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M M Howe

Publications and source records attributed to M M Howe.

At least 37 records · Page 2Linked to original sources
PubMed1990

Characterization of the C operon transcript of bacteriophage Mu.

Mu transcription occurs in three phases: early, middle, and late. Middle transcription occurs in the region of the C gene, which encodes the transactivator for late transcription. A middle promoter, Pm, was previously localized between 0.28 and 1.2 kilobase pairs upstream of C. We used S1 nuclease mapping with both unlabeled and radiolabeled capped RNAs from induced lysogens to characterize C transcription and identify its promoter. The C transcription initiation site was localized to a 4-base-pair region, approximately 740 base pairs upstream of C within the region containing Pm. Transcription of C was activated between 4 and 8 min after induction of cts and Cam lysogens and increased throughout the lytic cycle. Significant C transcription did not occur in replication-defective Aam lysogens. These kinetic and regulatory characteristics identify the C transcript as a middle RNA species and demonstrate that Pm is the C promoter. DNA sequence analysis of the Pm region showed a good -10, but poor -35, site homology to the Escherichia coli RNA polymerase consensus sequence. In addition, the sequence demonstrated that C is the distal gene in a middle operon containing several open reading frames. S1 mapping also showed an upstream transcript with a 3' end in the Pm region at a sequence strongly resembling a Rho-independent terminator. The regulatory characteristics of this RNA are consistent with this terminator, t9.2, being the early operon terminator.

Bacteriophage mu↗
PubMed1990

Identification of a positive regulator of the Mu middle operon.

Transcription of bacteriophage Mu occurs in a regulatory cascade consisting of three phases: early, middle, and late. The 1.2-kb middle transcript is initiated at Pm and encodes the C protein, the activator of late transcription. A plasmid containing a Pm-lacZ operon fusion was constructed. beta-Galactosidase expression from the plasmid increased 23-fold after Mu prophage induction. Infection of plasmid-containing cells with lambda phages carrying different segment of the Mu early region localized the Pm-lacZ transactivation function to the region containing open reading frames E16 and E17. Deletion and linker insertion analyses of plasmids containing this region identified E17 as the transactivator; therefore we call this gene mor, for middle operon regulator. Expression of mor under the control of a T7 promoter and T7 RNA polymerase resulted in the production of a single polypeptide of 17 kDa as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Insertion of a linker into mor substantially reduced the ability of Mu to form plaques. When growth of the mor mutant was assayed in liquid, lysis was delayed by about 50 min and the burst size was approximately one-fifth that of wild-type Mu. The mor requirement for plaque formation and normal growth kinetics was abolished when C protein was provided in trans, indicating that the primary function of Mor is to provide sufficient C for late gene expression. Comparison of the predicted amino acid sequence of Mor with other proteins revealed that Mor and C share substantial amino acid sequence homology.

Amino Acid Sequence↗
PubMed1990

Activation of the bacteriophage Mu lys promoter by Mu C protein requires the sigma 70 subunit of Escherichia coli RNA polymerase.

Bacteriophage Mu C protein, a product of the middle operon, is required for activation of the four Mu late promoters. To address its mechanism of action, we overproduced the approximately 16.5-kilodalton C protein from a plasmid containing the C gene under the control of a phage T7 promoter and ribosome-binding site. A protein fraction highly enriched for Escherichia coli RNA polymerase (E sigma 70) and made from the overproducing strain was able to activate transcription in vitro from both the tac promoter (Ptac) and a Mu late promoter, Plys. The behavior of Plys was similar in vivo and in vitro; under both conditions, transcription was C dependent and the RNA 5' ends were identical. When anti-sigma 70 antibody was added to C-dependent transcription reactions containing both Ptac and Plys templates, transcription from both promoters was inhibited; transcription was restored by the addition of excess E sigma 70. This result suggests that C-dependent activation of Plys requires sigma 70. Further supporting evidence was provided by a reconstitution experiment in which an E sigma 70-depleted fraction containing C was unable to activate transcription from Plys unless both purified sigma 70 and core polymerase were added. These results strongly suggest that C is not a new sigma factor but acts as an activator for E sigma 70-dependent transcription.

Bacteriophage mu↗
PubMed1989

Bacteriophage Mu late promoters: four late transcripts initiate near a conserved sequence.

Late transcription of bacteriophage Mu, which results in the expression of phage morphogenetic functions, is dependent on Mu C protein. Earlier experiments indicated that Mu late RNAs originate from four promoters, including the previously characterized mom promoter. S1 nuclease protection experiments were used to map RNA 5' ends in the three new regions. Transcripts were initiated at these points only in the presence of C and were synthesized in a rightward direction on the Mu genome. Amber mutant marker rescue analysis of plasmid clones and limited DNA sequencing demonstrated that these new promoters are located between C and lys, upstream of I, and upstream of P within the N gene. A comparison of the promoter sequences upstream from the four RNA 5' ends yielded two conserved sequences: the first (tA . . cT, where capital and lowercase letters indicate 100 and 75% base conservation, respectively), at approximately -10, shares some similarity with the consensus Escherichia coli sigma 70 -10 region, while the second (ccATAAc CcCPuG/Cac, where Pu indicates a purine), in the -35 region, bears no resemblance to the E. coli -35 consensus. We propose that these conserved Mu late promoter consensus sequences are important for C-dependent promoter activity. Plasmids containing transcription fusions of these late promoters to lacZ exhibited C-dependent beta-galactosidase synthesis in vivo, and C was the only Mu product needed for this transactivation. As expected, the late promoter-lacZ fusions were activated only at late times after induction of a Mu prophage. The C-dependent activation of lacZ fusions containing only a few bases of the 5' end of Mu late RNA and the presence of altered promoter sequences imply that C acts at the level of transcription initiation.

Bacteriophage mu↗
PubMed1989

Localization and regulation of bacteriophage Mu promoters.

Mu promoters active during the lytic cycle were located by isolating RNA at various times after induction of Mu prophages, radiolabeling it by capping in vitro, and hybridizing it to Mu DNA fragments on Southern blots. Signals were detected from four new promoters in addition to the previously characterized Pe (early), PcM (repressor), and Pmom (late) promoters. A major signal upstream of C was first observed at 12 min and intensified thereafter with RNA from cts and C amber but not replication-defective prophages; these characteristics indicate that this signal arises from a middle promoter, which we designate Pm. With 20- and 40-min RNA, four additional major signals originated in the C-lys, F-G-I, N-P, and com-mom regions. These signals were missing with RNA from C amber and replication-defective prophages and therefore reflected the activity of late promoters, one of which we presume was Pmom. Uninduced lysogens showed weak signals from five regions, one from the early regulatory region, three between genes B and lys, and one near the late genes K, L, and M. The first of these probably resulted from PcM activity; the others remain to be identified.

Bacteriophage mu↗
PubMed1986

Localization and DNA sequence analysis of the C gene of bacteriophage Mu, the positive regulator of Mu late transcription.

The C gene of bacteriophage Mu, required for transcription of the phage late genes, was localized by construction and analysis of a series of deleted derivatives of pKN50, a plasmid containing a 9.4 kb Mu DNA fragment which complements Mu C amber mutant phages for growth. One such deleted derivative, pWM10, containing only 0.5 kb of Mu DNA, complements C amber phages and transactivates the mom gene, one of the Mu late genes dependent on C for activation. The DNA sequence of the 0.5 kb fragment predicts a single long open reading frame coding for a 140 amino acid protein. Sequence analysis of DNA containing a C amber mutation located the base change to the second codon of this reading frame. Generation of a frameshift mutation by filling in a BglII site spanning codon 114 of this reading frame resulted in the loss of C complementation and transactivation activity. These results indicate that this open reading frame encodes the Mu C gene product. Comparison of the predicted amino acid sequence of the C protein with those of other transcriptional regulatory proteins revealed some similarity to a region highly conserved among bacterial sigma factors.

Amino Acid Sequence↗
PubMed1986

Isolation of point mutations in bacteriophage Mu attachment regions cloned in a lambda::mini-Mu phage.

Twenty-one derivatives of a lambda::mini-Mu phage containing point mutations in the Mu attachment regions were isolated after mutD mutagenesis and selection for relief from Mu-specific replicative interference of lambda growth. DNA sequence analysis revealed that the single left-end mutant had suffered a T----C transition at position 1 of the Mu sequence, while the remaining 20 right-end mutants contained single base-pair insertions or deletions within the terminal 19 base pairs. A genetic assay showed that the right-end mutations revealed by sequencing were necessary for relief of the replicative inhibition of lambda growth. The properties of these mutants suggest that the terminal 2-base-pair and subterminal 8-base-pair inverted repeats are important for Mu-specific replicative transposition.

Bacteriophage lambda↗
PubMed1986

Mutants of Escherichia coli defective for replicative transposition of bacteriophage Mu.

We isolated 142 Hir- (host inhibition of replication) mutants of an Escherichia coli K-12 Mu cts Kil- lysogen that survived heat induction and the killing effect of Mu replicative transposition. All the 86 mutations induced by insertion of Tn5 or a kanamycin-resistant derivative of Tn10 and approximately one-third of the spontaneous mutations were found by P1 transduction to be linked to either zdh-201::Tn10 or Tn10-1230, indicating their location in or near himA or hip, respectively. For a representative group of these mutations, complementation by a plasmid carrying the himA+ gene or by a lambda hip+ transducing phage confirmed their identification as himA or hip mutations, respectively. Some of the remaining spontaneously occurring mutations were located in gyrA or gyrB, the genes encoding DNA gyrase. Mutations in gyrA were identified by P1 linkage to zei::Tn10 and a Nalr gyrA allele; those in gyrB were defined by linkage to tna::Tn10 and to a gyrB(Ts) allele. In strains carrying these gyrA or gyrB mutations, pBR322 plasmid DNA exhibited altered levels of supercoiling. The extent of growth of Mu cts differed in the various gyrase mutants tested. Phage production in one gyrA mutant was severely reduced, but it was only delayed and slightly reduced in other gyrA and gyrB mutants. In contrast, growth of a Kil- Mu was greatly reduced in all gyrase mutant hosts tested.

Bacterial Proteins↗
PubMed1985

Morphogenetic structures present in lysates of amber mutants of bacteriophage Mu.

The Mu phage particle is structurally similar to that of the T-even phages, consisting of an icosahedral head and contractile tail. This study continues an analysis of the morphogenesis of the Mu phage particle by defining the structural defects resulting from mutations in specific Mu genes. Defective lysates produced by induction of 55 amber mutants, representing 24 essential genes, were examined in the electron microscope and categorized into eight classes based on the observed phage-related structures. (1) Mutations in genes lys, F and G, and some H mutations, did not cause a visible alteration in particle structure. (2) Mutants defective in genes A, B, and C produced no detectable phage structures, consistent with their lack of production of late RNA. (3) Extracts defective in genes L, M, Y, N, P, Q, V, W, and R contained only head structures, and these appeared normal. (4) K-defective mutants accumulated free heads as well as free tails which were longer than normal and variable in length. (5) Tails which appeared normal were the only structures found in T- and some I-defective extracts. (6) Free tails and empty heads accumulated in D-, E-, and some I- and H-defective extracts. These heads were as much as 16% smaller than normal heads. The heads found in some I amber lysates had a protruding neck-like structure and unusually thick shells suggestive of a scaffolding-like structure. (7) Defects in gene J resulted in the accumulation of unattached tails and full heads. (8) Previous analysis of lysates produced by inversion-defective gin mutants fixed in the G(+) orientation demonstrated that S and U mutants produced particles lacking tail fibers (F.J. Grundy and M.M. Howe (1984), Virology 134, 296-317). In these experiments with Gin+ phages S and U mutants produced apparently normal phage particles. Presumably the tail fiber defects were masked by the production of S' and U' proteins by G(-) phages in the population.

Chromosome Deletion↗
PubMed1985

Regulation and expression of the bacteriophage mu mom gene: mapping of the transactivation (dad) function to the C region.

Expression of the bacteriophage Mu mom gene is under tight regulatory control. One of the factors required for mom gene expression is the trans-acting function (designated Dad) provided by another Mu gene. To facilitate studies on the signals mediating mom regulation, we have constructed a mom-lacZ fusion plasmid which synthesizes beta-galactosidase only when the Mu Dad transactivating function is provided. lambda pMu phages carrying different segments of the Mu genome have been assayed for their ability to transactivate beta-galactosidase expression by the fusion plasmid. The results of these analyses indicated that the Dad transactivation function is encoded between the leftmost EcoRI site and the lys gene of Mu; this region includes the C gene, which is required for expression of all Mu late genes. Cloning of an approx. 800-bp fragment containing the C gene produced a plasmid which could complement MuC- phages for growth and could transactivate the mom-lacZ fusion plasmid to produce beta-galactosidase. These results suggest that the C gene product mediates the Dad transactivation function.

Bacteriophage lambda↗
PubMed1984

Multiple factors and processes involved in host cell killing by bacteriophage Mu: characterization and mapping.

The regions of bacteriophage Mu involved in host cell killing were determined by infection of a lambda-immune host with 12 lambda pMu-transducing phages carrying different amounts of Mu DNA beginning at the left end. Infecting lambda pMu phages containing 5.0 (+/- 0.2) kb or less of the left end of Mu DNA did not kill the lambda-immune host, whereas lambda pMu containing 5.1 kb did kill, thus locating the right end of the kil gene between approximately 5.0 and 5.1 kb. For the Kil+ phages the extent of killing increased as the multiplicity of infection (m.o.i.) increased. In addition, killing was also affected by the presence of at least two other regions of Mu DNA: one, located between 5.1 and 5.8 kb, decreased the extent of killing; the other, located between 6.3 and 7.9 kb, greatly increased host cell killing. Killing was also assayed after lambda pMu infection of a lambda-immune host carrying a mini-Mu deleted for most of the B gene and the middle region of Mu DNA. Complementation of mini-Mu replication by infecting B+ lambda pMu phages resulted in killing of the lambda-immune, mini-Mu-containing host, regardless of the presence or absence of the Mu kil gene. The extent of host cell killing increased as the m.o.i. of the infecting lambda pMu increased, and was further enhanced by both the presence of the kil gene and the region located between 6.3 and 7.9 kb. These distinct processes of kil-mediated killing in the absence of replication and non-kil-mediated killing in the presence of replication were also observed after induction of replication-deficient and kil mutant prophages, respectively.

Bacteriophage lambda↗
PubMed1984

Involvement of the invertible G segment in bacteriophage mu tail fiber biosynthesis.

The orientation [G(+) or G(-)] of the invertible G segment of bacteriophage Mu DNA determines the host range specificity of the phage particles. In this study the hypothesis that the G segment genes are involved in synthesis of Mu tail fibers has been tested. Serum blocking power (SBP) assays demonstrated that among Mu late gene mutants only those defective in genes S or U encoded by the G segment were defective in G(+) SBP and that they lacked the same antigens. Electron microscopy of lysates produced by inversion-defective gin mutants (isolated by their inability to complement a hin inversion-defective mutant of the Salmonella phase variation segment) showed that G(+) phages with amber mutations in S or U made tail-fiberless particles with contracted tail sheaths. Inversion of G to the G(-) orientation or suppression of the amber mutations restored the normal phage particle morphology. These experiments demonstrate that genes S and U are required for Mu G(+) tail fiber biosynthesis and/or attachment.

Antigens, Viral↗
PubMed1983

AvaII and BglI restriction maps of bacteriophage Mu.

The AvaII and BglI restriction maps of bacteriophage Mu were derived by restriction analysis of a series of plasmid clones containing segments of Mu DNA which, in combination, covered the entire Mu genome. The plasmids analyzed included pKN36, pKN54, pKN62, pKN50, pKN35, pKN27, pKN48, pKN82, and pKN56 from the collection of W. Schumann and E. G. Bade, and pCM02, a newly constructed plasmid containing the rightmost internal EcoRI-PstI fragment of Mu DNA. BglI cuts Mu DNA at 23 sites, producing 24 fragments which range in size from 0.05 kb up to the approximately 7-kb fragment derived from the right end. AvaII cuts Mu DNA at 17 sites (including 2 within the G segment), producing fragments which range in size from 0.17 to 8.9 kb. The derived maps were confirmed by results of hybridization of 32P-labeled, nick-translated plasmid DNA to AvaII- and BglI-digested Mu DNAs. Evidence for modification of one of the AvaII sites in E. coli was obtained.

Bacteriophage mu↗
PubMed1983

Mu dX, a derivative of Mu d1 (lac Apr) which makes stable lacZ fusions at high temperature.

We describe defective Mu phage Mu dX (Mu d1 Bx::Tn9 [lac Apr Cmr]) which is useful for insertion mutagenesis and for construction of lac operon fusions in vivo. Mu dX retains the insertion properties of Mu d1 but produces temperature-resistant lysogens and transposes at a reduced frequency. A method is described to convert existing Mu d1 insertions to Mu dX.

Bacteriophage mu↗