Relationship of vector insert size to homologous integration during transformation of Neurospora crassa with the cloned am (GDH) gene

Summary We used lambda and plasmid vectors containing the am ⁺ gene in an insert of from 2.7 to 9.1 kb, to transform am point mutant and deletion strains. A total of 199 transformants were examined with the potential to yield am ^– transformants by homologous recombination. When we used vectors that had 9.1 kb of homology with the chromosomal DNA, 30% of the transformants obtained were the result of homologous recombination regardless of whether the vector was a lambda molecule, a circular plasmid, or a plasmid that had been linearized prior to transformation. When vectors with up to 5.1 kb of homology were used, very few transformants (1 of 89 tested) resulted from homologous recombination. Of a sample of 29 ectopic integration events obtained by transformation with the 9.1 kb fragment cloned in a vector, 18 included a major part (usually almost all) of both arms of lambda with the entire Neurospora 9.1 kb insert between them. Four included only long arm sequence together with an adjacent segment of the insert containing the am gene. The remaining seven were the result of multiple integrations. There was no evidence of circularization of the vector prior to integration. All transformants that had multiple copies of the am gene appeared to be subject to the RIP process, which causes multiple mutations in duplicated sequences during the sexual cycle.     

Molecular cloning and nucleotide sequence of the Rhizobium phaseoli recA gene

Summary A recombinant phage carrying the recA gene of Rhizobium phaseoli was isolated from a R. phaseoli genomic library by complementation of the Fec^– phenotype of the recombinant phage in Escherichia coli. When expressed in E. coli, the cloned recA gene was shown to restore resistance to both UV irradiation and the DNA alkylating agent methyl methanesulphonate (MMS). The R. phaseoli recA gene also promoted homologous recombination in E. coli. The cloned recA gene was only weakly inducible in E. coli recA strains by DNA damaging agents. The DNA sequence of the R. phaseoli recA gene was determined and compared with published recA sequences. No LexA-binding site was detected in the R. phaseoli recA upstream region.     

A hotspot of spontaneous and UV-induced illegitimate recombination during formation ofλbio transducing phage

To study the mechanism of spontaneous and UV-induced illegitimate recombination, we examined the formation of thebio specialized transducing phage inEscherichia coli. Because mostbio transducing phages have double defects in thered andgam genes and have the capacity to form a plaque on anE. coli P2 lysogen (Spi^– phenotype), we selectedbio transducing phage by their Spi^– phenotype, rather than using thebio marker. We determined sequences of recombination junctions ofbio transducing phages isolated with or without UV irradiation and deduced sequences of parental recombination sites. The recombination sites were widely distributed onE. coli bio and DNAs, except for a hotspot which accounts for 57% of UV-inducedbio transducing phages and 77% of spontaneously inducedbio transducing phages. The hotspot sites onE. coli and DNAs shared a short homology of 9 bp. In addition, we detected direct repeat sequences of 8 by within and near both thebio and hotspots. ArecA mutation did not affect the frequency of the recombination at the hotspot, indicating that this recombination is not a variant ofrecA-dependent homologous recombination. We discuss a model in which the short homology as well as the direct repeats play essential roles in illegitimate recombination at the hotspot.     

Illegitimate recombination mediated by T4 DNA topoisomerase in vitro

Summary Illegitimate recombination dependent on T4 DNA topoisomerase in a cell-free system has recently been described. In that work, recombinants between two phage DNA molecules were produced by the topoisomerase alone, without an Escherichia coli extract. In this paper, it is shown that recombination between phage and circular plasmid DNA molecules can also be detected in the presence or absence of an E. coli extract but at frequencies two or three orders of magnitude lower than that observed in the phage-phage cross. The frequency is probably lower because multiple recombination is required in the case of the phage-plasmid cross.     

An improved selection method for λlacZ − phages based on galactose sensitivity

The determination of thelacZ mutant frequency in gt10lacZ phage vectors isolated from the transgenic mouse strain 40.6 (MutaMouse), requires the screening of large numbers of phages on -galactosidase activity. Existing methods rely on distinguishing a few white plaques on X-gal containing plates amongst a multide of blue ones which is both time-consuming and expensive. The new screening method described here employs the galactose sensitiveEscherichia coli C lacZ recA galE strain into which a multicopy plasmid has been introduced, which results in over-expression of thegalK andgalT genes. In the presence of phenyl--d-galactopyranoside, a substrate for -galactosidase, this leads to the suppression of lacZ ⁺ phage propagation without affecting the ability of lacZ ^– phages to form plaques. With this method it is possible to screen 1.5×10⁶ phages on a single 9-cm Petri dish. Furthermore, the need for blue/white screening has been eliminated.     

Site-directed insertion mutagenesis with cloned fragments in Escherichia coli by P1 phage transduction

Summary A cloned gene with an insertion, which was made by introducing cat, was ligated to the cloning site of the phage gt11. P1 phage grown on cells lysogenized with the recombinant phage could transduce the mutant gene into the original site on the Escherichia coli chromosome.     

Alleviation of EcoK DNA restriction in Escherichia coli and involvement of umuDC activity.

Summary The activity of the EcoK DNA restriction system of Escherichia coli reduces both the plating efficiency of unmodified phage and the transforming ability of unmodified pBR322 plasmid DNA. However, restriction can be alleviated in wild-type cells, by UV irradiation and expression of the SOS response, so that 10³-to 10⁴-fold increases in phage growth and fourfold increases in plasmid transformation occurred with unmodified DNA. Restriction alleviation was found to be a transient effect because induced cells, which initially failed to restrict unmodified plasmid DNA, later restricted unmodified phage . Although the SOS response was needed for restriction alleviation, constitutive SOS induction, elicited genetically with a recA730 mutation, did not alleviate restriction and UV irradiation was still needed. A hitherto unsuspected involvement of the umuDC operon in this alleviation of restriction is characterized and, by differential complementation, was separated from the better known role of umuDC in mutagenic DNA repair. The need for cleavage of UmuD for restriction alleviation was shown with plasmids encoding cleavable, cleaved, and non-cleavable forms of UmuD. However, UV irradiation was still needed even when cleaved UmuD was provided. The possibility that restriction alleviation occurs by a general inhibition of the EcoK restriction/modification complex was tested and discounted because modification of was not reduced by UV irradiation. An alternative idea, that restriction activity was competitively reduced by an increase in EcoK modification, was also discounted by the lack of any increase in the modification of Ral^–, a naturally undermodified phage. Other possible mechanisms for restriction alleviation are discussed.     

Mutant of the glutamine transfer RNA gene as UGA suppressor in Escherichia coli

Summary A UGA suppressor derived from a glutamine tRNA gene of Escherichia coli K 12 was isolated and characterized. Phages carrying the suppressor su⁺2_UGA could be obtained only from a hybrid transducing phage, h ⁸⁰ cI ₈₅₇psu ⁺2_oc, but not from the original transducing phage cI ₈₅₇psu ⁺2_oc. By DNA sequence analysis, it was found that the su ⁺2 UGA suppressor obtained has two mutations; one is in the anticodon (TTATCA), as expected, and the other (CT) is at the 7th position from the 3 end of tRNA ₂ ^Gln . The significance of these mutations and the lethal effect on phage of the increased amounts of UGA suppressor tRNAs are discussed.     

Molecular analysis of the recombination junctions of lambda bio transducing phages.

Summary To examine the mechanism of recombination involved in the formation of specialized transducing phage during the induction of bacteriophage we have determined the nucleotide sequences of the recombination junctions of bio phages. The results indicate that abnormal excision takes place at many sites on both bacterial and phage genomes and that the recombination sites have short regions of homology (5–14 bp). Some of the sequences of the recombination sites were similar to the consensus sequences of DNA gyrase-cleavage sites and repetitive extragenic palindromic (REP) sequences. These results showed that abnormal excision is a type of illegitimate recombination. The possible involvement of DNA gyrase in this recombination is discussed.     

Salmonella typhimurium LT2 metF operator mutations

Summary Using an Escherichia coli lac deletion strain lysogenized with lambda phage carrying a metF-lacZ gene fusion (Flac), in which -galactosidase levels are dependent on metF gene expression, cis-acting mutations were isolated that affect regulation of the Salmonella typhimurium metF gene. The mutations were located in a region previously defined as the metF operator by its similarity to the E. coli metF operator sequence. Regulation of the metF gene was examined by measuring -galactosidase levels in E. coli strains lysogenized with the wild-type Flac phage and mutant Flac phage. The results suggest that the mutations disrupt the methionine control system mediated by the metJ gene product, but not the vitamin B₁₂ control system mediated by the metH gene product. The results also demonstrate that negative control of the metF gene by the metH gene product and vitamin B₁₂ is dependent on a functional metJ gene product.Abbreviations Ap ampicillin - dNTP deoxyribonucleoside triphosphates - GM glucose minimal - Km kanamycin - L-agar Luria agar - LM lactose minimal - SAM s-adenosyl-L-methionine - TPEG phenylethyl -D-thiogalactoside - X-gal 5-bromo-4-chloro-3-indolyl -D-galactopyranoside - [] designates plasmid-carrier state - :: novel joint     

Cloning and sequencing of a cDNA encoding ascorbate peroxidase fromArabidopsis thaliana

A cDNA clone encoding ascorbate peroxidase (AP, EC 1.11.1.11) was isolated from a phage gt11 library of cDNA fromArabidopsis thaliana by immunoscreening with monoclonal antibodies against the enzyme, and then sequenced. The cDNA insert hybridized to a 1.1 kb poly(A)⁺ RNA from leaves ofA thaliana. Genomic hybridization suggests that the cDNA obtained here corresponds to a single-copy gene. The N-terminal amino acid sequence ofArabidopsis AP was determined by protein sequencing of the immunochemically purified enzyme, and proved to be homologous to the N-terminal amino acid sequence of the chloroplastic AP of spinach. The predicted amino acid sequence of the mature AP ofA. thaliana, deduced from the nucleotide sequence, consists of 249 amino acid residues, which is 34% homologous with cytochromec peroxidase of yeast, but less homologous with other plant peroxidases. Amino acid residues at the active site of yeast cytochromec peroxidase are conserved in the amino acid sequence ofArabidopsis AP. The poly(dG-dT) sequence, which is a potential Z-DNA-forming sequence, was found in the 3 untranslated region of the cDNA.     

Organization of the ribosomal RNA genes in Mycoplasma hyopneumoniae: the 5S rRNA gene is separated from the 16S and 23S rRNA genes

Summary In order to study the organization of the ribosomal RNA genes of Mycoplasma hyopneumoniae the rRNA genes were cloned in phage vectors EMBL3 and EMBL4. By subcloning the restriction fragments into various plasmids and analysing the resulting clones by Southern and Northern blot hybridization, a restriction map of the rRNA genes was generated and the organization of the rRNA genes was determined. The results show that the genes for the 16S and 23S rRNAs are closely spaced and occur only once in the genome, whereas the 5S rRNA gene is separated from the other two genes by more than 4 kb.     

Properties of RecA441 protein reveal a possible role for RecF and SSB proteins in Escherichia coli

Summary We examined the possibility that the recA441 mutation, which partially suppresses the UV sensitivity of uvr recF mutant bacteria, exerts its effect by coding for an altered RecA protein that competes more efficiently than the RecA⁺ protein with SSB for ssDNA in vivo. Using an assay measuring recombination between UV-damaged DNA and intact homologous DNA, we found that the introduction of the recA441 mutation partially suppressed the defects in recombination in bacteria lacking RecF activity but not in bacteria with excess SSB, although recombination was affected more in recF mutants than in bacteria overproducing SSB. These results therefore do not support the hypothesis that RecA441 protein, or RecA protein with the help of RecF protein, is required during recombination of UV-damaged DNA to compete with SSB for ssDNA.     

Homologous recombination between plasmid and chromosomal DNA in Bacillus subtilis requires approximately 70 bp of homology

Summary To determine the minimal DNA sequence homology required for recombination in Bacillus subtilis, we developed a system capable of distinguishing between homologous and illegitimate recombination events during plasmid integration into the chromosome. In this system the recombination frequencies were measured between is pE194 derivatives carrying segments of the chromosomal -gluconase gene (bglS) of various lengths and the bacterial chromosome, using selection for erythromycin resistance at the non-permissive temperature. Homologous recombination events, resulting in disruption of the bglS gene, were easily detected by a colorimetric assay for -gluconase activity. A linear dependence of recombination frequency on homology length was observed over an interval of 77 bp. It was found that approximately 70 bp of homology is required for detectable homologous recombination. Homologous recombination was not detected when only 25 by of homology between plasmid and chromosome were provided. The data indicate that homology requirements for recombination in B. subtilis differ from those in Escherichia coli.     

The bI4 RNA mitochondrial maturase of Saccharomyces cerevisiae can stimulate intra-chromosomal recombination in Escherichia coli

Summary When the bI4 RNA maturase, encoded by the fourth intron of the mitochondrial cytochrome b gene of Saccharomyces cerevisiae, was expressed in Escherichia coli, formation of intra-chromosomal Lac⁺ recombinants was stimulated threefold. This hyper-rec phenotype was recA as well as recBCD dependent. The most active form of the bI4 maturase stimulated homologous recombination whereas splicing deficient mutants of bI4 maturase were either deficient in or unable to stimulate homologous recombination.     

Lysis protein T of bacteriophage T4

Summary Lysis protein T of phage T4 is required to allow the phage's lysozyme to reach the murein layer of the cell envelope and cause lysis. Using fusions of the cloned gene t with that of the Escherichia coli alkaline phosphatase or a fragment of the gene for the outer membrane protein OmpA, it was possible to identify T as an integral protein of the plasma membrane. The protein was present in the membrane as a homooligomer and was active at very low cellular concentrations. Expression of the cloned gene t was lethal without causing gross leakiness of the membrane. The functional equivalent of T in phage is protein S. An amber mutant of gene S can be complemented by gene t, although neither protein R of (the functional equivalent of T4 lysozyme) nor S possess any sequence similarity with their T4 counterparts. The murein-degrading enzymes (including that of phage P22) have in common a relatively small size (molecular masses of ca. 18 000) and a rather basic nature not exhibited by other E. coli cystosolic proteins. The results suggest that T acts as a pore that is specific for this type of enzyme.     

Isolation and characterization of an auxin-inducible glutathione S-transferase gene of Arabidopsis thaliana

Genes homologous to the auxin-inducible Nt103 glutathione S-transferase (GST) gene of tobacco, were isolated from a genomic library of Arabidopsis thaliana. We isolated a clone containing an auxin-inducible gene, At103-1a, and part of a constitutively expressed gene, At103-1b. The coding regions of the Arabidopsis genes were highly homologous to each other and to the coding region of the tobacco gene but distinct from the GST genes that have been isolated from arabidopsis thusfar. Overexpression of a cDNA clone in Escherichia coli revealed that the AT103-1A protein had GST activity.     

RexAB proteins of bacteriophage lambda enhance the effect of photolyase-dimer complexes on lacZ gene expression in Escherichia coli.

Summary Expression of the lacZ gene in Escherichia coli is inactivated by exposure to ultraviolet light (UV). Inactivation is exceptionally effective when cells contain amplified levels of DNA photolyase (which forms complexes with pyrimidine dimers in the absence of light for actual photoreversal) and a prophage. Without amplified photolyase, the prophage or both, inactivation rates are similar and much lower. UV-inactivation of lacZ gene expression in the presence of both amplified photolyase and is even more effective if cI857 is used in place of the wildtype prophage but is wholly unexceptional if the prophage carries defects in the genes rexA or rexB. When Rex AB proteins are provided by expression from a plasmid and the cell also contains amplified photolyase, exceptional inactivation rates again obtain; in fact inactivation is most effective under these conditions. The data are considered to reveal a role for Rex AB proteins, which mediate superinfection exclusion, in the exceptional inactivation of gene expression by photolyase bound to pyrimidine dimers in DNA. Photolyase-dimer complexes may mimic the structure of certain complexes that arise during phage development and thus influence Rex A and/or B proteins, thereby shutting down cell metabolism.     

Role of the bacterial and phage recombination systems and of DNA replication in genetic recombination of UV-irradiated phage lambda

Summary In this paper are studied in E. coli K12 the influence of the bacterial Rec and phage Red recombination systems on the rescue of the O ⁺ gene from the prophage by a superinfecting O ^- phage, UV irradiated or not. In the absence of UV irradiation the Red system produces more recombinants that does the Rec system, and its action requires DNA replication. The presence of UV lesions in the DNA facilitates the action of the Rec system, which is more efficient in this instance than the Red system and can act in the absence of DNA replication. In all cases, there is a cooperation between the two generalized recombination systems.     

Construction and some properties of packageable plasmid F

Summary A derivative of plasmid F which is packageable in phage coat was constructed using techniques of in vitro recombination. This plasmid is composed of three DNA fragments generated by restriction enzyme EcoRI: a miniF fragment (fragment f5 of F'lac) which is able to replicate autonomously, a DNA fragment from Staphylococcus Plasmid that carries the -lactamase gene, and a portion of guaA (B) transducing phage DNA carrying cohesive ends (cos site) along with almost all the late genes but devoid of all those genes and sites that are needed for replication, regulation, and recombination. The hybrid plasmid has a molecular weight of 2.7×10⁷ daltons, about 84% size of phage genome, and can be packaged in coat when helper phage replicates in the plasmid-carrier cell. The package plasmid and the helper phage particles are separated by CsCl density gradient centrifugation. The replication characteristics of the recombinant plasmid are all those of F including the copy number, incompatibility, and curing with acidine orange. The packaged plasmid is injected into an F^- cell and establishes a plasmid state with normal efficiency. In F⁺ or Hfr cells, the resident F factor hinders this process.