Effect of nalidixic acid and novobiocin on pBR322 genetic expression in Escherichia coli minicells.

The effects of two deoxyribonucleic acid (DNA) gyrase inhibitors, nalidixic acid and novobiocin, on the gene expression of plasmid pBR322 in Escherichia coli minicells were studied. Quantitative estimates of the synthesis of pBR322-coded polypeptides in novobiocin-treated minicells showed that the synthesis of a polypeptide of molecular weight of 34,000 (the tetracycline resistance protein) was reduced to 11 to 20% of control levels, whereas the amount of a polypeptide of 30,500 (the beta-lactamase precursor) was increased to as much as 200%. Nalidixic acid affected the synthesis of the tetracycline resistance protein similarly to novobiocin, although to a lesser extent. The effects of nalidixic acid were not observed in a nalidixic-resistant mutant; those induced by novobiocin were only partially suppressed in a novobiocin-resistant mutant. The synthesis of one of the inducible tetracycline-resistant proteins (34,000) coded by plasmid pSC101 was also reduced in nalidixic acid- and novobiocin-treated minicells. These results suggest that the gyrase inhibitors modified the interaction of ribonucleic acid polymerase with some promoters, either by decreasing the supercoiling density of plasmid DNA or by altering the association constant of the gyrase to specific DNA sites.     

Identification by deletion analysis of an inducible protein required for plasmid pSC101-mediated tetracycline resistance.

Plasmids containing small deletions within a tetracycline (Tc) resistance gene(s) of plasmid pHA121 were isolated. Plasmid pHA121 was formed by ligating the EcoRI-digested Tc resistance plasmid pSC101 and similarly digested mini-ColE1 plasmid pHA105. The DNA deletion plasmids were constructed by digesting plasmid pHA121 DNA with the restriction endonucleases BamH1 and Sal1 and, in addition, λ exonuclease. Two plasmids, designated pJT131 and pJT133, had small deletions of approximately 0.64 to 0.8 kb and a comparison of the radioactive polypeptides synthesized in plasmid-containing minicells revealed that a 34-kdal polypeptide was not specified by either pJT131 or pJT133. We conclude that the 34-kdal polypeptide is required for the expression of Tc resistance and that its structural gene probably maps in the deleted region of pSC101 DNA.     

Restriction endonuclease mapping of pSC101 and pMB9

Summary A restriction endonuclease analysis of the plasmids pSC101 and pMB9 has allowed a determination of the alterations that occurred in the tetracycline resistance locus during the construction of pMB9 from pSC101. The genes for four of the polypeptides involved in tetracycline resistance have been positioned on the restriction endonuclease map of pSC101.     

Protein expression in E. coli minicells by recombinant plasmids.

The polypeptides synthesized in E. coli minicells from recombinant plasmids containing DNA fragments from cauliflower mosaic virus, Drosophila melanogaster, and mouse mitochondria were examined. Molecularly cloned fragments of cauliflower mosaic virus DNA directed the synthesis of high levels of three polypeptides, which were synthesized entirely from within the cloned virus DNA fragments independent of their insertion into the plasmid vehicles. Several fragments of D. melanogaster DNA were capable of initiating polypeptide synthesis; however, termination of these polypeptides was dependent upon the insertion into the plasmid vehicle. The majority of D. melanogaster DNA fragments examined did not direct the detectable synthesis of any polypeptides. Insertion of DNA into the Eco RI site of ColE1 and pSC101 plasmids resulted in the altered expression of plasmid-encoded polypeptides. In the case of ColE1, this site of insertion lies within the colicin E1 structural gene, and insertion of foreign DNA into the site results in the synthesis of an inactive truncated colicin E1 molecule. It is probable that the Eco RI site in pSC101 lies within the structural gene for a polypeptide involved in tetracycline resistance, and insertion of DNA into this site may also result in the synthesis of a truncated or elongated polypeptide.     

Altered tetracycline resistance in pSC101 recombinant plasmids

Summary Investigation of tetracycline resistance genetically determined by the plasmid pSC101 and several recombinants of pSC101 containing of EcoRI generated DNA fragments inserted at the EcoRI site has revealed significant differences in the phenotypic expression of that resistance. The altered phenotypes of the recombinant plasmids may be the result of the location of the EcoRI site of pSC101, which has been determined to be near the genetic elements involved with tetracycline resistance.     

Restriction endonuclease mapping and mutagenesis of the F sex factor replication region.

The plasmids pSC138 and pML31 each contain the EcoRI-generated f5 replicator fragment of the conjugative plasmid F in addition to an EcoRI fragment encoding antibiotic resistance: ampicillin resistance derived from Staphylococcus aureus in pSC138 and kanamycin resistance from Escherichia coli in pML31. We have mapped one HindIII and two BamHI restriction sites in the f5 region of these plasmids and one HindIII site in the antibiotic resistance region of each plasmid. The HindIII site in the Km region of pML31 occurs in the kan gene whereas the HindIII site in the Ap region of pSC138 appears to occur in an area important for the regulation of beta-lactamase production. By means of in vitro recombinant DNA manipulation of plasmids pML31 and pSC138, we have shown that approximately 1.9 X 10(6) daltons of the 6.0 X 10(6) dalton f5 fragment can be deleted without disrupting plasmid stability. In addition, we have used these same techniques to isolate a novel F-controlled Ap plasmid cloning vehicle which contains a single restriction site for each of the enzymes EcoRI, HindIII, and BamHI. This cloning vehicle has been linked via either its EcoRI or HindIII site to a ColE1 plasmid replicon to yield stable recombinants.     

Cloned DNA fragment specifying major outer membrane protein a in Escherichia coli K-12.

Plasmid pMC44 is a recombinant plasmid that contains a 2-megadalton EcoRI fragment of Escherichia coli K-12 DNA joined to the cloning vehicle, pSC101. The polypeptides specified by plasmid pMC44 were identified and compared with those specified by pSC101 to determine those that are unique to pMC44. Three polypeptides specified by plasmid pMC44 were localized in the cell envelope fraction of minicells: a Sarkosyl-insoluble outer membrane polypeptide (designated M2), specified by the cloned 2-megadalton DNA fragment, and two Sarkosyl-soluble membrane polypeptides specified by the cloning plasmid pSC101. Bacteria containing plasmid pMC44 synthesized quantities of M2 approximately equal to the most abundant E. coli K-12 outer membrane protein. Evidence is presented that outer membrane polypeptide M2, specified by the recombinant plasmid pMC44, is the normal E. coli outer membrane protein designated protein a by Lugtenberg and 3b by Schnaitman.     

Complete nucleotide sequence of pT181, a tetracycline-resistance plasmid from Staphylococcus aureus

pT181 is a naturally occurring Staphylococcus aureus plasmid, encoding inducible resistance to tetracycline. The plasmid has a copy number of about 20 per cell, and belongs to the incompatibility group inc3. The complete nucleotide sequence of pT181 has been determined and consists of 4437 bp. The nucleotide sequence contains 69.8% A-T and 30.2% G-C pairs. pT181 was found to contain four open reading frames capable of coding for polypeptides containing more than 50 amino acids. All the putative polypeptides are coded by one strand. The molecular weights of the four putative polypeptides are (in daltons): A, 37,500; B, 35,000; C, 23,000, and D, 18,000. Polypeptide A corresponds to the repC protein, earlier shown to be specifically required for pT181 replication. Polypeptide B (and possibly polypeptide D) are involved in tetracycline resistance. No role has yet been established for polypeptide C; deletion of the coding sequence for the C polypeptide has no detectable effect on any property of the pT181 plasmid. A region consisting of about 1200 bp contains information for the replication and copy number control of this plasmid. The sequencing results are discussed in relation to the replication properties and tetracycline resistance associated with the pT181 plasmid.     

Synthesis of proteins coded by plasmid vectors of pCV series (Apr, Tcr) and their recombinant derivatives (pDm) in E. coli minicells.

Polypeptide synthesis directed by vector plasmids of pCV series conferring ampicillin and tetracycline resistance (Apr, Tcr) and by recombinant plasmids (pDm) have been analyzed using the minicell system. It has been found that a polypeptide of 34 000 daltons is responsible for the Tcr phenotype and regulated from the promoter near the HindIII site. Cloning of DNA fragments into HindIII site allowed to conclude that DNA from Drosophila melanogaster contains nucleotide sequences which may act as promoters for a 34 000 dalton polypeptide gene. beta-Lactamase is expressed as five proteins of 24 000, 26 5000, 27 000, 28 500 and 29 500 daltons. Insertion of DNA fragments into PstI site prevents the synthesis of all five polypeptides. Recombinant clones Dm39 and Dm187 produce additional proteins of 19 000, 23 000, 24 000 and 27 000 daltons.     

Nuclear Involvement in the Appearance of a Chloroplast-Encoded 32,000 Dalton Thylakoid Membrane Polypeptide Integral to the Photosystem II Complex

The genetic locus for the high chlorophyll fluorescent photosystem II-deficient maize mutant hcf^*-3 has been definitively located to the nuclear genome. Fluorography of lamellar polypeptides labeled with [³⁵S]methionine in vivo revealed the specific loss of a heavily labeled 32,000 dalton thylakoid membrane polypeptide as well as its chloroplast encoded precursor species at 34,000 daltons. Examination of freeze-fractured mesophyll and bundle sheath thylakoids from hcf^*-3 revealed that both plastid types lacked the large EFs particles believed to consist of the photosystem II reaction center-core complex and associated light harvesting chlorophyll-proteins. The present evidence suggests that the synthesis or turnover/integration of the chloroplast-encoded 34,000 to 32,000 dalton polypeptide is under nuclear control, and that these polyipeptides are integral components of photosystem II which may be required for the assembly or structural stabilization of newly formed photosystem II reaction centers in both mesophyll and bundle sheath chloroplasts.     

Polypeptides encoded by the mer operon.

HgCl2-induced polypeptides synthesized by Escherichia coli minicells containing recombinant or natural HgR plasmids were labeled with [35S]methionine and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All plasmids examined encoded two heavily labeled, HgCl2-inducible polypeptides of 69,000 and 12,000 daltons. Most plasmids also encoded two additional HgCl2-inducible proteins in the 14,000- to 17,000-dalton range. Antiserum prepared against a purified mercuric ion reductase reacts with the 69,000-dalton polypeptide and a minor 66,000-dalton protein seen in several different HgR minicells. Recombinant plasmids constructed from portions of mer DNA from the IncFII plasmid NR1 were also analyzed in the minicell system. Five HgCl2-inducible polypeptides (69,000, 66,000, 15,100, 14,000, and 12,000 daltons) were synthesized in minicells carrying pRR130, a recombinant derivative containing the EcoRI-H and EcoRI-I restriction fragments of NR1. The EcoRI-H fragment of NR1 encodes the three small mer proteins of 15,100, 14,000, and 12,000 daltons and the amino-terminal 40,000 daltons of the mercuric ion reductase monomer.     

Heterogeneity of tetracycline resistance determinants

We have found that tetracycline resistance on different naturally occurring bacterial plasmids is encoded by more than one genetic determinant. Using restriction enzyme analyses and DNA-DNA hybridization to specific ³²P-labeled genetic probes, we can define at least four genetically distinct tetracycline resistance determiants: Class A (the determinant on prototype plasmid RP1), Class B (that on R222), and Class C (that on plasmid pSC101). At least one other determinant, encoded by plasmid RA1, belongs to none of these three groups and has been designated Class D. These genetic classes confirm phenotypic differences in expression of resistance to tetracycline and tetracycline analogs encoded by the different plasmids.     

Cloning and expression of R-factor mediated arsenate resistance in Escherichia coli

Summary The resistance transfer factor R773 confers inducible arsenate, arsenite and antimony resistance on Escherichia coli. The genes for these resistances were cloned into the EcoRi site of plasmid pBR322 to produce a 33 kilobase plasmid, pUM1. Bacterial strains transformed with pUM1 synthesized a polypeptide of the apparent molecular weight 64,000 daltons when induced with arsenite. This polypeptide could be visualized on sodium dodecyl sulfate polyacrylamide gels stained with Coomassie blue. It was observed both in the membrane and cytosol fractions but not among the periplasmic proteins present in osmotic shock fluid. Minicells isolated from strain JR410(pUM1) incorporated [³⁵S]methionine into an inducible 64,000 dalton polypeptide, as demonstrated on autoradiographs of electrophoresed [³⁵S]-labeled minicell lysates, confirming that this polypeptide is a plasmid gene product. A 4.3 kilobase HindIII fragment of pUM1 was subcloned into the HindIII site of pBR322, producing recombinant plasmid pUM3. This plasmid conferred constitutive resistance to arsenite and arsenate. Extensive synthesis of two polypeptides of 64,000 and 16,000 daltons was observed both in Coomassie stained gels of whole cells and autoradiographs of gels of [³⁵S]methionine-labeled minicells. Synthesis of both polypeptides was constitutive.     

Plasmid pSC101 replication mutants generated by insertion of the transposon Tn1000

A derivative of pSC101, pLC709, was constructed by ligation of the HincII-A fragment of pSC101 to the mini-colEI plasmid pVH51 and to a DNA fragment encoding resistance to the antibiotics streptomycin and spectinomycin. Insertions of the transposon Tn1000 (gamma-delta) into the pSC101 replication region of pLC709 were isolated following cotransfer of the plasmid with the sex factor F. The sites of insertion of the transposon were determined by restriction enzyme analysis and the replication and incompatibility properties of the insertion plasmids and DNA fragments cloned from them were analysed. The insertion mutations defined a locus, inc, of approximately 200 base-pairs that is responsible for pSC101-specific incompatibility. Two mutations adjacent to this region inactivate pSC101 replication but can be complemented in trans by a wild-type pSC101 plasmid, and thus define a trans-acting replication function, rep. The inc locus is within a larger region of some 450 base-pairs that is essential for pSC101 replication and that includes the origin of replication. This 450 base-pair segment can replicate in the presence of a helper plasmid that supplies the rep function in trans.     

Effect of dna mutations on the replication of plasmid pSC101 in Escherichia coli K-12.

Escherichia coli strains with mutations in genes dnaB, dnaC, and dnaG were tested for their capacity to replicate pSC101 deoxyribonucleic acid (DNA) at a nonpermissive temperature. Only a small amount of radioactive thymine was incorporated into pSC101 DNA in the dna mutants at 42 degrees C, whereas active incorporation into plasmid DNA took place in wild-type strains under the same conditions. The effects of the dnaB and dnaC mutations were greater on plasmid DNA synthesis than on host chromosomal DNA synthesis, suggesting that these gene products are directly involved in the process of pSC101 DNA replication. In dnaG mutants, both plasmid and chromosomal DNA synthesis were blocked soon after the shift to high temperature; although the extent of inhibition of the plasmid DNA synthesis was greater during the early period of temperature shift to 42 degrees C as compared with that of the host DNA synthesis, during the later period it was less. It was found that the number of copies of pSC101 per chromosome in dnaA and dnaC strains, grown at 30 degrees C, was considerably lower than that in wildtype strains, suggesting that the replication of pSC101 in these mutant strains was partially suppressed even under the permissive conditions. No correlation was found between the number of plasmid copies and the tetracycline resistance level of the host bacterium.     

Mapping of regions on cloned Saccharomyces cerevisiae 2-mum DNA coding for polypeptides synthesized in Escherichia coli minicells.

Summary Saccharomyces cerevisiae 2-m DNA and some of its restriction fragments were integrated in vector pCR1, pBR313 or pBR322 and their expression in Escherichia coli P678-54 minicells was analyzed. 2-m DNA inserted at the EcoRI site of pCR1 or pBR313 and at the PstI site of pBR322, promoted the synthesis of polypeptides of 48,000, 37,000, 35,000 and 19,000 daltons. The DNA regions coding for these polypeptides were mapped on the 2-m DNA molecule by insertion of single EcoRI or HindIII restriction fragments and comparison of the polypeptides produced. For the synthesis of the 37,000 dalton polypeptide, intact sites RIB and H3 were required. The disappearance of the 37,000 dalton polypeptide on interruption of one of these sites by insertion of the vector, was correlated with the appearance of a polypeptide of 22,000 or 23,500 daltons repectively. The DNA sequence coding for the 37,000 dalton polypeptide, therefore, has to be located in the S-loop region close to or overlapping with the sites RIB and H3. Assuming that the 22,000 and the 23,500 dalton polypeptides are truncated forms of the 37,000 dalton polypeptide, the last polypeptide can be exactly mapped. The polypeptide of 48,000 daltons was mapped to that half of the L-loop segment containing the sites H1 and H2. If, however, HindIII fragment H1-H2 was expressed, the 48,000 dalton polypeptide was lost and concomitantly a 43,000 dalton polypeptide appeared. We assume that this polypeptide results from early termination of the polypeptide of 48,000 daltons. The 35,000 and 19,000 dalton polypeptides were mapped to the S-loop region.The integrated inverted repeat sequence of yeast 2-m DNA did not induce any detectable insert-specific polypeptide synthesis.     

In vitro translation of mushroom tyrosinase

Mushroom tyrosinase was purified and antibodies prepared against the holo enzyme and a protein of 26,000 daltons. Both antibodies recognized the large subunit of the enzyme but only one recognized the 26,000 dalton protein. Poly A+ mRNA was isolated from mushrooms, translated in vitro, and a 41,000 dalton protein immunoprecipitated from the translation mix with either antibody. This 41,000 dalton protein presumably corresponds to the large subunit of the holoenzyme. Antibodies against the holoenzyme also immunoprecipitated another translation product with a molecular weight of 15,000 daltons corresponding to the small subunit of the holoenzyme. These results suggest that each subunit may be coded for by different genes and undergo posttranslational processing.     

Identification of a second tetracycline-inducible polypeptide encoded by Tn10.

Three Tn10 polypeptides were detected by analyzing the proteins synthesized in ultraviolet light-irradiated Escherichia coli cells after infection with lambda::Tn10. One of these polypeptides was the previously identified 36,000-dalton TET polypeptide. The other two had approximate sizes of 25,000 and 13,000 daltons. The syntheses of both the TET polypeptide and the 25,000-dalton polypeptide were inducible by tetracycline in lambda-immune hosts. Similarly, the synthesis of the TET polypeptide was inducible in nonimmune hosts. However, the synthesis of the 25,000-dalton polypeptide was constitutive in nonimmune hosts. An amber mutation in a gene required for tetracycline resistance on lambda::Tn10 was isolated that eliminated the synthesis of the TET polypeptide in sup+ hosts but not the synthesis of the 25,000-dalton or the 13,000-dalton polypeptides. The expression of tetracycline resistance from wild-type Tn10 was found to be anomalous in E. coli strains carrying the amber suppressors supD, supE, and supF. In general, strains containing these nonsense suppressors were less resistant to tetracycline.     

Phosphorylation of specific polypeptides in isolated murine splenocyte nuclei which is controlled by cyclic nucleotides

Murine splenocyte nuclei were phosphorylated with a less than 10(-5) M concentration of [gamma-32P]ATP at 0 degrees C and the phosphorylated nuclear proteins were analyzed by SDS-polyacrylamide gel slab electrophoresis and Sephadex gel filtration column chromatography. Two polypeptides of 10K and 11K daltons were predominantly phosphorylated. These polypeptides were likely linked by a disulfide bond to form a nonhistone protein of 21K daltons. Both phosphoserine and phosphothreonine were detected in the hydrolysate of the 10.5K dalton polypeptide, while phosphoserine was predominant in the 10K dalton polypeptide. Maximal activation of phosphorylation by cAMP of both polypeptides was shown at a concentration of 10(-6) M. On the contrary, cGMP activated phosphorylation of the 10K dalton polypeptide at 10(-8) M and at 10(-4) M. The phosphorylation of the 10.5K polypeptide was not activated by 10(-4) M cGMP and suppression of the phosphorylation was seen in both polypeptide chains by cAMP at higher concentrations.