High efficiency DNA mutagenesis mediated by using in vitro transcription, DNase I digestion, and RT-PCR

Several reliable methods to produce transgenic animals utilize the male genome. After penetration into oocyte, sperm DNA undergoes dramatic conformational changes that could represent a great opportunity for exogenous DNA to be integrated in the zygote genome. Among the enzymes responsible for sperm remodeling, a nuclease could be involved. The presence of a DNase I in oocytes has not been much investigated. To date, an immunolocalization of DNase I has been reported only in rat immature oocytes and the presence of nuclease activities has been shown in avian oocytes. The present study was conducted to verify whether a DNase-I like activity is present in MII mature pig oocytes. To do this, oocyte extracts were assessed for nuclease activity by a plasmid degradation assay and by zymography; these analyses evidenced a 33 kDa, Ca2+/Mg2+ dependent DNase I-like activity that was inhibited by Zn2+. A further identification of DNase I was achieved by Western blot, immunofluorescence and RT-PCR experiments. Moreover, the presence of the enzyme activity was confirmed by the rapid degradation of exogenous DNA microinjected into the ooplasm. Finally, the exogenous DNA transferred to oocyte by spermatozoa during sperm mediated gene transfer in vitro fertilisation protocol seemed to be protected from DNase I degradation and to persist in the ooplasm till 6 h. These results, together with the high efficiency of sperm based transgenesis methods, suggest that the association with spermatozoa protects exogenous DNA from nuclease activities.

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Transcriptional regulation of the human cytomegalovirus major immediate-early gene is associated with induction of DNase I-hypersensitive sites

Molecular and Cellular Biology ◽

10.1128/mcb.6.2.452-461.1986 ◽

1986 ◽

Vol 6 (2) ◽

pp. 452-461 ◽

Cited By ~ 2

Author(s):

J A Nelson ◽

M Groudine

Keyword(s):

Human Cytomegalovirus ◽

Structural Features ◽

In Vitro Transcription ◽

Dnase I ◽

Early Gene ◽

Immediate Early ◽

Dnase I Hypersensitive Sites ◽

Teratocarcinoma Cells ◽

Hypersensitive Sites

Human teratocarcinoma cells were used to examine structural features associated with expression of the major immediate-early (IE) gene of human cytomegalovirus. By immunofluorescence, comparison of RNA levels, and in vitro transcription of nuclei, we showed that the major IE gene is inactive in undifferentiated but active in differentiated cells. Therefore, the block in human cytomegalovirus replication in teratocarcinoma cells appears to be at the transcriptional level, in one of the initial genes transcribed. In addition, the in vitro transcription experiments demonstrated that in permissive infections the gene was transcriptionally inactive late in infection. A comparison of the structural features of the promoter region with the active and inactive IE genes showed the presence of constitutive and inducible DNase I-hypersensitive sites. The majority of the constitutive sites existed at -175, -275, -375, -425, and -525 relative to the cap site in an area which has been shown to be capable of simian virus 40 enhancer function. In contrast, the inducible DNase I sites were located outside this region at -650, -775, -875, and -975.

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Inhibition of Fas-mediated Apoptosis in Yac-1 Cell via Anti-Fas Ribozyme

Acta Biochimica et Biophysica Sinica ◽

10.1093/abbs/36.3.199 ◽

2004 ◽

Vol 36 (3) ◽

pp. 199-205

Author(s):

Min Zhang ◽

Fang Liu ◽

Wei He ◽

Yong You ◽

Ping Zou ◽

...

Keyword(s):

Flow Cytometry ◽

Proteolytic Activity ◽

Caspase 3 ◽

Hammerhead Ribozyme ◽

Annexin V ◽

In Vitro Transcription ◽

Rt Pcr ◽

Fas Mrna

Abstract To detect a new and more effective way against apoptosis mouse lymphomatic cell line-Yac-1 in which fas gene was expressed highly was used as a model for studying the effects of anti-Fas ribozyme on Fas-mediated apoptosis. A hammerhead ribozyme gene targeting the fas mRNA was synthesized and its in vitro transcription vector was constructed, which was transfected into Yac-1 cells using electroporation. Rz596 expression was detected using RT-PCR, and Fas expression in Yac-1 cells was detected using RT-PCR, Western blot and flow cytometry. After treated with anti-Fas antibody (JO2), Yac-1 cell viability was measured with MTT assay, caspase-3 proteolytic activity was detected, and cell apoptosis was measured according to annexin V apoptosis detecting kit. Anti-Fas ribozyme could cleave fas mRNA efficiently in vivo and in vitro. Fas expression in Yac-1 cells transfected with anti-Fas ribozyme was decreased remarkably and correlated with resistance to Fas-mediated apoptosis as determined by flow cytometry and caspase-3 proteolytic activity. Anti-Fas ribozyme was detected in cells transfected with pU6-RZ596 and pU6-dRZ596 and could remarkably decrease the Fas expression in Yac-1 cells, which made Yac-1 cells get rid of Fas-mediated apoptosis. Because of wide expression of fas in organs and tissues, our research was very useful for studying the inhibition of apoptosis of many organs and tissues in the future.

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Level of CYP4G19 Expression Is Associated with Pyrethroid Resistance inBlattella germanica

Journal of Parasitology Research ◽

10.1155/2010/517534 ◽

2010 ◽

Vol 2010 ◽

pp. 1-7 ◽

Cited By ~ 8

Author(s):

Guang-zhou Guo ◽

Yi-jie Geng ◽

Da-na Huang ◽

Cai-fang Xue ◽

Ren-li Zhang

Keyword(s):

Target Gene ◽

Pyrethroid Resistance ◽

Wild Strain ◽

Resistance Index ◽

In Vitro Transcription ◽

Rt Pcr ◽

Immunohistochemistry Analysis ◽

Long Time ◽

In The Wild

German cockroaches have become a large problem in the Shenzhen area because of their pesticide resistance, especially to pyrethroid. A pyrethroid called “Jia Chong Qing” to prevent pests for a long time were found to be resistant to “Jia Chong Qing” with resistance index of 3.88 measured using RT-PCR and immunohistochemistry analysis showed that both CYP4G19 mRNA and CYP4G19 protein expression levels in the wild strain were substantially higher than that of a sensitive strain. dsRNA segments derived from the target gene CYP4G19 were prepared using in vitro transcription and were microinjected into abdomens of the wild strain. Two to eight days after injection, the result showed that CYP4G19 mRNA expressions were significantly reduced in the groups injected with dsRNAs.

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Cyclic AMP Receptor Protein and RhaR Synergistically Activate Transcription from the l-Rhamnose-Responsive rhaSR Promoter in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.187.19.6708-6718.2005 ◽

2005 ◽

Vol 187 (19) ◽

pp. 6708-6718 ◽

Cited By ~ 17

Author(s):

Jason R. Wickstrum ◽

Thomas J. Santangelo ◽

Susan M. Egan

Keyword(s):

Escherichia Coli ◽

Cyclic Amp ◽

In Vitro Transcription ◽

Dnase I ◽

Receptor Protein ◽

Transcription Activator ◽

Transcription Start ◽

Dnase I Footprinting

ABSTRACT The Escherichia coli rhaSR operon encodes two AraC family transcription activator proteins, RhaS and RhaR, which regulate expression of the l-rhamnose catabolic regulon in response to l-rhamnose availability. RhaR positively regulates rhaSR in response to l-rhamnose, and RhaR activation can be enhanced by the cyclic AMP (cAMP) receptor protein (CRP) protein. CRP is a well-studied global transcription regulator that binds to DNA as a dimer and activates transcription in the presence of cAMP. We investigated the mechanism of CRP activation at rhaSR both alone and in combination with RhaR in vivo and in vitro. Base pair substitutions at potential CRP binding sites in the rhaSR-rhaBAD intergenic region demonstrate that CRP site 3, centered at position −111.5 relative to the rhaSR transcription start site, is required for the majority of the CRP-dependent activation of rhaSR. DNase I footprinting confirms that CRP binds to site 3; CRP binding to the other potential CRP sites at rhaSR was not detected. We show that, at least in vitro, CRP is capable of both RhaR-dependent and RhaR-independent activation of rhaSR from a total of three transcription start sites. In vitro transcription assays indicate that the carboxy-terminal domain of the alpha subunit (α-CTD) of RNA polymerase is at least partially dispensable for RhaR-dependent activation but that the α-CTD is required for CRP activation of rhaSR. Although CRP requires the presence of RhaR for efficient in vivo activation of rhaSR, DNase I footprinting assays indicated that cooperative binding between RhaR and CRP does not make a significant contribution to the mechanism of CRP activation at rhaSR. It therefore appears that CRP activates transcription from rhaSR as it would at simple class I promoters, albeit from a relatively distant position.

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Interaction of Bacillus subtilis CodY with GTP

Journal of Bacteriology ◽

10.1128/jb.01115-07 ◽

2007 ◽

Vol 190 (3) ◽

pp. 798-806 ◽

Cited By ~ 77

Author(s):

Luke D. Handke ◽

Robert P. Shivers ◽

Abraham L. Sonenshein

Keyword(s):

Bacillus Subtilis ◽

Transcriptional Repression ◽

Target Genes ◽

In Vitro Transcription ◽

Dnase I ◽

Gtp Hydrolysis ◽

Dnase I Footprinting ◽

Target Dna ◽

Branched Chain

ABSTRACT Many of the adaptive mechanisms that allow Bacillus subtilis to adjust to changes in nutrient availability are controlled by CodY. Binding of CodY to its target genes is stimulated by interaction with its effectors, GTP and the branched-chain amino acids (BCAAs). Upon nutrient limitation, intracellular pools of these effectors are depleted and CodY can no longer repress genes required for adaptation. In vitro studies reported here explored in more detail the interaction of CodY with GTP. DNase I footprinting experiments indicated that CodY has an affinity for GTP in the millimolar range. Further, CodY was shown to interact specifically with GTP and dGTP; no other naturally occurring nucleotides that were tested, including ppGpp and pppGpp, resulted in DNA protection. Two nonhydrolyzable analogs of GTP were fully able to activate CodY binding to target DNA, demonstrating that GTP hydrolysis is not necessary for CodY-dependent regulation. GTP and the BCAAs were shown to act additively to increase the affinity of CodY for DNA; increased protection was observed in DNase I footprinting experiments when both effectors were present, compared to either effector alone, and in in vitro transcription reactions, transcriptional repression by CodY was stronger in the presence of both GTP and BCAAs than of BCAAs alone. Thus, interaction of CodY with GTP is specific and results in increased affinity for its target genes. This increase in affinity is independent of GTP hydrolysis and is augmented in the presence of BCAAs.

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H-NS Antagonism in Shigella flexneri by VirB, a Virulence Gene Transcription Regulator That Is Closely Related to Plasmid Partition Factors

Journal of Bacteriology ◽

10.1128/jb.01813-06 ◽

2007 ◽

Vol 189 (9) ◽

pp. 3403-3413 ◽

Cited By ~ 37

Author(s):

Elizebeth C. Turner ◽

Charles J. Dorman

Keyword(s):

Transcription Factor ◽

Promoter Region ◽

Shigella Flexneri ◽

Virulence Gene ◽

In Vitro Transcription ◽

Dnase I ◽

Intracellular Pathogen ◽

Transcription Regulator ◽

Plasmid Partition

ABSTRACT The VirB protein of Shigella flexneri is a positive regulator of the major virulence operons of this enteroinvasive intracellular pathogen. VirB resembles no other transcription factor but is strongly homologous to plasmid partition proteins. We found that the binding of the VirB protein to the promoter region of the icsB virulence gene induced hypersensitivity to cleavage by DNase I over a region to which the H-NS repressor protein binds and completely abolished the protection of this sequence from DNase I by H-NS. In the absence of H-NS, the VirB protein had no additive effect on the ability of the icsB promoter to form an open transcription complex, indicating that VirB is not involved in the recruitment of RNA polymerase to the promoter or in open complex formation. Similarly, VirB did not stimulate promoter function in an in vitro transcription assay but acted as an antagonist of H-NS-mediated repression. A sequence located upstream of the icsB promoter and related to cis-acting elements involved in plasmid partitioning was required for promoter derepression by VirB. Alterations to one heptameric motif within this DNA sequence attenuated VirB binding and derepression of icsB transcription.

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DNase I footprinting, DNA bending and in vitro transcription analyses of ClcR and CatR interactions with the clcABD promoter: evidence of a conserved transcriptional activation mechanism

Molecular Microbiology ◽

10.1046/j.1365-2958.1997.4041763.x ◽

1997 ◽

Vol 24 (5) ◽

pp. 965-976 ◽

Cited By ~ 19

Author(s):

Sally M. McFall ◽

Thomas J. Klem ◽

Nobuyuki Fujita ◽

Akira Ishihama ◽

A. M. Chakrabarty

Keyword(s):

Transcriptional Activation ◽

Dna Bending ◽

In Vitro Transcription ◽

Dnase I ◽

Activation Mechanism ◽

Dnase I Footprinting

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Specifically Deoxyribozyme of the PTPRO Gene as a Potential Gene Therapy Means for Human Hepatocellular Carcinoma

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.781-784.1203 ◽

2013 ◽

Vol 781-784 ◽

pp. 1203-1208

Author(s):

Chun Nian Su ◽

Min Yu

Keyword(s):

Tyrosine Phosphatase ◽

In Vitro Transcription ◽

Tumor Suppressor Function ◽

Rt Pcr ◽

Recombinant Plasmids ◽

Design And Synthesis ◽

Cleavage Activity ◽

Reverse Transcription Pcr ◽

Catalytic Cleavage

Protein tyrosine phosphatase receptor-type O (PTPRO) has been described in several forms of cancer as a new member of the PTP family. The tumor suppressor function of PTPRO was evaluated by design and synthesis the 10-23 deoxyribozyme (DRz), thio-modified DRz (DRz-s) and antisense oligonucleotide (asON) of the PTPRO genomic mRNA to detect the catalytic cleavage activity. Firstly, the cDNA fragment of PTPRO gene was amplified from total cellular RNA of the HepG2.2.15 cells by reverse transcription PCR (RT-PCR). Subsequently, the fragments were cloned to pcDNA3.1(+) plasmids and generated a recombinant plasmids, then sifted the positive recombinant plasmids out to amplify. The expression vector of PTPRO mRNA was obtained in vitro transcription by using T7 RNA polymerase. The results of transfection indicated that when PTPRO mRNA gamyed with deoxyribozyme which activity enhanced, so DRz-s were detected with more intensive specific catalytic cleavage activity than DRz by cells transfecting. And the asON wasn't detected with the property.

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