Yoshimasa Sakakibara

Regulation of transcription of the lambda bacteriophage genome

Abstract More than ten species of λ mRNA have been characterized by zone sedimentation and by hybridization with the separated λ and λdg DNA strands. Five species of mRNA were transcribed from the λl strand (l mRNA), five from the left half of the λr strand (rL mRNA) and four from the λdgA-Jr strand (rR mRNA). The summation of the length of these classes, l, rL, and rR, of mRNA species corresponds roughly to that of the regions of genes cI-intA(aa′), A-J, and x-R of a λ chromosome, respectively. All these classes of mRNA species consist of both large (>16 S) and small (

Formation of catenated molecules by replication of colicin E1 plasmid DNA in cell extracts.

Catenated molecules consisting of two interlocked closed circular monomeric units are formed as a minor product of colicin E1 plasmid DNA synthesis in extracts of Escherichia coli . The interlocked units of catenanes are mostly monomeric molecules that have completed semiconservative replication in the extract. Pulse and chase studies revealed that the catenanes are derived from two types of precursors: those consisting of two open circular monomeric units and of an open circular and a closed circular monomeric unit. Most of the latter type of precursors contain an interruption in the newly synthesized “heavy” strand and those of the former type contain an interruption in both of the newly synthesized strands. It is concluded that a catenance consisting of two closed circular units is formed by sequential sealing of the progeny strands of a catenane consisting of two open circular units. The sealing of the progeny strands of both types of precursors is blocked by addition of 10 m m -nicotinamide mononucleotide. Catenanes once formed are stable in extracts and their conversion to monomeric molecules, if it occurs, is insignificant. Formation of catenanes from monomeric molecules that do not participate in replication and/or that have formed in extracts is also insignificant. It is concluded that catenances are formed by an event associated with replication rather than by recombination and that replication of a monomeric molecule results in the formation of two monomeric molecules or a catenane. Formation of a catenane, in the process of segregation of daughter molecules at the final stage of replication is discussed.

dnaR function of the prs gene of Escherichia coli in initiation of chromosome replication

A new Escherichia coli mutant named dnaR, which was temperature sensitive in initiation of DNA replication, has been characterized through identification of the mutant gene. A 1.65 x 10(3) base-pair chromosomal DNA fragment isolated from wild-type cells, but not the corresponding fragment from the dnaR mutant, exhibited an activity that reversed temperature-sensitive growth of the mutant. The DNA fragment was found to include the entire prs-coding sequence and specify a 34,000 M(r) protein with phosphoribosylpyrophosphate synthetase activity. The dnaR mutation resided within the prs-coding segment and made the synthetase thermolabile. The coding segment for the dnaR product was determined, by introduction of various mutations into the cloned fragment, to be the same as that for the synthetase. The dnaR function of the prs gene product in DNA replication is discussed on the basis of an observation that thermal treatment of the dnaR mutant caused a delay in initiation of chromosome replication after the downshift, despite the presence of the synthetase activity at the preheat level.

Gene N and Membrane Association of Lambda DNA

The expression of the early and late genes of λ is regulated by the N gene function (Dove, 1968; Echols and Joyner, 1968). Regulator gene N has a pleiotrophic effect on RNA transcription (Skalka et al., 1967; Taylor et al., 1967; Cohen and Hurwitz, 1968) and DNA replication (Eisen et al., 1966; Joyner et al., 1966; Ogawa and Tomizawa, 1968). Hallick et al. (1969) have presented evidence that N gene function is required for the association of λ DNA with membrane (a cellular structure which sedimented very rapidly after gentle lysis) and proposed that gene N may carry out its regulation of RNA transcription and DNA replication through direct control of membrane association. Replication and membrane association of the DNA of λ operator-and promoter-mutants, virCR, virL (Horiuchi et al., 1969), c 17 (Pereira da Silva and Jacob, 1968), and ri c 5b (Dove et al., 1969), were studied in the presence of represser. The results show that the λ gene product participates indirectly rather than directly in DNA-membrane association through its regulation of transcription. INDIRECT ROLE OF N GENE FUNCTION ON MEMBRANE ASSOCIATION Operator mutants virCR and virL express the operons on the right and left sides of the immunity region in the presence of represser (Eshima et al., in preparation), as v 1 v 3 and v 2 do (Ptashne and Hopkins, 1968). virCR but not virL shows constitutive DNA replication in λ lysogens, as shown in Table 1. Membrane association of the parental DNA of these mutants in λ lysogens was examined essentially according to...

Discontinuous replication of colicin E1 plasmid DNA in a cell extract containing thermolabile DNA ligase.

A cell extract prepared from the lig-ts7 mutant of Escherichia coli is able to carry out a complete round of DNA replication of colicin E1 plasmid at 25 °C. However, the apparent rate of elongation of the progeny strands at this temperature is much smaller than in an extract from the thermoresistant revertant cells. Chain elongation in the lig-ts extract is depressed by raising the incubation temperature from 25 °C to 32 °C, whereas that in the lig+ revertant extract is not. The rate of closure of the progeny strands of newly formed open circular molecules is also reduced in the lig-ts extract, even at 25 °C. The DNA pulse-labelled with the lig-ts extract for 30 seconds at 32 °C contains a large amount of short DNA fragments of approximately 7 S, in addition to DNA chains of various sizes between 7 S and 17 S (unit length). Most of these replicating molecules are converted to completely replicated closed circular molecules upon chasing with a lig+ extract. DNA-DNA hybridization experiments show that molecules replicated to various extents contain 7 S DNA fragments of both strands, but more of the L-strand component, whose 5′-to-3′ direction corresponds to the overall direction of unidirectional replication. The longer DNA chains are enriched in the H-strand component. The cell extracts used for the plasmid DNA replication have an activity which converts alkali-labile closed circular plasmid DNA containing apurinic sites to alkali-stable closed circular molecules. Addition of nicotinamide mononucleotide leads to conversion of the alkali-labile DNA to open circular molecules. In the replication system with the cell extract, however, the compound does not interfere with elongation of progeny strands. Chain elongation in the lig-ts extract at 25 °C is not significantly affected by nicotinamide mononucleotide. Thus, the 7 S DNA fragments formed with the lig-ts extract are unlikely to be generated as a result of incomplete repair of misincorporated nucleotides. We conclude that both strands of colicin E1 plasmid DNA replicate discontinuously.

Novel escherichia coli mutant, dnaR, thermosensitive in initiation of chromosome replication

A newly isolated Escherichia coli mutant thermosensitive in DNA synthesis had an allele named dnaR130, which was located at 26.3 minutes on the genetic map. The mutant was defective in initiation of chromosome replication but not in propagation at a high temperature. This mutant was capable of growing in the absence of the rnh function at the high temperature by means of a dnaA-independent replication mechanism. In the mutant exposed to the high temperature, an oriC plasmid was able to replicate, although at a lower rate than at the low temperature. The plasmid replication at the high temperature depended on the dnaA function essential for the initiation of replication from oriC. The mutant lacking the rnh function persistently maintained the oriC plasmid at the high temperature in a dnaA-dependent manner. Thus, the dnaR function was required for initiation of replication of the bacterial chromosome from oriC but not the oriC plasmid. This result reveals that a dnaR-dependent initiation mechanism that is dispensable for oriC plasmid replication operates in the bacterial chromosome replication.

Regulation of transcription of lambda bacteriophage operator mutants

Abstract λvirL preferentially synthesizes l mRNA over r mRNA in λcIts lysogens and fails to synthesize large rR mRNA (from the region including Q-R genes) and rL mRNA (from the A–J region). λvirCR preferentially synthesizes r mRNA over l mRNA in the lysogens and fails to synthesize large l mRNA (from the cIII-b2 region). The mRNA synthesis by these mutants in the lysogens is subject to positive regulation of the N gene. The following mechanisms of regulation of transcription of the λ genome in the presence of repressor were found: (1) The virCR and virL operators are responsible for the repression of transcription of the right and left sides of immunity region respectively. (2) The normal virL operator of λvirCR does not permit efficient transcription of the cI-N region and allows no transcription of the cIII-b2 region. The normal virCR operator of λvirL permits efficient transcription from the x-P region but no transcription of the region including Q-R genes. However, λvirL does not express the gene functions from the right operon. (3) The N gene product participates in the stimulation of transcription of the cI-N and x-P regions and in the induction of transcription of the cIII-b2 region and the region including Q-R genes. Transcription of the latter regions is inefficiently induced by the N gene product. The N gene-dependent l mRNA synthesis is reduced in trans by certain product(s) from the right operon without correlation with the onset of DNA replication.