Restriction map, partial cloning and localization of 9S and 12S kinetoplast RNA genes on the maxicircle component of the kinetoplast DNA of Leishmania tarentolae.

We have constructed a restriction map of the maxicircle component of the kinetoplast DNA of Leishmania tarentolae for the enzymes EcoRI, Bam HI, HaeIII, HpaII, SalI, BglII and HindIII. The 9 and 12S kinetoplast RNAs were localized on this map. Two fragments of this maxicircle molecule were cloned in the bacterial plasmid, pBR322, including a 4.4 . 10(6) dalton EcoRI/BamHI fragment which contains the 9 and 12S RNA genes.     

Cloning the genetic determinant of alpha-hemolysin and obtaining a series of insertional mutations in this determinant by the transposon Tn1000

Functionally active genetic determinant of alpha-hemolysin was cloned. Hemolytic plasmid pHly195 was used as a donor of the determinant and pBR322 plasmid served as recipient. Cloning was done with a help of HindIII restriction endonuclease. The recombinant plasmid obtained represents pBR322 plasmid with the built-in fragment of 7.4 kb containing genes of functionally active determinant of alpha-hemolysin. Restriction map was constructed using HindIII, EcoRI, BamHI and SalI restriction endonucleases. Insertional mutagenesis was carried out with the help of the Tn1000 transposon. Plasmid DNAs were isolated from insertional mutants of Hly- phenotype and treated with EcoRI, SalI and BamHI. On the basis of the sizes of restriction fragments of the mutant plasmid DNAs localization and orientation of insertions of Tn1000 into the cloned determinant of alpha-hemolysin were determined.     

Versatile kanamycin-resistance cartridges for vector construction in Escherichia coli

To generate polylinker sequences which can be transferred together with an adjacent selectable marker, two plasmids (pWW-84 and pWW-97) were constructed which contain a kanamycin-resistance gene (KmR) flanked by various restriction sites. From these plasmids KmR-cartridges can be obtained as EcoRI, BamHI, SalI, AccI or HincII fragments for insertion into the appropriate restriction site of any plasmid. The following restriction sites can be introduced with these cartridges: BamHI, SalI (AccI, HincII), EcoRI, SacI, SphI and KpnI (Asp718) all adjacent to KmR, XhoI and HindIII, both within KmR. If desired, KmR can be removed by PstI digestion and religation, creating a single PstI site and leaving all adjacent sites intact.     

Refined, Circular Restriction Map of the Bacillus thuringiensis subsp. israelensis Plasmid Carrying the Mosquito Larvicidal Genes

All the genetic elements responsible for the mosquito larval toxicity of Bacillus thuringiensis subsp. israelensis are located on one of its largest plasmids, nicknamed pBtoxis. Two linkage groups (with sizes of about 75 and 55 kb) have previously been mapped partially with respect to SacI and BamHI restriction sites (Ben-Dov et al., 1996), but linking them to a single circular plasmid unambiguously was impossible with the available data. To finalize the plasmid map, another rare cutting restriction endonuclease, AlwNI, was used in addition. The two linkage groups and the fragments generated by AlwNI were aligned on the circular plasmid, and known insertion sequences were localized on the refined map. Pulsed-field electrophoresis revealed that the total size of pBtoxis (137 kb) was larger than thought before.     

Physical map of the Agrobacterium rhizogenes strain 8196 virulence plasmid

Virulence of Agrobacterium rhizogenes, agent of hairy root disease, is conferred by large plasmids called Ri (root-inducing) plasmids. We have determined the BamHI fragment map of pRi8196, MW 143 Mda, principally by analysis of recombinant plasmids containing overlapping BamHI partial-digest fragments. Clones containing solitary BamHI inserts of remaining unmapped fragments were used to probe a series of Southern-blotted, pRi8196-derived EcoRI, PstI, HindIII, SalI, or SmaI digests. Continguous hybridized bands represented complements of EcoRI, PstI, HindIII, SalI, or SmaI fragments which bridged the unmapped BamHI fragments. We present, in addition, a detailed map of the core T-DNA region with respect to the restriction endonucleases SalI, EcoRI, HpaI, and HindIII.     

Restriction map of a capsule plasmid of Bacillus anthracis

The capsule plasmid pTE702 of Bacillus anthracis has been physically mapped with the restriction endonucleases HindIII, PstI, BamHI, SalI, and XhoI. A HindIII fragment map of pTE702 (96.5 kb) was obtained by analysis of the recombinant plasmids and cosmids containing overlapping fragments partially digested with HindIII. The physical map for PstI, BamHI, SalI, and XhoI was obtained by double digestion mapping of these sites in relation to the HindIII sites. The replication region of pTE702 was determined by in vitro genetic replicon labeling in B. subtilis.     

Construction of plasmid vectors that facilitate subcloning and recovery of yeast and Escherichia coli DNA fragments

We have constructed a plasmid vector (pNF2) which is a derivative of the multicopy yeast cloning vehicle YEp24. This derivative contains a single BamHI site flanked immediately on each side by SalI sites. The latter site was selected because it appears to be infrequent in yeast nuclear DNA. Thus, DNA fragments produced by partial digestion with enzymes (such as Sau3A) that cut at frequent intervals and leave single-stranded ends that have sequence homology with BamHI sites, can be conveniently subcloned into this site. Such fragments can then be excised intact by digestion with SalI enzyme. Plasmid pNF2 also contains the kanamycin-resistance (kanR) gene derived from Tn903 and confers resistance in yeast to the antibiotic G418. pNF2 was converted into an integrating vector (pNF3) by deleting a 2.2-kb EcoRI fragment containing a sequence that determines autonomous replication in yeast. Further deletion of a HindIII fragment containing the yeast URA3 gene converts the plasmid into one containing only pBR322 sequences plus the kanR gene (pNF4).     

The effect of Z-conformation on enzymatic activity of restriction endonucleases

Recombinant plasmid pGC20 containing (GC)9-insert into SmaI site of pUC19 has been used to study the inhibition of cleavage by six restriction endonucleases; KpnI, SacI, EcoRI and also BamHI, XbaI and SalI, due to Z-DNA formation in negatively supercoiled plasmid. The recognition sites of these enzymes were located at different distances on both sides of the (CG)10-sequence. It was shown that the inhibition of the cleavage by KpnI, SacI and EcoRI was decreased in this series as fast as the distance between recognition site and B-Z junction was increased, and no inhibition of cleavage by EcoRI was found. However, such a correlation was not found in the series of BamHI, XbaI and SalI. In contrast with EcoRI the cleavage by SalI was inhibited completely. These results indicate the difference for "sensitivity" of restriction endonucleases to the structural perturbations of DNA associated with B-Z junctions. It seems to depend on features of the enzyme-substrate interaction mechanisms and also on recognition and flanking sequences of DNA. Consequently, experiments with the inhibition of the cleavage by any enzyme can not help to determine the dimension of the region of DNA with altered structure.     

Location of the structural genes for glycoproteins gD and gE and for other polypeptides in the S component of herpes simplex virus type 1 DNA.

To map the structural genes for the gD and gE polypeptides and for other viral products encoded in the S component of herpes simplex virus type 1 DNA, we selected mRNAs capable of hybridizing to cloned viral DNA fragments and translated the mRNAs in vitro to determine which polypeptides were encoded therein. The gD and gE polypeptides were identified by immunoprecipitation with appropriate monoclonal and monospecific antibodies, whereas the other polypeptides were characterized only by their electrophoretic mobilities in polyacrylamide gels. We found that gD mRNA hybridized to a single SacI subfragment of BamHI fragment J, whereas gE mRNA hybridized to an adjacent SacI subfragment of BamHI fragment J and also to BamHI fragment X. These and other results permit the conclusion that the structural gene for gD is located between map coordinates 0.911 and 0.924, and the gene for gE is between map coordinates 0.924 and 0.951. We also found that mRNAs for polypeptides of 55,000, 42,000, 33,000, and 22,000 molecular weight hybridized to DNA fragments spanning the regions from map coordinates 0.911 to 0.924, 0.897 to 0.911, 0.939 to 0.965, and 0.939 to 0.965, respectively. Finally, in accord with the results of others, we found that mRNA for a 68,000-molecular-weight polypeptide hybridized to the two noncontiguous BamHI fragments N and Z, which share a reiterated DNA sequence.     

Organization of rRNA genes in Mycobacterium bovis BCG.

The number of rRNA genes in Mycobacterium bovis BCG was examined by Southern hybridization of end-labeled 5S, 16S, and 23S rRNAs with BamHI, PstI, and SalI digests of M. bovis BCG DNA. Each RNA probe gave only one radioactive band with three kinds of DNA digest. These results suggest that M. bovis BCG chromosomes may carry only a minimum set of rRNA genes. Hybridization of randomly labeled rRNAs with BamHI, PstI, SalI, BglII, and PvuII digests of DNA from the same organism supported these conclusions. The 6.4-kilobase-pair SalI fragment containing the entire structural genes for both 16S and 23S rRNAs was cloned into pBR322. The cloned fragment was characterized by restriction endonuclease mapping, DNA-RNA hybridization analysis, and the R-loop technique. The results indicated that the fragments contained rRNA genes in the following order: 16S, 23S, and 5S rRNA genes. No tRNA gene was detected in the spacer region between the 16S and 23S rRNA genes, but one was found downstream of the 23S rRNA and 5S rRNA genes.     

羊抑制素α亚基N端1-33序列肽段多聚体的构建及其表达纯化

为了提高短肽的免疫原性以制备短肽基因工程疫苗 ,将羊抑制素α亚基N端 1-33氨基酸残基片段的基因序列插入表达质粒pRSET A的BamHⅠ \SacⅠ位点之间构建重组质粒pR INH ,然后利用质粒中的一对同尾酶位点BamHⅠ \BglⅡ和下游的另一酶切位点HindⅢ ,经过简单的酶切后 ,将产物按各种组合连接 ,构建了抑制素串联 2至 6聚体基因。含3至6聚体基因的重组质粒pR 3INH、pR 4INH、pR 5INH和pR 6INH经IPTG诱导均能在大肠杆菌 (E .coli)BL21(DE3)中表达目的蛋白 ,分别占菌体蛋白的 6 %、6 %、7%和 8% ,且都以包涵体形式存在。结果说明 ,利用同尾酶切技术可以快速正确地构建短肽片段的串联多聚体重组质粒 ,为构建短肽半抗原的高免疫原性基因工程疫苗提供了新的思路。     

A physical map of the genome of temperate phage phi 3T.

A physical map of the genome of Bacillus subtilis bacteriophage phi 3T was constructed by ordering the fragments produced by cleavage of phi 3T DNA with restriction endonucleases AvaII (2 fragments), BglI (2 fragments), SmaI (3 fragments), BamHI (6 fragments), SalI (7 fragments), AvaI (7 fragments), SacI (12 fragments), PstI (14 fragments), and BglII (26 fragments). Two techniques were used to order the fragments: (1) Sets of previously ordered restriction fragments were isolated and redigested with the endonuclease whose cleavage sites were to be mapped. (2) Fragments located near the ends of the genome or near the ends of other restriction fragments were ordered by treating the DNA with lambda exonuclease prior to restriction endonuclease cleavage. The susceptibility of phi 3T DNA to 15 other restriction endonucleases is also reported.     

Comparative restriction analysis of HindIII-F-fragments of DNA from 4 strains of vaccinia virus

The localization of KpnI, SacI, XhoI, AvaI, PstI, BglI, BamHI, EcoRI, PmiI, SalI, BglII, restriction endonuclease cleavage sites in HindIII-F-fragments of DNA from vaccinia strains WR, Copenhagen, LIVP and neurovaccine has been detected. The fragments have been shown to differ in the number of AvaI, EcoRI and BamHI sites. The fragments also differ from the analogue of Tian Tan vaccinia strain in the pattern of restriction by AvaI, XhoI, PstI, EcoRI and BamHI endonucleases.     

Physical and functional map of supervirulent Agrobacterium tumefaciens tumor-inducing plasmid pTiBo542.

Agrobacterium tumefaciens strains carrying pTiBo542 induce large, fast-appearing tumors and have an unusually wide host range. A clone bank was made from this 250-kilobase plasmid in a wide-host-range vector, and restriction maps were determined for BamHI and SalI. The virulence genes, transferred DNA genes, plasmid incompatibility region, and a region that inhibits growth of certain A. tumefaciens strains were localized. The six virulence genes and two tms genes were highly homologous to the genes of pTiA6, but the tmr gene was not. Mutations in each of the six vir loci of pTiA6 were complemented by clones from the vir region of pTiBo542.     

Design and use of synthetic oligonucleotide probes in the cloning of delta-endotoxin genes from Bacillus thuringiensis.

A detailed protocol is described for the design and use of synthetic oligonucleotide probes for screening DNA libraries from Bacillus thuringiensis var. kurstaki (strain HD191) for copies of the gene (tox) encoding the insecticidal delta-endotoxin. Two homologous tox genes were identified in this organism; one of these was located on a 75-kb plasmid and the other on a second large plasmid or the bacterial chromosome. A tox gene was isolated as a 6.5-kb HindIII fragment of B. thuringiensis plasmid DNA.     

Molecular cloning and characterization of an rRNA operon in Streptomyces lividans TK21.

The number of rRNA genes in Streptomyces lividans was examined by Southern hybridization. Randomly labeled 23 and 16S rRNAs were hybridized with BamHI, BglII, PstI, SalI, or XhoI digests of S. lividans TK21 DNA. BamHi, BglII, SalI and XhoI digests yielded six radioactive bands each for the 23 and 16S rRNAs, whereas PstI digests gave one band for the 23S rRNA and one high-intensity band and six low-density bands for the 16S rRNA. The 7.4-kilobase-pair BamHI fragment containing one of the rRNA gene clusters was cloned into plasmid pBR322. The hybrid plasmid, pSLTK1, was characterized by physical mapping, Southern hybridization, and electron microscopic analysis of the R loops formed between pSLTK1 and the 23 and 16S rRNAs. There were at least six rRNA genes in S. lividans TK21. The 16 and 23S rRNA genes were estimated to be about 1.40 and 3.17 kilobase pairs, respectively. The genes for the rRNAs were aligned in the sequence 16S-23S-5S. tRNA genes were not found in the spacer region or in the context of the rRNA genes. The G + C content of the spacer region was calculated to be approximately 58%, in contrast to 73% for the chromosome as a whole.     

Versatile cloning vector for Pseudomonas aeruginosa.

A pBR322:RSF1010 composite plasmid, constructed in vitro, was used as a cloning vector in Pseudomonas aeruginosa. This nonamplifiable plasmid, pMW79, has a molecular weight of 8.4 X 10(6) and exists as a multicopy plasmid in both P. aeruginosa and Escherichia coli. In P. aeruginosa strain PAO2003, pMW79 conferred resistance to carbenicillin and tetracycline. Characterization of pMW79 with restriction enzymes revealed that four enzymes (BamHI, SalI, HindIII, and HpaI) cleaved the plasmid at unique restriction sites. Cloning P. aeruginosa chromosomal deoxyribonucleic acid fragments into the BamHI or SalI site of pMW79 inactivated the tetracycline resistance gene. Thus, cells carrying recombinant plasmids could be identified by their carbenicillin resistance, tetracycline sensitivity phenotype. Deoxyribonucleic acid fragments of approximately 0.5 to 7.0 megadaltons were inserted into pMW79, and the recombinant plasmids were stably maintained in a recombination-deficient (recA) P. aeruginosa host.     

Cloning and expression of the lepidopteran toxin produced by Bacillus thuringiensis var. thuringiensis in Escherichia coli

The Bacillus thuringiensis var. thuringiensis strain 3A produces a proteinaceous parasporal crystal toxic to larvae of a variety of lepidopteran pests including Spodoptera littoralis (Egyptian cotton leaf worm), Heliothis zeae, H. virescens and Boarmia selenaria. By cloning of individual plasmids of B. thuringiensis in Escherichia coli, we localized a gene coding for the delta-endotoxin on the B. thuringiensis plasmid of about 17 kb designated pTN4. Following partial digestion of the B. thuringiensis plasmid pTN4 and cloning into the E. coli pACYC184 plasmid three clones were isolated in which toxin production was detected. One of these hybrid plasmids pTNG43 carried a 1.7-kb insert that hybridized to the 14-kb BamHI DNA fragments of B. thuringiensis var. thuringiensis strains 3A and berliner 1715. This BamHI DNA fragment of strain berliner 1715 has been shown to contain the gene that codes for the toxic protein of the crystal (Klier et al., 1982). No homologous sequences have been found between pTNG33 and the DNA of B. thuringiensis var. entomocidus strain 24, which exhibited insecticidal activity against S. littoralis similar to that of strain 3A.