DNA amplification in tunicamycin-resistant Leishmania mexicana. Multicopies of a single 63-kilobase supercoiled molecule and their expression

Tunicamycin-resistant variants of Leishmania mexicana were found to contain elevated activity of N-acetylglucosamine-1-phosphate transferase and amplified DNA (Kink, J. A., and Chang, K.-P. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1253-1257). Complete digestion of their DNA with restriction endonucleases produced discrete ethidium bromide-staining bands after agarose gel electrophoresis. All four BamHI fragments of the amplified DNA were cloned separately into pBR322 and found to share no substantial sequence homology. DNA complementary to each of the cloned fragments is 64-128-fold more abundant in the variants than in the wild type cells. The amplified DNA appears to originate from a single chromosomal region of 63 kilobases. Individual copies of the 63 kilobases are each circularized at the newly formed junction site producing multiple extrachromosomal supercoiled molecules in the drug-resistant cells. There is overproduction of RNA ranging in size from 1.9 to 6.6 kilobases complementary to the amplified DNA in these cells.     

Study on rRNA genes in Mycobacterium smegmatis

The number of rRNA genes in Mycobacterium smegmatis was examined by hybridization of BamHI and SalI digests of chromosomal DNA with 3'-end-labeled 5S, 16S, 23S rRNA and tRNA. Each RNA probe gave two hybridization bands. The PstI fragments of 6.6 kilobases were cloned to pBR322. The cloned DNA was characterized by restriction endonuclease mapping, DNA-RNA hybridization, and the R-loop technique.     

Cloning and identification of the product of the dnaE gene of Escherichia coli

We successively subcloned the dnaE gene of Escherichia coli into pBR322, resulting in a plasmid that contains 4.6 kilobases of E. coli DNA. This plasmid can complement a dnaE temperature-sensitive mutation. A restriction map of the dnaE gene and the surrounding 10.7-kilobase region of the E. coli chromosome was determined. A unique HindIII restriction endonuclease site within the cloned segment of DNA was identified as a site required for expression of the dnaE gene. By using the maxicell plasmid-directed protein synthesizing system, we demonstrated that dnaE codes for the alpha subunit of DNA polymerase III.     

Physical map of Salmonella typhimurium LT2 DNA in the vicinity of the proA gene.

More than 55 kilobases of chromosomal DNA of Salmonella typhimurium LT2, including the gpt, proA, ataA, and newD genes, were cloned in plasmid vector pULB113. The locations of the genes and selected restriction endonuclease cleavage sites were established, and some of the restriction enzyme fragments were subcloned in plasmid vector pBR322.     

Cloning and mapping of the genetic determinants for microcin B17 production and immunity.

Plasmid pMccB17 (70 kilobases [kb]) codes for the production of microcin B17, a peptide that inhibits DNA synthesis, and for microcin B17 immunity. A BamHI-EcoRI fragment of 5.1 kb from pMccB17 was cloned into pBR322 in two steps. The resulting plasmid (pMM102) overproduced microcin B17 and expressed immunity against microcin. Mcc- and Mcc- Imm- mutants were isolated on plasmids pMccB17 and pMM102 by deleting various DNA fragments and by inserting different translocatable elements. Physical and phenotypic characterization of these mutants showed that a DNA region of 3.0 to 3.5 kb is required to produce microcin B17, whereas an adjacent region of about 1.0 kb is required to express microcin B17 immunity.     

Cloning of the Streptococcus thermophilus beta-galactosidase gene and its expression in Escherichia coli and Bacillus subtilis cells

The beta-galactosidase gene from the chromosome of Streptococcus thermophilus, strain 6 kb, has been cloned on a vector plasmid pBR322. The corresponding gene has been found to be located on the Pst1 DNA fragment. The restriction map of this 6 kb fragment has been constructed. The shortening of the DNA fragment carrying the beta-galactosidase gene has been achieved by digestion of the recombinant derivative of pBR322 by the restriction endonuclease Sau3A under the conditions of incomplete hydrolysis. The obtained fragments have been cloned into the BamHI site in the berepliconed shuttle vector pCB20 for grampositive and gramnegative bacteria. The obtained recombinant plasmids contained the beta-galactosidase gene in the inserted fragments of different length. Expression of the cloned beta-galactosidase gene in Escherichia coli and Bacillus subtilis cells has been studied.     

Cloning and physical mapping of the dnaA region of the Escherichia coli chromosome.

The dnaA gene of Escherichia coli K-12, supposedly present in the deoxyribonucleic acid (DNA) of specialized transducing phase lambda i21 dnaA-2, was cloned onto plasmid pBR322. The new plasmid was named pMCR501. Physical analyses of DNAs of lambda i21 dnaA-2 and pMCR501 revealed the following. The lambda i21 dnaA-2 DNA retained the delta sr I lambda 1-2 and ninR5 deletions and imm21 substitution which were originally present in the parental phage. The size reduction was compensated for by the insertion-substitution segment (tna-dnaA region) in lambda i21 dnaA-2 DNA. The fractional size of this segment was approximately 7 megadaltons (Md), or 10 kilobases, which was found to be the sum of the tna insertion subsegment of ca. 3.5 Md and the dnaA substitution subsegment of ca. 3.5 Md. Phage P1-mediated transductional mapping between the dnaA46 and tna mutations gave a cotransduction frequency of 84%, corresponding to approximately 5 kilobases. Thus, it is strongly suggested that the dnaA gene resides in the lambda i21 dnaA-2 DNA. Cleavage mapping with the restriction endonuclease of pMCR501 DNA confirmed that it was constructed by excising a BamHI fragment of 4.29 Md, containing the 3.5-Md dnaA substitution segment, from the lambda i21 dnaA-2 DNA, inserting it into the sole BamHI cleavage site on pBR322.     

Escherichia coli glycerol kinase. Cloning and sequencing of the glpK gene and the primary structure of the enzyme

The glpK gene, which codes for Escherichia coli K-12 glycerol kinase (EC 2.1.7.30, ATP:glycerol 3-phosphotransferase), has been cloned into the HindIII site of pBR322. The gene was contained in a 2.8-kilobase DNA fragment which was obtained from a lambda transducing bacteriophage, lambda dglpK100 (Conrad, C.A., Stearns, G.W., III, Prater, W.E., Rheiner, J.A., and Johnson, J.R. (1984) Mol. Gen. Genet. 195, 376-378). The DNA sequence of 2 kilobases of the cloned HindIII fragment was obtained using the dideoxynucleotide method. The start of the open reading frame for the glpK gene was identified from the N-terminal sequence of the first 22 amino acid residues of the purified enzyme, which was determined by automated Edman degradation. The open reading frame codes for a protein of 502 amino acids and a molecular weight of 56,106 which is in good agreement with the value previously determined by sedimentation equilibrium. The primary structure of the protein as deduced from the gene sequence was corroborated by the isolation and sequencing of four tryptic peptides, which were found to occur at the following amino acid locations: 173-177, 203-211, 279-281, 464-468. The N-terminal sequence of the purified enzyme shows that the enzyme undergoes post-translational processing. Restriction digestion as well as DNA sequencing of the supercoiled plasmid shows that the HindIII fragment is inserted into pBR322 such that the glpK gene is transcribed in a counterclockwise direction. Examination of the upstream DNA sequence reveals two possible promoters of essentially the same efficiency: the P1 promoter of pBR322 and a hybrid promoter which contains both bacterial and pBR322 DNA sequences.     

Plasmid vectors for positive galactose-resistance selection of cloned DNA in Escherichia coli

Insertion of a HindIII-EcoRI fragment carrying part of the gal operon from lambda gal+ into pBR322 yields a plasmid (pAA3) which confers strong galactose sensitivity on E. coli strains deleted for the gal operon. Sensitivity to galactose is caused by the expression of kinase and transferase (but not epimerase) genes from a promoter located in the tet gene of pBR322. Insertion of a DNA fragment carrying Tn9 at the HindIII junction blocks gal expression and produces a galactose-resistant phenotype. Hence, galactose resistance can be used to select DNA fragments cloned at the HindIII site. The system was used efficiently for cloning lambda, yeast, and human DNA. The cloned fragments can be screened directly for the presence of promoters by testing for tetracycline resistance. Alternatively, these plasmids can be used as cosmids for cloning large fragments of DNA at a number of sites. Construction of several related vectors is described.     

Lysogenic conversion induced by phages phi 80. I. A description of the phenomenon and the cloning of the conversion gene

Escherichia coli cells lysogenic for coliphage phi 80 stop adsorbing the superinfecting phi 80 phage after having been kept under anaerobic conditions for a long time, which conferred on these cells the TonA phenotype. To determine the location of the gene for lysogenic conversion (cor), BamHI fragments of phi 80 DNA were cloned in pBR322 plasmid. The cells transformed with the recombinant plasmid pDK01 = pBR322 + phi 80 BamHI fragment 1 immediately acquire the TonA phenotype. So, the cor gene(s) is contained in the central phi 80 BamHI fragment (fragment 1) which includes gene 13, the b2 region and the att site.     

Isolation of cis-acting vaccinia virus DNA fragments promoting the expression of herpes simplex virus thymidine kinase by recombinant viruses.

Recombinant TK- vaccinia viruses containing the pBR322 sequence inserted in either orientation within the coding sequence of the viral thymidine kinase gene were constructed. They were characterized by genomic analysis, hybridization studies, reversion to wild-type virus by in vivo recombination, and rescue from their genomes of plasmids which contained all or parts of the pBR322 sequence. TK- cells were infected with one of these recombinant viruses and then transfected with pools of chimeric plasmids composed of a cloned herpes simplex virus thymidine kinase gene which contained upstream inserts of different vaccinia DNA fragments prepared by restriction or sonication. Recombination between homologous pBR322 sequences within infected cells generated selectable recombinant viruses in which expression of the herpes simplex virus thymidine kinase gene was promoted by the upstream vaccinia insert. These viruses were characterized by genomic analysis, hybridization, and in vivo or in vitro phosphorylation of (5-[125I]deoxycytidine as a specific assay for the expressed herpes simplex virus thymidine kinase. Vaccinia DNA inserts were isolated conveniently for transfer to bacteria by rescuing appropriate plasmids from the genome of recombinant viruses. The sequence of 100 nucleotides adjacent to the upstream region of the herpes simplex virus gene was determined in nine different inserts measuring 0.17 to 1.07 kilobase pairs.     

Cloning and delimiting one chloroplast DNA replicative origin of Chlamydomonas.

The EcoR1 restriction fragments containing D-loops which marked the replication origin of chloroplast DNA were identified in two different species of Chlamydomonas. Each fragment was cloned in the E. coli plasmid pBR325. The cloned fragments were compared by restriction endonuclease analyses and by heteroduplex analyses in the electron microscope. The relative position of the D-loop regions and the homologous regions between the 2 fragments was determined. The D-loops were located within one short homologous region of 0. 42kb in length between the 2 cloned restriction fragments. The homologous region was subcloned in pBR322. Closed circular plasmid DNAs containing the short homologous region showed preferred denaturation in the D-loop region under physiological salt concentration which suggested that D-loop region was AT rich. Sequence divergence was detected at both ends of the D-loop region. Southern blot analyses indicated the presence of species-specific repetitive sequences within the divergent regions.     

Cloning and expression of the staphylokinase gene of Staphylococcus aureus in Escherichia coli

Restriction fragments of DNA from bacteriophage S phi-C of Staphylococcus aureus which carries the gene for staphylokinase, one of the plasminogen activators, were cloned onto plasmid pBR322. Recombinant plasmids carrying the 2.5 kilobase pair segment of S phi-C DNA confer on Escherichia coli cells the capacity to synthesize staphylokinase. The enzyme is synthesized in amounts comparable to that found in S. aureus, and irrespective of the orientation of cloned fragments and their insertion site on pBR322. The active enzyme produced by E. coli cells is preferentially recovered from the periplasmic space and in part excreted into the culture medium. It is indistinguishable from the enzyme produced by S. aureus in molecular weight, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and in antigenicity, as determined by the micro-Ouchterlony precipitation test.     

Structure of a yeast non-initiating methionine-tRNA gene.

4 to 8 kb Hind III fragments of yeast DNA were cloned into pBR322. One of these clones (pY6m3) containing a single tRNA3Met gene has been characterized in detail. The DNA sequence of the structural gene is colinear with the tRNA sequence, which means that in this case no intervening sequence is present. The 5'-leader and 3'-trailer sequences have also been determined. The 5'-flanking region can be folded up into possible secondary structures.     

Genomic integration system based on pBR322 sequences for the cyanobacterium Synechococcus sp. PCC7942: transfer of genes encoding plastocyanin and ferredoxin.

Synechococcus sp. PCC7942 recipient strains were constructed for the chromosomal integration of DNA fragments cloned in any pBR322-derived vector, which carries the ampicillin resistance (ApR) marker. The construction was based on the incorporation of specific recombination targets, the so-called 'integration platforms', into the chromosomal metF gene. These platforms consist of an incomplete bla gene (ApS) and the pBR322 ori separated from each other by a gene encoding an antibiotic (streptomycin or kanamycin) resistance (SmR or KmR). Recombination between a pBR322-derived donor plasmid and such a chromosomal platform results with high frequency in restoration of the bla gene and replacement of the chromosomal marker (SmR or KmR) by the insert of the donor plasmid. The integration into the platform depends on recombination between pBR322 ori and bla sequences only and is therefore independent of the DNA insert to be transferred. The desired recombinants are found by selection for a functional bla gene (ApR) and subsequent screening for absence of the chromosomal antibiotic marker. Gene transfer with this integration system was found to occur efficiently and reliably. Furthermore, the presence of the pBR322 ori in the platform allowed for 'plasmid rescue' of integrated sequences. The system was applied successfully for the transfer of the gene encoding plastocyanin (petE1) from Anabaena sp. PCC7937 and for the integration of an extra copy of the gene encoding ferredoxin I (petF1) from Synechococcus sp. PCC7942 itself.     

Autonomous replicating sequences from intron of human ras gene in a simian virus 40 T antigen dependent system

Of the several DNA fragments present in the human lung cancer gene, 1.1 and 2.0 kilobase (kb) fragments corresponding to the intron of this gene were hybridized to a half part of the 27 nucleotides perfect palindrome present in the initiation part of replication in simian virus 40 (SV40) DNA. These two fragments cloned in pBR322 had good template activity, and the initiation of DNA replication started from the region of these fragments in an in vitro system, in which the initiation of DNA replication occurs on cloned DNA containing SV40 origin of DNA replication as described previously. Furthermore, these two clones could replicate autonomously in nuclei of SV40 transformed Cos cells, producing SV40 T antigen constitutively when the clones were transfected into Cos cells. These results show that functional SV40 origin-like sequences are present in human genomes, and they can replicate autonomously within the cells which are producing SV40 T antigen.