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.     

Shuttle cloning vectors for the cyanobacterium Anacystis nidulans.

Hybrid plasmids capable of acting as shuttle cloning vectors in Escherichia coli and the cyanobacterium Anacystis nidulans R2 were constructed by in vitro ligation. DNA from the small endogenous plasmid of A. nidulans was combined with two E. coli vectors, pBR325 and pDPL13, to create vectors containing either two selectable antibiotic resistance markers or a single marker linked to a flexible multisite polylinker. Nonessential DNA was deleted from the polylinker containing plasmid pPLAN B2 to produce a small shuttle vector carrying part of the polylinker (pCB4). The two polylinker-containing shuttle vectors, pPLAN B2 and pCB4, transform both E. coli and A. nidulans efficiently and provide seven and five unique restriction enzyme sites, respectively, for the insertion of a variety of DNA fragments. The hybrid plasmid derived from pBR325 (pECAN1) also transforms both E. coli and A. nidulans, although at a lower frequency, and contains two unique restriction enzyme sites.     

A hybrid plasmid is a stable cloning vector for the cyanobacterium Anacystis nidulans R2.

Anacystis nidulans R2 is a highly transformable strain which is suitable as a recipient for molecular cloning in cyanobacteria. In an effort to produce an appropriate cloning vector, we constructed a hybrid plasmid molecule, pSG111, which contained pBR328 from Escherichia coli and the native pUH24 plasmid of A. nidulans. pSG111 replicated in and conferred ampicillin and chloramphenicol resistance to both hosts. It contained unique sites for the restriction enzymes EcoRI, SalI, SphI, and XhoI, which could be used for the insertion of exogenous DNA. To demonstrate that a molecule like pSG111 could serve as a shuttle vector for the cloning of A. nidulans genes, we constructed a hybrid plasmid, pRNA404, containing an A. nidulans rRNA operon. This recombinant molecule was genetically and structurally stable during passage through A. nidulans and E. coli. The stability of the hybrid plasmid and the inserted rRNA operon demonstrates the feasibility of cloning in A. nidulans with hybrid vectors, with the subsequent retrieval of cloned sequences.     

Restriction endonuclease mapping and cloning of Mycobacterium intracellulare plasmid pLR7

A restriction map of Mycobacterium intracellulare plasmid pLR7 was developed. This 15.3-kb plasmid had unique sites for BamHI, HindIII, and XbaI. Various large fragments of pLR7 were cloned into pBR322 or pHP34 and propagated in Escherichia coli. A hybrid pLR7 ::pBR322 plasmid carrying the complete pLR7 sequence was constructed by joining the plasmids at their HindIII sites. The construction of these hybrids will facilitate the analysis and manipulation of pLR7 and may allow the development of this plasmid as a model system for genetic analysis in mycobacteria.     

Decreased DNA damage by acid and increased repair of acid-damaged DNA in acid-habituated Escherichia coli

A study of the conjugal transfer of ColV,I-K94 tn10 from acid-treated donors suggested that acid-habituated recipients repair acid-damaged plasmid DNA better than those that are not habituated. The presence of an increased repair activity for acid-damaged DNA in habituated cells was confirmed by isolating pBR322 from acid-treated organisms; habituated cells produced more transformants when transformed by it than did non-habituated ones. Additionally, agarose gel electrophoretic studies of pBR322 DNA isolated from acid-damaged cells and tests of its transforming activity both indicated that plasmid DNA in habituated cells is less damaged by extreme acidity than is that in non-habituated organisms.     

Transformation by integration in Aspergillus nidulans

DNA-mediated genetic transformation of Aspergillus nidulans has been achieved by incubating protoplasts from a strain of A. nidulans carrying a deletion in the acetamidase structural gene with DNA of derivatives of plasmid pBR322 containing the cloned structural gene for acetamidase [Hynes et al., Mol. Cell. Biol. 3 (1983) 1430-1439; p3SR2] in the presence of polyethylene glycol and CaCl2. The highest frequency obtained was 25 transformants per microgram of DNA. No enhancement of the transformation frequency was observed when DNAs of plasmids carrying either a fragment of the A. nidulans ribosomal repeat (p3SR2rr) or a fragment containing a possible A. nidulans mitochondrial origin of replication (p3SR2mo) in addition to the acetamidase gene were used. Both pBR322 and acetamidase gene sequences become integrated into the genome of A. nidulans in transformant strains. Integration events into the residual sequences adjacent to the deletion in the acetamidase gene, and probably (for p3SR2rr and p3SR2mo) into the ribosomal repeat unit are described.     

Decreased DNA damage by acid and increased repair of acid-damaged DNA in acid-habituated Escherichia coli

A study of the conjugal transfer of ColV,I-K94 tn 10 from acid-treated donors suggested that acid-habituated recipients repair acid-damaged plasmid DNA better than those that are not habituated. The presence of an increased repair activity for acid-damaged DNA in habituated cells was confirmed by isolating pBR322 from acid-treated organisms; habituated cells produced more transformants when transformed by it than did non-habituated ones. Additionally, agarose gel electrophoretic studies of pBR322 DNA isolated from acid-damaged cells and tests of its transforming activity both indicated that plasmid DNA in habituated cells is less damaged by extreme acidity than is that in non-habituated organisms.     

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.     

Role of the rom protein in copy number control of plasmid pBR322 at different growth rates in Escherichia coli K-12

The copy number per cell mass of plasmid pBR322 and a rom- derivative was measured as a function of generation time. In fast growing cells the copy number per cell mass was virtually identical for rom+ and rom- derivatives. However, the copy number of pBR322 only increased 3- to 4-fold from a 20- to 80-min generation time, whereas the copy number of the rom- derivative increased 7- to 10-fold. The copy number stayed constant for the rom+ and rom- plasmids at generation times longer than 80-100 min. Thus, the presence of the rom gene decreased the copy number of plasmid pBR322 in slowly growing cells at least 2-fold when compared with the rom- plasmid. To study the effect of the rom gene in trans we cloned the gene into the compatible P15A-derived rom- plasmid pACYC184. In cells carrying both pACYC184 rom+ and pBR322 rom- the presence of the rom gene in trans had little effect on the copy number of pBR322 rom- at fast growth, but it decreased its copy number at slow growth to the same level as found for pBR322, i.e., complemented the pBR322 rom- plasmid. The pACYC184 plasmid and its rom+ derivatives showed copy numbers similar to those of pBR322 rom- and pBR322 itself, respectively, at fast and slow growth. We conclude that the rom gene product-the Rom protein-is an important element in copy number control of ColE1-type plasmids especially in slowly growing cells.     

Cloning and characterization of a plasmid DNA from anacystis nidulans 6301

A plasmid DNA of Anacystis nidulans 6301 was isolated by CsCl-EtBr centrifugation. The Mr of the plasmid, named pBA1, was estimated to be 5.04 +/- 0.26 X 10(6) by electron microscopic analysis and 5.2 X 10(6) by agarose gel electrophoresis. The pBA1 DNA was opened at a unique site with BamHI and cloned in pBR322 vector propagated in Escherichia coli HB101 cells. The recombinant plasmid, named pBAS18, was digested with various restriction endonucleases and its cleavage map was constructed. Based on this result, the cleavage map of the pBA1 plasmid is presented.     

Function of the N-terminal half of RepA in activation of Rts1 ori.

The RepA protein of the Rts1 plasmid, consisting of 288 amino acids, is a trans-acting protein essential for replication. A mutant repA gene, repA delta C143, carrying a deletion that removed the 143 C-terminal amino acids of RepA, could transform, but at a low frequency, an Escherichia coli polA strain, JG112, when repA delta C143 was cloned into pBR322 with Rts1 ori in the natural configuration. The transformation was less efficient without the dyad DnaA box in the ori region, and no transformation occurred at 42 degrees C, characteristic of Rts1 replication. A fusion of the 3'-terminal half of repA of the P1 plasmid to repA delta C143 yielded a pBR322 chimeric plasmid that contained Rts1 ori through hybrid (Rts1-P1) repA. This plasmid was maintained much more stably in JG112 at 37 degrees C. At 42 degrees C, however, it was quite unstable. The overproduced hybrid RepA protein showed interference with mini-Rts1 replication in trans and also exhibited an autorepressor function, although both activities were decreased. These findings suggest that the N-terminal half of the RepA molecule of Rts1 is involved in the activation of the replication origin.     

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.     

Structure of chi hotspots of generalized recombination

Chi recombinational hotspots are sites around which the rate of Rec-promoted recombination in bacteriophage λ is elevated. Examination of a derivative of λ into which the plasmid pBR322 was inserted reveals that pBR322 lacks Chi sites. Using this λ-pBR322 hybrid, we obtained mutations creating Chi sites at three widely separated loci within pBR322. Nucleotide sequence analysis reveals that the mutations are single base-pair changes creating the octamer 5′ GCTGGTGG 3′. This sequence is present at three previously analyzed Chi sites in λ, and all analyzed mutations creating or inactivating these Chi sites occur within this octamer. We conclude that Chi is 5′ GCTGGTGG 3′, or its complement, or both.     

Cloning of a gene responsible for the biosynthesis of glutathione in Escherichia coli B

A gene (gshI) responsible for gamma-glutamylcysteine synthetase (GSH-I) activity was cloned to construct an Escherichia coli B strain having high glutathione synthesizing activity. For this purpose, two E. coli B mutants (strains C912 and RC912) were used. C912 was deficient in GSH-I activity. RC912, a revertant of C912, had a GSH-I activity that was desensitized to feedback inhibition of reduced glutathione. To clone gshI, chromosomal DNAs of RC912 and plasmid vector pBR322 were digested with various restriction endonucleases and then ligated with T4 DNA ligase. The whole ligation mixture was used to transform C912, and the transformants were selected as tetramethylthiuramdisulfide-resistant colonies. Of about 20 resistant colonies, 2 or 3 became red when treated with nitroprusside and showed appreciably high GSH-I activities. The chimeric plasmid DNA, designated pBR322-gshI, was isolated from the strain having the highest GSH-I activity and transformed into RC912. The structure and molecular size of pBR322-gshI in RC912 were determined. The molecular size of this plasmid was 6.2 megadaltons, and the plasmid contained a 3.4-megadalton segment derived from RC912 chromosomal DNA, which included gshI gene. The GSH-I activity of RC912 cells containing pBR322-gshI was fourfold higher than that of RC912 cells without pBR322-gshI.     

Cloning of a gene responsible for the biosynthesis of glutathione in Escherichia coli B.

A gene (gshI) responsible for gamma-glutamylcysteine synthetase (GSH-I) activity was cloned to construct an Escherichia coli B strain having high glutathione synthesizing activity. For this purpose, two E. coli B mutants (strains C912 and RC912) were used. C912 was deficient in GSH-I activity. RC912, a revertant of C912, had a GSH-I activity that was desensitized to feedback inhibition of reduced glutathione. To clone gshI, chromosomal DNAs of RC912 and plasmid vector pBR322 were digested with various restriction endonucleases and then ligated with T4 DNA ligase. The whole ligation mixture was used to transform C912, and the transformants were selected as tetramethylthiuramdisulfide-resistant colonies. Of about 20 resistant colonies, 2 or 3 became red when treated with nitroprusside and showed appreciably high GSH-I activities. The chimeric plasmid DNA, designated pBR322-gshI, was isolated from the strain having the highest GSH-I activity and transformed into RC912. The structure and molecular size of pBR322-gshI in RC912 were determined. The molecular size of this plasmid was 6.2 megadaltons, and the plasmid contained a 3.4-megadalton segment derived from RC912 chromosomal DNA, which included gshI gene. The GSH-I activity of RC912 cells containing pBR322-gshI was fourfold higher than that of RC912 cells without pBR322-gshI.