Expression plasmid vectors containing Escherichia coli tryptophan promoter transcriptional units lacking the attenuator

Two DNA fragments which contain the Escherichia coli tryptophan promoter-operator region but lack the attenuator have been used in the construction of a series of pAT153 based plasmids suitable for the regulated expression of foreign genes in E. coli. The first, a 139-bp HhaI fragment includes 59 bp of the trp leader sequence, ending within the “attenuator peptide” coding sequence, eleven codons from the N-terminus. A fusion-type expression plasmid incorporating this fragment has been constructed. The second, a 99-bp HaeIII-TaqI fragment contains no coding sequence but includes the “attenuator peptide” SD site situated 4 bp upstream of the TaqI site. This fragment has been incorporated in expression vectors which result in the direct expression of cloned gene sequences. To further maximise expression, plasmids with directly repeating trp promoter HaeIII-TaqI units have been constructed.     

Molecular cloning,characterization and expression of a nitrofuran reductase gene ofescherichia coli

Mini-mu derivatives carrying plasmid replicons can be used to clone genesin vivo. This method was adopted to generate phasmid clones which were later screened for their ability of restore nitrofurantoin sensitivity of a nitrofuran-resistant host by eliciting nitroreductase activity. One phasmid-derived clone (pAJ101) resulted in considerable increase in nitroreductase activity when introduced into a nitrofurantoin-resistant mutant ofEscherichia coli with reduced nitroreductase activity. Subsequently, a 1.8 kb fragment obtained from pAJ101 by partial digestion with 5au3A, was subcloned into pUC18 to yield pAJ102. The nitroreductase activity attributable to pAJ102 was capable of reducing both nitrofurantoin and nitrofurazone. The polypeptides encoded by pAJ102 were identified by the minicell method. A large, well-defined band corresponding to 37 kDa and a smaller, less-defined band corresponding to 35 kDa were detected. Tnl000 mutagenesis was used to delineate the coding segment of the 1.8 kb insert of pAJ102. A 0.8 kb stretch of DNA was shown to be part of the nitroreductase gene. The gene was mapped at 19 min on theEscherichia coli linkage map.     

Degradation of Substituted Phenylurea Herbicides by Arthrobacter globiformis Strain D47 and Characterization of a Plasmid-Associated Hydrolase Gene, puhA

Arthrobacter globiformis D47 was shown to degrade a range of substituted phenylurea herbicides in soil. This strain contained two plasmids of approximately 47 kb (pHRIM620) and 34 kb (pHRIM621). Plasmid-curing experiments produced plasmid-free strains as well as strains containing either the 47- or the 34-kb plasmid. The strains were tested for their ability to degrade diuron, which demonstrated that the degradative genes were located on the 47-kb plasmid. Studies on the growth of these strains indicated that the ability to degrade diuron did not offer a selective advantage to A. globiformis D47 on minimal medium designed to contain the herbicide as a sole carbon source. The location of the genes on a plasmid and a lack of selection would explain why the degradative phenotype, as with many other pesticide-degrading bacteria, can be lost on subculture. A 22-kb EcoRI fragment of plasmid pHRIM620 was expressed in Escherichia coli and enabled cells to degrade diuron. Transposon mutagenesis of this fragment identified one open reading frame that was essential for enzyme activity. A smaller subclone of this gene (2.5 kb) expressed in E. coli coded for the protein that degraded diuron. This gene and its predicted protein sequence showed only a low level of protein identity (25% over ca. 440 amino acids) to other database sequences and was named after the enzyme it encoded, phenylurea hydrolase (puhA gene).     

Identification of replication and stability functions in the complete nucleotide sequence of plasmid pUH24 from the cyanobacterium Synechococcus sp. PCC 7942

The complete nucleotide sequence is presented for pUH24, the small plasmid of Synechococcus sp. PCC 7942. pUH24 consists of 7835bp and has a G+C content of 59%. The distribution of translation start and stop codons in the sequence allows 36 open reading frames that potentially encode polypeptides of 50 or more amino acids. We postulate that eight of these open reading frames are actual coding sequences. A region has been identified, by experiment, that contains two functions, designated pmaA and pmaB, involved in the segregational stability of the plasmid. The minimal region of pUH24 fully capable of supporting autonomous replication consists of a 3.6kb DNA fragment, which is almost entirely occupied by two overlapping genes most likely coding for essential replication proteins (repA and repB).     

Cloning of the REP-region of plasmids from a broad circle of pM3 hosts (INC P-9)]

The rep-region of a broad host range plasmid pM3 (IncP-9) has been cloned by connecting the 7.2 kb PstI fragment of the plasmid with the 1.4 kb PstI fragment of the pUC4K vector plasmid. The latter includes the gene for kanamycin resistance and is unable to replicate by itself. The obtained minireplicon designated pMT2 contains rep-genes permitting its inheritance in the different gram-negative cells and a mob-site coding for incompatibility and temperature instability characteristic of the parent plasmid pM3.     

Cloning and Expression of OX-DAPRO Degrading Genes from Soil Microbe

The strain BYK 1, capable ot utilizing DAP or OX-DAPRO as a sole source ot nitrogen and carbon was Identitied as Pseudomonas stutzeri by various microbial and biochemical tests. Transtormation experiments showed that the OX-DAPRO utilization property Is encoded by the plasmid. Restriction of the plasmid DNA toll owed by cloning ot fragments and screening on OX-DAPRO medium led to Isolation of a clone with DNA Insert of ～ 2.2 kb (designated as BSKS) which encoded OX-DAPRO degrading gel)e. The growth and OX-DAPRO utilization with this fragment was similar to wild type strain.     

The organization of sea urchin histone genes

Sucrose gradient analysis of total sea urchin DNA cleaved with theEcoRI andHind III restriction endonucleases and identification of histone coding gene sequences by hybridization with histone mRNA have elucidated the basic organization of the histone gene repeat unit. These data, plus results obtained by electrophoretic analysis of purified endonuclease-cleaved sea urchin histone DNA and hybridization with cRNA transcribed from the eucaryotic segment of constructed plasmid chimeras cloned in E. coli, show that the several DNA sequences coding for individual histone proteins are intermingled in a 7 kilobase (kb) repeat unit. Cleavage of total sea urchin DNA withEcoRI produces 2.2 and 4.8 kb fragments which are homologous with the two cloned fragments, and which are contained in a 7 kbHind III fragment. Cleavage with both enzymes reveals that the 2.2 kbEcoRI fragment contains aHind III site 0.15–0.2 kb from an end. RNA · DNA hybridization between chimeric plasmid DNA and purified individual mRNAs isolated from sea urchin embryo polyribosomes has been used to assign coding sequences to either the 2.2 or 4.8 kb region of the histone DNA repeat unit. A map of the histone genes is proposed.     

小花棘豆(Oxytripis glabra DC)毒素基因的cDNA克隆及序列测定

在完成小花棘豆毒素 95 %氨基酸序列的基础上 ,根椐已知的氨基酸序列 ,设计合成了特异简并引物 .以小花棘豆总RNA为模板 ,逆转录合成cDNA第一链 ,用置换法合成双链cDNA .用特异引物对此双链cDNA进行PCR扩增 ,将扩增后的目的基因与用SmaⅠ酶切的质粒pUC 18连接 ,转化大肠杆菌JM10 7.筛选阳性克隆进行序列分析 ,获得了OXY基因的全部序列 .经测序后测得基因序列与原氨基酸序列对照完全一致 .GenBnak数据检索说明 ,OXY基因编码序列确定是一个从未报道的序列 .此研究结果对该毒素的应用研究奠定了基础 .     

Homology of pCS1 Plasmid Sequences with Chromosomal DNA in Clavibacter michiganense subsp. sepedonicum: Evidence for the Presence of a Repeated Sequence and Plasmid Integration

Restriction fragments of pCS1, a 50.6-kilobase (kb) plasmid present in many strains of Clavibacter michiganense subsp. sepedonicum (“Corynebacterium sepedonicum”), have been cloned in an M13mp11 phage vector. Radiolabeled forms of these cloned fragments have been used as Southern hybridization probes for the presence of plasmid sequences in chromosomal DNA of this organism. These studies have shown that all tested strains lacking the covalently closed circular form of pCS1 contain the plasmid in integrated form. In each case the site of integration exists on a single plasmid restriction fragment with a size of 5.1 kb. Southern hybridizations with these probes have also revealed the existence of a major repeated sequence in C. michiganense subsp. sepedonicum. Hybridizations of chromosomal DNA with deletion subclones of a 2.9-kb plasmid fragment containing the repeated sequence indicate that the size of the repeated sequence is approximately 1.3 kb. One of the copies of the repeated sequence is on the plasmid fragment containing the site of integration.     

Localization of an origin of replication in Corynebacterium diphtheriae broad host range plasmid pNG2 that also functions in Escherichia coli

Subcloning and protoplast transformation studies identified a 2.6 kb fragment of Corynebacterium diphtheriae plasmid pNG2 which contains an origin of replication (oriR). Molecular combination of the 2.6 kb oriR cartridge with Escherichia coli plasmid pUC18CmR enabled the E. coli cloning vector to replicate within several species of Corynebacterium host cells. A 2.6 kb plasmid formed from the oriR cartridge alone is capable of replicating in E. coli. This suggests that a single origin could be used in vectors shuttling between Corynebacterium spp. and E. coli.     

Molecular cloning of a new endoglucanase gene from Clostridium cellulolyticum and its expression in Escherichia coli

Summary Two genes coding for endoglucanase activity in Clostridium cellulolyticum were cloned and expressed in Escherichia coli by using plasmid pUC18. The sizes of two fragments harbouring endoglucanase genes are 4.4 kb and 2.0 kb, respectively. The 2.0-kb fragment was identical with a reported DNA fragment encoding an endoglucanase of C. cellulolyticum. The 4.4-kb fragment was obtained first in this study. Deletion analysis showed that a 1.3-kb portion of the 4.4-kb fragment is necessary for the endoglucanase expression by its own promoter. The 4.4-kb fragment hybridized with several different fragments of the genomic DNA in C. cellulolyticum.Offprint requests to: T. Kodama     

降解2,4-二氯酚微生物的分离及其2,4-二氯酚羟化酶基因的克隆和表达

从土壤中分离到一株降解2,4-二氯酚能力较强的细菌菌株GT241-1,经鉴定该菌株属于假单胞菌属。菌株GT241-1在最适条件下能在48h内将90mg/L的2,4-DCP降解91%,能利用2,4-二氯酚、2,4-二氯苯氧乙酸、苯甲酸和儿茶酚为唯一碳源生长。采用Southern杂交对2,4-二氯酚羟化酶基因（dcpA）定位后构建基因组文库,再用斑点杂交筛选目的转化子,克隆了该菌株的dcpA。序列测定得知含dcpA的亚克隆片段全长2389bp,其中dcpA基因编码区1797bp。核苷酸和氨基酸序列分析表明,dcpA与已在GenBank登记的相关基因有一定的差异。dcpA基因能够在大肠杆菌转化子中成功地表达有生物活性的酶。     

柞蚕核型多角体病毒(ApNPV)转移载体质粒pAp M2614的组建

自从美国科学家G.Smith等首次建立苜蓿尺蠖核型多角体病毒(AcNPV)转移载体表达系统以来,已被广泛用于外源基因的表达,成为世界上一新的具有巨大潜力的载体表达系统。为了进一步提高表达产量,降低成本,日本科学家前田进建立了家蚕核型多角体病毒(BmNPV)载体表达系统,并获得了高效表达。柞蚕是我国特产,以蛹滞育越冬,保存时间长,个体大,可工厂化生产。因此,组建柞蚕NPV转移载体,进而建立该载体表达系统,是目前利用昆虫活体为宿主进行外源基因表达较理想的昆虫杆状病毒载体表达系统。     

Cloning and characterization of a linear 2.3 kb mitochondrial plasmid of maize

Summary A linear 2.3 kb DNA molecule found in maize mitochondria was cloned into pUC8. A natural deletion of this plasmid, found in cmsT and some N (fertile) types of maize plants, was mapped to one end of the plasmid. A minor sequence homology to S-2, another linear mitochondrial plasmid, was detected, as well as more significant sequence homology with chloroplast and maize nuclear DNA. Hybridization to teosinte mitochondrial DNA (mtDNA) revealed the presence of part of the maize plasmid in the high molecular weight mtDNA of the maize relatives. RNA dot hybridization indicates that the plasmid is transcribed in mitochondria. The termini of the 2.3 kb linear plasmid contain inverted repeated sequences; of the first 17 nucleotides of the termini, 16 are identical to the terminal inverted repeats of the linear S plasmids found in the mitochondria of cmsS maize plants.     

Molecular cloning and expression of the Cellulomonas flavigena cell-associated amylase gene in E. coli and Cellulomonas flavigena

Summary The gene encoding the inducible cell-associated amylase activity was cloned on a 1.5kb Pst1 fragment into pUC8 in E.coli giving the recombinant plasmid pJA 871, and subcloned onto a shuttle vector, pJA85, and transferred into Cellulomonas flavigena AP1(amy^-). Expression was observed in both organisms with increased levels being observed from the recombinant in Cellulomonas compared to the parent strain. The 1.5kb fragment was reoriented in pJA871 and the same level of expression observed in both orientations. Tn1000 insertions into the cloned fragment revealed the location of the coding region. Nucleotide sequencing of both ends of the cloned fragment revealed one open reading frame preceded by a putative control region.