Structure and function of the genome of coliphage T5. 1. The physical structure of the chromosome of T5 + .
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Biomedical subjects
Publications and source records attributed to H Bujard.
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The positions of three single-strand interruptions in the DNA of phage T5(+) have been located by electron microscopy. All three interruptions were found in the same strand. Uneven base composition along the molecule is indicated by the preferential melting of certain regions. The data suggest a model according to which (1) the first-step-transfer DNA section is separated by a single-strand interruption from the rest of the phage genome, (2) the phage carries only one such section and therefore transfers the asymmetrical DNA molecule always in the same direction into the host cell, and (3) single-strand interruptions are points of preferred breakage.
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A simple method is described for the isolation of bovine papilloma virus and its deoxyribonucleic acid (DNA). As found with other representatives of this virus group, this DNA preparation contains two components, I and II, as shown by sedimentation and electron microscopic studies. Component I is a fast-sedimenting, twisted, circular DNA molecule and represents usually 70 to 90% of the DNA in the mixture. The direction of the twist in the superstructure is right-handed. Component II originates from I by one or more single-strand breaks and is the "relaxed" circular from of the viral DNA.
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BACKGROUND: Tight control of gene activity has been achieved in cells and transgenic organisms using the Tet regulatory systems. Unregulated basal transcription can, however, be observed whenever integration of target genes driven by promoters responsive to tetracycline controlled transcriptional activators (tTA, rtTA) does not occur at suitable chromosomal sites. Moreover, in viral vectors containing both the tTA coding sequence and the regulated target gene, proximity of the enhancer element driving tTA/rtTA expression to the responsive unit will lead to elevated background levels. Similarly when tTA/rtTA responsive transcription units are in a non-integrated state as e.g., during transient expression, intrinsic residual transcription persists in their 'off' state, which can differ in intensity among different cell types. METHODS: To efficiently repress such background activities we generated tetracycline controlled transcriptional silencers (tTS) that bind promoters responsive for rtTA in absence of the effector doxycycline (Dox). Addition of Dox prevents binding of tTS thus relieving repression, promotes binding of rtTA and thereby switches the promoter from an actively repressed to an activated state. RESULTS: Of several tTS--fusions between a modified Tet repressor and transcriptional silencing domains--tTSKid was found to be most effective in reducing the activity of two target promoters. Ten to 200 fold repression is seen in transient expression whereas in stably transfected HeLa cells the regulatory range of the rtTA system was increased by three orders of magnitude. CONCLUSIONS: The new system appears particularly suited for the transfer of toxic genes into appropriate chromosomal sites as well as for tight regulation of genes carried by viral or episomal vectors.
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