Hisao Uchida

Initiation of the DNA replication of bacteriophage lambda in Escherichia coli K12

A selective system was designed for the isolation of Escherichia coli mutants which can grow normally at 37 or 42 °C but cannot support replication of bacteriophage λ. One kind of these grp mutants maps at the dnaB locus and the other three kinds map at sites different from all other previously identified dna loci. The grpC, D and E loci map at approximately 0, 71 and 52 to 62 minutes on the E. coli 100-minute map, respectively. The grpC mutation is at a site presently identified as new dna locus (dnaK) by a tsAB756 mutation. In all the grp hosts tested, λ DNA remains superhelical after infection, indicating that the initiation step is blocked. Different classes of λ reg mutants were isolated which are able to replicate either in some or in all grp hosts. Two reg mutations were mapped and found to be located within the ori-O-P region of the λ genome. Some reg mutant phages showed simultaneous temperature sensitivity in their growth on grp+ cells. Both the temperature sensitivity and the reg character were found to be caused by a single mutation in the P gene. Moreover, λ phages harboring typical π mutations in the P gene (Georgopoulos & Herskowitz, 1971) can multiply on some of the grp mutants, and some of the reg mutant phages can replicate on the groP mutant cells. The grp mutations are recessive to grp+ but reg mutations are dominant over reg+ in all cases studied. The present data are consistent with the replisome model in which several gene products participate in the replication apparatus.

Genetic analysis of two genes, dnaJ and dnaK, necessary for Escherichia coli and bacteriophage lambda DNA replication

SummaryWe show that a collection of 93 E. coli mutations which map between thr and leu and which block phage lambda DNA replication define two closely linked cistrons. Work published in the accompanying paper shows that these mutations also affect host DNA replication, so we designate them dnaJ and dnaK; the gene order is thr-dnaK-dnaJ-leu. Demonstration of two cistrons was possible with the isolation of lambda transducing phages carrying one or the other or both of the dna genes. These phages were employed in phage vs bacterial complementation studies which unambiguously show that dnaK and dnaJ are different cistrons.

DNA sequencing of the Escherichia coli ribonuclease III gene and its mutations

SummaryA 0.7 kb DNA fragment of the Escherichia coli K12 chromosome was shown to contain the structural gene for RNAse III (rnc). The DNA sequence of the gene was determined and its alteration in an RNAse III defective mutant, AB301-105, was identified. DNA sequence analysis also showed that a secondary-site suppressor of a temperature-sensitive mutation in the E. coli ribosomal protein gene, rpsL, occurred within the rnc gene, providing genetic evidence for the interaction of ribosomal proteins with RNAse III, which in turn acts on the nascent ribosomal RNA during assembly of ribosomes in E. coli.

An F factor based cloning system for large DNA fragments

An effective technique using an Escherichia coli plasmid system was developed to clone fragments of exogenous DNA of as large as 100 kilobase pairs. The characteristic features of this technique are the use of a low copy number (one to two) mini-F based plasmid vector and the introduction of artificial lambda cosR ends into the termini of DNA sources and then of the cosL ends into those of linearized vector molecules. This terminal modification greatly facilitated the formation of active large recombinant molecules, which was rarely achieved when the modification was omitted. The efficiency with which large recombinant clones can be generated is high enough to allow construction of a comprehensive library of higher organisms. All analyses of the plasmids recovered have revealed that the inserts were faithful replicas of the human DNAs used as sources.

Late steps in the assembly of 30 S ribosomal proteins in vivo in a spectinomycin-resistant mutant of Escherichia coli☆

Abstract The late steps in ribosome assembly in vivo were studied by characterizing mutations which suppress the cold-sensitivity of a spectinomycin-resistant mutant of Escherichia coli . The results obtained indicated that the cold-sensitivity could be relieved by secondary alterations in either the S2, S3 or S5 protein of the 30 S ribosomal subunit. The gene controlling the alteration of S2 protein was closely linked to the polC gene located at about 3.5 minutes on the genetic map of E. coli , whereas S3 and S5 suppressor genes were linked to the str-spc region at 64 minutes. A possible model in which the S2, S3 and S5 proteins constitute a sub-assembly pathway in the assembly of 30 S subunits in vivo is discussed.

Organization and function of the tail of bacteriophage T4: II. Structural control of the tail contraction☆

Abstract The interaction of proteins, during their operation in the mature T4 bacteriophage particle, was studied by examining the function of various mutant phage particles incorporating genetically altered protein(s) or those that lack the component protein(s). Thus, the contraction behavior of the tail of the phage was studied by heating phage particles which carried various combinations of heat-sensitive (hs) mutations and structural defects produced by amber (am) mutations. The following phenomena were observed: 1. (1) Elimination of the long tail fibers from the viral particle completely suppressed tail contraction in many situations, provided the phage particle retained protein P9 ‡ . 2. (2) Elimination of P12, the short tail fibers, induced tail contraction after heat treatment at 55 °C, which does not affect intact phage particles. 3. (3) Heat denaturation of the carboxyl terminus of the short tail fiber inhibited the process of tail contraction. The inhibition was relaxed by superimposed denaturation of P5, as indicated by the behavior of double mutants hs in genes 5 and 12. 4. (4) Major constituent proteins of the tail, including P12 and P9, were conserved during tail contraction. Based on these findings, a possible model of the baseplate was constructed in which P6, P7, P8, P10, P25, P27 and P29 were visualized to constitute one functional unit acting as the skeleton of the baseplate whose hexagon-hexagram transition was controlled by “the outer proteins” which included P5, P9, P11 and both the short and long tail fibers.

Organization and function of bacteriophage T4 tail: I. Isolation of heat-sensitive T4 tail mutants

Abstract Phage T4 mutants which produce viral particles more sensitive to heating at 55°C than the wild type T4 have been isolated. They map in T4 tail genes 5, 6, 7, 10, 12, 18, 25, 27, and 48. According to the types of morphological alterations of the heat-inactivated particles, they were classified into three groups: 1. A: genes 5 and 12. No obvious change. 2. B: genes 18, 25, and 48. Sheath contracts, but baseplate is left. 3. C: genes 6, 7, 10, 25, and 27. Baseplate attached to the contracted sheath. Heat-sensitivities of Type C mutants only were suppressed by the presence of indole, whereas lower pH suppressed every mutants including Type A and Type B mutants. Heat-inactivated gene 5 mutant particles contract their sheath when mixed with the host cells, whereas gene 12 inactive phage remained unchanged. The analyses of heat-sensitive mutants may identify functional interactions between proteins in the phage particle.

Connection of the right-hand terminus of DNA to the proximal end of the tail in bacteriophage lambda

Abstract Lambda phage particles were disrupted by treatment with 50–60% formamide and subsequent spreading on a meniscus of ammonium acetate solution and analyzed by electron microscopy. The predominant product of this treatment is a phage DNA molecule of which the “right-hand” end is attached to the proximal end of a tail.

Suppressors of temperature-sensitive mutations in a ribosomal protein gene, rpsL (S12), of Escherichia coli K12.

SummaryTemperature-sensitive (ts) mutations were isolated within a ribosomal protein gene (rpsL) of Escherichia coli K12. Mutations were mapped by complementation using various transducing phages and plasmids carrying the rpsL gene, having either a normal or a defective promoter for the rpsL operon. One of these mutations, ts118, resulted in a mutant S12 protein which behaved differently from the wild-type S12 on CM-cellulose column chromatography. Suppressors of these ts mutations were isolated and characterized; one was found to be a mutation of a nonribosomal protein gene which was closely linked to the RNAase III gene on the E. coli chromosome. This suppressor, which was recessive to its wild-type allele, was cloned into a transducing phage and mapped finely. A series of cold-sensitive mutations, affecting the assembly of ribosomes at 20°C, was isolated within the purL to nadB region of the E. coli chromosome and one group, named rbaA, mapped at the same locus as the suppressor mutation, showing close linkage to the RNAase III gene.

Temperature-sensitive mutations in the α subunit gene of Escherichia coli RNA polymerase☆

Abstract Two temperature-sensitive mutations in the α subunit gene of Escherichia coli RNA polymerase ( rpoA ) were isolated by localized mutagenesis of the aroE - strA region of the chromosome, selecting for strains defective in RNA synthesis at non-permissive temperature. These mutations were mapped within a narrow segment assigned to rpoA , rpsD (S4) and rpsK (S11) genes. RNA polymerase purified from one of the mutants ( ts 112) was more thermolabile than the wild-type enzyme, but after a cycle of reversible dissociation and re-assembly, the thermolabile enzyme became even more thermostable than the wild-type enzyme. On the other hand, RNA polymerase purified from another mutant ( ts 101) was more thermostable to start with than the wild-type enzyme. By reversible dissociation and cross-assembly of these purified enzymes, mutational alterations were demonstrated to have occurred within the α subunit of the enzyme for both of the mutant strains. Transcriptional fidelity of these mutant RNA polymerases was greatly impaired, and frequent misincorporations of adenine and uracil in place of guanine and cytosine were noted in in vitro reactions at 30 °C and 42 °C. Nevertheless, the mutant strains showed no indication of erroneous transcription in vivo .