SSB Protein—Its Interaction with DNA and Use as a Tool in Studying Genome Structure

1981 ◽  
Vol 39 ◽  
pp. 448-451
Author(s):  
Susan Chrysegelos ◽  
Kathi Dunn ◽  
Jack Griffith ◽  
Marcia Manning ◽  
Claire Moore
Keyword(s):  
Genome Structure ◽  
Duplex Dna ◽  
Single Stranded Dna ◽  
Functional Roles ◽  
E Coli ◽  
Gene 5 Protein ◽  
Nucleoprotein Complex ◽  
Infected Cells ◽  
Gene 32

A protein which binds tightly to single stranded DNA but not duplex DNA was first isolated from Escherichia coli (E. coli) by Sigal et. al and is called SSB for single stranded DNA binding protein. Together with SSB the gene 32 protein of T4 infected E. coli cells, and the gene 5 protein of phage M13 infected cells, are the best characterized members of the helix destabilizing family of proteins. They all share the properties (reviewed by Kbrnberg) of binding very tightly and cooperatively to single stranded DNA, of binding somewhat less well to single stranded RNA, and of binding poorly if at all to duplex DNA or RNA. In binding single stranded polynucleotides these proteins disrupt all secondary structure yielding a linear nucleoprotein complex. The details of binding however are very different from one protein to another and must reflect their functional roles in vivo.Physical studies of SSB have showi it to exist as a 75,000 dalton tetramer in solution which is assumed to be the active unit.

The Mechanism of Replication of Single-Stranded DNA. VI. Requirements for Ribonucleoside Triphosphates and DNA Polymerase III

1973 ◽  
Vol 51 (12) ◽  
pp. 1588-1597 ◽  
Author(s):  
David T. Denhardt ◽  
Makoto Iwaya ◽  
Grant McFadden ◽  
Gerald Schochetman
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerases ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Dna Polymerase Iii ◽  
Single Stranded Dna ◽  
E Coli ◽  
Polymerase Iii

Evidence is presented that in Escherichia coli made permeable to nucleotides by exposure to toluene, the synthesis of a DNA chain complementary to the infecting single-stranded DNA of bacteriophage [Formula: see text] requires ATP as well as the four deoxyribonucleoside triphosphates. This synthesis results in the formation of the parental double-stranded replicative-form (RF) molecule. The ATP is not required simply to prevent degradation of the ribonucleoside or deoxyribonucleoside triphosphates; it can be partially substituted for by other ribonucleoside triphosphates.No single one of the known E. coli DNA polymerases appears to be uniquely responsible in vivo for the formation of the parental RF. Since [Formula: see text] replicates well in strains lacking all, or almost all, of the in-vitro activities of DNA polymerases I and II, neither of these two enzymes would seem essential; and in a temperature-sensitive E. coli mutant (dnaEts) deficient in DNA polmerase-I activity and possessing a temperature-sensitive DNA polymerase III, the viral single-stranded DNA is efficiently incorporated into an RF molecule at the restrictive temperature. In contrast, both RF replication and progeny single-stranded DNA synthesis are dependent upon DNA polymerase III activity.

Reduced synthesis of β-galactosidase in Escherichia coli infected with phage ϕX174

1977 ◽  
Vol 23 (8) ◽  
pp. 1069-1077 ◽  
Author(s):  
Amit Ghosh ◽  
Ramendra K. Poddar
Keyword(s):  
Escherichia Coli ◽  
Single Stranded Dna ◽  
E Coli ◽  
Infected Cells ◽  
Specific Mrna

The synthesis of β-galactosidase (EC 3.2.1.23; β-D-galactoside galactohydrolase) in E. coli was repressed as a result of infection with single-stranded DNA phage [Formula: see text]. Evidence is presented to show that this repression was not due to the restricted entry of the inducer molecules into the infected cells but to some phage-specified product(s). It was further shown that either the infected cells synthesized a fewer number of enzyme-specific mRNA or all such molecules were translated with a reduced efficiency; the half-lives of the mRNA's remained more or less unaffected.

Homologous recombination in prokaryotes: enzymes and controlling sites

Genome ◽  
1989 ◽  
Vol 31 (2) ◽  
pp. 520-527 ◽  
Author(s):  
Gerald R. Smith
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Duplex Dna ◽  
Single Stranded Dna ◽  
E Coli ◽  
D Loop ◽  
Enzymatic Mechanisms ◽  
Λ Red

A common step in prokaryotic recombination appears to be the synapsis of the 3′-end of single-stranded DNA with duplex DNA to form a D-loop. The enzymatic mechanisms by which 3′-ends are produced and by which D-loops are converted into recombinant molecules are illustrated by proposed mechanisms of recombination by the Escherichia coli RecBCD pathway and the phage λ Red pathway. The enzymes promoting recombination and the special DNA sites at which they act are emphasized. Recombination by other E. coli pathways and in other prokaryotes is compared with these mechanisms.Key words: Escherichia coli RecBCD pathway, phage λ Red pathway, Chi and cos sites, recombination enzymes.

The rep mutation. VI. Purification and properties of the Escherichia coli rep protein, DNA helicase III

1979 ◽  
Vol 57 (6) ◽  
pp. 855-866 ◽  
Author(s):  
Seishi Takahashi ◽  
Christian Hours ◽  
Alan Chu ◽  
David T. Denhardt
Keyword(s):  
Escherichia Coli ◽  
Cell Extract ◽  
Dna Helicase ◽  
Protein Product ◽  
Duplex Dna ◽  
Replicative Form ◽  
Single Stranded Dna ◽  
Rep Protein ◽  
E Coli

The protein product of the rep gene of Escherichia coli is required for the replication of certain bacteriophage genomes ([Formula: see text], fd, P2) and for the normal replication of E. coli DNA. We have used a specialized transducing phage, λp rep+, which complements the defect of rep mutants, to identify the rep protein. The rep protein has been purified from cells infected with λp rep+ phage; it has a molecular weight of about 70 000 and appears similar to the protein found in normal cells. Stimulation of [Formula: see text] replicative form DNA synthesis in vitro was observed when highly purified rep protein was supplied to a cell extract derived from [Formula: see text]-infected E. coli rep cells and supplemented with replicative form DNA. The purified protein has a single-stranded DNA-dependent ATPase activity and is capable of sensitizing duplex DNA to nucleases specific for single-stranded DNA. For this reason we propose the enzyme be called DNA helicase III. We infer that the rep protein uses the energy of hydrolysis of ATP to separate the strands of duplex DNA; the E. coli DNA binding protein need not be present. The rep3 mutant appeared to make a limited amount of active rep protein.

scholarly journals Transformation inEscherichia coli: studies on the nature of donor DNA after uptake and integration

1980 ◽  
Vol 35 (3) ◽  
pp. 279-289 ◽  
Author(s):  
W. P. M. Hoekstra ◽  
H. E. N. Bergmans ◽  
E. M. Zuidweg
Keyword(s):  
Genetic Analysis ◽  
Uv Irradiation ◽  
Inescherichia Coli ◽  
Single Stranded Dna ◽  
Dna Integration ◽  
E Coli ◽  
Dna Restriction ◽  
P1 Transduction

SUMMARYChromosomalE. coliDNA appears to be sensitive towardsin vivoDNA restriction when transformed to a restrictiveE. colirecipient. It is therefore concluded that transforming chromosomal donor DNA is present in a double-stranded form immediately after uptake.Genetic analysis ofE. colitransformants, obtained with UV-irradiated donor DNA under conditions that exclude photorepair, show, especially in auvrBrecipient, loss of donor DNA information compared with the situation where DNA was not subjected to UV-irradiation. Similar conclusions were arrived at after genetic analysis of transductants obtained with UV-irradiated particles of the generalized transducing phage P1. The processing inE. coliof DNA after P1 transduction is thus similar to that of transforming DNA. The observations are discussed and a possible explanation based on single-stranded DNA integration is presented in detail.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

Production and Preliminary In Vivo Evaluations of a Novel in silico-designed L2-based Potential HPV Vaccine

2020 ◽  
Vol 21 (4) ◽  
pp. 316-324
Author(s):  
Manica Negahdaripour ◽  
Navid Nezafat ◽  
Reza Heidari ◽  
Nasrollah Erfani ◽  
Nasim Hajighahramani ◽  
...  
Keyword(s):  
In Silico ◽  
Group Versus ◽  
Tlr Agonists ◽  
E Coli ◽  
Metal Affinity ◽  
Th1 And Th2 Cytokines ◽  
Ifn Γ ◽  
Humoral Responses ◽  
Future Work

Background: L2-based Human Papillomavirus (HPV) prophylactic vaccines, containing epitopes from HPV minor capsid proteins, are under investigation as second-generation HPV vaccines. No such vaccine has passed clinical trials yet, mainly due to the low immunogenicity of peptide vaccines; so efforts are being continued. A candidate vaccine composed of two HPV16 L2 epitopes, flagellin and a Toll-Like Receptor (TLR) 4 agonist (RS09) as adjuvants, and two universal T-helper epitopes was designed in silico in our previous researches. Methods: The designed vaccine construct was expressed in E. coli BL21 (DE3) and purified through metal affinity chromatography. Following mice vaccination, blood samples underwent ELISA and flow cytometry analyses for the detection of IgG and seven Th1 and Th2 cytokines. Results: Following immunization, Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-5, IL-10) type cytokines, as well as IgG, were induced significantly compared with the PBS group. Significant increases in IFN-γ, IL-2, and IL-5 levels were observed in the vaccinated group versus Freund’s adjuvant group. Conclusion: The obtained cytokine induction profile implied both cellular and humoral responses, with a more Th-1 favored trend. However, an analysis of specific antibodies against L2 is required to confirm humoral responses. No significant elevation in inflammatory cytokines, (IL-6 and TNF-α), suggested a lack of unwanted inflammatory side effects despite using a combination of two TLR agonists. The designed construct might be capable of inducing adaptive and innate immunity; nevertheless, comprehensive immune tests were not conducted at this stage and will be a matter of future work.

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