Deoxyribonucleic acid modifications and restriction endonuclease production in Neisseria gonorrhoeae.

Modification of gonococcal deoxyribonucleic acid (DNA) was investigated, and the relationship with endonuclease production was explored. Both chromosomal and plasmid DNA from different gonococcal strains, irrespective of their plasmid content, was poorly cleaved by the restriction endonucleases HaeII, HaeIII, SacII, and BamHI. The fragment pattern of the Tn3 segment present on the 7.2-kilobase gonococcal resistance plasmid, when compared to its known DNA sequence, allowed us to conclude that the HaeIII and BamHI resistance was due to modification of these sites. A comparison of the fragment pattern of the resistance plasmid, when isolated from Escherichia coli or Neisseria gonorrhoeae, revealed that the resistance of HaeII must also be due to modification of its recognition sequence. Isoschizomers of HaeII and HaeIII can be found in isolates of N. gonorrhoeae (NgoI and NgoII, respectively). A new restriction endonuclease in gonococci, NgoIII, with a specificity similar to SacII, is reported here. High-pressure liquid chromatography of gonococcal DNA showed the presence of 5-methylcytosine. It is suggested that the methylation of cytosine residues in the HaeII (NgoI), HaeIII (NgoII), and SacII (NgoIII) recognition sites is the basis for the resistance of gonococcal DNA to cleavage by these enzymes. This methylation may be part of a host restriction modification system. In two out of five gonococcal strains the sequence -GATC- was modified. One strain unable to modify this sequence was a spontaneous mutant of a strain carrying such a modifying function.     

Specificity of cleavage by restriction nuclease from Bacillus subtilis.

The restriction nuclease from B. subtilis (Bsu) which cleaves in the middle of the tetra-nucleotide sequence 5'-GGCC-3' 3'-CCGG-5' has been found to decrease its substrate specificity at high nuclease concentrations. There are special conditions, high pH, low ionic strength, and high glycerol content, which strongly enhance splitting with decreased specificity and also lead to splitting of single-stranded DNA. By sequence analyses it is shown that the reduction in specificity of Bsu corresponds to cleavage predominantly at 5'-GC-3' 3'-CG-5' sequences. No comparable change in specificity has been observed in a restriction nuclease from Haemophilus aegyptius (HaeIII), and isoschizomer of Bsu.     

Cloning the KpnI restriction-modification system in Escherichia coli

The genes encoding the KpnI restriction and modification (R-M) system from Klebsiella pneumoniae, recognizing the sequence, 5'-GGTAC decreases C-3', were cloned and expressed in Escherichia coli. Although the restriction endonuclease (ENase)- and methyltransferase (MTase)-encoding genes were closely linked, initial attempts to clone both genes as a single DNA fragment in a plasmid vector resulted in deletions spanning all or part of the gene coding for the ENase. Initial protection of the E. coli host with MTase expressed on a plasmid was required to stabilize a compatible plasmid carrying both the ENase- and the MTase-encoding genes on a single DNA fragment. However, once established, the MTase activity can be supplied in cis to the kpnIR gene, without an extra copy of kpnIM. A chromosomal map was generated localizing the kpnIR and kpnIM genes on 1.7-kb and 3.5-kb fragments, respectively. A final E. coli strain was constructed, AH29, which contained two compatible plasmids: an inducible plasmid carrying the kpnIR gene which amplifies copy number at elevated temperatures and a pBR322 derivative expressing M.KpnI. This strain produces approx. 10 million units of R.KpnI/g of wet-weight cells, which is several 1000-fold higher than the level of R.KpnI produced by K. pneumoniae. In addition, DNA methylated with M.KpnI in vivo does not appear to be restricted by the mcrA, mcrB or mrr systems of E. coli.     

Isolation and characterization of the M.EaeI modification methylase.

The modification enzyme (M.EaeI) corresponding to the restriction endonuclease EaeI was partially purified from Enterobacter aerogenes PW201. The M.EaeI enzyme methylates the innermost cytosine residue in each strand of the family of related sequences that constitute the EaeI recognition site to give: 5'-Y-G-G-5mC-C-R-3' where 5mC is 5-methylcytosine. M.EaeI protects these sites against cleavage by HaeIII, and protects overlapping 5'-C-C-G-G-3' sites against cleavage by both HpaII and MspI.     

Cloning of CviPII nicking and modification system from chlorella virus NYs-1 and application of Nt.CviPII in random DNA amplification

The cloning and expression of the CviPII DNA nicking and modification system encoded by chlorella virus NYs-1 is described. The system consists of a co-linear MTase encoding gene (cviPIIM) and a nicking endonuclease encoding gene (cviPIINt) separated by 12 nt. M.CviPII possesses eight conserved amino acid motifs (I to VIII) typical of C5 MTases, but, like another chlorella virus MTase M.CviJI, lacks conserved motifs IX and X. In addition to modification of the first cytosine in CCD (D = A, G or T) sequences, M.CviPII modifies both the first two cytosines in CCAA and CCCG sites as well. Nt.CviPII has significant amino acid sequence similarity to Type II restriction endonuclease CviJI that recognizes an overlapping sequence (RG--CY). Nt.CviPII was expressed in Escherichia coli with or without a His-tag in a host pre-modified by M.CviPII. Recombinant Nt.CviPII recognizes the DNA sequence CCD and cleaves the phosphodiester bond 5' of the first cytosine while the other strand of DNA at this site is not affected. Nt.CviPII displays site preferences with CCR (R = A or G) sites preferred over CCT sites. Nt.CviPII is active from 16 to 65 degrees C with a temperature optimum of 30-45 degrees C. Nt.CviPII can be used to generate single-stranded DNAs (ssDNAs) for isothermal strand-displacement amplification. Nt.CviPII was used in combination with Bst DNA polymerase I large fragment to rapidly amplify anonymous DNA from genomic DNA or from a single bacterial colony.     

A complex family of class-II restriction endonucleases, DsaI-VI, in Dactylococcopsis salina

A series of class-II restriction endonucleases (ENases) was discovered in the halophilic, phototrophic, gas-vacuolated cyanobacterium Dactylococcopsis salina sp. nov. The six novel enzymes are characterized by the following recognition sequences and cut positions: 5'-C decreases CRYGG-3' (DsaI); 5'-GG decreases CC-3' (DsaII); 5'-R decreases GATCY-3' (DsaIII); 5'-G decreases GWCC-3' (DsaIV); 5'-decreases CCNGG-3' (DsaV); and 5'-GTMKAC-3' (DsaVI), where W = A or T, M = A or C, K = G or T, and N = A, G, C or T. In addition, traces of further possible activity were detected. DsaI has a novel sequence specificity and DsaV is an isoschizomer of ScrFI, but with a novel cut specificity. A purification procedure was established to separate all six ENases, resulting in their isolation free of contaminating nuclease activities. DsaI cleavage is influenced by N6-methyladenine residues [derived from the Escherichia coli-encoded DNA methyltransferase (MTase) M.Eco damI] within the overlapping sequence, 5'-CCRYMGGATC-3'; DsaV hydrolysis is inhibited by a C-5-methylcytosine residue in its recognition sequence (5'-CMCNGG-3'), generated in some DsaV sites by the E. coli-encoded MTase, M.Eco dcmI.     

Intrinsic bending in GGCC tracts as probed by fluorescence resonance energy transfer

Double-stranded oligonucleotides containing the sequence 5'-GGCC-3' can be intrinsically bent, according to X-ray crystallography and gel electrophoresis mobility studies. We have performed fluorescence resonance energy transfer (FRET) experiments with dye-labeled oligonucleotides to further investigate the solution structure of this sequence. We find that 5'-GGCC-3'-containing oligonucleotides bring 5'-attached donor and acceptor dyes much closer together than a comparable "straight" sequence that contains 5'-GCGC-3'. The bend angle for the 5'-GGCC-3' sequence is estimated to be approximately 70 degrees, much larger than the crystallographically observed 23 degrees kink but in agreement with other FRET work. In contrast to gel electrophoresis studies, divalent metal ions do not promote increased kinking in 5'-GGCC-3' above background as judged by FRET. Thus, sequence-dependent structural effects in DNA may be a complicating feature of FRET experiments.     

Fine cleavage map of a small colicin E1 plasmid carrying genes responsible for replication and colicin E1 immunity.

A small plasmid (pAO2, 1 megadalton) carrying genes responsible for replication and colicin E1 immunity has been constructed from colicin E1 plasmid (A. Oka, K. Sugimoto, and M. Takanami, Proc. Mol. Biol. Jpn., p. 113-115, 1976). pAO2 DNA was cleaved into unique fragments with seven restriction endonucleases (R.HaeII,R.HaeIII,R.HapII,R.HhaI,R.AluI,R.HgaI, and R.HinfI). R.HaeII cleaved pAO2 DNA at two sites, R.HaeIII at four sites, R.HapII at nine sites, R.HhaI at eight sites, R-AluI at nine sites, R.HgaI at two sites, and R.HinfI at four sites, respectively. The order of HaeIII fragments of pAO2 was deduced from the physical map of colicin E1 plasmid previously reported (A. Oka and M. Takanami, Nature (London) 264:193-196, 1976). HapII, HhaI, and AluI fragments of pAO2 were assigned by analyzing overlapping sets of fragments arising upon digestion of individual HaeIII fragments with one of R.HapII, R.HhaI, or R.AluI, and upon their reciprocal digestion. The cleavage sites for R.HaeII, R.HgaI, and R.HinfI were localized on HapII, HhaI, and AluI fragments by combined digestion. On the basis of these data and estimates of the size of each fragment, a fine cleavage map of pAO2 was constructed.     

Cloning and organization of genes for 5S ribosomal RNA in the sea urchin. Lytechinus variegatus

A 1.35-kb EcoRI fragment of Lytechinus variegatus DNA containing a single 5S rRNA gene has been cloned into the plasmid vector pACYC184. Four clones from different transformation experiments contain 5S rDNA inserts of about the same size and have the same restriction enzyme digestion patterns for the enzymes HaeIII, HinfI, HhaI, and AluI. One EcoRI site near the HindIII site of the plasmid vector pACYC184 is missing in all the four clones. By DNA sequencing, the missing EcoRI ws found to be EcoRI site, d(AAATTN)d(TTTAAN) in pLu103, one of the four 5S rDNA clones. The structure of pLu103 was determined by restriction mapping and blot hybridization. Three restriction fragments, 1.0-kb HaeIII/HaeIII, 0.375-kb AluI/AluI and 0.249-kb MboII/MboII, which contain the 5S rRNA coding region, have been subcloned into the EcoRI site of the plasmid pACYC184. The organization of 5S rRNA genes in the sea urchin genome was also investigated. It was found that restriction endonuclease HaeIII has a single recognition site within each 5S rDNA repeat, and yields two fragment lengths, 1.2 and 1.3 kb. The behavior of these 5S rRNA genes when total L. variegatus DNA is partially digested with HaeIII is consistent with an arrangement of 5S rRNA genes in at least two tandemly repeated, non-interspersed families. Both the coding region and spacer region of the 5S rRNA gene in pLu103 hybridize to 1.2 and 1.3-kb rDNA families. This indicates that the cloned EcoRI fragment of 5S rDNA in pLu103 represents one single repeat of 5S rDNA in the genome.     

Purification, cloning and sequence analysis of RsrI DNA methyltransferase: lack of homology between two enzymes, RsrI and EcoRI, that methylate the same nucleotide in identical recognition sequences.

RsrI DNA methyltransferase (M-RsrI) from Rhodobacter sphaeroides has been purified to homogeneity, and its gene cloned and sequenced. This enzyme catalyzes methylation of the same central adenine residue in the duplex recognition sequence d(GAATTC) as does M-EcoRI. The reduced and denatured molecular weight of the RsrI methyltransferase (MTase) is 33,600 Da. A fragment of R. sphaeroides chromosomal DNA exhibited M.RsrI activity in E. coli and was used to sequence the rsrIM gene. The deduced amino acid sequence of M.RsrI shows partial homology to those of the type II adenine MTases HinfI and DpnA and N4-cytosine MTases BamHI and PvuII, and to the type III adenine MTases EcoP1 and EcoP15. In contrast to their corresponding isoschizomeric endonucleases, the deduced amino acid sequences of the RsrI and EcoRI MTases show very little homology. Either the EcoRI and RsrI restriction-modification systems assembled independently from closely related endonuclease and more distantly related MTase genes, or the MTase genes diverged more than their partner endonuclease genes. The rsrIM gene sequence has also been determined by Stephenson and Greene (Nucl. Acids Res. (1989) 17, this issue).     

Gene structure and nucleotide sequence for rat cytochrome P-450c

Two clones from rat genomic libraries that contain the entire gene for rat cytochrome P-450c have been isolated. lambda MC4, the first clone isolated from an EcoR1 library, contained a 14-kb insert. A single 5.5-kb EcoR1 fragment from lambda MC4, the EcoR1 A fragment, hybridized to a partial cDNA clone for the 3' end of the cytochrome P-450c mRNA. This fragment was sequenced using the dideoxynucleotide chain termination methodology with recombinant M13 bacteriophage templates. Comparison of this sequence with the complete cDNA sequence of cytochrome P-450MC [Yabusaki et al. (1984) Nucleic. Acids Res. 12, 2929-2938] revealed that the EcoR1 A fragment contained the entire cytochrome P-450c gene with the exception of a 90-bp leader sequence. The gene sequence is in perfect agreement with the cDNA sequence except for two bases in exon 2. A second genomic clone, lambda MC10, which was isolated from a HaeIII library, contains the missing leading sequence as well as 5' regulatory sequences. The entire gene is about 6.1 kb in length with seven exons separated by six introns, all of the intron/exon junctions being defined by GT/AG. Amino- and carboxy-terminal information are contained in exons 2 and 7, respectively. These exons contain the highly conserved DNA sequences that have been observed in other cytochrome P-450 species. Potential regulatory sequences have been located both 5' to the gene as well as within intron I. A comparison of the coding information for cytochrome P-450c with the sequence of murine cytochrome P3-450 and rat cytochrome P-450d revealed a 70% homology in both the DNA and amino acid sequence, suggesting a common ancestral gene. Genomic blot analyses of rat DNA indicated that the 3-methylcholanthrene-inducible family of cytochrome P-450 isozymes is more limited in number compared to the phenobarbital-inducible isozymes. Cross-hybridization studies with human DNA suggest a high degree of conservation between rat cytochrome P-450c and its human homolog although gross structural differences do exist between the two genes.     

稻瘟病菌(Magnaporthe grisea)诱导的水稻早期反应基因ER1的5' 端cDNA片段克隆及序列分析

研究高等生物基因表达与调控的一个重要方面是分离基因的编码区及其上游的调控序列（ＤｅＶｅｅｒ等１９９７）,这需要获得一个基因的ｃＤＮＡ全长及从植物基因组获取全基因。在前文（周建明等１９９９）中曾经分离了稻瘟病菌侵染诱导的水稻早期反应基因ＥＲ１的ｃＤＮＡ片段,但是运用ｍＲＮＡ差异显示技术分离的ｃＤＮＡ片段往往只有近ｍＲＮＡ３’端的一部分,难以反映基因的结构及功能特点,因此,必须进一步分离其５’端的部分才有可能比较全面地了解此基因的特点。ＲＡＣＥ（ｒａｐｉｄａｍｐｌｉｆｉｃａｔｉｏｎｏｆｃＤＮＡｅｎ…     

In-vivo-modified gonococcal plasmid pJD1. A model system for analysis of restriction enzyme sensitivity to DNA modifications

The 4207-bp cryptic plasmid (pJD1) of Neisseria gonorrhoeae has 5-methylcytosine bases present at several positions in the DNA sequence. Fortuitously, these modified bases lie in the recognition sequences of many restriction enzymes. This feature makes the cryptic plasmid a model system for assaying the effect of these modified cytosines on the activities of the following restriction endonucleases and their isoschizomers: R X AvaII, R X BamHI, R X BglI, R X Fnu4HI, R X HaeII, R X HaeIII, R X HhaI, R X HpaII, R X KpnI, R X MspI, R X NaeI, R X NarI, R X NciI, R X NgoI, R X NgoII, and R X Sau96I. Of particular interest was the finding that methylation of one of the external cytosines of the palindrome 5'-CCGG-3' prevented its cleavage by R X MspI, but not by R X HpaII as had been suggested by Walder et al. [J. Biol. Chem. (1983) 258, 1235-1241].     

桑泛素延伸蛋白基因片段的克隆及序列分析

目的:利用3’RACE技术克隆植物泛素基因,是进一步研究其功能的基础。方法:本研究从桑树(丰驰桑)(Morus bomby-cis)幼叶中提取总RNA,反转录成cDNA,根据已报道的泛素基因序列设计1条正向引物,利用3’RACE(Rapid Amplification of cDNAEnd)技术进行扩增。结果:扩增出1条690 bp的泛素基因片段。该片段5’端为编码156个氨基酸残基的阅读框,3’末端有219bp的非翻译区。结论:同源分析表明,此cDNA序列为泛素延伸蛋白基因(Genebank登录号为DQ839403)。用Genedoc软件对该片段编码的氨基酸序列进行同源性分析的结果表明:桑树泛素延伸蛋白与马铃薯、烟草、陆地棉、黄瓜的泛素延伸蛋白以及苜蓿的核糖体S27A蛋白的同源性都在96%以上。     

DsaV methyltransferase and its isoschizomers contain a conserved segment that is similar to the segment in Hhai methyltransferase that is in contact with DNA bases.

The methyltransferase (MTase) in the DsaV restriction--modification system methylates within 5'-CCNGG sequences. We have cloned the gene for this MTase and determined its sequence. The predicted sequence of the MTase protein contains sequence motifs conserved among all cytosine-5 MTases and is most similar to other MTases that methylate CCNGG sequences, namely M.ScrFI and M.SsoII. All three MTases methylate the internal cytosine within their recognition sequence. The 'variable' region within the three enzymes that methylate CCNGG can be aligned with the sequences of two enzymes that methylate CCWGG sequences. Remarkably, two segments within this region contain significant similarity with the region of M.HhaI that is known to contact DNA bases. These alignments suggest that many cytosine-5 MTases are likely to interact with DNA using a similar structural framework.     

金龟子绿僵菌中性海藻糖酶基因的克隆及其表达特性分析

根据中性海藻糖酶NTL基因的同源序列设计引物,PCR扩增出杀蝗专一菌株———金龟子绿僵菌CQMa102NTL基因片段,利用5′_RACE和3′_RACE扩增出NTLcDNA的5′和3′端序列,经拼接得到CQMa102NTL基因cDNA全长。根据其全长cDNA序列,设计引物PCR扩增出CQMa102NTL的完整基因。为了解该基因的上游调控信息,采用PanhandlePolymeraseChainReactionAmplification方法扩增其上游序列。序列分析表明,CQMa102NTL全长DNA3484bp,cDNA全长2385bp,编码737个氨基酸的蛋白,推测蛋白分子量为83.1kD;含有3个内含子,包含一个依赖于cAMP的磷酸化作用位点(RRGS)和一个钙附着位点(DTDGNMQITIED);上游序列含有一个压力反应元件(CCCCT);与金龟子绿僵菌广谱性菌株ME1NTL的核苷酸序列和氨基酸序列分别具有93%和99%同源性,由此确定该序列为金龟子绿僵菌中性海藻糖酶基因序列。Southern杂交表明,NTL基因在CQMa102基因组中为单拷贝。Northern杂交表明,NTL基因转录出约2.5kb的mRNA单带,在液体培养条件下,对数生长前期表达水平最高,对数生长后期降到最低,进入稳定生长期后表达水平又有所提高。金龟子绿僵菌CQMa102中性海藻糖酶基因DNA全长和cDNA全长登录GenBank,登录号分别为:AY557613,AY557612。