Filamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in M13 replicative form in vitro.

Messing, Joachim; Gronenborn, Bruno; Müller‐Hill, Benno; Hopschneider, P Hans

doi:10.1073/pnas.74.9.3642

Cited by 602 publications

(138 citation statements)

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“…was used to synthesize a radioactive probe by extension from a synthetic 24-bp primer (Bethesda Research Laboratories) which had been annealed to the singlestrand M13 phage DNA containing the 36-bp 5S DNA insert. Hybridization of this probe to DNA blotted onto diazobenzyloxymethyl paper was performed in 37.5% formamide-5 x SSCP-10% dextran sulfate-calf thymus DNA (250 ,ug/ml) at 42°C for 48 to 72 h. Singlestranded and replicative form DNA were prepared by procedures previously described (20,37).…”

mentioning

confidence: 99%

Regular arrangement of nucleosomes on 5S rRNA genes in Xenopus laevis.

Young¹,

Carroll²

1983

Mol. Cell. Biol.

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The chromatin structure of the oocyte-type 5S RNA genes in Xenopus laevis was investigated. Blot hybridization analysis of DNA from micrococcal nuclease digests of erythrocyte nuclei showed that 5S DNA has the same average nucleosome repeat length, 192 ± 4 base pairs, as two Xenopus satellite DNAs and bulk erythrocyte chromatin. The positions of nuclease-sensitive regions in the 5S DNA repeats of purified DNA and chromatin from erythrocytes were mapped by using an indirect end-labeling technique. Although most of the sites cleaved in purified DNA were also cleaved in chromatin, the patterns of intensities were strikingly different in the two cases. In 5S chromatin, three nuclease-sensitive regions were spaced approximately a nucleosome length apart, suggesting a single, regular arrangement of nucleosomes on most of the 5S DNA repeats. The observed nucleosome locations are discussed with respect to nucleotide sequences known to be important for expression of 5S RNA. Because the preferred locations appear to be reestablished in each repeating unit, despite spacer length heterogeneity, we suggest that the regular chromatin structure reflects the presence of a sequence-specific DNA-binding component on inactive 5S RNA genes.

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confidence: 99%

Regular arrangement of nucleosomes on 5S rRNA genes in Xenopus laevis.

Young¹,

Carroll²

1983

Mol. Cell. Biol.

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“…and fused to the leader sequence of the Erwinia carotovora pelB gene in plasmid pHEN-1 (Hoogenboom et al, 199 1 ), thus obtaining the expression plasmid pPEcSod81A. The enzyme was overexpressed in the E. coli strain BMH 71/18 (Messing et al, 1977). Cells were grown at 37 "C in LB medium containing 100 mg/mL ampicillin and 0.25 mM CuS04 and 2.5 pM ZnS04.…”

Section: Resultsmentioning

confidence: 99%

Crystallization and preliminary X‐ray analysis of the monomeric Cu, Zn superoxide dismutase from Escherichia coli

et al. 1996

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Abstract:The Cu,Zn superoxide dismutase (Cu,Zn SOD) originally isolated from the periplasmic space of Escherichia coli has been cloned and overexpressed in the E. coli strain BMH 71/18. The protein has been purified as a single component of 17,000 Da, corresponding to one subunit of the common dimeric eukaryotic Cu,Zn SODs. Large crystals of the purified protein have been grown in the presence of polyethylene glycol 4,000 at pH 8.5; the crystals belong to the monoclinic space group P2,, with unit cell constants a = 33.1 A, b = 52.6 A, c = 43.3 A, P = I 11.4 degrees.One SOD subunit is contained in the asymmetric unit, yielding a V,,, value of 2.1 A3/Da; the crystals diffract X-rays beyond 2.0 A resolution.Keywords: crystals; enzyme structure; Escherichia coli; metal proteins; monomer-dimer equilibrium Cu,Zn superoxide dismutases (SODs) are a class of metalloenzymes involved in the physiologic response to oxygen toxicity by their ability to catalyze the dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide (Bannister et al., 1987). All the eukaryotic Cu,Zn SODs so far characterized are homodimeric enzymes containing one atom of copper and one atom of zinc per subunit. The catalytic mechanism entails alternate reduction and reoxidation of the Cu2+ ion, during successive encounters with the substrate, in a process that is thought to be dominated by an evolutionarily conserved distribution of electrostatic charges around the active site (Getzoff et al., 1983; Desideri et al., 1992). Analysis of the known crystal structure of oxidized Cu,Zn SODs from ox (Tainer et al., 1982), spinach (Kitagawaet al., 1991), yeast (Djinovic et al., 1992a), human (Parge et al. 1992), and Xenopus luevis (DjinovicReprint requests to: Alessandro Desideri, Department of Biology, University of Rome "Tor Vergata," Via della Ricerca Scientifica, 0 0 1 33 Rome, Italy; e-mail: desideri@utovrm.it. Carugo et al., 1996), as well as of the semisynthetic Cu,Co bovine SOD (Djinovic et al., 1992b) and the reduced SOD from the same source (Rypnieswski et al., 1995), have demonstrated that all these eukaryotic isoenzymes possess a nearly superimposible threedimensional fold, based on a flattened Greek-key eight-stranded P-barrel, with only minor local differences (Bordo et al., 1994). Such an exceptionally high degree of structural conservation is highlighted by the discovery (Deng et al., 1993; Rosen et al., 1993) that different point-mutations potentially able to influence the subunit fold, the dimeric association, or the active site structure of human Cu,Zn SOD are associated with the neurologic degeneration observed in familial amyotrophic lateral sclerosis (FALS).Cu,Zn SODs have also been isolated from the periplasmic space of some bacterial species. The primary sequences of the bacterial Cu,Zn SODs display a variable pattern of insertions and deletions in the loops connecting the P-strands that are expected to result in a modified subunit interface and in an altered organization of the active site channel with respect to the...

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“…1) which still retained the correct frame for expression of the foreign protein. Recombinant plasmids were transformed into the E. coli host BMH71-18 (Messing et al, 1977).…”

Section: Methodsmentioning

confidence: 99%

Epitope Mapping of Monoclonal Antibodies to Keratin 19 Using Keratin Fragments, Synthetic Peptides and Phage Peptide Libraries

Böttger

Stasiak

Harrison

et al. 1995

European Journal of Biochemistry

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To generate tools for monitoring processing and folding in keratin intermediate filaments, a group of monoclonal antibodies reacting with the intermediate filament protein keratin 19 were studied using different approaches to define the structure and localization of their epitopes. The binding pattern to bacterially expressed human keratin 19 fragments allowed the definition of minimal amino acid sequences required for antibody binding. The screening of overlapping 15-residue peptides confirmed and further specified the epitope locations for a subset of the tested antibodies. In addition, the epitope of an antibody with apparent species-restricted specificity (LE64) was revealed by isolating and characterizing a fulllength keratin 19 clone from a PtK2 cDNA library. Taken together with species cross-reactivity of individual antibodies and sequence information obtained by probing a phage display library, specific amino acid residues could be highlighted as likely to be involved in the antibody binding.

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Filamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in M13 replicative form in vitro.

Cited by 602 publications

References 24 publications

Regular arrangement of nucleosomes on 5S rRNA genes in Xenopus laevis.

Regular arrangement of nucleosomes on 5S rRNA genes in Xenopus laevis.

Crystallization and preliminary X‐ray analysis of the monomeric Cu, Zn superoxide dismutase from Escherichia coli

Epitope Mapping of Monoclonal Antibodies to Keratin 19 Using Keratin Fragments, Synthetic Peptides and Phage Peptide Libraries

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