genome of the phage L ( Salmonella typhimurium ) has been constructed with five restriction endonucleases, Eca I, Eco RI, Bam HI, Bgl I, and Pst I. The Eco RI fragments of phage-L DNA were cloned into pACYC184, and the resulting recombinant plasmids pL1, pL2,…,pL7 were introduced into Salmonella typhimurium . The genes present on the fragments cloned were identified by the marker rescue experiments with the L-phage amber mutants. A physical gene map of the L genome obtained in this way was compared with that of P22.     

Comparative restriction analysis of chromosomal DNA of strains of Photobacterium leiognathi

Chromosomal DNA in 5 hereditary variants occurring in Photobacterium leiognathi population was subjected to restriction analysis. The variants differed in the levels and regulation of luminescence and colony morphology. Agarose electrophoresis of DNA fragments isolated after exposure to Hind II, Bam HI, Bgl I and Pst I restriction endonucleases revealed respectively 38, 28, 35 and 29 fragments equally distributed by their molecular weights. Electrophoregrams of the 5 strains were absolutely identical. After exposure of DNA of all the strains to PVu II, Xho II, Sal GI and Eco RI restriction endonucleases there were detected no fragments. The pleoiotropic genetic variation in these strains was not associated with large deletions or amplification of chromosomal DNA regions.     

Physical mapping of genes on yeast mitochondrial DNA: Localization of antibiotic resistance loci, and rRNA and tRNA genes

Summary We have physically mapped the loci conferring resistance to antibiotics that inhibit mitochondrial protein synthesis (erythromycin, chloramphenicol and paromomycin) or respiration (oligomycin I and II), as well as the 21s and 14s rRNA and tRNA genes on the restriction map of the mitochondrial genome of the yeast Saccharomyces cerevisiae. The mitochondrial genes were localized by hybridization of labeled RNA probes to restriction fragments of grande (strain MH41-7B) mitochondrial DNA (mtDNA)¹ generated by endonucleases EcoRI, HpaI, BamHI, HindIII, SalI, PstI and HhaI. We have derived the HhaI restriction fragment map of MH41-7B mit DNA, to be added to our previously reported maps for the six other endonucleases.The antibiotic resistance loci (ant ^R) were mapped by hybridization of ³H-cRNA transcribed from single marker petite mtDNA's of low kinetic complexity to grande restriction fragments. We have chosen the single Sal I site as the origin of the circular physical map and have positioned the antibiotic loci as follows: C (99.5-1.Ou)-P(27-36.Ou)-O_II (58.3-62u)-O_I (80-84u)-E (94.4-98.4u). The 21s rRNA is localized at 94.4-99.2u, and the 14s rRNA is positioned between 36.2-39.8u. The two rRNA species are separated by 36% of the genome. Total mitochondrial tRNA labeled with ¹²⁵I hybridized primarily to two regions of the genome, at 99.5-11.5u and 34-44u. A third region of hybridization was occasionally detected at 70-76u, which probably corresponds to seryl and glutamyl tRNA genes, previously located to this region by petite deletion mapping.Supported by USPHS Training Grant T32-GM-07197.Supported by USPHS Training Grant 5-T01-GM-0090-19.The Franklin McLean Memorial Research Institute is operated by the University of Chicago for the U. S. Energy Research and Development Administration under Contract EY-76-C-02-0069.     

Cloning of a Vero toxin (VT2) gene from a VT2-converting phage isolated from Escherichia coli 0157: H7

Abstract A Vero toxin (VT2 or Shiga-like toxin II)-converting phage was isolated from Escherichia coli 0157: H7 strain J-2. Nontoxigenic E. coli C600 produced VT2 when lysogenized with the toxin-converting phage. Eco RI fragments of the phage DNA were ligated with Eco RI-digested pBR322 or pUC118 and were transformed into E. coli MC1061 or MV1184. Transformants exhibiting VT2 production commonly contained a 4.6 kb Eco RI fragment. It was found that a 2.3 kb Kpn I- Sph I fragment coded VT2 production and that this fragment hybridized weakly with the 2.1 kb fragment encoding VT1.     

The gene for the large subunit of ribulose-1,5-bisphosphate carboxylase in Euglena gracilis chloroplast DNA: location, polarity, cloning, and evidence for an intervening sequence

The gene for the large subunit (LS) of ribulose-1,5,-bisphosphate carboxylase of Euglena gracilis Z chloroplast DNA has been mapped by heterologous hybridization with DNA restriction fragments containing internal sequences from the Zea mays and Chlamydomonas reinhardii LS genes. The Euglena LS gene which has the same polarity as the Euglena rRNA genes has been located with respect to Pst I, Pvu I, and HindIII sites within the Eco RI fragment Eco A. The region of Euglena chloroplast DNA complementary to an 887 bp internal fragment from the Chlamydomonas chloroplast LS gene is interrupted by a 0.5-1.1 kbp non-complementary sequence. This is the first chloroplast protein gene located on the Euglena genome, and the first evidence for an intervening sequence within any chloroplast protein gene.     

A restriction endonuclease cleavage map of mitochondrial DNA from transformed hamster cells.

Mitochondrial DNA from cultured C13/B4 hamster cells was cleaved by the restriction endonucleases Hpa II, Hind III, Eco RI and Bam HI into 7, 5, 3 and 2 unique fragments, respectively. The summed molecular weights of fragments obtained from electrophoretic mobilities in agarose-ethidium bromide gels (with Hpa I-cleaved T7 DNA as standard) and electron microscopic analysis of fragment classes isolated from gels (with SV40 DNA as standard) were in good agreement with the size of 10.37 +/- 0.22 x 10(6) daltons (15,700 +/- 330 nucleotide pairs) determined for the intact circular mitochondrial genome. Cyclization of all Hind III, Eco RI and Bam HI fragments was observed. A cleavage map containing the 17 restriction sites (+/- 1% s.d.) was constructed by electrophoretic analysis of 32P-labeled single- and double-enzyme digestion products and reciprocal redigestion of isolated fragments. The 7 Hpa II sites were located in one half of the genome. The total distribution of the 17 cleavages around the genome was relatively uniform. The position of the D-loop was determined from its location and expansion on 3 overlapping restriction fragments.     

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。     

Physical map of Aspergillus nidulans mitochondrial genes coding for ribosomal RNA: an intervening sequence in the large rRNA cistron

Summary A detailed map of the 32 kb mitochondrial genome of Aspergillus nidulans has been obtained by locating the cleavage sites for restriction endonucleases Pst I, Bam H I, Hha I, Pvu II, Hpa II and Hae III relative to the previously determined sites for Eco R I, Hind II and Hind III. The genes for the small and large ribosomal subunit RNAs were mapped by gel transfer hybridization of in vitro labelled rRNA to restriction fragments of mitochondrial DNA and its cloned Eco R I fragment E3, and by electron microscopy of RNA/DNA hybrids.The gene for the large rRNA (2.9 kb) is interrupted by a 1.8 kb insert, and the main segment of this gene (2.4 kb) is separated from the small rRNA gene (1.4 kb) by a spacer sequence of 2.8 kb length.This rRNA gene organization is very similar to that of the two-times larger mitochondrial genome of Neurospora crassa, except that in A. nidulans the spacer and intervening sequences are considerably shorter.     

Stable heterogeneity of mitochondrial DNA in grande and petite strains of S. cerevisiae.

Several instances of mitochondrial DNA heterogeneity in grande and petite strains of Saccharomyces cerevisiae were examined. We have detected heterogeneity in the mtDNA from some of the progeny strains of a cross between two grande strains (D273-10B, MH41-7B) which differ in genome size and restriction cleavage pattern of their mtDNA. The progeny strains transmit restriction fragments characteristic of both parental strains from homologous regions of the mitochondrial genome, and this sequence heterogeneity is not eliminated by additional subcloning. Sequence diversity is more common in the mtDNA of petite than of grande strains of yeast. We have examined subclones of one petite strain to identify the origin of this variability. Many of the submolar restriction fragments persist in independent subclones of this petite after 15 and 30 cell divisions; some submolar fragments disappear, and some new fragments appear. We conclude that the observed sequence heterogeneity is due to molecular heterogeneity, i.e., to differences in the multiple copies of the petite mitochondrial genome, as well as to clonal heterogeneity. It is likely that tandem repeats on the same mtDNA molecule also differ, i.e., that there is intramolecular heterogeneity, and that this accounts for the stability of the heterogeneity. Continuing deletion is probably responsible for the appearance of “new” fragments in petite subclones.