Sequence arrangement of tRNA genes on a fragment of Drosophila melanogaster DNA cloned in E. coli.

A plasmid with the vector Col E1 attached to an insert of Drosophila melanogaster DNA carrying four tRNA genes has been cloned in E. coli. Some features of the sequence arrangement and the positions of the tRNA genes have been determined by electron microscopic methods and by restriction endonuclease mapping. tRNA genes were mapped at 1.4, 4.7, 5.9 and 8.6 kb from one of the Drosophila/Col E1 junctions in the Drosophila insert of total length 9.34 kb. There are several secondary structure features consisting of inverted repeat sequences of length about 70-100 nucleotide pairs, some with and some without intervening loops, irregularly distributed on the insert. Cross-hybridization of tRNAs isolated by hybridization to separated restriction fragments indicate that the tRNA genes at 4.7, 5.9 and 8.6 kb are identical and differ from the one at 1.4 kb. Thus the positions of the genes, of the secondary structure features and of the restriction endonuclease sites all indicate that the spacers between the genes are not identical tandem repeats. In situ hybridization with cRNA transcribed from the plasmid showed localization at region 42A of chromosome 2R.     

Nonallelic histone gene clusters of individual sea urchins (Lytechinus pictus): Polarity and gene organization

We have analyzed the histone genes from the sea urchin Lytechinus pictus. Examination of native DNA from individuals reveals four major Eco RI restriction endonuclease histone gene DNA fragments which have been labeled A (6.0 kb), B (4.1 kb), C (3.1 kb) and D (1.2 kb). The fragments A, B and C have been cloned into E. coli plasmids (pLpA, pLpB and pLpC). These histone gene fragments display length and sequence heterogeneity in different individuals. The plasmid pLpA contains the coding regions for H1, H4, H2B and H3 histones, and we determined that the DNA fragment D is tandem to A in native DNA and that it contains the H2A gene. The plasmids pLpB and pLpC contain the histone genes H2A-H1-H4 and H2B-H3, respectively, and together contain the sequences for the five major histones. Restriction analysis of native L. pictus DNA reveals that B and C are tandem to each other but not intermingled with the A-D-type repeat units, and are thus in separate clusters with a repeat length of 7.2 kb. Since the two cluster types do not segregate, they are not alleles. Hybridization of histone mRNA to exonuclease III-digested linear DNA demonstrated an identical polarity of the histone genes in the A-D- and B-C-type repeat units. This result revealed that the L. pictus histone genes have a polarity which is the same as other sea urchin histone genes examined to date—that is, 3′ H1-H4-H2B-H3-H2A 5′. Restriction endonuclease cleavage patterns of the cloned segments indicate that considerable sequence heterogeneity exists between the two types of histone gene repeat units.     

絮凝基因的克隆和在工业啤酒酵母菌株中表达

The partial genomic library of Saccharomyce cerevisiae FL189 possessing strong flocculation ability was constructed using Yeast-E.coli shuttle plasmid YCp50 as vector.Recombinant plasmid containing flocculation gene was obtained by screening the growth of transformants on the selective medium and measurement flocculation,designated as pCF1.pCF1 was introduced into industrial yeast strain PJ208-5-15.Six transformants PJ208-5-15-1(pCF1)～PJ208-5-15-6(pCF1)possessing strong flocculation ability were obtained.Th…     

Positions of sea urchin (Strongylocentrotus purpuratus) histone genes relative to restriction endonuclease sites on the chimeric plasmids pSp2 and pSp17.

The positions of the several sea urchin histone genes on the eukaryotic fragments of the chimeric plasmids pSp2 and pSp17 have been mapped relative to the Eco RI and Hind III restriction endonuclease sites on the plasmids. Two principal mapping methods using the electron microscope have been used: (a) the R-loop procedure and a new modification thereof to map the genes on duplex DNA; (b) the gene 32-ethidium bromide technique to visualize RNA-DNA hybrids on single strands of DNA. It is known that there are two histone genes, H3 and H2A, on pSp17. There are two Eco RI sites at the two junctions of the procaryotic segment with the eucaryotic segment on the plasmid. We show, by an electron microscope method, that for H2A, with a length of 0.52 kilobases (kb), one end of the gene is situated 0.02 to 0.03 kb from one RI site, and that there is a Hind III site within this gene at about 0.13 kb from the end phe other RI site of this plasmid. The H4 gene lies between H2B and H1. The ms the incubation temperature is raised up to a temperature just below that at which strand dissociation of the duplex DNA occurs.     

The replicator region of the Rhizobium leguminosarum cryptic plasmid pRL8JI

Abstract The replicator region of the cryptic plasmid pRL8JI from Rhizobium leguminosarum strain 3841 was cloned and sequenced. The recombinant plasmid (pYK3) was selected by function from a partial Eco RI library of total DNA cloned in pSUP202 and shows incompatibility with plasmid pRL8JI when conjugated into R. leguminosarum strains 3841 and its derivative 1062. The cloned insert (∼ 10.5 kb) comprises five Eco RI fragments none of which confers replicative stability when cloned individually. A single 5.0-kb Bam HI fragment, that spans all five Eco RI fragments and confers replicative stability on pSUP202 in R. leguminosarum , has been sequenced. This replicator region shows organisational and sequence similarity to the replicator regions of the Agrobacterium plasmids pTiB6S3 and pRiA4b. It has three open reading frames ( repA, repB, repC ) and a conserved intergenic sequence.     

Isolation and characterization of a cryptic plasmid from Erwinia citreus ATCC 31623

A multiple-copy plasmid pPZG500 (3·8 kb) was isolated from a phytopathogenicbacterium Erwinia citreus ATCC 31623. This is the smallest plasmid so far isolated fromthe genus Erwinia . The plasmid was partially characterized by a set of restriction enzymesand the unique restriction sites were mapped for Hin dIII, Eco RI, Eco RVand XBa I, while three sites were found for Bgl II. Nineteen other enzymes did notcut pPZG500. By deletion analyses minimal regions required for replication ( ori ) andsegregational stability ( par ) were localized on 1·4 kb Eco RV/ Bgl IIand 0·7 kb Bgl /II/ Eco RI fragments, respectively. The erythromycin resistancemarker (Em^r) was cloned into pPZG500 and two plasmid derivatives, pPZG502 andpPZG503, were constructed expressing erythromycin resistance as a good selective marker forrecombinant selection in Erw. citreus and Escherichia coli . The segregationalstability of both constructed plasmids during 90 generations in E. coli JM109 and Erw.citreus C-4 showed that plasmid pPZG503 lacking the presumptive par region was lost fromthe population at a higher rate. The results of this study demonstrate that plasmid pPZG500 andderivatives are suitable prerequisites for the construction of useful cloning vector(s) in the genus Erwinia .     

Molecular cloning and characterization of form I antigen genes of Shigella sonnei.

Sau3AI-generated DNA fragments of the Shigella sonnei large plasmid encoding the form I antigen were cloned into Escherichia coli with cosmid vector pHSG262. One resulting plasmid, designated pJK1137, was studied further. Restriction endonuclease mapping and analysis of transposon Tn3 insertion mutants demonstrated that the form I antigen genes were located within a region of about 12.6 kb consisting of the two contiguous HindIII fragments of 1.26 kb and 12.4 kb. The results of complementation studies between Tn3 insertion mutants of pJK1137 and recombinant plasmids carrying different parts of the form I antigen genes indicated that the 12.6 kb DNA sequence contained at least four gene clusters, regions A, B, C and D. Analysis of radioactively labelled proteins in minicells demonstrated that the DNA sequence of about 12.6 kb coded for at least four specific proteins of 42, 23, 48 and 39 kDa. The former two were coded by region A, the latter two by region D.     

A family of moderately repetitive sequences in mouse DNA.

When mouse DNA is digested to completion with restriction endonuclease Eco R1, a distinct band of 1.3 kb segments comprising about 0.5-3% of the genome is observed upon agarose gel electrophoresis. This DNA is not tandemly repeated in the genome and is not derived from mouse satellite DNA. Restriction endonuclease analysis suggested that the 1.3 kb segments are heterogeneous. Specific sequences were selected from the 1.3 kb segments and amplified by cloning in plasmid pBR322. Southern transfer experiments indicated that three separately cloned mouse DNA inserts hybridized predominantly to the Eco R1 1.3 kb band and to the conspicuous subsegments generated by secondary restriction endonuclease cleavage of the sucrose gradient purified 1.3 kb segments. Segments were also excised by Hha I (Hha I segments) from the chimeric plasmids containing mouse DNA inserts and subjected to restriction endonuclease and cross-hybridization analysis. It was found that the three Hha I segments were different, although two of them exhibited partial sequence homology. Cot analysis indicated that each of the Hha I segments are repeated about 10(4) times in the mouse genome. These findings indicate that a family of related but non-identical, moderately repetitive DNA sequences, rather than a single homogeneous repeat, is present in the 1.3 kb Eco R1 band.     

Purification and properties of the Eco57I restriction endonuclease and methylase--prototypes of a new class (type IV).

The Eco57I restriction endonuclease and methylase were purified to homogeneity from the E.coli RR1 strain carrying the eco57IRM genes on a recombinant plasmid. The molecular weight of the denaturated methylase is 63 kDa. The restriction endonuclease exists in a monomeric form with an apparent molecular weight of 104-108 kDa. R.Eco57I also possesses methylase activity. The methylation activities of both enzymes modify the outer A residue in the target sequence 5'CTGAAG yielding N6-methyladenine. M.Eco57I modifies both strands of the substrate while R.Eco57I modifies only one. Only the methylase enzyme is stimulated by Ca2+. The restriction endonuclease shows an absolute requirement for Mg2+ and is stimulated by AdoMet. ATP has no influence on either activity of the enzymes. The subunit structure and enzymatic properties of the Eco57I enzymes distinguish them from all other restriction-modification enzymes that have been described previously. Therefore, RM.Eco57I may be regarded as a representative of a novel class of restriction-modification systems, and we propose to classify it as type IV.     

Cloning and endonuclease restriction analysis of argF and of the control region of the argECBH bipolar operon in Escherichia coli.

A 1.8 kb DNA fragment, liberated by endonuclease HindIII, contains the control region of the argECBH bipolar operon near one end and the weak secondary promoter of argH at the other extremity; it has been cloned in plasmid pBR322. The same plasmid vector has been used to clone the argF gene liberated from the chromosome by endonuclease BamHI. Restriction patterns for the two hybrid plasmids have been determined, using enzymes AluI, BglI, EcoRI, HaeIII, HincII, HindIII, HpaI and II, PstI and SalI. Two AluI sites situated on either side of and close to a HincII target delineate two short fragments covering the whole of the argECBH control region. The argF control elements are located in a region accessible to further dissection by BamHI, EcoRI, PstI and HindIII. Carriers of the argF plasmid produce extremely high amounts of ornithine carbamoyltransferase, a feature useful for purification of this enzyme.     

Cloning of pectate-lyase genes of Erwinia chrysanthemi in Escherichia coli cells

Erwinia chrysanthemi DNA fragment digested by restriction endonuclease EcoRI and carrying the gene EC16 determining the synthesis of pectatelyase with Rf 0.20 and mol. mass 40kD has been cloned in plasmid pUC 9 plasmid in Escherichia coli HB101 cells. Three genes for pectatelyases of Erwinia chrysanthemi ENA49 have been cloned in vector phage lambda 47.1 in Escherichia coli cells. Two genes determining the synthesis of pectatelyases with Rf 0.06 and 0.19 and mol. masses 40 kD and 39 kD have been cloned as a part of an 7 kb Eco RI-fragment, that suggested their close location on the chromosome of Erwinia chrysanthemi ENA49. All of the cloned pectatelyase genes are expressed constitutively with pectatelyases accumulating in periplasm and being unable to secret into the cultural medium.     

Cloning and sequence analysis of the genes coding for Eco57I type IV restriction-modification enzymes.

A 6.3 kb fragment of E.coli RFL57 DNA coding for the type IV restriction-modification system Eco57I was cloned and expressed in E.coli RR1. A 5775 bp region of the cloned fragment was sequenced which contains three open reading frames (ORF). The methylase gene is 1623 bp long, corresponding to a protein of 543 amino acids (62 kDa); the endonuclease gene is 2991 bp in length (997 amino acids, 117 kDa). The two genes are transcribed convergently from different strands with their 3'-ends separated by 69 bp. The third short open reading frame (186 bp, 62 amino acids) has been identified, that precedes and overlaps by 7 nucleotides the ORF encoding the methylase. Comparison of the deduced Eco57I endonuclease and methylase amino acid sequences revealed three regions of significant similarity. Two of them resemble the conserved sequence motifs characteristic of the DNA[adenine-N6] methylases. The third one shares similarity with corresponding regions of the PaeR7I, TaqI, CviBIII, PstI, BamHI and HincII methylases. Homologs of this sequence are also found within the sequences of the PaeR7I, PstI and BamHI restriction endonucleases. This is the first example of a family of cognate restriction endonucleases and methylases sharing homologous regions. Analysis of the structural relationship suggests that the type IV enzymes represent an intermediate in the evolutionary pathway between the type III and type II enzymes.     

Isolation and characterization of cloned human fetal globin genes.

Three clones containing both the human G gamma and A gamma globlin genes have been isolated and characterized from a library of DNA fragments generated by partial Eco RI digestion of cellular DNA using charon 4A phage as vector. Two of the clones (NY 2 and 3) are identical and have an insert of 14.0 kb. The third clone (NY 1) has a 15.4 kb insert by virtue of an extra 1.4 kb Eco RI fragment at its 5' most end. This clone also has a Kpn I site not present in the other two suggesting it is the product of the gamma gene on the opposite chromosome. Restriction analysis of the three clones indicates that the G gamma and A gamma genes are linked on a single continuous piece of DNA and are separated by 3.5 kb and each contains at least one large intervening sequence of 0.85 kg between the Bam HI and Eco RI sites. These findings in cloned DNA provide direct evidence for linkage and organization of the gamma genes in man.     

茶尺蠖核型多角体病毒多角体蛋白基因核苷酸序列及其在Baculoviridae中的地位(英文)

茶尺蠖核型多角体病毒（ＥｏＳＮＰＶ）基因组的ｐｏｌｈ和ｅｇｔ基因区约１４．２ｋｂ的酶切图谱被构建．ｅｇｔ基因位于ｐｏｌｈ基因上游约４．８ｋｂ处,但转录方向与ｐｏｌｈ基因相反．ＥｃｏＲⅤ－Ｌ片段ｐｏｌｈ基因及其旁侧的１１２５核苷酸序列被测定．ｐｏｌｈ基因编码区长７３８核苷酸,可编码２４６氨基酸的多肽．起始密码子ＡＴＧ上游是一个富含ＡＴ（ＡＴ占７１．２％）的启动子区,在－５２核苷酸处有杆状病毒晚期基因启动子转录起始基序ＡＴＡＡＧ．在终止密码子下游２０８核苷酸有一个ｐｏｌｙ（Ａ）信号,ＡＡＴＡＡＡ．但ＥｏＳＮＰＶｐｏｌｈ基因起始密码子ＡＴＧ相邻核苷酸序列为ＧＴＡＡＴＧＴ,其－３是个Ｇ,这与已知的１６种其它杆状病毒ｐｏｌｈ基因－３位置均是Ａ不相同．在分析了ＥｏＳＮＰＶ和ＨａＳＮＰＶ多角体蛋白基因核苷酸序列的基础上,通过ＭＡＬＩＧＮ程序,比较了目前已发表的２６种杆状病毒包涵体蛋白的序列,ＥｏＳＮＰＶ与黄杉毒蛾核型多角体病毒（ＯｐＳＮＰＶ）的同源性为最高,核苷酸序列的同源性为８３．０％,氨基酸序列达９４．７％;与其它２０种鳞翅目ＮＰＶ的同源性也很高,核苷酸序列同源性为７２．６％～８１．９％,氨基酸序列为８３．７％～９３     

Structural analysis of a mutational hot-spot in the EcoRV restriction endonuclease: a catalytic role for a main chain carbonyl group.

Following random mutagenesis of the Eco RV endonuclease, a high proportion of the null mutants carry substitutions at Gln69. Such mutants display reduced rates for the DNA cleavage step in the reaction pathway, yet the crystal structures of wild-type Eco RV fail to explain why Gln69 is crucial for activity. In this study, crystal structures were determined for two mutants of Eco RV, with Leu or Glu at residue 69, bound to specific DNA. The structures of the mutants are similar to the native protein and no function can be ascribed to the side chain of the amino acid at this locus. Instead, the structures of the mutant proteins suggest that the catalytic defect is due to the positioning of the main chain carbonyl group. In the enzyme-substrate complex for Eco RV, the main chain carbonyl of Gln69 makes no interactions with catalytic functions but, in the enzyme-product complex, it coordinates a metal ion bound to the newly liberated 5'-phosphate. This re-positioning may be hindered in the mutant proteins. Molecular dynamics calculations indicate that the metal on the phosphoryl oxygen interacts with the carbonyl group upon forming the pentavalent intermediate during phosphodiester hydrolysis. A main chain carbonyl may thus play a role in catalysis by Eco RV.     

Cloning of the restriction-modification genes of Bacillus centrosporus in Escherichia coli

A genomic library of Bacillus centrosporus was obtained using pBR327 as a vector. The total plasmid DNA of the library was cleaved by the BcnI restriction endonuclease and then transformed in Escherichia coli RR1. Two clones possessing restriction and DNA modification profiles of BcnI were identified among the transformants. Their respective plasmids were 13.3 and 9.05 kbp in size. Restriction mapping of both plasmids showed each of them to contain two sites for HindIII and one for both Eco31I and Eco47III, located at the same distance. This was assumed to be the location region of the BcnI restriction-modification genes. Confirmation of the assumption was obtained by deletion mapping of the recombinant plasmids. Special features concerning cloning of the restriction-modification genes are discussed on the basis of the results obtained.