A physical map of bacteriophage T4 including the positions of strong promoters and terminators recognized in vitro

Gram, Hermann; Liebig, Hans‐Dieter; Hack, Alfons; Niggemann, Elisabeth; Rüger, Wolfgang

doi:10.1007/bf00383522

Cited by 35 publications

(11 citation statements)

References 38 publications

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“…2, 3 promoter (39,46). Hilfinger, using Northern blot analysis with probes from segments of either nrdA or nrdB, has found that mRNA from phage-infected cultures reached sizes of about 6 kb (unpublished data), supporting our previous findings (46), those of Gram et al (25), and those of Ruckman and co-workers (42). That the frd and td genes overlap slightly (14,40) and that the translational frames of nrdB and denA are separated by only 30 bp (43) argue against promoters in these interfaces.…”

Section: Discussionsupporting

confidence: 88%

Bacteriophage T4 nrdA and nrdB genes, encoding ribonucleotide reductase, are expressed both separately and coordinately: characterization of the nrdB promoter

Tseng

Hilfinger

et al. 1990

J Bacteriol

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We examined the expression of the bacteriophage T4 nrdA and nrdB genes, which encode the a2 and 02 subunits, respectively, of ribonucleoside diphosphate reductase, the first committed enzyme in the pathway of synthesis of the deoxyribonucleoside triphosphates. T4 nrdA, located 700 bp upstream from nrdB, has been shown previously to be transcribed by two major transcripts: a prereplicative, polycistronic message, Tu, originating at an immediate-early promoter, PEI that is 3.5 kb upstream from nrdA, and a postreplicative message commencing from a late promoter in its 5' flank. We have found a third promoter initiating a transcript at 159 nucleotides upstream from the reading frame of nrdB. PrdB functions only in the presence of the T4 motA gene product, which is required for middle (time) promoters, and therefore the onset of nrdB transcription is delayed more than 2 min after infection. Because of the distance of nrdA from PEI the inception of nrdA transcription (delayed early) coincides closely with that of nrdB. An apparent termination site, tA, occurs about 80 bp downstream from nrdA. Some of the polycistronic mRNA reading through the site after 5 min contributes to nrdB transcription. nrdA and nrdB genes in an uninfected host have been reported to be transcribed only coordinately. In contrast, T4 nrdA and nrdB are initially transcribed separately onto the PE and PnrdB transcripts, respectively, but at about 5 min after infection are transcribed both coordinately and on separate transcripts. Evidence is presented that Tu coordinately transcribes a deoxyribonucleotide operon in the order: frd, td, gene 'Y,' nrdA, nrdB. Since the 02 subunit is known to be formed after the a2 subunit, the expression of the nrdB gene determines the onset of deoxyribonucleoside triphosphate synthesis and thus of T4 DNA replication.

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Section: Discussionsupporting

confidence: 88%

Bacteriophage T4 nrdA and nrdB genes, encoding ribonucleotide reductase, are expressed both separately and coordinately: characterization of the nrdB promoter

Tseng

Hilfinger

et al. 1990

J Bacteriol

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“…amN55 x 5 resulted from a G + A exchange in a TGG codon (Trp) and amC87 from a C + T exchange in a CAG codon (Gln). The transcription of gene 42 apparently can be started from the early promoter, as already shown under in vitro conditions [34]. The early mode of transcription includes the imm gene.…”

Section: Open Readingjrumes Transcription and Translation Signalsmentioning

confidence: 55%

Deoxycytidylate hydroxymethylase gene of bacteriophage T4

Wang

Mathews

et al. 1988

European Journal of Biochemistry

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We describe two approaches to cloning and over-expressing gene 42 of bacteriophage T4, which encodes the early enzyme deoxycytidylate hydroxymethylase. In Bochum a library of sonicated fragments of wild-type phage DNA cloned into M13mpl8 was screened with clones known to contain parts of gene 42. Two overlapping fragments, each of which contained one end of the gene, were cleaved at a HincII site and joined, to give a fragment containing the entire gene. In Corvallis a 1.8-kb fragment of cytosine-substituted DNA, believed to contain the entire gene, was cloned into pUC18 and shown to express the enzyme at low level. The cloned fragment bore an amber mutation in gene 42. From the DNA sequence of gene 42, the cloned gene was converted to the wild-type allele by site-directed mutagenesis. Both gene-42-containing fragments were cloned into the pT7 expression system and found to be substantially overexpressed.dCMP hydroxymethylase purified from one of the over-expressing strains had a turnover number similar to that of the enzyme isolated earlier from infected cells. In addition, the N-terminal20 amino acid residues matched precisely the sequence predicted from the gene sequence. The amino acid sequence of gp42 bears considerable homology with that of thymidylate synthase of either host or T4 origin. The gene 42 nucleotide sequences of bacteriovhages T2 and T6 were determined and found to code for amino acid sequences nearly identical to that of T4 gp42. The enzyme has played an important role in the modern history of biochemistry. Its discovery in 1957 [l] represented the first realization that virus infection can direct the development of novel metabolic pathways in infected cells. In this case the pathway led to the substitution of 5-hydroxymethylcytosine for cytosine in phage DNA. Later this enzyme was used for the first demonstration that virus-induced enzymes are synthesized de novo after infection [2], and it was one of the first essential gene products in T4 to have its structural gene identified and mapped [3].Recent studies of dCMP hydroxymethylase have focused upon its role in determining replication fidelity [4, 51 and its role as an element of a multienzyme complex that synthesizes deoxyribonucleoside triphosphates (dNTPs) from distal precursors, and perhaps channels them into replicating DNA [6, 71. Interest in the mechanism of the reaction catalyzed has been rekindled with the proposal of Santi et al. [8] that all reactions involving electrophilic substitution at carbon 5 of a pyrimidine proceed by a common mechanism. These reactions

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“…The expression of pinA in the Q43 and L512 lysogens is probably different from that which occurs during T4 infection. First, three strong promoters, approximately 4, 7, and 9 kb upstream of pinA, are recognized in vitro by unmodified RNA polymerase (15). Although a termination sequence may be present between these promoters and the early promoter immediately upstream of pinA (J. Tomaschewski and W. Ruger, unpublished data), its effects on transcription in vivo are unknown.…”

Section: Resultsmentioning

confidence: 99%

A bacteriophage T4 gene which functions to inhibit Escherichia coli Lon protease

et al. 1988

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In Escherichia coli, proteins with abnormal conformations, protein fragments, and some foreign proteins encoded by cloned genes are turned over rapidly as compared with normal cellular proteins (10,12,17,20,40). The degradation of such abnormal proteins is ATP dependent (12), and the Lon protease, a tetramer of 94-kilodalton subunits with ATP-dependent proteolytic activity, has a major role in this process (5,6,11,14,34

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A physical map of bacteriophage T4 including the positions of strong promoters and terminators recognized in vitro

Cited by 35 publications

References 38 publications

Bacteriophage T4 nrdA and nrdB genes, encoding ribonucleotide reductase, are expressed both separately and coordinately: characterization of the nrdB promoter

Bacteriophage T4 nrdA and nrdB genes, encoding ribonucleotide reductase, are expressed both separately and coordinately: characterization of the nrdB promoter

Deoxycytidylate hydroxymethylase gene of bacteriophage T4

A bacteriophage T4 gene which functions to inhibit Escherichia coli Lon protease

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