In Vitro Recombination and Inverted Terminal Repeat Binding Activities of theMcmar1Transposase

Biochemistry ◽  
2010 ◽  
Vol 49 (17) ◽  
pp. 3534-3544 ◽  
Author(s):  
Sylvaine Renault ◽  
Marie-Véronique Demattéi ◽  
Hichem Lahouassa ◽  
Yves Bigot ◽  
Corinne Augé-Gouillou
Keyword(s):  
Terminal Repeat ◽  
Inverted Terminal Repeat ◽  
In Vitro Recombination

scholarly journals Random-priming in vitro recombination: An effective tool for directed evolution

1998 ◽  
Vol 26 (2) ◽  
pp. 681-683 ◽  
Author(s):  
Z. Shao ◽  
H. Zhao ◽  
L. Giver ◽  
F. H. Arnold
Keyword(s):  
Directed Evolution ◽  
In Vitro Recombination ◽  
Random Priming

Binding of a cellular protein to the gibbon ape leukemia virus enhancer

1987 ◽  
Vol 7 (8) ◽  
pp. 2735-2744
Author(s):  
J P Quinn ◽  
N Holbrook ◽  
D Levens
Keyword(s):  
Long Terminal Repeat ◽  
Specific Binding ◽  
Leukemia Virus ◽  
Cellular Protein ◽  
Terminal Repeat ◽  
Enhancer Activity ◽  
Repeated Element ◽  
Gibbon Ape Leukemia Virus

The gibbon ape leukemia virus (GALV) contains enhancer activity within its long terminal repeat. In the GALV Seato strain this activity resides in a 48-base-pair (bp) repeated element. We demonstrate the existence of a cellular protein which binds in this region of the Seato strain. A sensitive method for enriching protein-DNA complexes from crude extracts coupled with exonuclease and DNase footprint analysis revealed the specific binding of this protein to a 21-bp region within each repeated element. A 22-bp oligonucleotide fragment defined solely by the 21-bp footprint binds a protein in vitro and displays enhancer activity in vivo, suggesting that this protein is a major determinant of GALV enhancer activity. The protein is present in three cell lines which are positive for enhancer activity and is not detected in Jurkat cells, which are negative for enhancer activity. Only GALV long-terminal-repeat variants which support high levels of enhancer activity in vivo compete with this protein for specific binding in vitro, suggesting a potential role for the protein in determining enhancer activity. This protein binding is not inhibited by competition with heterologous retroviral enhancers, demonstrating that it is not a ubiquitous retroviral enhancer binding protein.

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.

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