Nucleotide sequence of the thymidylate synthetase gene (thyP3) from the Bacillus subtilis phage phi 3T

The thyP3 gene, encoding thymidylate synthetase, from the Bacillus subtilis phage phi 3T has been cloned and the nucleotide sequence determined. The derived amino acid sequence indicates a subunit Mr of 32 748. The primary amino acid sequence is compared with the sequences of the analogous proteins specified by Escherichia coli (thyA), Lactobacillus casei, (thyA) and phage T4 (td). Extensive conservation exists in all four sequences implying a shared tertiary structure.     

Genetic Analysis of Recombination-Deficient Mutants of Escherichia coli K-12 Carrying rec Mutations Cotransducible with thyA

The rec mutations carried by 20 strains of Escherichia coli K-12 which are defective in genetic recombination and sensitive to ultraviolet light and X rays, and whose lambda lysogens show spontaneous phage production, have been mapped near thyA. In 15 of the strains, the rec mutation fails to complement recB21 but complements rec-22. The other five strains carry a rec mutation which complements recB21 but not rec-22. These mutations map closer to thyA than those which fail to complement recB21. They therefore appear to be defective in a different recombination gene, denoted recC. The order of recB and recC on the linkage map of E. coli K-12 is thyA-recC-recB-argA.     

The cloning, DNA sequence, and overexpression of the gene araE coding for arabinose-proton symport in Escherichia coli K12

A lambda placMu1 insertion was made into araE, the gene for arabinose-proton symport in Escherichia coli. A phage containing an araE'-'lacZ fusion was recovered from the lysogen and its restriction map compared with that of the 61-min region of the E. coli genome to establish the gene order thyA araE orf lysR lysA galR; araE was transcribed toward orf. A 4.8-kilobase SalI-EcoRI DNA fragment containing araE was subcloned from the phage lambda d(lysA+ galR+ araE+) into the plasmid vector pBR322. From this plasmid a 2.8-kilobase HincII-PvuII DNA fragment including araE was sequenced and also subcloned into the expression vector pAD284. The araE gene was 1416-base pairs long, encoding a hydrophobic protein of 472 amino acids with a calculated Mr of 51,683. The amino acid sequence was homologous with the xylose-proton symporter of E. coli and the glucose transporters from a human hepatoma HepG2 cell line, human erythrocytes, and rat brain. The overexpressed araE gene product was identified in Coomassie-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of cell membranes as a protein of apparent Mr 35,000 +/- 1,150. Arabinose protected this protein against reaction with N-ethylmaleimide.     

Uracil-DNA glycosylase causes 5-bromodeoxyuridine photosensitization in Escherichia coli K-12.

An Escherichia coli uracil-DNA glycosylase-defective mutant (ung-1 thyA) was more resistant than its wild-type counterpart (ung+ thyA) to the killing effect of UV light when cultured in medium containing 5-bromouracil or 5-bromo-2'-deoxyuridine (BrdUrd). The phenotype of resistance to BrdUrd photosensitization and the uracil-DNA glycosylase deficiency appeared to be 100% cotransduced by P1 phage. During growth with BrdUrd, both strains exhibited similar growth rates and 5-bromouracil incorporation into DNA. The resistant phenotype of the ung-1 mutant was observed primarily during the stationary phase. In cells carrying 5-bromouracil-substituted DNA, mutations causing resistance to rifampin and valine were induced by UV irradiation at a higher frequency in the wild type than in the ung-1 mutant. This Ung-dependent UV mutagenesis required UmuC function. These results suggest that the action of the uracil-DNA glycosylase on UV-irradiated 5-bromouracil-substituted DNA produces lethal and mutagenic lesions. The BrdUrd photosensitization-resistant phenotype allowed us to develop a new, efficient method for enriching and screening ung mutants.     

Construction of a dihydrofolate reductase-deficient mutant of Escherichia coli by gene replacement.

The dihydrofolate reductase (fol) gene in Escherichia coli has been deleted and replaced by a selectable marker. Verification of the delta fol::kan strain has been accomplished using genetic and biochemical criteria, including Southern analysis of the chromosomal DNA. The delta fol::kan mutation is stable in E. coli K549 [thyA polA12 (Ts)] and can be successfully transduced to other E. coli strains providing they have mutations in their thymidylate synthetase (thyA) genes. A preliminary investigation of the relationship between fol and thyA gene expression suggests that a Fol- cell (i.e., a dihydrofolate reductase deficiency phenotype) is not viable unless thymidylate synthetase activity is concurrently eliminated. This observation indicates that either the nonproductive accumulation of dihydrofolate or the depletion of tetrahydrofolate cofactor pools is lethal in a Fol- ThyA+ strain. Strains containing the thyA delta fol::kan lesions require the presence of Fol end products for growth, and these lesions typically increase the doubling time of the strain by a factor of 2.5 in rich medium.     

Further characterization of a non-essential mutator gene in Escherichia coli K-12.

The properties of mutR, a mutator closely linked to thyA, have been further characterized. We have found that the mutator gene is carried on a specialized transducing phage (lambdapcI857 thyA) generated by the excision of lambdacI857 integrated at a secondary attachment site between lysA and thyA. We present three lines of evidence indicating that mutR is a nonessential gene. (i) Deletions of the mutator can be found amoung survivors of heat induction of lambdacI857 when the phage is integrated between lysA and thyA. (ii) Mutations in mutR can be induced with the frameshift mutagen ICR-191. (iii) An amber mutant in mutR has been found. Viable strains could be made by combining the mutator with polB, polA polR, ligts7, and uvrA mutations. The mutator was still able to increase the spontaneous mutation frequency in these genetic backgrounds. When the reversion patterns of a series of well-characterized trpA mutations were analyzed, the results suggested that mutR is more efficient at causing transitions than transversion mutations.     

Purification and properties of T4 phage thymidylate synthetase produced by the cloned gene in an amplification vector

We have introduced the T4 thymidylate synthetase gene, resident in a 2.7-kilobase EcoRI restriction fragment, into an amplification plasmid, pKC30. By regulating expression of this gene from the phage lambda pL promoter within pKC30 in a thyA host containing a temperature-sensitive lambda repressor, the T4 synthetase could be amplified about 200-fold over that after T4 infection. At this stage, a 20-fold purification was required to obtain homogeneous enzyme, mainly by an affinity column procedure. The purified plasmid-amplified T4 synthetase appeared to be identical with the T2 phage synthetase purified from phage-infected Escherichia coli in molecular weight, amino end group analysis, and immunochemical reactivity. The individual nature of the phage and host proteins was revealed by the fact that neither the T2 nor the T4 enzyme reacted with antibody to the E. coli synthetase, nor did antibody to the phage enzymes react with the E. coli synthetase. These differences were corroborated by DNA hybridization experiments, which revealed the absence of apparent homology between the T4 and E. coli synthetase genes. The techniques and genetic constructions described support the feasibility of employing similar amplification methods to prepare highly purified thymidylate synthetases from other sources.     

通过大肠杆菌thyA染色体-质粒平衡致死系统构建无抗性基因表达载体

克隆了大肠杆菌和霍乱弧菌胸腺嘧啶合成酶基因thyA ,并以pcDNA3质粒为基础 ,分别用两种来源的thyA基因替代其氨苄抗性基因Amp,构建了不含抗性基因 ,且可在thyA营养缺陷型大肠杆菌中基于染色体 质粒平衡致死系统稳定传代的真核表达载体。该载体可有效表达红色荧光蛋白报告基因。为核酸疫苗的制备提供一个无抗性的表达载体系统     

Mutation induction by thymidine deprivation in Escherichia coli B/r. I. Influence of caffeine.

The addition of caffeine to the plating medium after thymine deprivation of E. coli WP2 uvr+ thyA or WP2 uvrA thyA had no influence on survival. Caffeine, however, reduced the frequency of mutants. The hypothesis is presented that the reduced mutagenesis is due to the sensitivity to caffeine of an inducible error-prone repair mechanism operating during thymine deprivation and after the re-addition of thymine.     

Loss of an Essential Function of Escherichia coli by Deletions in the thyA Region

In an attempt to obtain deletions in the thyA gene, an abnormal lysogen of lambda having the prophage inserted between the thyA and lysA genes was induced, and the surviving cured cells were examined for Thy(-) and Lys(-) mutants. In nearly 10,000 cured cells, 184 Lys(-) but no Thy(-) mutants were found. At the same time, the induced lambda phage contained an approximately equivalent number of lambdathyA(+) and lambdalysA(+) transducing particles. By contrast, in a strain with the genotype F' thyA(-)lysA(+)/ thyA(+)lysA(+), induction of the abnormal lambda lysogen gave rise to many Thy(-) mutants in the cells cured of the prophage. In these Thy(-) mutants it was not possible to eliminate the episome with acridine orange, although the episome could be removed in control cultures with a thyA(+) allele in the resident gene. Therefore, it was suggested that deletion of a gene in the region of the chromosome from the position of the insertion of the lambda prophage through the thyA gene caused loss of an essential and diffusible function.     

Thymidylate synthase gene from Lactococcus lactis as a genetic marker: an alternative to antibiotic resistance genes

The potential of the thymidylate synthase thyA gene cloned from Lactococcus lactis subsp. lactis as a possible alternative selectable marker gene to antibiotic resistance markers has been examined. The thyA mutation is a recessive lethal one; thyA mutants cannot survive in environments containing low amounts of thymidine or thymine (such as Luria-Bertani medium) unless complemented by the thyA gene. The cloned thyA gene was strongly expressed in L. lactis subsp. lactis, Escherichia coli, Rhizobium meliloti, and a fluorescent Pseudomonas strain. In addition, when fused to a promoterless enteric lac operon, the thyA gene drove expression of the lac genes in a number of gram-negative bacteria. In transformation experiments with thyA mutants of E. coli and conjugation experiments with thyA mutants of R. meliloti, the lactococcal thyA gene permitted selection of transformants and transconjugants with the same efficiency as did genes for resistance to ampicillin, chloramphenicol, or tetracycline. Starting from the broad-host-range plasmid pGD500, a plasmid, designated pPR602, was constructed which is completely free of antibiotic resistance genes and has the lactococcal thyA gene fused to a promoterless lac operon. This plasmid will permit growth of thyA mutant strains in the absence of thymidine or thymine and has a number of unique restriction sites which can be used for cloning.     

Thymidylate synthase gene from Lactococcus lactis as a genetic marker: an alternative to antibiotic resistance genes.

The potential of the thymidylate synthase thyA gene cloned from Lactococcus lactis subsp. lactis as a possible alternative selectable marker gene to antibiotic resistance markers has been examined. The thyA mutation is a recessive lethal one; thyA mutants cannot survive in environments containing low amounts of thymidine or thymine (such as Luria-Bertani medium) unless complemented by the thyA gene. The cloned thyA gene was strongly expressed in L. lactis subsp. lactis, Escherichia coli, Rhizobium meliloti, and a fluorescent Pseudomonas strain. In addition, when fused to a promoterless enteric lac operon, the thyA gene drove expression of the lac genes in a number of gram-negative bacteria. In transformation experiments with thyA mutants of E. coli and conjugation experiments with thyA mutants of R. meliloti, the lactococcal thyA gene permitted selection of transformants and transconjugants with the same efficiency as did genes for resistance to ampicillin, chloramphenicol, or tetracycline. Starting from the broad-host-range plasmid pGD500, a plasmid, designated pPR602, was constructed which is completely free of antibiotic resistance genes and has the lactococcal thyA gene fused to a promoterless lac operon. This plasmid will permit growth of thyA mutant strains in the absence of thymidine or thymine and has a number of unique restriction sites which can be used for cloning.     

Cloning and nucleotide sequence of a negative regulator gene for Klebsiella aerogenes arylsulfatase synthesis and identification of the gene as folA.

A negative regulator gene for synthesis of arylsulfatase in Klebsiella aerogenes was cloned. Deletion analysis showed that the regulator gene was located within a 1.6-kb cloned segment. Transfer of the plasmid, which contains the cloned fragment, into constitutive atsR mutant strains of K. aerogenes resulted in complementation of atsR; the synthesis of arylsulfatase was repressed in the presence of inorganic sulfate or cysteine, and this repression was relieved, in each case, by the addition of tyramine. The nucleotide sequence of the 1.6-kb fragment was determined. From the amino acid sequence deduced from the DNA sequence, we found two open reading frames. One of them lacked the N-terminal region but was highly homologous to the gene which codes for diadenosine tetraphosphatase (apaH) in Escherichia coli. The other open reading frame was located counterclockwise to the apaH-like gene. This gene was highly homologous to the gene which codes for dihydrofolate reductase (folA) in E. coli. We detected 30 times more activity of dihydrofolate reductase in the K. aerogenes strains carrying the plasmid, which contains the arylsulfatase regulator gene, than in the strains without plasmid. Further deletion analysis showed that the K. aerogenes folA gene is consistent with the essential region required for the repression of arylsulfatase synthesis. Transfer of a plasmid containing the E. coli folA gene into atsR mutant cells of K. aerogenes resulted in repression of the arylsulfatase synthesis. Thus, we conclude that the folA gene codes a negative regulator for the ats operon.     

Genetic system for analyzing Escherichia coli thymidylate synthase.

Random in vitro mutagenesis of the thyA gene is being used to delineate its regulatory elements as well as the functional domains of its product, thymidylate synthase (EC 2.1.1.45). Streamlined procedures have been developed for the isolation and characterization of the mutants. Positive selection for synthase-deficient thyA Escherichia coli permitted the isolation of 400 mutants, which are being categorized by phenotypic and genetic criteria. An in situ 5-fluorodeoxyuridylate binding assay was devised to rapidly probe the substrate binding domain, whereas facile mapping procedures, based on pBR322- or M13-borne thyA deletion derivatives, were developed to localize mutations. The sequence changes of one amber mutation and another mutation that abolishes catalysis while maintaining substrate binding activity are presented. The orientation of the thyA gene on the E. coli chromosome was established.     

Activity of Chi Recombinational Hotspots in SALMONELLA TYPHIMURIUM

Chi sites have previously been shown to stimulate homologous recombination by the Escherichia coli RecBC pathway. To test the activity of Chi in another organism, bacteriophage lambda crosses were carried out in Salmonella typhimurium strains bearing the E. coli lambda receptor protein. Chi is active in these crosses in S. typhimurium, but is less active than in the same crosses carried out in E. coli. The lower Chi activity in S. typhimurium appears to be intrinsic to the S. typhimurium RecBC enzyme, since the Chi activity in E. coli-S. typhimurium hybrids depends on the species of origin of their RecBC enzyme. For these studies we constructed and F' factor and a pBR322-derived plasmid carrying the thyA+ recC+ recB+ argA+ region of the S. typhimurium chromosome.     

The construction in vitro of transducing derivatives of phage lambda

Summary Methods are described for the construction of plaque-forming, transducing derivatives of phage lambda, using appropriate receptor genomes and fragments of DNA generated by the restriction enzymes endo R.EcoRI and endo R.HindIII. The general properties of the transducing derivatives are described and discussed. Plaque-forming phages carrying the E. coli trp, his, cysB, thyA, supD, supE, supF, hsd, tna and lig genes have been isolated.     

Suppression by thymidine-requiring mutants of Escherichia coli K-12.

Thymidine-requiring strains of Escherichia coli isolated by trimethoprim selection often simultaneously acquire the ability to suppress bacteriophage T4 nonsense mutations. Suppression is lost in Thy+ revertants and recombinants, but is sometimes retained in thyA plasmid-bearing transformants. Suppression is restricted in Strr derivatives of the Thy- mutants, indicating that suppression occurs at the level of translation.     

Genetic and physiological characterization of a spontaneous mutant of Escherichia coli B/r with aberrant control of deoxyribonucleic acid replication.

Strain TJK16, a low-thymine-requiring thyA deoB derivative of Escherichia coli B/r A, was found to have an increased initiation mass due to a mutation in a gene affecting the control of initiation of deoxyribonucleic acid replication. In contrast to temperature-sensitive initiation mutants, initiation in TJK16 was not temperature sensitive. By phage P1 transduction, it was found that the mutation lies within a small region of the chromosome between dnaA and gyrB; this region includes dnaN and recF. Coumermycin-resistant derivatives of B/r and TJK16 had the same initiation mass as their coumermycin-sensitive parents, and TJK16 had the same sensitivity to coumermycin as the B/r parent, suggesting that the initiation mutation is not in gyrB.