DNA mismatch repair detected in human cell extracts

1987 ◽  
Vol 7 (1) ◽  
pp. 218-224
Author(s):  
P M Glazer ◽  
S N Sarkar ◽  
G E Chisholm ◽  
W C Summers
Keyword(s):  
Hela Cell ◽  
Mismatch Repair ◽  
Plasmid Dna ◽  
Coli Strain ◽  
Dna Polymerization ◽  
E Coli ◽  
Cell Extracts ◽  
Repair Activity ◽  
Supf Gene

A system to study mismatch repair in vitro in HeLa cell extracts was developed. Preformed heteroduplex plasmid DNA containing two single base pair mismatches within the SupF gene of Escherichia coli was used as a substrate in a mismatch repair assay. Repair of one or both of the mismatches to the wild-type sequence was measured by transformation of a lac(Am) E. coli strain in which the presence of an active supF gene could be scored. The E. coli strain used was constructed to carry mutations in genes associated with mismatch repair and recombination (mutH, mutU, and recA) so that the processing of the heteroduplex DNA by the bacterium was minimal. Extract reactions were carried out by the incubation of the heteroduplex plasmid DNA in the HeLa cell extracts to which ATP, creatine phosphate, creatine kinase, deoxynucleotides, and a magnesium-containing buffer were added. Under these conditions about 1% of the mismatches were repaired. In the absence of added energy sources or deoxynucleotides, the activity in the extracts was significantly reduced. The addition of either aphidicolin or dideoxynucleotides reduced the mismatch repair activity, but only aphidicolin was effective in blocking DNA polymerization in the extracts. It is concluded that mismatch repair in these extracts is an energy-requiring process that is dependent on an adequate deoxynucleotide concentration. The results also indicate that the process is associated with some type of DNA polymerization, but the different effects of aphidicolin and dideoxynucleotides suggest that the mismatch repair activity in the extracts cannot simply be accounted for by random nick-translation activity alone.

scholarly journals DNA mismatch repair detected in human cell extracts.

1987 ◽  
Vol 7 (1) ◽  
pp. 218-224 ◽  
Author(s):  
P M Glazer ◽  
S N Sarkar ◽  
G E Chisholm ◽  
W C Summers
Keyword(s):  
Hela Cell ◽  
Mismatch Repair ◽  
Plasmid Dna ◽  
Coli Strain ◽  
Dna Polymerization ◽  
E Coli ◽  
Cell Extracts ◽  
Repair Activity ◽  
Supf Gene

A system to study mismatch repair in vitro in HeLa cell extracts was developed. Preformed heteroduplex plasmid DNA containing two single base pair mismatches within the SupF gene of Escherichia coli was used as a substrate in a mismatch repair assay. Repair of one or both of the mismatches to the wild-type sequence was measured by transformation of a lac(Am) E. coli strain in which the presence of an active supF gene could be scored. The E. coli strain used was constructed to carry mutations in genes associated with mismatch repair and recombination (mutH, mutU, and recA) so that the processing of the heteroduplex DNA by the bacterium was minimal. Extract reactions were carried out by the incubation of the heteroduplex plasmid DNA in the HeLa cell extracts to which ATP, creatine phosphate, creatine kinase, deoxynucleotides, and a magnesium-containing buffer were added. Under these conditions about 1% of the mismatches were repaired. In the absence of added energy sources or deoxynucleotides, the activity in the extracts was significantly reduced. The addition of either aphidicolin or dideoxynucleotides reduced the mismatch repair activity, but only aphidicolin was effective in blocking DNA polymerization in the extracts. It is concluded that mismatch repair in these extracts is an energy-requiring process that is dependent on an adequate deoxynucleotide concentration. The results also indicate that the process is associated with some type of DNA polymerization, but the different effects of aphidicolin and dideoxynucleotides suggest that the mismatch repair activity in the extracts cannot simply be accounted for by random nick-translation activity alone.

Localization of active promoters for eucaryotic RNA polymerase II in the long terminal repeat of avian sarcoma virus DNA

1983 ◽  
Vol 3 (5) ◽  
pp. 811-818
Author(s):  
S A Mitsialis ◽  
J L Manley ◽  
R V Guntaka
Keyword(s):  
Rna Polymerase ◽  
Hela Cell ◽  
Long Terminal Repeat ◽  
Rna Polymerase Ii ◽  
Terminal Repeat ◽  
Avian Sarcoma Virus ◽  
Cell Extracts ◽  
Sarcoma Virus ◽  
Avian Sarcoma

The nucleotide sequences in the long terminal repeat of avian sarcoma virus that are recognized in vitro by HeLa cell RNA polymerase II have been identified. For this purpose, various 5' and 3' deletions were introduced into a cloned long terminal repeat fragment. The effects of these deletions on transcription initiation in HeLa whole-cell extracts were then studied. Three specific transcripts have been identified. The major transcript is initiated at nucleotide +1 (relative to the cap site). Deletion of the upstream sequence between -299 and -55 has no effect on the level of transcription from this start site, whereas deletion of the sequence downstream of -14 drastically reduces the levels of transcription. In contrast, deletion of the sequence downstream from the TATA box has no effect on the initiation or efficiency of synthesis of the two minor RNA species, which are initiated at around nucleotides -260 and -105. The transcription of these RNA products, however, is abolished by an upstream deletion between -299 and -55. These results suggest that HeLa cell RNA polymerase II recognizes in vitro more than one promoter site present in the long terminal repeat of the avian sarcoma virus genome and defines the sequences required for initiation of the major transcript.

scholarly journals The Production of the Recombinant Protein of Anthrax Bacteriolysine PlyPH and In Vitro Study of the Lytic Properties of the Protein Coded for them Against Bacillus anhracis Microbial Cells

2019 ◽  
pp. 5-22
Author(s):  
Onuchina N.V., Soybanov V.D.
Keyword(s):  
In Vitro Study ◽  
Coli Strain ◽  
Vitro Study ◽  
Chromosomal Dna ◽  
Lytic Enzymes ◽  
Microbial Cells ◽  
Phage Gene ◽  
E Coli ◽  
Species Specific

The causative agent of anthrax - Bacillus anthracis, due to the prevalence of its natural foci in Russia, high virulence for humans and most mammals, the unique resistance of spore forms to environmental factors and repeated use in terrorist acts, is an extremely dangerous biological agent. Therefore, the search for new effective drugs for the diagnosis and treatment of anthrax, including diseases caused by antibiotic-resistant strains of B. anthracis is necessary. The use of lytic enzymes of species-specific bacteriophages is a new trend in the diagnosis, prevention and treatment of infectious diseases. The goal of this work is the cloning of the anthrax bacteriolysin PlyPH gene as part of the pTrcHis2C vector in Escherichia coli and the in vitro study of the lytic properties of the protein encoded by it against B. anthracis microbial cells. According to the complete sequencing of the B. anthracis genomes of the Ames, Stern 34F2 and JB17 strains, a prophage was found in their chromosomal DNA, which lost part of the structural genes necessary for its replication, but retained a gene with a high degree of homology with the bacteriolysin γ phage gene. For amplification and subsequent cloning of the PlyPH gene, we developed primers containing EcoRI and BamHI restriction enzyme recognition sites. Amplification of the PlyPH gene in a polymerase chain reaction (PCR) with a developed pair of primers was performed using the Stern 34F2 strain of the anthrax microbe as a template. Based on the obtained amplification products and the pTrcHis2C vector, we constructed a recombinant plasmid containing the bacteriolysin synthesis PlyPH gene and stably functioning in the cells of the recombinant E. coli strain. In the course of research, it has been established that microbial cells of the E. coli recombinant TOP10 strain provide for the production of the bacteriolysin of the anthrax prophage, PlyPH , which has the ability to in vitro lyse the vegetative cells of the STI-1 vaccine strain of B. anthracis

scholarly journals Synthesis of silver nanoparticles from extracts of Scytonema geitleri HKAR-12 and their in vitro antibacterial and antitumor potentials

2019 ◽  
Vol 8 (3) ◽  
pp. 576-585
Keyword(s):  
Silver Nanoparticles ◽  
Coli Strain ◽  
X Ray Diffraction ◽  
Xrd Pattern ◽  
E Coli ◽  
Vis Spectroscopy ◽  
Shape Structure ◽  
Actual Size

In the present study silver nanoparticles (AgNPs) have been synthesized through the cell-free extracts of the rooftop dwelling cyanobacterium Scytonema geitleri HKAR-12. UV-VIS spectroscopy, FTIR, X-ray diffraction, SEM and TEM were used for the determination of morphological, structural and optical properties of synthesized AgNPs. Extracts of Scytonema geitleri HKAR-12 have the ability to reduce AgNO3 to Ag0. Sharp peak at 422 nm indicated the rapid synthesis of AgNPs. FTIR results showed the presence of different groups responsible for the reduction of AgNO3 to AgNPs. XRD pattern confirmed the crystalline nature of AgNPs. SEM showed the bead shape structure of AgNPs. TEM confirmed the actual size of AgNPs to be ranging between 9-17 nm. AgNPs showed antibacterial activity against Pseudomonas aeruginosa, Escherichia coli strain1 and E. coli strain 2 and 11 μg/mL of AgNPs effectively inhibited the growth of MCF-7 cells. Hence, Scytonema geitleri HKAR-12, isolated from the rooftop could serve as a desirable biological candidate for convenient and cheap production of AgNPs having antimicrobial and anti-cancerous properties.

A cell-specific factor represses stimulation of transcription in vitro by transcriptional enhancer factor 1

1994 ◽  
Vol 14 (8) ◽  
pp. 5290-5299
Author(s):  
S Chaudhary ◽  
C Brou ◽  
M E Valentin ◽  
N Burton ◽  
L Tora ◽  
...  
Keyword(s):  
Hela Cell ◽  
Binding Protein ◽  
Activation Function ◽  
Tata Binding Protein ◽  
Lymphoid Cells ◽  
Specific Factor ◽  
Transcriptional Enhancer ◽  
Cell Extracts ◽  
A Cell

Transcription in HeLa cell extracts in vitro was stimulated 8- to 10-fold by a recombinant chimera, GAL-TEF-1, consisting of the DNA-binding domain of GAL4 and the activation function of the HeLa cell activator TEF-1. In contrast, only a 2- to 3-fold stimulation was obtained with GAL-TEF-1 in extracts from BJA-B lymphoid cells. Stimulation by GAL-TEF-1 in BJA-B extracts was dramatically increased by the addition of immunopurified HeLa cell TFIID, suggesting that BJA-B TFIID lacks or contains lower quantities of a TATA-binding-protein-associated factor(s) required for the activity of the TEF-1 activation function. However, chromatography, immunopurification, and transcriptional reconstitution experiments indicated that BJA-B extracts did not lack the previously identified TATA-binding-protein-associated factors required for TEF-1 activity but rather contained a negatively acting factor(s) which inhibited transactivation by GAL-TEF-1. These results indicate that the relative lack of activity of the TEF-1 activation function in vitro in BJA-B cell extracts does not result from the absence of positively acting factors from the presence of a cell-specific negatively acting factor(s).

scholarly journals 1127. Genomic Analysis of Biofilm-Forming Enteroinvasive E. coli Emergent Pathogen

2018 ◽  
Vol 5 (suppl_1) ◽  
pp. S337-S338
Author(s):  
Oscar Gomez-Duarte ◽  
Julio Guerra ◽  
Ricky Ko
Keyword(s):  
Virulence Factors ◽  
Dna Sequences ◽  
Horizontal Transfer ◽  
Antibiotic Resistance Genes ◽  
Genomic Analysis ◽  
Coli Strain ◽  
Virulence Plasmid ◽  
E Coli ◽  
Query Coverage

Abstract Background Enteroinvasive Escherichia coli (EIEC) are involved in dysenteric diarrhea among children in low- and middle-income countries. EIEC strains isolated in Colombia, South America were shown to form biofilms and to be invasive in vitro. The O96:H19 serotypes and biofilm formation (BF) are not common phenotypes among EIEC, and the role they may play in diarrhea is at present unknown. The main goal of this study was to identify virulence and BF genes from EIEC genomic data. We hypothesize that EIEC O96:H19 strain 52.1 originated from horizontal transfer of a Shigella-like virulence plasmid into a non-EIEC pathogenic E coli strain. Methods WGS was performed on the BF-EIEC 52.1 strain using NextGen Illumina and Pacific Biosciences (PacBio) platforms. Publically available genomes from other EIEC O96H19 and Shigella genomes previously published were analyzed using online available software and databases including NCBI, BLAST, Mauve, among others. This analysis was tailored to identify virulence factors from the virulence factor database (VFDB). BLASTn was used to determine identity and query coverage of genes encoding the Shigella virulence factors. EIEC and Shigella genomes were analyzed on a multiple genome alignment software (Mauve) to verify results from BLASTn and to determine pseudogenes. Results The genome of EIEC O96:H19 strain 52.1 was 5,193,449 bp in size, containing 5,050 coding DNA sequences (CDSs). O96:H19 strain 52.1 carries three plasmids, the invasion plasmid (pINV) contains all type 3 secretion system (TTSS) and TTSS effectors genes previously described for Shigella and EIEC O96:H19 CFSAN029787 Italian strain. Non-TTSS virulence genes were also identified, including: long polar fimbrial gene (IpfA), enterotoxin (senB), and antibiotic resistance genes. Conclusion The EIEC O96:H19 strain 52.1 genome carries TTSS genes within a virulence plasmid, protein effector genes, and enterotoxin genes known to be associated with EIEC virulence. The EIEC O96:H19 stain 52.1 is an emergent diarrheagenic pathogen likely derived from an E. coli O96:H19 strain that acquired a Shigella-like virulence plasmid by horizontal transfer. Disclosures All authors: No reported disclosures.

scholarly journals Esterases in Serum-Containing Growth Media Counteract Chloramphenicol Acetyltransferase Activity In Vitro

1999 ◽  
Vol 43 (3) ◽  
pp. 655-660 ◽  
Author(s):  
Charles D. Sohaskey ◽  
Alan G. Barbour
Keyword(s):  
Human Serum ◽  
Horse Serum ◽  
Chloramphenicol Acetyltransferase ◽  
Rabbit Serum ◽  
Growth Media ◽  
Chloramphenicol Resistance ◽  
E Coli ◽  
Cell Extracts

ABSTRACT The spirochete Borrelia burgdorferi was unexpectedly found to be as susceptible to diacetyl chloramphenicol, the product of the enzyme chloramphenicol acetyltransferase, as it was to chloramphenicol itself. The susceptibilities of Escherichia coli and Bacillus subtilis, as well as that ofB. burgdorferi, to diacetyl chloramphenicol were then assayed in different media. All three species were susceptible to diacetyl chloramphenicol when growth media were supplemented with rabbit serum or, to a lesser extent, human serum. Susceptibility ofE. coli and B. subtilis to diacetyl chloramphenicol was not observed in the absence of serum, when horse serum was used, or when the rabbit or human serum was heated first. In the presence of 10% rabbit serum, a strain of E. colibearing the chloramphenicol acetyltransferase (cat) gene had a fourfold-lower resistance to chloramphenicol than in the absence of serum. A plate bioassay for chloramphenicol activity showed the conversion by rabbit, mouse, and human sera but not bacterial cell extracts or heated serum of diacetyl chloramphenicol to an inhibitory compound. Deacetylation of acetyl chloramphenicol by serum components was demonstrated by using fluorescent substrates and thin-layer chromatography. These studies indicate that esterases of serum can convert diacetyl chloramphenicol back to an active antibiotic, and thus, in vitro findings may not accurately reflect the level of chloramphenicol resistance by cat-bearing bacteria in vivo.

scholarly journals Localization of active promoters for eucaryotic RNA polymerase II in the long terminal repeat of avian sarcoma virus DNA.

1983 ◽  
Vol 3 (5) ◽  
pp. 811-818 ◽  
Author(s):  
S A Mitsialis ◽  
J L Manley ◽  
R V Guntaka
Keyword(s):  
Rna Polymerase ◽  
Hela Cell ◽  
Long Terminal Repeat ◽  
Rna Polymerase Ii ◽  
Terminal Repeat ◽  
Avian Sarcoma Virus ◽  
Cell Extracts ◽  
Sarcoma Virus ◽  
Avian Sarcoma

The nucleotide sequences in the long terminal repeat of avian sarcoma virus that are recognized in vitro by HeLa cell RNA polymerase II have been identified. For this purpose, various 5' and 3' deletions were introduced into a cloned long terminal repeat fragment. The effects of these deletions on transcription initiation in HeLa whole-cell extracts were then studied. Three specific transcripts have been identified. The major transcript is initiated at nucleotide +1 (relative to the cap site). Deletion of the upstream sequence between -299 and -55 has no effect on the level of transcription from this start site, whereas deletion of the sequence downstream of -14 drastically reduces the levels of transcription. In contrast, deletion of the sequence downstream from the TATA box has no effect on the initiation or efficiency of synthesis of the two minor RNA species, which are initiated at around nucleotides -260 and -105. The transcription of these RNA products, however, is abolished by an upstream deletion between -299 and -55. These results suggest that HeLa cell RNA polymerase II recognizes in vitro more than one promoter site present in the long terminal repeat of the avian sarcoma virus genome and defines the sequences required for initiation of the major transcript.

Comparison of Vibrio and Firefly Luciferases as Reporter Gene Systems for Use in Bacteria and Plants

1996 ◽  
Vol 23 (1) ◽  
pp. 75 ◽  
Author(s):  
SR Mudge ◽  
WR Lewis-Henderson ◽  
RG Birch
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Ccd Camera ◽  
Reporter Gene Expression ◽  
In Vitro Assays ◽  
E Coli ◽  
Cell Extracts ◽  
Cooled Ccd

Luciferase genes from Vibrio harveyi (luxAB) and firefly (luc) were introduced into E. coli, Agrobacteriurn, Arabidopsis and tobacco. Transformed bacteria and plants were quantitatively assayed for luciferase activity using a range of in vitro and in vivo assay conditions. Both lux and luc proved efficient reporter genes in bacteria, although it is important to be aware that the sensitive assays may detect expression due to readthrough from distant promoters. LUX activity was undetectable by liquid nitrogen-cooled CCD camera assays on intact tissues of plants which showed strong luxAB expression by in vitro assays. The decanal substrate for the lux assay was toxic to many plant tissues, and caused chemiluminescence in untransformed Arabidopsis leaves. These are serious limitations to application of the lux system for sensitive, non-toxic assays of reporter gene expression in plants. In contrast, LUC activity was readily detectable in intact tissues of all plants with luc expression detectable by luminometer assays on cell extracts. Image intensities of luc-expressing leaves were commonly two to four orders of magnitude above controls under the CCD camera. Provided adequate penetration of the substrate luciferin is obtained, luc is suitable for applications requiring sensitive, non-toxic assays of reporter gene expression in plants.

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