麦迪霉素产生菌具有启动功能的DNA片段的克隆和分析

利用启动子探针质粒载体pIJ486从麦迪霉素产生菌总DNA中克隆得到了一段具有启动功能的DNA片段.通过限制性酶酶切分析,测定插入DNA片段大小为2.3kb.又利用载体pIJ486和pIJ487的新霉素抗性结构基因上游有多酶切点方向相反的性质,分析了插入片段在两个不同方向上的启动能力.结果表明,在两个方向上均有启动功能,但强弱相差六倍.其中在XbaI-HindIII方向上具有较强的启动能力,在变铅青链霉菌中新霉素抗性水平可达20mg/ml以上.进一步对插入片段的三个BamHI小片段进行分析的结果表明,较强启动子区域集中在BamHI-BamHI 0.79kb DNA片段上.     

Rapid preparation of vector-free hybridization probes suitable for screening recombinant libraries

A procedure is described to rapidly prepare radioactively labeled DNA inserts from crude recombinant plasmid DNA preparations. These probes can subsequently be used to identify homologous nucleotide sequences in bacteria containing recombinant plasmids by colony hybridization. In a single procedure, crude recombinant plasmid DNA is both ³²P-labeled and fragmented by nick-translation in the presence of sufficient pancreatic DNase I to produce radioactive DNA of about 0.2–0.3-kb single-strand length. At this DNA fragment length the majority of the vector and insert sequences are on different DNA fragments. The insert DNA can then be separated from vector and contaminating Escherichia colt host chromosomal DNA by the following method. The DNA fragment population is first denatured and renatured under conditions such that the recombinant plasmid DNA reassociates but host DNA does not. The renatured plasmid DNA fragments are separated from the denatured host DNA by hydroxylapatite chromatography. The plasmid DNA fragments are then denatured and renatured with an excess of insert-free vector DNA. Conditions are chosen such that the insert DNA remains single-stranded while the vector DNA becomes double-stranded. The single-stranded insert DNA can be separated from the double-stranded vector DNA on hydroxylapatite and used directly for colony hybridization.     

在枯草杆菌中有启动子功能的噬菌体T5 DNA片段的克隆

用启动子探针质粒pTG 402为载体,用鸟枪射击法克隆了噬菌体T5 DNA的片段。克隆中的2％含有具启动子功能的插入片段,其中pTG 402-20含有启动功能最强的插入片段。DNA-DNA分子杂交实验结果表明,pTG 402-20的插入片段能与T5 DNA Hind Ⅲ酶切的G和B片段杂交。这个插入片段的大小约为0.84kb。     

Preparation of milligram amounts of 21 deoxyribonucleic acid restriction fragments

Twenty-one DNA restriction fragments ranging in size from 12 to 880 base pairs (bp) were purified to homogeneity in milligram amounts. The developments which facilitated this work were (a) procedures for the rapid preparation of gram quantities of pure recombinant plasmid DNAs, (b) selective poly(ethylene glycol) (PEG) precipitation of DNAs according to broad classes of lengths, and (c) large-scale high-pressure liquid chromatography on RPC-5 for the purification of fragments to homogeneity. The 95- and 301-bp sequences from the lactose control region of Escherichia coli were cloned into the single EcoRI site of pVH51 in up to four copies per plasmid. These tandem inserts are separated by EcoRI sites and have a head to tail orientation in all cases. A total of 50 and 90 mg of th 95- and 301-bp fragments, respectively, were prepared from 300-L fermentations of E. coli cells transformed with these plasmids. A rapid and improved method, which can easily be scaled up, for the purification of plasmids and DNA restriction fragments was developed. Also, the linear pVH51 vector DNA was digested with HaeIII to yield fragments ranging in size from 12 to 880 bp. The five smaller fragments (from 12 to 180 bp) were purified quantitatively by a selective PEG precipitation enrichment step followed by RPC-5 column fractionation. The larger fragments (245-880 bp) were prepared in milligram amounts. Ten subfragments from the 301-bp lac fragment were prepared by HpaII, HinfI, or HaeIII/AluI digestions followed by separation of the reaction products on RPC-5.     

Overproduction and large-scale preparation of deoxyuridine triphosphate nucleotidohydrolase from Escherichia coli

A recombinant plasmid, pHW1, directing the overproduction of the enzyme deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase, EC 3.6.1.23) from Escherichia coli has been constructed. A 1900-base DNA fragment carrying the structural gene for the enzyme (dut) has been recloned into a runaway replication vector that also carries the strong leftward promoter (pL) of bacteriophage lambda. Upon temperature shift, an E. coli strain carrying the new plasmid gives an increase in dUTPase activity of about 600-fold in rich medium compared to wild-type bacteria. The 64-kDa protein corresponding to the mature form of the enzyme reaches 20% of the total protein content of the bacterial cell. Using this strain, a simplified procedure has been developed for the purification of dUTPase. The purification steps consist of extraction of the cytoplasmic proteins, ammonium sulfate precipitation, anion-exchange chromatography and gel filtration on FPLC. The new overproducing plasmid and the simplified purification procedure developed will make it possible to purify dUTPase in sufficient amounts for detailed characterization studies.     

Identification in vivo of promoter activity in the left site of the att region of bacteriophage lambda DNA

The promoter-probing vector (pSK plasmid) was explored for cloning of the fragments from lambda cI857 and lambda b2 DNAs containing different regions of the att site. We have constructed all-tet fusions where the fusions are: 1) HindIII/BamHI-491 base pairs (b. p.) fragment of lambda cI857 DNA containing POP' site (plasmid pSK-PP'); 2) AluI-242 b. p. fragment of lambda cI857 DNA containing the left arm of the POP' site (plasmid pSK-P); 3) AluI-242 b. p. fragment of lambda cI857 DNA with opposite orientation (plasmid pSK-P); 4) EcoRI/BamHI-750 b. p. fragment of lambda b2 DNA containing the right arm of the POP' site (plasmid pSK-P'). These fusions permit us to analyse the effect of various pieces of the attachment site on the expression tet gene as the result of reparation of this gene promoter. We find that expression of tet (tetracycline resistant phenotype) takes place in the pSK-PP' and pSK-P but not in the pSK-P' and pSK-P. These facts permit us to conclude that the left arm of the att site contains a rightward promoter functioning in vivo. We postulate that this promoter activity might correspond to the promoter patt, which was described in previous experiments in vitro.     

Structure of the cutinase gene and detection of promoter activity in the 5''-flanking region by fungal transformation.

The cutinase gene from Fusarium solani f. sp. pisi (Nectria hematococa) was cloned and sequenced. Sau3A fragments of genomic DNA from the fungus were cloned in a lambda Charon 35 vector. When restriction fragments generated from the inserts were screened with 5' and 3' probes from cutinase cDNA, a 5.5-kilobase SstI fragment hybridized with both probes, suggesting the presence of the entire cutinase gene. A 2,818-base pair segment was sequenced, revealing a 690-nucleotide open reading frame that was identical to that found in the cutinase cDNA with a single 51-base pair intron. Transformation vectors were constructed containing a promoterless gene for hygromycin resistance, which was translationally fused to flanking sequences of the cutinase gene. When protoplasts and mycelia were transformed with these vectors, hygromycin-resistant transformants were obtained. Successful transformation was assessed by Southern blot analysis by using radiolabeled probes for the hygromycin resistance gene and the putative promoter. The results of Southern blot analysis indicated that the plasmid had integrated into the Fusarium genome and that the antibiotic resistance was a manifestation of the promoter activity of the cutinase flanking sequences. Transformation of Colletotrichum capsici with the same construct confirmed the promoter activity of the flanking region and the integration of the foreign DNA. Transformation and deletion analysis showed that promoter activity resided within the 360 nucleotides immediately 5' to the cutinase initiation codon.     

Construction of a long hairpin RNA expression library using Cre recombinase

A large number of genome-wide screens using RNA interference (RNAi) libraries have been utilized to determine the function of individual gene products involved in a variety of biological processes. In this study, we describe a new method to enzymatically generate a long hairpin RNA (lhRNA) expression library from a cDNA plasmid library using a nicking endonuclease, BcaBEST DNA polymerase, and Cre recombinase without excising the inserted DNA fragment from the plasmid vector. This method involves 5 steps: (1) conversion of an inserted DNA fragment in a plasmid into a direct repeat (DR); (2) purification of the plasmid containing the DR; (3) subcloning a lox71 cassette into the plasmid; (4) conversion of the DR in the plasmid into an inverted repeat (IR) using Cre recombinase; and (5) purification of the plasmid containing the IR. We also established an efficient method for inserting DNase I-digested DNA fragments into expression plasmids to enable construction of a cDNA plasmid library suitable as source materials to construct the lhRNA expression library. We confirmed that each of the lhRNA expression plasmids constructed using this method induced strong RNAi in a silkworm cell line, NIAS-Bm-oyanagi2.     

Construction and mapping of recombinant plasmids used for the preparation of DNA fragments containing the Escherichia coli lactose operator and promoter.

Three DNA restriction fragments of established sequence containing the Escherichia coli lac genetic controlling regions were cloned. In each case a recombinant plasmid was constructed which was suitable for the subsequent large scale purification of the lac fragment. A 789-base pair HindII fragment, containing the lac operator, promoter, and cyclic AMP receptor protein binding site, was ligated into the single HindII site of the amplifiable plasmid minicolicin E1 DNA (pVH51). A 203-base pair Hae III fragment containing the same genetic sites was ligated into the single Eco RI site of pVH51 which had been "filled in" by the Micrococcus luteus DNA polymerase. Thus, the lac fragment was inserted between two Eco RI sites. Plasmids containing multiple copies of this Eco RI fragment were then constructed. A 95-base pair Alu I fragment containing the lac promoter and operator was cloned similarly. Also, the 203-base pair fragment was cloned into the Eco RI site of pVH51 using a 300-base pair linker fragment (isolated by RPC-5 column chromatography) which permitted retention of its Hae III ends. Mapping studies on pVH51 DNA with a number of DNA restriction endonucleases, including Alu I, Taq I, and Hpa II, are described.     

Construction of fowlpox virus vectors with intergenic insertions: expression of the beta-galactosidase gene and the measles virus fusion gene.

A DNA fragment from fowlpox virus cloned on a plasmid vector was modified to contain foreign DNA inserts within an intergenic region. In a first step, a 32-base-pair intergenic region from the fowlpox virus genome corresponding to the position of the thymidine kinase locus in the vaccinia virus genome was enlarged to 55 base pairs by site-directed mutagenesis. A unique restriction endonuclease site introduced upstream of the intergenic region was then used to insert various foreign DNA fragments. The lacZ gene encoding beta-galactosidase and the measles virus gene encoding the fusion protein were positioned downstream of two vaccinia virus p7.5 promoter elements in either a direct repeat or inverted repeat orientation. Foreign DNA inserts contained within the fowlpox virus sequence were transferred to the viral genome by homologous recombination occurring in cells infected with a fowlpox virus temperature-sensitive mutant and transfected with both wild-type viral DNA and plasmid DNA. Recombinant viruses were selected for the expression of beta-galactosidase activity by screening for blue plaques in the presence of a chromogenic substrate. Stable recombinants expressing both the lacZ gene and the unselected measles gene were obtained when the p7.5 promoter was present as an inverted repeat. However, when the p7.5 promoter was in the direct repeat orientation, viral recombinants which initially expressed both gene inserts readily deleted the lacZ gene flanked by the promoter repeat. The methods described enable precise insertion and deletion of foreign genes in the fowlpox virus genome and could be applied to other intergenic regions of the same virus as well as other poxviruses.     

Overproduction of the Bacillus sphaericus R modification methylase in Escherichia coli and its purification to homogeneity

A DNA fragment containing the information coding for the GGCC-specific Bacillus sphaericus R modification methylase, BspR, was inserted into plasmid vector pKK223-3 under the control of the strong and inducible tac promoter, and transformed into Escherichia coli HB101. Upon induction this strain accumulated the methylase enzyme (while cell growth was inhibited) up to several percent of total cellular protein. Homogeneous methylase could be prepared in three purification steps.     

Construction and characterization of three yeast-Escherichia coli shuttle vectors designed for rapid subcloning of yeast genes on small DNA fragments

We have constructed three new subcloning plasmid vectors, pRC1, pRC2, and pRC3, derived from pKC7, which allow the rapid, single-step subcloning of yeast genes. Subcloning with these vectors utilizes a partial digestion with Sau3A to generate a quasi-random set of DNA fragments from the original plasmid. All three vectors contain a kanamycin resistance gene. Therefore, if the original cloned yeast DNA fragment is present in a vector that does not specify kanamycin resistance, the subclone pool can be propagated in Escherichia coli in the presence of kanamycin to select against parent plasmids that escaped restriction by Sau3A. Selection by complementation in yeast yields a collection of plasmids with smaller yeast DNA inserts containing the gene of interest. In the vectors pRC2 and pRC3, constructed from pRC1, the unique BamHI site is located within an intact tetracycline resistance gene, thus making it possible to screen bacterial transformants for those containing recombinant plasmid molecules. Vectors pRC2 and pRC3 also contain the yeast 2 micrometers DNA replication origin, and thus are more stable than plasmids carrying only the TRP1-associated replicator (ars1).     

Plasmid vector for cloning in Streptococcus pneumoniae and strategies for enrichment for recombinant plasmids

A new plasmid, pLS101, was constructed for use as a vector for cloning in Streptococcus pneumoniae. This plasmid carries two selectable genes, tet and malM, each of which contains two or more restriction sites for cloning. Insertional inactivation of the malM gene allowed direct selection of TcRMal- clones containing recombinant plasmids. Other means of enriching a recipient population for cells containing recombinant plasmids were examined. The effect of removing vector terminal phosphate in attempts to clone heterogeneous DNA fragments, such as those from chromosomal DNA, was to abolish recombinant plasmid establishment altogether, presumably because donor DNA processing during entry into the cell prevented establishment of the hemiligated molecule. However, with homogeneous DNA fragments, such as those from plasmid or viral DNA, vector phosphate removal allowed enrichment for recombinant plasmids. In the cloning of heterogeneous DNA that was homologous to the recipient chromosome (i.e. chromosomal DNA from S. pneumoniae), recovery of recombinant plasmids could be enriched tenfold (relative to the regenerated vector) by the process of chromosomal facilitation of plasmid establishment. This involved an additional passage of the mixed plasmids in which interaction with the chromosome of plasmids containing chromosomal DNA inserts (i.e. recombinant plasmids) increased their frequency of establishment relative to the vector plasmid. An overall strategy for cloning in S. pneumoniae, depending on the nature of the fragment to be cloned, is proposed.     

A rapid method for determining the relationship between cDNA clones

We describe a technique for rapidly screening the inserts of plasmids for homology to each other by using DNA fragments isolated in agarose gels to probe Southern blots of DNA prepared by the "miniprep" alkaline lysis method. The procedure includes a technique for labeling DNA fragments in agarose gel slices without further purification. The protocol results in a significant savings in time and expense and a considerable increase in fragment yield over methods involving fragment purification from polyacrylamide or agarose gels.     

Molecular genetic studies of the DNA promotor regions in Bacillus thuringiensis subsp. galleriae 69-6. II. Increased level of gene expression resulting from IS1 element integration

The 1.45 kb promoter containing HindIII fragment of Bacillus thuringiensis DNA promotes the expression of the tet gene of recombinant pPBT9 plasmid in Escherichia coli cells. Spontaneous mutants of this plasmid were isolated and analysed. They are responsible for an increase in the level of tetracycline resistance. This 3-fold increase resulted from integration of IS1 element into the bacillar promoter containing HindIII fragment, which led to formation of a mutant pPBT9::IS1 plasmid. The IS sequence integrated was defined as an IS1 element of the E. coli HB101 chromosomal DNA. The integration site of IS1 was localized.     

CdCl2对质粒的生态效应及质粒在其宿主抗镉性中的作用

用ＣｄＣｌ２处理体外或大肠杆蓖体内质粒ｐＷＨ５８后,通过琼脂糖电泳和限制性内切酶分析,研究了Ｃｄ对质粒ＤＮＡ结构的影响。通过比较带质粒与不带质粒大肠杆菌在含不同浓度ＣｄＣｌ２的氨苄ＬＢ与无抗ＬＢ培养液中的生长量,研究了Ｃｄ对大肠杆菌体内质粒的影响及质粒在其宿主Ｃｄ耐性的作用。结果表明,体外、体内ＣｄＣｌ２处理对质粒ｐＷＨ５８ ＤＮＡ结构无明显诱变性。Ｃｄ胁迫下大肠杆菌体内质粒ｐＷＨ５８可进行复制传     

扣囊拟内孢霉α-淀粉酶基因的克隆和在酿酒酵母中表达

以穿梭质粒pCN60为载体,大肠杆菌C600为受体构建了扣囊拟内孢霉(Endomycopsis fibuligera)G45 Sau 3A基因文库。从基因文库中提取重组质粒DNA并转化酿酒酵母(Saccharomyces cerevisiae)BJ1991,选出四个具有o-淀粉酶活性的转化子,琼脂糖凝胶电泳结果证实插入的DNA片段为9.0kb。对插入DNA片段亚克隆,确定。-淀粉酶基因位于PstI-Sall 3.9kb片段上,启动子位于PstI-EcoRI 的1.3kb片段上。用亚克隆PGK11.9kb片段置换。-淀粉酶启动子区,其转化子的e-淀粉酶活性有明显提高。     

Instability of Tn5 in a plasmid cloning vector for the cyanobacterium Anacystis nidulans.

Transposon Tn5 was used to produce insertions within the region of a cyanobacterial shuttle vector previously identified as necessary for transformation of Anacystis nidulans. These transposon-containing plasmids were used to transform a plasmid-cured derivative of Anacystis strain R2 and tested for structural stability of the transforming plasmid. The transposon DNA was deleted from all the plasmids containing Tn5 within the cyanobacterial replication region. Inserts in the vector DNA were physically stable and expressed the kanr gene. The internal Tn5 HindIII fragment was also cloned into each of the three HindIII sites in the shuttle plasmid. Inserts in two of these sites were stable, whereas inserts into the third site were not.