Location of the multivalent control site for the ilvEDA operon of Escherichia coli

A strain of Escherichia coli K-12 containing a deletion extending from early in the ilvE gene toward the ilvG gene was shown to exhibit a higher expression of the downstream genes, ilvD and ilvA, than did an ilv+ strain. The elevated expression was under apparently normal ilv-specific control, however. The deletion was transferred to the ilv region of lamba h80dilv and shown by restriction endonuclease and heteroduplex analysis to extend through the deoxyribonucleic acid (DNA) shown, in the preceding paper (C. S. Subrahmanyam, G. M. McCorkle, and H. E. Umbarget, J. Bacteriol 142:547--555, 1980), to contain the ilvO determinant. The deletion was also transferred to an ilv-lac fusion strain and shown to cause an increase in beta-galactosidase formation while allowing retention of ilv-specific control. Transducing phages excised from these fusion strains with and without the ilvO determinant were compared. The phage carrying the ilvO+ determinant contained ilv DNA extending only into but not through the ilvG gene. It did not exhibit an ilv-specific control of beta-galactosidase formation. The phage carrying the deletion of ilvO but containing ilv DNA extending beyond the ilvG gene exhibited ilv-specific control of beta-galactosidase formation. It was concluded that the multivalently controlled ilv-specific promoter affecting ilv operon expression lies upstream from ilvG and that the ilvO region in the wild-type K-12 strain is a region of polarity preventing ilvG expression and reducing ilvEDA expression.     

Absence of significant membrane localization of the proteins coded by the ilvGEDAC genes of Escherichia coli K-12.

We previously characterized a set of lambda dilv phages by genetic, restriction enzyme, and heteroduplex analyses and tentatively correlated isoleucine-valine gene products with specific ilv DNA segments by using cloned ilv segments in maxicells and lambda dilv phage infection of UV-irradiated cells. In this work, the identity of the ilvC gene product, alpha-acetohydroxy acid isomeroreductase, was confirmed by demonstrating its induction by the physiological inducers alpha-acetolactate and alpha-acetohydroxybutyrate. The identity of the ilvE gene product, transaminase, B, was confirmed by antibody precipitation of the purified enzyme. Phage derivatives with ilv regulatory mutations were found to have the predicted effect upon the ilvGEDA and ilvC protein products. The distribution of the ilvGEDA and ilvC gene products in the soluble, periplasmic, inner membrane, and outer membrane fractions was examined, and no significant membrane association was observed. The expression of the ilv genes in the lambda dilv phage from ilv and phage lambda promoters was compared in order to determine the fractional contribution of each to ilv gene expression. An additional protein of 54,000 daltons that was not detected in the previous analysis was observed to be coded by a bacterial gene but was produced only by readthrough from phage promoters.     

Identification of a protein of 15,000 daltons related to isoleucine-valine biosynthesis in Escherichia coli K-12.

The effect of the ilvG671, ilvG468, and ilvG603 mutations (phenotype, IlvG+ Valr; formerly ilvO) upon proteins synthesized was determined by infection of irradiated Escherichia coli K-12 cells, using specifically constructed derivatives of lambda dilv phage. These ilvG alleles are similar to the previously studied ilvG2096(Valr) allele in that they activate the latent ilvG gene which is present in the wild-type strain, leading to the synthesis of a 62,000-dalton protein. In addition, all of these ilvG (Valr) alleles increase the synthesis of a 15,000-dalton protein. To localize the gene coding for the 15,000-dalton protein, the proteins produced in maxicells containing plasmids with specific deletions of ilv and rrnX DNA segments were analyzed. The gene coding for the 15,000-dalton protein was located within a region about 1,000 base pairs long between ilv and trpT. The function of the 15,000-dalton protein is not known.     

Genetic analysis of the Escherichia coli K-12 srl region.

Specialized transducing lambda derivatives, deletion mapping, and Plkc transductional crosses have been used to analyze the genetic organization and regulation of the srl genes. Transducing phages obtained from a secondary site lambda insertion in srlA are of two types: lambdapsrlC1 and lambdaprecA are substituted in the b2 region of the lambda chromosome (galtype) and carry the srlC gene but not srlD; lambdapsrlD is substituted in the early region of the phage deoxyribonucleic acid (biotype) and carries the srlD gene but not srlC. The lambdapsrlC1 phage, which lysogenizes at attlambda, complements srlC mutants in trans, indicating that this gene codes for a diffusable positive regulatory element. The srl genes have been ordered relative to the cysC, recA, and alaS genes by two- and three-factor P1kc crosses. The order, cysC...srlD-srlA-srlC-recA-alaS, has been obtained. The srlA and srlD genes comprise an operon with srlD operator distal. From the secondary site lysogen, it has been possible to obtain deletion mutants of this region that are sensitive to ultraviolet light and are recombination deficient. Genetic evidence suggests that these deletions extend from srl into the recA gene.     

λ噬菌体和Cosmid重组DNA克隆的快速限制性内切酶图谱分析方法

cosmld克隆的线性化用λcos末端酶来完成,线性的cosmid或λDNA经部份限制性内切酶酶解后,分别与已标记的cos顺序探针杂交(探针为分别与λ的左端或右端的cos顺序互补的12核苷酸单链片段),杂交后的部份酶解片段经电泳分离和自显影后,酶切点位置可直接在X-底片上读出。在本实验室条件下,可一次完成二个克隆包括5—6种限制性内切酶的图谱分析,分析和作图可通过计算机或手工进行。     

Relative map location of the rep and rho genes of Escherichia coli.

The rep gene of Escherichia coli was mapped between ilvC and rho by three-factor P1 transductional crosses and also by complementation with a set of lambda transducing phages that contain known amounts of bacterial DNA linked to ilvC. The physical distance between ilvC and rep and between rep and rho were calculated with an accuracy of +/- 0.4 kilobase to be 0 less than or equal to ilvC-rep less than or equal to 3.4 kilobases and 2.0 less than or equal to rep-rho less than or equal to 6.0 kilobases. It was shown that rho-15 is Gro+ for phage ST-1. An ilv::Tn10 mutation was located in ilvY.     

Complementation Between Different Mutations in the ilvA Gene of Escherichia coli K-12

An ilvA mutation carried by a ?80i(lambda)dilv transducing phage complemented some ilvA mutations and did not complement others. Complementation was accompanied by appearance of threonine deaminase activity in vivo. These results divided the ilvA mutations into two sets which formerly appeared to define two cistrons.     

Fine-scale mapping in Neurospora crassa by using genome-wide knockout strains

Fine-scale genetic mapping is often hindered by the lack of adequate markers surrounding the locus of interest. In the filamentous ascomycete Neurospora crassa, the genome has been sequenced and an effort has been made to generate genome-wide deletion strains for the entire gene set. Accordingly, the hygromycin-resistant marker in each deletion strain can be used as a mapping locus in a classical three-point cross, along with the mapping target and a standard marker. We have demonstrated the feasibility of this fine-scale mapping approach in N. crassa by refining the location of r(Sk-2).     

New transducing phage with RNA polymerase beta- and beta'-subunit genes derived from a hybrid phage lambda att80: isolation, genetic analysis and physical mapping]

A hybrid lambda att 80 phage with the genetic structure lambda (A-J) phi 80 (att-int-xis) imm lambda..cI857s7 is shown to be a convenient vector for creating transducing phages. On the one hand, the restriction analysis indicates that it has 3 restriction sites for EcoRI in comparison with 5 and 9 sites for parental phages lambda and phi 80 respectively. On the other hand, its buoyant density is less than that of phage lambda and under centrifugation it is easier separated from the phage transducing particles. When lambda att 80 prophage was excluded from the bfe locus of Escherichia coli, transducing phages with genes of two RNA polymerase beta-subunits (rpoB and rpoC) were isolated. To identify the latter, a convenient genetic test was worked out. A physical map of lambda att 80 drifd 35 transducing phage, carrying rpoB and rpoC genes has been constructed using endonucleases EcoRI and HindIII. A comparison of this map and the corresponding maps of transducing phages lambda drifd 18 and lambda drifd 47, studied earlier, led to the discovery of two integration sites of phage lambda within the locus bfe spaced apart by about 1800 nucleotide pairs. At all the sites both phages (lambda and lambda att 80) have integrated in the locus bfe in the counter clockwise order.     

Conditional lethal phosphatidylserine decarboxylase mutants of Escherichia coli

Summary The final step in the biosynthesis of phosphatidylethanolamine, the major membrane lipid of Escherichia coli, is catalyzed by the membrane-bound enzyme, phosphatidylserine decarboxylase. A variation of a procedure for localized mutagenesis (Hong and Ames, 1971) was employed to generate conditional lethal mutants in phosphatidylserine decarboxylase. In our modification, an episome carrying the psd gene closely linked to purA ⁺ was heavily mutagenized in vivo in a strain also lysogenic for phage P1 CMclr100. After induction of a phage lytic cycle, the purA ⁺ marker was transduced to a purA ^- recipient. A majority of the Pur⁺ transductants thus contained a psd gene originating from the heavily mutagenized episomal strain. Three mutants were isolated in which temperature-sensitive growth is caused by thermosensitive phosphatidylserine decarboxylase activity that is defective in vivo at the non-permissive temperature. All 3 mutations were mapped at the same location as psd1, being cotransduced with melA, purA, and ampA. The gene order in this region, as determined by a phage P1-mediated, three-factor cross is ampA-psd-purA. psd ⁺ is dominant to the psd mutant alleles.     

Characterization and in Vivo Cloning of prlC, a Suppressor of Signal Sequence Mutations in Escherichia coli K12

The prlC gene of E. coli was originally identified as an allele, prlC1, which suppresses certain signal sequence mutations in the genes for several exported proteins. We have isolated six new alleles of prlC that also confer this phenotype. These mutations can be placed into three classes based on the degree to which they suppress the lamB signal sequence deletion, lamBs78. Genetic mapping reveals that the physical location of the mutations in prlC correlates with the strength of the suppression, suggesting that different regions of the gene can be altered to yield a suppressor phenotype. We also describe an in vivo cloning procedure using lambda placMu9H. The procedure relies on transposition and illegitimate recombination to generate a specialized transducing phage that carries prlC1. This method should be applicable to any gene for which there is a mutant phenotype.     

A simple and efficient procedure for the isolation of high-quality phage lambda DNA using a DEAE-cellulose column

A simple and rapid procedure for purifying large quantities of bacteriophage lambda particles and DNA is described. The procedure involves DEAE-cellulose column chromatography of the phage particles and elution of the phage particles from the column with a low-ionic-strength buffer. The resulting phage were well separated from RNA, DNA, and proteins derived from Escherichia coli host cells. The lambda DNA was prepared from the purified phage particles by the conventional method of phenol extraction and ethanol precipitation. This procedure did not use nucleases, proteases, detergents, or CsCl density gradient centrifugation. The lambda DNA obtained by this method was equivalent in purity to the material prepared by CsCl density gradient centrifugation and amenable to restriction enzyme digestion, ligation, radiolabeling, and double-stranded DNA sequencing. A detailed protocol is described for obtaining 0.5 to 1.0 mg DNA from a 1-liter liquid lysate in less than 5 h. This procedure is simple, inexpensive, and timesaving, and is particularly suitable for large-scale isolation of lambda DNA.     

Genetic characterization of a gene for prolipoprotein signal peptidase in Escherichia coli

A mutation (lspA, prolipoprotein signal peptidase) rendering the prolipoprotein signal peptidase temperature-sensitive in Escherichia coli has been analyzed. The mutation was mapped in the dnaJ-rpsT-ileS-dapB region by interrupted mating with various Hfr strains and P1 phage transduction. lambda transducing phage lambda ddapB2 that carries the rpsT-ileS-dapB region was shown to complement the lspA mutation. Plasmid pLC3-13 which had been isolated from Clarke and Carbon's collection as a plasmid carrying the lspA locus was shown to carry the dnaJ and rpsT loci. Complementation analysis with plasmids carrying various DNA fragments derived from pLC3-13 showed that the lspA locus is between the rpsT and ileS loci. The wildtype allele was dominant over the lspA allele.     

Deletion mapping of the ilvGOEDAC genes of Escherichia coli K-12.

Summary A set of dilv phage has been examined that carry overlapping segments of isoleucine-valine structural and regulatory genes derived from the ilv cluster at 83 min on the Escherichia coli K-12 chromosome. The ilv genes present in these phage, and their order, have been determined by transduction of auxotrophs, escape synthesis, and deletion mapping. The order of ilv genes in the phage, and hence the order in the host chromosome, was found to be ilvG-ilvO-ilvEDA-ilvC. Lysogens containing dilv phage were constructed for dominance analysis of regulatory mutations in the ilvO and ilvA genes. The ilvO671 allele is cis-dominant to ilvO ⁺, while the ilvA538 allele is trans-recessive to ilvA ⁺. Thus, the ilvO gene, that is identified by cis-dominant regulatory mutations that result in increased ilvG and ilvEDA expression, is situated between and may be contiguous with ilvG and ilvEDA.     

Mutant of Escherichia coli that instantaneously loses the ability to adsorb lambda bacteriophage upon exposure to high temperature.

lad (lambda adsorption), an Escherichia coli mutant that loses the ability to adsorb lambda phage immediately after a shift to high temperature (e.g., 42 C), was isolated. This property for phage adsorption is irreversible and has been observed with phage lambda and 21 but not with phages 434, phi 170, and phi 80. A crude receptor preparation, extracted from lad cells will cholate-ethylenediaminetetraacetic acid by the procedure of Randall-Hazelbauer and Schwartz (1973), inactivated the phage lambda only at low temperature.