A High-Transformation-Efficiency Cloning Vector for Thermus thermophilus

The cloning vector pMK18 was developed through the fusion of the minimal replicative region from an indigenous plasmid of Thermus sp. ATCC27737, a gene cassette encoding a thermostable resistance to kanamycin, and the replicative origin and multiple cloning site of pUC18. Plasmid pMK18 showed transformation efficiencies from 10⁸ to 10⁹ per microgram of plasmid in Thermus thermophilus HB8 and HB27, both by natural competence and by electroporation. We also show that T. thermophilus HB27 can take pMK18 modified by the Escherichia coli methylation system with the same efficiency as its own DNA. To demonstrate its usefulness as a cloning vector, a gene encoding the β-subunit of a thermostable nitrate reductase was directly cloned in T. thermophilus HB27 from a gene library. Its further transfer to E. coli also proved its utility as a shuttle vector.     

F plasmid replication and the division cycle of Escherichia coli B/r

A terminal stage in the duplication of many bacterial plasmids involves the transient formation of catenated molecules containing two interlocked monomeric plasmid units. This property of plasmid replication was exploited to examine the relationship between F replication and the division cycle of Escherichia coli B/r cells growing in undisturbed, exponential-phase cultures. Various cultures of F′lac- or FKm^r-containing cells were briefly exposed to [³H]thymidine, and then the transfer of radioactivity into, and out of, a catenated dimer consisting of two closed circular monomers was measured during a chase period. The fraction of plasmid molecules present in this dimer form was determined by separating cellular DNA in alkaline sucrose gradients. In addition, plasmid replication was studied in synchronously growing cultures by measuring both [³H]thymidine incorporation into covalently closed circular DNA and β-galactosidase inducibility. The results suggest that replication of F plasmids can take place throughout the cell division cycle, with the probability of replication increasing toward the end of the cycle. The presence of DNA homologous to the chromosome on the F′lac did not alter the replication pattern of the plasmid during the division cycle.     

Genomic Organization and Chromosomal Assignment of the Human Voltage-Gated Na Channel β1 Subunit Gene (SCN1B)

Voltage-gated sodium (Na⁺) channels are essential for the generation and propagation of action potentials in striated muscle and neuronal tissues. Biochemically, Na⁺ channels consist of a large α subunit and one or two smaller β subunits. The α subunit alone can exhibit all of the functional attributes of a voltage-gated Na⁺ channel, but requires a β₁ subunit for normal inactivation kinetics. While genetic mutations in the skeletal muscle Na⁺ channel α-subunit gene can cause human disease, it is not known whether hereditary defects in the β₁ subunit underlie any inherited syndromes. To help explore this further, we have carried out an analysis of the detailed structure of the human β₁ subunit gene (SCN1B) including the delineation of intron-exon boundaries hy genomic DNA cloning and sequence analysis. The complete coding region of SCN1B is found in 9.0 kb of genomic DNA and consists of five exons (72 to 749 bp) and four introns (90 bp to 5.5 kb). Using a 15.9-kb genomic SCN1B clone, we assigned the gene to the long arm of chromosome 19 (19q13.1-q13.2) by fluorescence in situ hybridization. An intragenic polymorphic (TTA)_n repeat that is positioned between two tandem Alu repetitive sequences was also characterized. The (TTA)_n repeat exhibits 5 distinct alleles and a heterozygosity index of 0.59. This information should be useful in evaluating SCN1B as a candidate gene for hereditary disorders affecting membrane excitability.     

Characterization of a region of the Enterococcus faecalis plasmid pAMβ1 which enhances the segregational stability of pAMβ1-derived cloning vectors in Bacillus subtilis

The nucleotide sequence of a 2.13-kb EcoRI-HindIII, pAMβ1-derived fragment, isolated from the gram-positive cloning vector pHV1431, has been determined and shown to encode two ORFs. ORF H encodes for a protein of 23,930 Da which exhibits substantial homology to bacterial site-specific recombinases, particularly the resolvases of the gram-positive transposons Tn917 (30.3% identity) and Tn552 (31.6% identity) and the clostridial plasmid pIP404 (27.1% identity). The second ORF (I) is incomplete and encodes a polypeptide which has significant homology with Escherichia coli topoisomerase I (26.0% identity). Insertion of either the entire 2.13-kb EcoRI-HindIII fragment or a 0.73-kb EcoRI-DraI subfragment encoding only the resolvase into the pAMβ1-based cloning vector pMTL500E causes a significant enhancement of segregational stability (from 6.5 × 10⁻² to 3.0–4.0 × 10⁻³ plasmid loss per cell per generation). Improved segregational stability is mirrored by a reduction in plasmid polymerization. The introduction of a stop codon into the resolvase coding region negates its ability to promote segregational stability. It is proposed that the identified determinant stabilizes pAMβ1-based vectors in Bacillus subtilis by maintaining the plasmid population in the monomeric state, thereby reducing the chances of producing plasmid-free segregants.     

A bifunctional vector system for controlled expression and subsequent release of the cloned gene product by øX174 lysis protein-E

Summary A new bifunctional Escherichia coli cloning vector, pSB50, is presented. The plasmid allows controlled expression of a gene of interest under control of the lac promoter and the subsequent release of the cloned product by the use of bacteriophage øX174 lysis protein-E, the gene of which is under control of the phage Lambda pL promoter. To ensure optimal repression of the Lambda pL promoter and the lac promoter in plasmid pSB50, E. coli strain UB89-1 was constructed which carries a chromosomal copy of the lambda cl857 repressor allele and the lacl ^q1 allele, respectively. Here, we employ the E-based lysis system to release human prourokinase to the culture medium. Offprint requests to: U. Bläsi     

Cloned structural gene (ompA) for an integral outer membrane protein of Escherichia coli K-12

Summary pTU 100 is a hybrid plasmid constructed by cloning a 7.5 Kb EcoRI fragment (carrying the wildtype ompA gene) onto pSC 101 (Henning et al., 1979). This plasmid confers sensitivity to phages Tull^* and K3h1 when present in an ompA host strain, due to the expression of the phage receptor protein II^* from the plasmid ompA ⁺ gene. Plasmid mutants have been isolated that have become resistant to one or both of these phages. Restriction endonuclease analysis and DNA-sequencing studies in these plasmids demonstrate that a BamHI site and two PvuII sites are located within the ompA gene. BamHI cuts the gene at a site corresponding to residue 227 within a total of 325 amino acid residues.Neither the wildtype ompA gene nor the BamHI fragment encoding the NH₂-terminal part of the protein (residues 1–227) could be transferred to a high copy number plasmid, presumably due to lethal overproduction of the protein or its NH₂-terminal fragment. However, the NH₂-terminal fragment derived from one of the ompA mutants of pTU100 could be transferred to the high copy number plasmid pBR322, and was expressed in the presence of the amber suppressors supD or supF. Under these conditions two new envelope proteins with apparent molecular weights of 30,000 and 24,000 were synthesized, and the cells became sensitive to phage TuII^*, indicating the presence of phage receptor activity in the outer membrane. The major, 24,000 dalton protein has the molecular weight expected of a protein comprising residues 1–227 of protein II^*. DNA-sequencing studies demonstrated that no termination codons are present in the DNA region immediately downstream from the BamHI site at residue 227 in this hybrid plasmid, and it is therefore likely that the 24,000-dalton protein arises from the posttranslational proteolytic cleavage of a larger polypeptide. The 30,000-dalton protein is a likely candidate for such a larger polypeptide. These results also demonstrate that the 98 CO₂H-terminal residues of wildtype protein II^* (resisdues 228–325) are not required either for the activity of the protein as a phage receptor or for its incorporation into the outer membrane.     

Non-random distribution of transduction termini in transductants from the integrated R plasmid, R100-1

Summary Tra ⁺and tra ^–derivatives of drug resistance plasmid, R100-1, were isolated by phage P1 from an Hfr donor with integrated R100-1 and then analyzed by complementation tests with tra ^–point mutants of Flac. Tra ⁺derivatives of R100-1 carrying tetracycline resistance alone and those carrying all six drug-resistance genes could support transfer of tra ^–point mutants of Flac except Flac traJ, whereas all of tra ^–derivatives of R100-1 failed to complement any one of tra ^–point mutants of Flac. This suggests that these tra ^–derivatives of R100-1 carrying tetracycline resistance gene are deleted for all the transfer genes impaired in the Flac point mutants tested. We assume a hot point, probably a specific base sequence similar to an IS element, at the left of the tetracycline gene (Fig. 1) becomes a transduction terminus in transduction of the integrated R100-1 by phage P1. Complementation analysis of tra ^–derivatives carrying five resistance genes except the tetracycline gene led us to a supposition that a gene(s), probably analogous to traJ of the F plasmid, is located on R100-1 near the tetracycline gene which plays an important regulatory role for self-transfer as well as for the complementation of tra ^–Flac mutants.     

Regulation of the CYP27B1 5′-flanking region by transforming growth factor-beta in ROS 17/2.8 osteoblast-like cells

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), regulates osteoblast proliferation and differentiation. Production of 1,25(OH)₂D₃ is catalysed by the enzyme 25-hydroxyvitamin D₃-1α-hydroxylase (CYP27B1). Though highly expressed in the kidney, the CYP27B1 gene is also expressed in non-renal tissues including bone. It is hypothesised that local production of 1,25(OH)₂D₃ by osteoblasts plays an autocrine or paracrine role. The aim of this study was to investigate what factors regulate expression of the CYP27B1 gene in osteoblast cells. ROS 17/2.8 osteoblast cells were transiently transfected with plasmid constructs containing the 5′-flanking sequence of the human CYP27B1 gene fused to a luciferase reporter gene. Cells were treated with either parathyroid hormone (PTH), 1,25(OH)₂D₃, transforming growth factor-beta (TGF-β) or insulin-like growth factor-1 (IGF-1) and luciferase activity was measured 24 h later. The results showed that 1,25(OH)₂D₃ did not alter expression of the reporter construct, however treatment with PTH, IGF-1 and TGF-β decreased expression by 18, 53 and 58% respectively. The repressive action of TGF-β was isolated to the region between −531 and −305 bp. These data suggest that expression of the 5′-flanking region for the CYP27B1 gene in osteoblast cells may be regulated differently to that previously described in kidney cells.     

The maintenance affinities of KLF-1 proximal F merogenotes inEscherichia coli

Summary In conjugation with donor strains carrying proximal F merogenotes of KLF-1 type about 100-fold lower frequency ofLeu ⁺ orLac ⁺ recombinants was found. The determination of the level of β-galactosidase synthesis during the initial period of mating indicated that the transfer process of plasmid DNA was not impaired. Among the recombinants selected a large fraction have not expressed the plasmic fertility functions. This phenomenon was found to be replicon specific and was observed only with proximal F merogenotes but not with classical F'lac and F'ORF-1 elements or RI-19 plasmid. The expression of KLF-1 plasmid functions in the cell seems to be affected by a chromosomal gene of the proximal F merogenote closely linked toleu marker.     

Presence of the bacterial hemoglobin gene improves α-amylase production of a recombinantEscherichia coli strain

A recombinant plasmid (pMK57) was constructed by cloning theBacillus stearothermophilus α-amylase gene into pUC8; plasmid pMK79 was then derived from pMK57 by inserting the bacterial (Vitreoscilla) hemoglobin gene into the latter plasmid. Both pMK57 and pMK79 were transformed intoEscherichia coli strain JM103 to make strains MK57 and MK79, respectively. Both MK57 and MK79 produced α-amylase and MK79 produced hemoglobin. MK79 outgrew MK57 in shake flasks in LB medium, the advantage of the former appearing in late log phase. MK79 produced more α-amylase than MK57, on both per cell and per volume bases, in both mid and late log phases; the maximum advantage of MK79 (on a per volume basis) occurred in late log phase, at which time it produced 3.3 times as much α-amylase as MK57. The numbers of copies per cell of both pMK57 and pMK79 were significantly lower than that of pUC8.     

The Origin of Mutants Under Selection: How Natural Selection Mimics Mutagenesis (Adaptive Mutation)

Selection detects mutants but does not cause mutations. Contrary to this dictum, Cairns and Foster plated a leaky lac mutant of Escherichia coli on lactose medium and saw revertant (Lac⁺) colonies accumulate with time above a nongrowing lawn. This result suggested that bacteria might mutagenize their own genome when growth is blocked. However, this conclusion is suspect in the light of recent evidence that revertant colonies are initiated by preexisting cells with multiple copies the conjugative F′lac plasmid, which carries the lac mutation. Some plated cells have multiple copies of the simple F′lac plasmid. This provides sufficient LacZ activity to support plasmid replication but not cell division. In nongrowing cells, repeated plasmid replication increases the likelihood of a reversion event. Reversion to lac⁺ triggers exponential cell growth leading to a stable Lac⁺ revertant colony. In 10% of these plated cells, the high-copy plasmid includes an internal tandem lac duplication, which provides even more LacZ activity—sufficient to support slow growth and formation of an unstable Lac⁺ colony. Cells with multiple copies of the F′lac plasmid have an increased mutation rate, because the plasmid encodes the error-prone (mutagenic) DNA polymerase, DinB. Without DinB, unstable and stable Lac⁺ revertant types form in equal numbers and both types arise with no mutagenesis. Amplification and selection are central to behavior of the Cairns–Foster system, whereas mutagenesis is a system-specific side effect or artifact caused by coamplification of dinB with lac. Study of this system has revealed several broadly applicable principles. In all populations, gene duplications are frequent stable genetic polymorphisms, common near-neutral mutant alleles can gain a positive phenotype when amplified under selection, and natural selection can operate without cell division when variability is generated by overreplication of local genome subregions.     

Plasmid Copy Number Underlies Adaptive Mutability in Bacteria

The origin of mutations under selection has been intensively studied using the Cairns-Foster system, in which cells of an Escherichia coli lac mutant are plated on lactose and give rise to 100 Lac⁺ revertants over several days. These revertants have been attributed variously to stress-induced mutagenesis of nongrowing cells or to selective improvement of preexisting weakly Lac⁺ cells with no mutagenesis. Most revertant colonies (90%) contain stably Lac⁺ cells, while others (10%) contain cells with an unstable amplification of the leaky mutant lac allele. Evidence is presented that both stable and unstable Lac⁺ revertant colonies are initiated by preexisting cells with multiple copies of the F′lac plasmid, which carries the mutant lac allele. The tetracycline analog anhydrotetracycline (AnTc) inhibits growth of cells with multiple copies of the tetA gene. Populations with tetA on their F′lac plasmid include rare cells with an elevated plasmid copy number and multiple copies of both the tetA and lac genes. Pregrowth of such populations with AnTc reduces the number of cells with multiple F′lac copies and consequently the number of Lac⁺ colonies appearing under selection. Revertant yield is restored rapidly by a few generations of growth without AnTc. We suggest that preexisting cells with multiple F′lac copies divide very little under selection but have enough energy to replicate their F′lac plasmids repeatedly until reversion initiates a stable Lac⁺ colony. Preexisting cells whose high-copy plasmid includes an internal lac duplication grow under selection and produce an unstable Lac⁺ colony. In this model, all revertant colonies are initiated by preexisting cells and cannot be stress induced.