Molecular cloning of the T4 genome; Organization and expression of the frd-DNA ligase region

Mileham, Alan J.; Revel, Helen R.; Murray, Noreen E.

doi:10.1007/bf00425449

Cited by 47 publications

(27 citation statements)

References 43 publications

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“…The thin line to the right of the EcoRI site (RI) of the polylinker designates the right arm of the lambda genome, including a functional attachment site (att), the phage 21 immunity region substitution (mm21) and the nin5 deletion. This right arm, although directly donated by XTL25, was originally derived from XNM616 (29). The sequence of the polylinker-RNaseIIl region is presented in Fig.…”

Section: Methodsmentioning

confidence: 99%

Improved vector system for constructing transcriptional fusions that ensures independent translation of lacZ

1990

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An improved vector system has been developed for the in vitro construction of transcriptional fusions to lacZ. The principal feature is an RNaseIII cleavage site inserted between the polylinker cloning site and the promoterless lacZ gene. When these vectors are used to construct transcriptional fusions, the subsequent cleavage of the hybrid mRNA at the RNaseIH site generates an unchanging 5' end for the lacZ mRNA. In contrast to earlier vectors, this feature helps to ensure independent translation of the lacZ mRNA and, thus, the level of ,l-galactosidase produced should accurately reflect the frequency of transcription of the upstream DNA sequences. Additional modifications of the vectors include removal of a weak transcriptional terminator between the cloning site and lacZ, insertion of a terminator downstream of lac, and alteration of restriction endonuclease cleavage sites to facilitate the in vitro construction of fusions. Both multicopy plasmid (pTL61T) and single-copy lambda (ATL61) vectors have been assembled. These vectors should be generally useful in scanning for transcriptional regulatory signals.Gene fusions have become important tools for the analysis of gene regulation in both procaryotic and eucaryotic systems. In general, the attachment of the regulatory sites of a given gene upstream of a reporter gene that encodes an easily assayed enzyme facilitates the analysis of expression of the affixed gene. The reporter gene may be used in either transcriptional (operon) fusions, where it retains its own translational start site but is dependent on the attached DNA for transcription, or in translational (protein) fusions, where both its transcription and translation are dependent on signals in the attached upstream DNA. The history and a wide variety of specific applications of gene fusions have been reviewed by Silhavy and Beckwith (45).Although a variety of reporter genes have been used in the construction of gene fusions, the most common is lacZ of Escherichia coli. The product, 3-galactosidase, is stable in many cellular backgrounds and can be precisely quantitated by a very simple and sensitive colorimetric assay. Moreover, the construction of fusions to lacZ extends all the experimental convenience of the lac genetic system to any given gene. That is, by using the available lac technology, one can readily screen and select for altered levels of f-galactosidase produced from the gene fusion. This greatly facilitates the regulatory analysis of the upstream attached gene.Numerous vector systems have been described for the construction of gene fusions to lacZ (45). Both multicopy (plasmid) and single-copy (bacteriophage) vectors have been used to construct gene fusions by either in vivo or in vitro manipulations. We previously described a vector system for the in vitro construction of transcriptional fusions to lacZ which had some advantages over the then currently available vectors (26). Both plasmid (pTL25) and phage (XTL25) versions were assembled, but we have used primarily the single-copy vector...

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Section: Methodsmentioning

confidence: 99%

Improved vector system for constructing transcriptional fusions that ensures independent translation of lacZ

1990

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“…In turn, the onset of phage DNA replication, which occurs at about 5 min after infection, is dependent on the turning on of deoxyribonucleotide synthesis (13,33,45,51). Accordingly, workers in our laboratory are investigating the control of expression of the T4 nrdA and nrdB genes, which encode the a2 and 2 subunits, respectively, of ribonucleotide reductase (4,5,16,36,39,43,46). nrdC, which encodes phage thioredoxin, is unlikely to be limiting in the initiation of ribonucleotide reductase synthesis (7,46).…”

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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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“…1) containing the 3' end of nrdA, exon I, and the 5' half of the intron of the nrdB gene was excised from Xtd652. The 3' half of the intron, exon II of the nrdB gene, the denA gene and a 5' segment of gene 63 were contained in the 1.7-kb EcoRI fragment isolated from T4D deoxycytidylate DNA (14). These two fragments were inserted into pTZ19R to obtain pMTnrdB.…”

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confidence: 99%

“…E. coli N4830 and the expression plasmids PPL-P and pKK223-3 were from Pharmacia LKB Biotechnology. Xtd30 (a hybrid of phages X and T4D) and Xtd652 (a hybrid of phages X and T4D) were described by Mileham et al (14). Materials and chemicals were as previously reported (23,24).…”

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Tandem cloning of bacteriophage T4 nrdA and nrdB genes and overproduction of ribonucleoside diphosphate reductase (alpha 2 beta 2) and a mutationally altered form (alpha 2 beta 2(93))

Tseng

Hilfinger

et al. 1992

J Bacteriol

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To investigate the role of ribonucleoside diphosphate reductase in the deoxyribonucleoside triphosphate synthetase multienzyme complex induced by bacteriophage T4 infection and to study the expression of the T4 nrdA and nrdB genes, we have constructed separate plasmid expression strains overproducing their respective a2 and jP2 protein products. Because complementation of the two proteins to form an active a2132 enzyme presented complications, nrdA and nrdB, each with its own tac promoter, were also cloned in tandem into a single expression vector by the 39 nrdB93 mutant, the a219 3 complex is formed at wild-type levels and is enzymatically active. However, after exposure to 41°C, the P39' subunit, though still synthesized, loses its fragile catalytic structure and its ability to bind to the a2 subunit (7). We have cloned both the nrdA and the nrdB genes into a * Corresponding author.single expression vector, pnrdAB. On the basis of the identification of the position of the nrdB93 mutation, the nrdB93 gene was formed by site-directed mutagenesis of nrdB and cloned into the combined expression vector, pnrdAB93. Upon induction of the promoter syrstem, the simultaneously synthesized a2 and 2 (or I ) subunits formed active enzyme that constituted more than 20% of the soluble proteins, and any difficulties in complementation of the subunits of separately cloned genes were overcome. The purifications of the several proteins have been outlined. Strains and materials. E. coli MV1304, plasmid pTZ19R, and the helper phage, M13K07 (8), were purchased from United States Biochemical Corporation. E. coli N4830 and the expression plasmids PPL-P and pKK223-3 were from Pharmacia LKB Biotechnology. Xtd30 (a hybrid of phages X and T4D) and Xtd652 (a hybrid of phages X and T4D) were described by Mileham et al. (14). Materials and chemicals were as previously reported (23,24). The aminophenyl-yphosphate-linked dATP-Sepharose was prepared and generously donated to us by Sue Cook (6). DNA preparation and cloning techniques were as described in earlier reports (23,24). Phage T4D deoxycytidylate DNA was the kind gift of Larry Snyder, Michigan State University, E. Lansing, Mich.Construction of expression vectors. (i) nrdB. A plasmid (pMTnrdB) containing the entire nrdB gene was constructed from Xtd652 (14) and from T4 deoxycytidylate DNA. The SphI-to-EcoRI DNA fragment (Fig. 1) containing the 3' end of nrdA, exon I, and the 5' half of the intron of the nrdB gene was excised from Xtd652. The 3' half of the intron, exon II of the nrdB gene, the denA gene and a 5' segment of gene 63 were contained in the 1.7-kb EcoRI fragment isolated from T4D deoxycytidylate DNA (14). These two fragments were inserted into pTZ19R to obtain pMTnrdB. denA28 carries a missense mutation (nd28), and its nuclease product is inactive. To form the nrdB gene expression vector, pnrdB-2, the 3.05-kb SphI-to-ClaI DNA fragment containing the nrdB and denA genes was excised from pMTnrdB and cloned into pKK223-3. Cultures of MV1304 containing pnrdB-2 overpro-5740

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Molecular cloning of the T4 genome; Organization and expression of the frd-DNA ligase region

Cited by 47 publications

References 43 publications

Improved vector system for constructing transcriptional fusions that ensures independent translation of lacZ

Improved vector system for constructing transcriptional fusions that ensures independent translation of lacZ

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

Tandem cloning of bacteriophage T4 nrdA and nrdB genes and overproduction of ribonucleoside diphosphate reductase (alpha 2 beta 2) and a mutationally altered form (alpha 2 beta 2(93))

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