Molecular cloning of part of the mitochondrial DNA of Drosophila melanogaster

We report the construction of recombinant plasmids containing part of the mitochondrial DNA of $\text{Drosophila}\text{melanogaster}$. Of the four fragments of this DNA generated by the restriction endonuclease HindIII, two were successfully cloned into the HindIII site of the plasmid pCM2. Unexpectedly the other two fragments could not be isolated by cloning into the HindIII site of either pCM2 or pBR322. Part of a third fragment, containing the gene for the large ribosomal RNA, was incorporated into the PstI site of pBR322. We show that this recombinant plasmid contains sequences complementary to an abundant RNA species which is present in $\text{Drosophila}$ embryos and which binds to oligo-dT-cellulose.     

Identification of two cDNA clones coding for androgen-dependent polypeptides in rat ventral prostate

cDNA clones were obtained by transformation of $\text{E. coli}$ x1776 with pBR322 containing insert of ds cDNA synthesized from total rat prostate poly(A) RNA. Two prostate-specific cDNA clones were isolated by colony hybridization and identified by message selection/translation as encoding polypeptides of M_r: 13,500 and 9,300. Hybridization of poly(A) RNA from normal and castrated rat prostates to the cloned cDNAs indicated that the levels of mRNAs coding for M_r: 13,500 and 9,300 polypeptides are regulated by testosterone.     

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.     

Cloning of two type II methylase genes that recognise asymmetric nucleotide sequences: FokI and HgaI

Summary The modification genes of Flavobacterium okeanokoites and Haemophilus galinarum have been cloned into the vector pBR322 and expressed in Escherichia coli cells. FokI methylase gene is contained on a 3.80 kb piece of F. okeanokoites DNA. Plasmid constructs carrying this fragment of DNA are resistant to digestion by FokI restriction endonuclease but are sensitive to cleavage by HindIII, EcoRI and PstI. Unmodified DNA molecules, exposed in vitro to cell extracts prepared from cells habouring this plasmid, became resistant to digestion by FokI.The smallest HgaI methylase clone carries the pBR322 plasmid containing a 3.50 kb piece of H. galinarum DNA. This plasmid is resistant to digestion by HgaI.Neither the FokI nor the HgaI restriction endonuclease was detected in either clone. This is the first report of cloning modification genes whose protein products recognise asymmetric nucleotide sequences.     

Identity of a Chi site of Escherichia coli and Chi recombinational hotspots of bacteriophage λ

Chi sites in bacteriophage λ stimulate recombination promoted by the RecBC pathway of Escherichia coli. We have located a Chi site within the E. coli lacZ gene by deletion mapping and have isolated a mutation inactivating this Chi. Sequence analysis showed that the mutation arose by a single base-pair transition $\text{G}\text{C}\text{?}\text{→}\text{A}\text{T}\text{?}$ within an eight base-pair sequence (5′ G-C-T-G-G-T-G-G 3′) identical to that found at Chi sites in λ and in plasmid pBR322.     

Expression in Escherichia coli of urokinase antigenic determinants

Hybrid genes were constructed between the β-lactamase gene of plasmid pBR322 and enzymatically synthesized DNA sequences coding for urokinase-like material. The recombinant plasmids, pULB1013 and pULB1010, contain DNA inserts of 220 and 535 base pairs respectively, which appear to be in the correct reading frame. The hybrid genes are expressed in $\text{Escherichia coli}$ and their products are specifically immunoprecipitated with antiserum to urokinase. In addition injection into a rabbit of a crude extract from the clone pULB1010 leads to the production of specific antibodies against urokinase.     

Cloning and sequencing of cDNA for mouse liver metallothionein-I

Metallothionein mRNA was purified from liver of mice injected with cadmium. The corresponding double-stranded cDNA was prepared and inserted into the PstI site of plasmid pBR322. The resulting recombinant DNA was used to transform the RR1 strain of $\text{Escherichia}\text{coli}$. Clones resistant to tetracycline and sensitive to ampicillin were screened for the presence of metallothionein-specific restriction fragments in their plasmids. One plasmid, called M135, contains a cDNA insert covering the entire length of the mRNA for mouse liver metallothionein-I, except for the first 18 bases at the 5′ end.     

Guanosine-5′-diphosphate-3′-diphosphate inhibits the in, vitro synthesis of β-lactamase from pBR322 DNA

The $\text{in}\text{,}\text{vitro}$ synthesis of β-lactamase directed by pBR322 DNA is inhibited by guanosine-5′-diphosphate-3′-diphosphate.     

Direct cloning of cDNA inserts from λgt11 phage DNA into a plasmid vector by a novel and simple method

Bacteriophage λgt11 has been used quite extensively for producing cDNA libraries. The cDNA inserts are usually subcloned into a plasmid vector for large scale production and analysis. However, isolating the recombinant DNA of interest from the phage clones can be a tedious task. Since the E. coli strain Y1088 used for λgt11 phage infection carries a pBR322-derived plasmid endogenously, we reasoned that this endogenous plasmid could be used directly for cloning the cDNA phage insert. In this report, we describe a method in which cDNA inserts from λgt11 phage were cloned directly into the pBR322 plasmid vector, by-passing the time-consuming procedures of preparing plasmid DNA as a subcloning vector. This method is likely to be extended to the cloning of DNA inserts derived from other phage λ vectors when bacteria containing endogenous pBR322 are used as host cells.     

Stability of plasmid pBR 322 in escherichia coli nf 161 (rela+) and nf 162 (rel a) grown in a methionine limited chemostat

The stability of the plasmid pBR 322 was measured in E. coli NF 161 (rel A⁺) and NF 162 (rel A) grown in a methionine limited chemostat. A significant plasmid loss occurred only in E. coli NF 162 at a low dilution rate and if the fermenter had been inoculated with cells from the stationary phase. Obviously, this behaviour resulted from the high expression of β-lactamase due to amplification of the pBR 322 DNA in the inoculum cells of E. coli NF162.     

Molecular cloning of the N-acetylneuraminate lyase gene in Escherichia coli K-12

Summary The gene for N-acetylneuraminate lyase [N-acetylneuraminate pyruvate-lyase; NPL] of Escherichia coli C600 was cloned onto pBR322 as a 9.8 kilobase HindIII fragment of chromosomal DNA and the hybrid plasmid was designated pMK2. The gene in the hybrid plasmid was subcloned in pBR322 as a 1.2 kilobase HindIII-EcoRI fragment and the resultant hybrid plasmid was designated pMK6. NPL activity level was increased more than 5-fold in the pMK6-bearing strain compared with that of the wild type, when the cells were grown on a medium containing inducer (N-acetylneuraminate: NANA). The transformants harbouring pMK6 also showed higher activity even in the absence of inducer. The NPL produced by pMK6-bearing cells was structurally and immunologically the same as that purified from E. coli C600.     

Studies on invitro DNA synthesis: II. Isolation of a protein which stimulates deoxynucleotide incorporation catalyzed by DNA polymerases of E.coli

Purified RNA polymerase, DNA polymerase III and unwinding protein of $\text{Escherichia}\text{coli}$ catalyze limited rifampicin sensitive fd or ØX 174 DNA-dependent DNA synthesis. A protein has been partially purified from $\text{E.}\text{coli}$ which stimulates rifampicin sensitive dXMP incorporation in this system 20 to 30 fold. This protein also stimulates DNA synthesis catalyzed by DNA polymerases I and II; the stimulation occurs in reactions primed with natural and synthetic DNAs as well as RNA-DNA hybrids. The protein is not a product of the known dna genes. In contrast to the above system of purified enzymes, rifampicin sensitive dXMP incorporation in crude extracts of $\text{E.}\text{coli}$ is specifically dependent on fd but not ØX 174 DNA. An additional factor has been isolated from extracts of $\text{E.}\text{coli}$ which restores specificity to the purified rifampicin sensitive system by preventing ØX 174 DNA from serving as a template.     

Cytosine-specific DNA modification interferes with plasmid establishment in Escherichia coli K12: involvement of rglB

Summary Several chimeric pBR322/328 derivatives containing genes for cytosine-specific DNA methyltransferases (Mtases) can be transformed into the Escherichia coli K12/E. coli B hybrid strains HB101 and RR1 but not into other commonly used E. coli K12 strains. In vitro methylation of cytosine residues in pBR328 and other unrelated plasmids also reduces their potential to transform such methylation sensitive strains, albeit to a lesser degree than observed with plasmids containing Mtase genes. The extent of reduced transformability depends on the target specificity of the enzyme used for in vitro modification. The role of a host function in the discrimination against methylated plasmids was verified by the isolation of K12 mutants which tolerate cytosine methylated DNA. The mutations map in the vicinity of the serB locus. This and other data indicate that the host rglB function is involved in the discrimination against modified DNA.     

The nucleotide sequence recognized by the Escherichia coli K12 restriction and modification enzymes.

The sites recognized by the Escherichia coli K12 restriction endonuclease were localized to defined regions on the genomes of phage φXsK1, φXsK2, and G4 by the marker rescue technique. Methyl groups placed on the genome of plasmid pBR322 by the E. coli K12 modification methylase were mapped in HinfI fragments 1 and 3, and HaeIII fragments 1 and 3. A homology of seven nucleotides in the configuration: 5′-A-A-C .. 6N .. G-T-G-C-3′, where 6N represents six unspecified nucleotides, was found among the DNA sequences containing the five EcoK sites of φXsK1, φXsK2, G4, and pBR322. Three lines of evidence indicate that this sequence constitutes the recognition site of the E. coli K12 restriction enzyme. The C in 5′-A-A-C and the T in 5′-G-T-G-C are locations of mutations leading to loss or gain of the site and thus are positions recognized by the enzyme. This sequence does not occur on φXam3cs70, simian virus 40 (SV40), and fd DNAs which do not possess EcoK sites, and occurs only once on φXsK1, φXsK2, and G4 DNAs, and twice on pBR322 DNA. In order to prove that all seven conserved nucleotides are essential for the recognition by the E. coli K12 restriction enzyme, the nucleotide sequences of φX174, G4, SV40, fd, and pBR322 were searched for sequences differing from the sequence 5′-A-A-C .. 6N .. G-TG-C-3′ at only one of the specified positions. It was found that sequences differing at each of the specified positions occur on DNA sequences that do not contain the EcoK sites. Thus, the recognition site of the E. coli K12 restriction enzyme has the same basic structure as that of the EcoB site (Lautenberger et al., 1978). In each case there are two domains, one containing three and the other four specific nucleotides, separated by a sequence of unspecified bases. However, the unspecified sequence in the EcoK site must be precisely six bases instead of the eight found in the EcoB site. Alignment of the EcoK and EcoB sites suggests that four of the seven specified nucleotides are conserved between the sequences recognized by these two allelic restriction and modification systems.     

Cloning of restriction and modification genes in E. coli: The HhaII system from Haemophilus haemolyticus

The genes for a Class II restriction-modification system (HhaII) from Haemophilus haemolyticus have been cloned in Escherichia coli. The vector used for cloning was plasmid pBR322 which confers resistance to tetracycline and ampicillin and contains a single endonuclease R·PstI site, (5′)C-T-G-C-A^↓-G (3′), in the ampicillin gene. The procedure developed by Bolivar et al. (1977) was used to form DNA recombinants. H. haemolyticus DNA was cleaved with PstI endonuclease and poly(dC) extensions were added to the 3′-OH termini using terminal deoxynucleotidyl transferase. Circular pBR322 DNA was cleaved to linear molecules with PstI endonuclease and poly(dG) extensions were added to the 3′-OH termini, thus regenating the PstI cleavage site sequence. Recombinant molecules, formed by annealing the two DNAs, were used to transfect a restriction and modification-deficient strain of E. coli (HB101 r^?m^?recA). Tetracycline-resistant clones were tested for acquisition of restriction phenotype (as measured by growth on plates seeded with phage λcI·O). A single phage-resistant clone was found. The recombinant plasmid, pDI10, isolated from this clone, had acquired 3 kilobases of additional DNA which could be excised with PstI endonuclease. In addition to the restriction function, cells carrying the plasmid expressed the HhaII modification function. Both activities have been partially purified by single-stranded DNA-agarose chromatography. The cloned HhaII restriction activity yields cleavage patterns identical to HinfI. A restriction map of the cloned DNA segment is presented.     

Cloning of a cDNA complementary to rat preputial gland β-glucuronidase mRNA

Messenger RNA was isolated from rat preputial glands by guanidine HCl extraction, ethanol and salt precipitation, followed by chromatography on oligo(dT) cellulose. Double-stranded cDNA was synthesized from the mRNA and inserted into the Pst 1 site of the plasmid pBR322 by the poly(dG)·poly(dC) tailing and annealing procedure. The hybrid plasmids were used to transform $\text{E. coli}$ HB101. Recombinant clones were screened for those containing cDNA inserts complementary to β-glucuronidase mRNA by a hybridization-selection procedure. One clone, containing an insert of about 1.2 kilobases, hybridized to preputial gland mRNA which, when translated $\text{in vitro}$, gave a product that migrated with the β-glucuronidase subunit on polyacrylamide gels.     

Stabilization by the Mini-F Fragment of a pBR322 Derivative Bearing the Tryptophan Operon in Escherichia coli

In an Escherichia coli K-12 strain (trpA trpE tnd) cultured in LB broth without selective pressure, a pBR322 derivative bearing the E. coli tryptophan Operon (pBR322-trp) was rapidly lost: after 27 cell-number doublings, only 7% cells retained both tryptophan prototrophy (Trp⁺) and ampicillin resistance (Ap^r), and 17% were Ap^r but Trp^?. Insertion of the mini-F DNA from F factor into this plasmid effectively suppressed both the plasmid loss and the discoordinate loss of Trp⁺: the percentage of Trp^? cells per cell-number doubling was decreased more than 100-fold. Partial derepression of the trp operon due to 3-indole acrylic acid further decreased the stability of the pBR322-trp but not that of the mini-F-inserted pBR322-trp.     

Cloning of a gene from the fission yeast S. pombe which complements E. coli pyrB, the gene for aspartate transcarbamylase

Summary DNA fragments of the fission yeast Schizosaccharomyces pombe that can complement pyrB mutations in Escherichia coli have been cloned into pBR322. Two contiguous HindIII fragments which are 2.7 kb and 1.5 kb in size are essential for this complementation. The cloned fragments have been proved to derive from yeast DNA by the use of Southern hybridization techniques. They apparently carry the structural gene for aspartate transcarbamylase [EC 2.1.3.2] and a promoter signal that is functional in E. coli. S. pombe is now the fourth eukaryote with a gene shown to be functionally expressed in E. coli. The DNA fragments cloned in this work will be useful in molecular-cloning studies in S. pombe.