Cloning of human mitochondrial DNA in Escherichia coli

In order to determine its nucleotide sequence, human mitochondrial DNA (mtDNA) purified from term placentae was cloned in Escherichia coli using the plasmid vector pBR322. The products of an mtDNA MboI digestion (23 fragments ranging in size from 2800 to 25 base-pairs (bp)) were ligated with BamHI-cut pBR322. The ampicillin-resistant tetracycline-sensitive colonies obtained upon transformation of E. coli χ1776 were screened by agarose gel electrophoresis of colony lysates, colony hybridization and restriction analysis. All but MboI fragment 2 were obtained in this way. MboI fragments 5 and 8 were each found only once among the 705 clones screened. All other MboI fragments were approximately equally represented in the population of clones except for a slight bias towards smaller fragments. MboI fragment 2 overlaps with the mtDNA BamHI/EcoRI (1.7 kb³) and the 0.9 kb HinIII fragments. These were cloned in similarly restricted pBR322 to provide a set of clones covering most of the mtDNA molecule. Clones representative of each MboI fragment were shown to be complementary to mtDNA by hybridization to Southern blots of mtDNA digests and were thereby partially mapped. Further mapping was obtained by restriction analysis of mtDNA sequentially degraded by exonuclease III. A collection of recombinant clones has thus been obtained using the mtDNA isolated from a single placenta and is now being used to obtain a complete nucleotide sequence of human mtDNA.     

Amplified expression of ribulose bisphosphate carboxylase/oxygenase in pBR322-transformants of Anacystis nidulans

Prior research suggested that the genes for large (L) and small (S) subunits of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) are amplified in ampicillin-resistant pBR322-transformants of Anacystis nidulans 6301. We now report that chromosomal DNA from either untransformed or transformed A. nidulans cells hybridizes with nick-translated [³²P]-pBR322 at moderately high stringency. Moreover, nick-translated [³²-P]-pCS75, which is a pUC9 derivative containing a PstI insert with L and S subunit genes (for RuBisCO) from A. nidulans, hybridizes at very high stringency with restriction fragments from chromosomal DNA of untransformed and transformed cells as does the ³²P-labeled PstI fragment itself. The hybridization patterns suggest the creation of two EcoRI sites in the transformant chromosome by recombination. In pBR322-transformants the RuBisCO activity is elevated 6- to 12-fold in comparison with that of untransformed cells. In spite of the difference in RuBisCO activity, pBR322-transformants grow in the presence of ampicillin at a similar initial rate to that for wild-type cells. Growth characteristics and RuBisCO content during culture in the presence or absence of ampicillin suggest that pBR322-transformants of A. nidulans 6301 are stable. The data also collectively suggest that a given plasmid in the transformed population replicates via a pathway involving recombination between the plasmid and the chromosome.     

Molecular Cloning and Physical Mapping of Restriction Endonuclease Fragments of Autographa californica Nuclear Polyhedrosis Virus DNA

A restriction fragment library containing Autographa californica nuclear polyhedrosis virus (AcNPV) DNA was constructed by using the pBR322 plasmid as a vector. The library, which is representative of more than 95% of the viral genome, consists of 2 of the 7 BamHI fragments, 12 of the 24 HindIII fragments, and 23 of the 24 EcoRI fragments. The cloned fragments were characterized and used to generate physical maps of the genome by hybridizing nick-translated recombinant plasmid to Southern blots of AcNPV DNA digested with SmaI, BamHI, XhoI, PstI, HindIII, and EcoRI restriction endonucleases. This information was used to define our strain of AcNPV (HR3) with respect to other strains for which physical maps have been previously published. The hybridization data also indicate that reiteration of DNA sequences occurs at the HindIII-L and -Q regions of the genome.     

Escherichia coli DNA topoisomerase I mutants: Increased supercoiling is corrected by mutations near gyrase genes

Bacterial chromosomes and plasmid (pBR322) DNA from topoisomerase I-defective Escherichia coli strains have been characterized with respect to superhelical density. The topoisomerase I defect results in increased negative superhelical density of both the bacterial chromosome and pBR322. Thus topoisomerase I is involved in determining the level of supercoiling in bacteria. Three of the topoisomerase I-defective strains we studied carry secondary mutations that decrease superhelical density; these additional mutations are closely linked to the gyrB locus in two of the strains and to the gyrA locus in the third strain.     

Cloning of tryptophanase gene of Alcaligenes faecalis for effective production of l-tryptophan

For the effective production of l-tryptophan from indole and l-serine by the action of tryptophanase from Alcaligenes faecalis, cloning of the enzyme gene (tna) was studied. A. faecalis was transformed not only by broad host range plasmid pKT231 but also by pLG338, pACYC177, and pBR322 derivatives. A recombinant tna plasmid was isolated by shotgun experiments with Escherichia coli K-12, and the isolated tna gene located on the 3.2 kb DNA fragment. Tryptophanase activity of A. faecalis transformed by the tna recombinant plasmid was about 4-fold higher than that of wild cells.     

A T4 DNA fragment containing genes for the baseplate central plug: Endonuclease restriction, gene expression and cell wall changes

Summary A fragment of Escherichia coli bacteriophage T4D DNA, containing 6.1 Kbp which included the six genes (genes 25, 26, 51, 27, 28 and 29) coding for the tail baseplate central plug has been partially characterized. This DNA fragment was obtained originally by Wilson et al. (1977) by the action of the restriction enzyme EcoRI on a modified form of T4 DNA and was inserted in the pBR322 plasmid and then incorporated into an E. coli K12 strain called RRI. This plasmid containing the phage DNA fragment has now been reisolated and screened for cleavage sites for various restriction endonucleases. Restriction enzymes Bgl 11 and Xbal each attacked one restriction site and the enzyme Hpa 1 attacked two restriction sites on this fragment. The combined digestion of the hybrid plasmid containing the T4 EcoRI DNA fragment conjugated to the pBR322 plasmid with one of these enzymes plus Bam H1 restriction enzyme resulted in the localization of the restriction site for Bgl 11, Xba 1 and Hpa 1. Escherichia coli strain B cells were transformed with this hybrid plasmid and found to have some unexpected properties. E. coli B cells, which are normally restrictive for T4 amber mutants and for T4 temperature sensitive mutants (at 44°) after transformation, were permissive for 25am, 26am and 26^Ts, 51am, and 51^Ts, 27^Ts, and 28^Ts T4 mutants. Extracts from the transformed E. coli cells were found in complementation experiments to contain the gene 29 product, as well as the gene 26 product, the gene 51 product, and the gene 27 product. The complementation experiments and the permissiveness of the transformed E. coli B cells to the various conditional lethal mutants clearly showed that the six T4 genes were producing all six gene products in these transformed cells. However, these cells were not permissive for T4 amber mutants in genes 27, 28, and 29. The transformed E. coli B cells, as compared to untransformed cells, were found to have altered outer cell walls which made them highly labile to osmotic shock and to an increased rate of killing by wild type T4 and all T4 amber mutants except for T4 am29. The change in cell walls of the transformed cells has been found to be due to the T4 baseplate genes on the hybrid plasmid, since E. coli B transformed by the pBR322 plasmid alone does not show the increase in osmotic sensitivity.     

Stabilization of anE. coli plasmid by a mini-F fragment of DNA

Summary In anEscherichia coli K-12 strain (trpA trpE tnaA) cultured in LB broth without selective pressure, a pBR322 derivative containing the gene for tryptophan synthase (pBR322-trpBA) was found to be unstable. After 70 cell-number doublings, only 50% of the host cells retained the gene for ampicillin resistance (Ap^r). Insertion of the mini-F fragment of F factor DNA into this plasmid could effectively reduce the plasmid loss. Partial derepression of the tryptophan promotor-operator by 3-indopleacrylic acid further decreased the stability of the pBR322-trpBA but not that of the mini-F inserted plasmid (pBR322F-trpBA) The vector pBR322F-trpBA could be maintained at high copy number in the culture after 100 generations of growth; the culture was able to overproduce tryptophan synthase in the presence of 3-indoleacrylic acid.l-Tryptophan was produced from indole andl-serine using andE. coli host transformed with.pBR322F-trpBA DNA. After 8 h of incubation, the expression level was approximately 180 g/l.     

The cloning of Aspergillus nidulans mitochondrial DNA in Escherichia coli on plasmid pBR322

Summary We report the cloning of almost the entire mitochondrial DNA of Aspergillus nidulans on plasmid pBR322 in Escherichia coli. Only one fragment containing about 11% of the mitochondrial genome has not been cloned. We believe that this fragment cannot be maintained in E. coli on the pBR322 plasmid.     

Molecular cloning of the ADE1 gene of Saccharomyces cerevisiae and stability of the transformants

Plasmid YEp(ADE1)1a, containing a 2.7-kb Sau3A fragment of Saccharomyces cerevisiae DNA inserted at the BamHI site of the yeast shuttle vector pBTI-1 (Morris et al., 1981), results in high frequency, unstable transformation of ade1 yeast strains. A second plasmid, YRp(ADE1)2, containing adjacent 0.5-kb and 3.0-kb BamHI fragments in pBR322 gave three types of yeast transformants: (1) transformants carrying extrachromosomal copies of the plasmid which indicate the presence of a functional ars sequence, (2) transformants indistinguishable from ade1 strains by hybridization analyis, and (3) a transformant carrying a multimeric form of YRp(ADE1)2. Cells transformed with either of the plasmids are free of the red pigment characteristic of ade1 mutants and indicate potential for direct colour-based selection of yeast transformants using ADE1 plasmids.     

Construction and characterization of new cloning vehicles V. Mobilization and coding properties of pBR322 and several deletion derivatives including pBR327 and pBR328

A DNA sequence essential for the R64drd11 + ColK-mediated conjugal transfer of pBR322 has been located in a 540 bp HaeIII fragment (HaeIII-2) between the vegetative origin of replication and the tetracycline resistance (Tc^r) gene of this vector. The pBR322 derivatives pBR327 and pBR328 lack this DNA sequence and are not mobilized by conjugation. Two derivatives of pBR328 were constructed by re-inserting the HaeIII-2 fragment in both orientations into the chloramphenicol-resistance gene of the same vector. One orientation of the HaeIII-2 fragment permitted mobilization by conjugation while the opposite orientation prevented mobilization. Further examination of pBR322 and derivatives revealed that the region between the origin of replication and Tc^r gene also plays a role in regulating plasmid copy number.     

Cloning and expression of the yeast galactokinase gene in an Escherichia coli plasmid.

This report describes the construction and isolation of a plasmid, derived from pBR322, which carries a BglII restriction fragment of DNA containing the galactokinase gene from Saccharomyces cerevisiae. This was accomplished by the following procedure: (1) Purified galactokinase mRNA, labelled with 125I, was hybridized to BglII digests of yeast DNA employing Southern's filter transfer technique to identify a restriction fragment containing the galactokinase gene. (2) This fragment was partially purified by agarose gel electrophoresis, ligated into the BamHI site of pBR322 and transformed into Escherichia coli to generate a clone bank containing the galactokinase gene. (3) This bank was screened by in situ colony hybridization with galactokinase mRNA resulting in the identification of a plasmid carrying this gene. This plasmid DNA hybridized with the galactokinase mRNA to the same extent in the presence of absence of a large excess of unlabelled mRNA from cells that were not induced for galactokinase synthesis, while the same amount of unlabelled galactose-induced mRNA reduced the hybridization by 95%. When this plasmid was introduced into an E. coli strain deleted for the galactose operon it caused the synthesis of low levels of yeast galactokinase activity.     

DNA polymerase I in constitutive stable DNA replication in Escherichia coli.

We examined the effects of mutations in the polA (encoding DNA polymerase I) and polB (DNA polymerase II) genes on inducible and constitutive stable DNA replication (iSDR and cSDR, respectively), the two alternative DNA replication systems of Escherichia coli. The polA25::miniTn10spc mutation severely inactivated cSDR, whereas polA1 mutants exhibited a significant extent of cSDR. cSDR required both the polymerase and 5'-->3' exonuclease activities of DNA polymerase I. A similar requirement for both activities was found in replication of the pBR322 plasmid in vivo. DNA polymerase II was required neither for cSDR nor for iSDR. In addition, we found that the lethal combination of an rnhA (RNase HI) and a polA mutation could be suppressed by the lexA(Def) mutation.     

Transfer of plasmid pTO1 from Escherichia coli to various representatives of the order Actinomycetales by intergeneric conjugation

Plasmid pTO1 containing the oriT fragment from RK2, the Escherichia coli replication function from pBR322, and a DNA fragment of actinophage φC31 with the attachment site was transferred from E. coli S17-1 to strains of the genera Actinomadura, Arthrobacter, Micromonospora, Nocardia, Rhodococcus, and to 16 strains of the genus Streptomyces. The frequency of conjugant formation was 1×10⁻³–1×10⁻⁵ depending on the strain. Hybridization experiments demonstrated that plasmid pTO1 integrates into chromosomes of a number of the recipient strains examined.     

Amplification of alpha-fetoprotein complementary DNA by insertion into a bacterial plasmid.

A fragment of the α-fetoprotein (AFP) structural gene was purified and amplified by bacterial cloning techniques. Double-stranded DNA_AFP was constructed from a cDNA copy of greater than 95% pure mRNA_AFP and inserted into E. coli plasmid pBR322 by poly(dA-dT)-linkers. Chimeric plasmid DNA isolated from transformants of E. coli strain χ1776 have been shown to contain α-fetoprotein sequences by hybridization to labeled mRNA_AFP. One clone, designated pA5 (chimeric plasmid pBR322 containing a cDNA_AFP sequence isolated from clone 5), has been studied in more detail. The inserted sequence of approximately 950 nucleotide pairs was positively identified by a hybridization-translation procedure. Hybridization of [³H]uridine-labeled poly(A)-containing RNA from an AFP-secreting cell line to excess pA5 DNA immobilized on nitrocellulose filters was used to show the selectivity of this probe for detecting expression of the AFP gene.     

Deletion analysis of the cloned replication origin region from bacteriophage M13.

A cloned 270-nucleotide fragment from the origin region of the M13 duplex replicative form DNA confers an M13-dependent replication mechanism upon the plasmid vector pBR322. This M13 insert permits M13 helper-dependent replication of the hybrid plasmid in polA cells which are unable to replicate the pBR322 replicon alone. Using in vitro techniques, we have constructed several plasmids containing deletions in the M13 DNa insert. The endpoints of these deletions have been determined by DNA sequence analysis and correlated with the transformation and replication properties of each plasmid. Characterization of these deletion plasmids allows the following conclusions. (i) The initiation site for M13 viral strand replication is required for helper-dependent propagation of the chimeric plasmid. (ii) A DNA sequence in the M13 insert, localized between 89 and 129 nucleotides from the viral strand initiation site, is necessary for efficient transformation of polA cells. A chimeric plasmid containing the viral strand initiation site, but lacking this additional 40 nucleotide M13 sequence, transforms helper-infected cells at a frequency approximately 10(4)-fold less than that of plasmids containing this additional DNA segment. (iii) The entire M13 complementary strand origin can be deleted without affecting M13-dependent transformation by the hybrid plasmids. We propose a model in which replication of one strand of duplex chimera initiates by nicking at the gene II protein nicking site in the viral strand of the M13 insert, followed by asymmetric single-strand synthesis. Initiation of the complementary strand possibly occurs within plasmid sequences.     

Cloning of rpsO, the gene for ribosomal protein S15 of Escherichia coli

Summary The gene for Escherichia coli ribosomal protein S15 (rpsO) was cloned on the vector pBR322 from F-prime JCH55 DNA. The recombinant plasmid was transformed to Serratia marcescens cells and it was proved that E. coli S15 was synthesized and incorporated into ribosome particles in S. marcescens cells. A DNA fragment containing rpsO was also inserted into the vector pRF3, which changes its copy number depending on the growth temperature in a temperature-sensitive polA host. By use of this recombinant plasmid it was shown that the relative synthesis rate of S15 increased about twice even when the copy number of the plasmid increased more than twenty-fold.     

Mutagenesis and mutation transfer induced by ultraviolet light in plasmid-cloned DNA

We describe here simple techniques for increasing the frequency of UV-induced mutations in a DNA fragment cloned in plasmid pBR322. Irradiation of both the host and the plasmid DNA before transformation is necessary to produce new mutations in the plasmid DNA, presumably because the UV-damaged pBR322 replicon cannot efficiently induce the error-prone repair pathway of Escherichia coli. In contrast, U V irradiation of the plasmid DNA alone before transformation primarily causes the transfer of preexisting mutations from the host chromosome to homologous DNA present in the plasmid. The only other kind of mutants obtained were large deletions of the plasmid DNA. Two chromosomal mutations from the host galK gene and one from the lacZ gene have been transferred to the plasmid by UV irradiation of the plasmid DNA alone. The technique can thus be of general use.     

Stability of the hybrid plasmid pIM138 and its curing by some eliminating agents

Hybrid plasmid pIM138 was constructed by insertion of a chromosomal fragment with the threonme operon fromEscherichia coli into the pBR322 vector. Molar mass of pIM138 was 2.8 Mg/mol. Heteroduplexes between pBR322 vector and pIM138 hybrid DNA molecules were prepared. The hybrid plasmid shows a high stability against the curing effect of rifampicin and clorobiocm inE. coli SK1590thr host.     

含SRSV U3区的重组Moloney小鼠白血病病毒的构建及其致病特性

质粒pSF11含有小鼠白血病病毒(SRSV)基因组5kb Bam H Ⅰ片段。应用限制性内切酶分析及Southern印迹杂交技术,确定了长末端重复区(Long terminal repeats,LTR)在pSF11上的位置。pSF11经Bg1 Ⅱ酶解,Klenow酶补平5’粘性末端,碱性磷酸酯酶脱磷后与EcoR Ⅰ Linker连接,转化大肠杆菌C600,经EcoRⅠ酶切筛选得到重组子 pER 1。pER 1经ClaⅠ+EcoR Ⅰ酶切,电泳回收含SRSV LTR的2.55kbDNA,与p63-2(含Moloney小鼠白血病病毒(leukemia virus,M-MuLV)完整基因组)的ClaⅠ+EcoRⅠ14.1kbDNA连接,转化C600受体菌,得到一个M-MuLV 3’LTR被SRSV LTR取代的重组质粒pMS。纯化后转染NIH 3T3细胞,经XC合胞试验及逆转录酶活性测定证明,转染细胞含有M-MuLV与SR-SV的重组病毒MSV,将MSV经皮下接种到新生的NIH Swiss小鼠,通过对小鼠发病阶段血像、大体解剖以及组织病理学检查,发现该重组病毒MSV具有与SRSV极其相似的致病特征。