The Escherichia coli peptide methionine sulfoxide reductase gene (msrA) encodes a single-subunit polypeptide of 212 amino acid residues (M. A. Rahman, H. Nelson, H. Weissbach, and N. Brot, J. Biol. Chem. 267:15549-15551, 1992). RNA blot analysis showed that the gene is transcribed into an mRNA of about 850 nucleotides. The promoter region was characterized, and the transcription initiation site was identified by primer extension. The synthesis of the MsrA protein increased about threefold in a growth-phase-dependent fashion. In an attempt to define the in vivo role of msrA, a chromosomal disruption was constructed. This mutant was more sensitive to oxidative stress, suggesting that oxidation of methionine in proteins plays an important role in oxidative damage.The enzyme peptide methionine sulfoxide reductase (MsrA) catalyzes the reduction of methionine sulfoxide [Met(O)] residues in proteins to methionine (1,5,12). The presence of Met(O) residues in proteins may arise during aerobic metabolism as a consequence of the oxidation of methionine by such reagents as hydrogen peroxide, hydroxyl radicals, and hypochlorite and superoxide ions (reviewed in reference 4). It has also been reported that oxidized linolenic acid is capable of oxidizing methionine residues in proteins in vitro (20). In many cases, the oxidation of a specific methionine residue leads to the loss of biological activity (4), which can be restored upon incubation of the oxidized protein with MsrA (1,5,12,17,27).The gene for the enzyme from Escherichia coli has recently been cloned and sequenced (24). The coding region of the gene is 636 nucleotides long and encodes a single-subunit protein with a calculated molecular weight of 23,316. In addition, the reductase has been overexpressed in E. coli and purified to homogeneity (23). In this report, we examine the regulation of expression of this gene and show that an msrA mutant is more sensitive to oxidative damage than wild-type cells. MATERIALS AND METHODS Materials.Restriction enzymes, T4 polynucleotide kinase, and T4 DNA ligase were purchased from New England Biolabs (Beverly, Mass.). Avian myeloblastosis virus reverse transcriptase was from Promega (Madison, Wis.). Radioisotopes were from either Amersham (Arlington Heights, Ill.) or Dupont/NEN (Boston, Mass.). Immobilon N transfer membranes were from Millipore (Bedford, Mass.). All chemicals were from Sigma (St. Louis, Mo.) or Boehringer Mannheim Biochemicals (Indianapolis, Ind.). Purified MsrA and antiserum against the protein were prepared as previously described (23).Northern (RNA) analysis. Total RNA was isolated as described previously (3) from 100-ml cultures (A 600 of 0.6) of either E. coli XL1-Blue or E. coli XL1-Blue/pAR100, which contains a plasmid bearing a genomic copy of the msrA gene (24). The coding region of the gene was amplified by PCR using two synthetic primers. The DNA was purified by agarose gel electrophoresis and the GeneClean (Bio 101, La Jolla, Calif.) procedure. Five hundred nanograms of the DNA was used for random prim...

A system is described for assembling infectious bacteriophage 46 nucleocapsids in vitro. Procapsids encoded by cDNA copies of genomic segment L in Escherichia coli were used to package and replicate viral RNA segments.The resulting filed particles were shown to be capable of infecting host cell spheroplasts after incubation with purified nucleocapsid shell protein P8. The infected spheroplasts yielded infectious virions. A modified cDNA-derived RNA segment was inserted into virions by this method. The resulting infectious virions contained the same 4-base-pair deletion as the modified cDNA. These findings support the contention that the preformed procapsids are the "machine" that replicates the 46 genome, by showing that the cDNA-derived procapsids are competent to package and replicate RNA properly.Bacteriophage 46 is a double-stranded RNA (dsRNA) virus of Pseudomonas phaseolicola (1). Its segmented genome, consisting of three separate pieces (2, 3), is associated with a complex isometric nucleocapsid (NC) structure (4, 5). The NC has five protein components, one of which (protein P8) forms the outer shell of the particle (4, 5). The other four proteins (P1, P2, P4, and P7) comprise an inner particle that displays the viral transcriptase and replicase activities (6-10). The 06 NC is enclosed in a lipid-protein envelope, the components of which have vital functions in the initial steps of the phage infection (1,(11)(12)(13).The internal NC proteins, P1, P2, P4, and P7, are encoded by the large (L) genomic segment (14, 15) and are synthesized early in 06 infection (5). Particles (procapsids) consisting of these four proteins and displaying RNA polymerase activity can be purified from Escherichia coli or P. phaseolicola cells harboring cDNA copies of the L segment in an expression vector (9, 10). Protein P1 forms the dodecahedral framework of the procapsids, to which the other proteins are attached (9,17,18). Defective particles lacking protein P2 have no polymerase activity, suggesting that this protein forms the active site of the enzyme. The cDNA-derived procapsids are capable of packaging and replicating viral (+)-strand RNA to double-stranded genomic segments, as well as of producing transcripts using the dsRNA formed as template (10).Recently, a technique for infecting P. phaseolicola spheroplasts with the purified 06 NC has been developed (19). Bacteriophage 06 (1) was used for the preparation of phage NCs used in single-stranded RNA (ssRNA) production and as the source of the NC coat protein, P8.The expression plasmid pLM450 (10) harboring 06 L-segment cDNA was used for the production of phage procapsids in E. coli JM109. Strain JM109 was the host for the propagation of recombinant plasmids. E. coli strain CJ236 is a dut, ung derivative that is used for the generation of uracilcontaining DNA in phagemids (21 tTo whom reprint requests should be addressed. 9173The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertiseme...

The genome of the lipid-containing bacteriophage +6 contains three segments of double-stranded RNA (dsRNA). We prepared cDNA copies of the viral genome and cloned this material in plasmids that replicate in Escherichia coli and Pseudomonas phaseolicola, the natural host of 406. These plasmids direct the formation of viral proteins and the assembly of structures similar to viral procapsids containing proteins P1, P2, P4, and P7. We found that these particles are capable of taking up viral single-stranded RNA and synthesizing the minus strands to produce dsRNA structures. Once the dsRNA is formed, it is then used as a template for the production of viral plus strands in a reaction that resembles normal transcription. The particles were also capable of directly transcribing exogenous dsRNA. The replicase reactions were specific for 46 RNA, were specific for procapsids, and resulted in substantial incorporation of product dsRNA into particles. These results offer strong support to a model in which genomic packaging is done by preformed procapsids.

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