DNA rearrangements generating artificial promoters

The promoter-cloning plasmid pBRH4 (a derivative of pBR322 with a partially deleted promoter of the tet gene) is shown to contain a sequence which is located near the EcoRI site and can operate as an effective Pribnow box, but is not the remainder of the deletion-inactivated tet promoter of pBR322. If there is a sequence homologous to the ‘-35’ promoter region at the border of the DNA fragment inserted at the EcoRI site, then a compound promoter arises and activates the tet gene. Point mutations in the nonfunctional -35 region of pBRH4 also activate the cryptic Pribnow box. Several compound promoters were obtained through deleting small portions of DNA around the HindIII site of pBR322; the deletions moved various sequences that could operate as Pribnow boxes towards the -35 region of the tet promoter.Показано, що плазміда pBRH4 (похідна pBR322 із частково вилученим промотором гена tet) містить послідовність, здатну функціонувати в якості блока Прібнова, але яка не є залишком частково вилученого промотору гена tet. Якщо в EcoRI-сайт клонується фрагмент ДНК, що містить послідовність, гомологічну "-35"-ій ділянці промотора, виникає штучний промотор, що активує tet-ген. Точкові мутації в нефункціонуючому - 35-му районі pBRH4 також активують блок Прібнова. За допомогою делецій у районі сайту HindIII плазміди pBR322 отриманий ряд складних промоторів; ці делеції змістили різні послідовності, здатні функціонувати в якості блока Прібнова, до - 35-го району tet-промотора. // Русск. версия: Показано, что плазмида pBRH4 (производная pBR322 с частично удалённым промотором гена tet) содержит последовательность, способную функционировать в качестве блока Прибнова, но которая не является остатком частично удаленного промотора гена tet. Если в EcoRI-сайт клонируется фрагмент ДНК, содержащий последовательность, гомологичную "-35"-му участку промотора, возникает искусственный промотор, активирующий tet-ген. Точковые мутации в нефункционирующем -35-м районе pBRH4 также активируют блок Прибнова. С помощью делеций в районе сайта HindIII плазмиды pBR322 получен ряд составных промоторов; эти делеции сместили различные последовательности, способные функционировать в качестве блока Прибнова, к -35-му району tet-промотора. При цитировании документа, используйте ссылку http://essuir.sumdu.edu.ua/handle/123456789/267

An Inducible and Reversible Mouse Genetic Rescue System

Inducible and reversible regulation of gene expression is a powerful approach for uncovering gene function. We have established a general method to efficiently produce reversible and inducible gene knockout and rescue in mice. In this system, which we named iKO, the target gene can be turned on and off at will by treating the mice with doxycycline. This method combines two genetically modified mouse lines: a) a KO line with a tetracycline-dependent transactivator replacing the endogenous target gene, and b) a line with a tetracycline-inducible cDNA of the target gene inserted into a tightly regulated (TIGRE) genomic locus, which provides for low basal expression and high inducibility. Such a locus occurs infrequently in the genome and we have developed a method to easily introduce genes into the TIGRE site of mouse embryonic stem (ES) cells by recombinase-mediated insertion. Both KO and TIGRE lines have been engineered for high-throughput, large-scale and cost-effective production of iKO mice. As a proof of concept, we have created iKO mice in the apolipoprotein E (ApoE) gene, which allows for sensitive and quantitative phenotypic analyses. The results demonstrated reversible switching of ApoE transcription, plasma cholesterol levels, and atherosclerosis progression and regression. The iKO system shows stringent regulation and is a versatile genetic system that can easily incorporate other techniques and adapt to a wide range of applications

Human Immunodeficiency Virus Type 1 (HIV-1) Accessory Protein Vpr Induces Transcription of the HIV-1 and Glucocorticoid-Responsive Promoters by Binding Directly to p300/CBP Coactivators

The accessory Vpr protein of human immunodeficiency virus type 1 (HIV-1) is a promiscuous activator of viral and cellular promoters. We report that Vpr enhances expression of the glucocorticoid receptor-induced mouse mammary tumor virus (MMTV) promoter and of the Tat-induced HIV-1 long terminal repeat promoter by directly binding to p300/CBP coactivators. In contrast, Vpr does not bind to p/CAF or to members of the p160 family of nuclear receptor coactivators, such as steroid receptor coactivator 1a and glucocorticoid receptor (GR)-interacting protein 1. Vpr forms a stable complex with p300 and also interacts with the ligand-bound glucocorticoid receptor in vivo. Mutation analysis showed that the C-terminal part of Vpr binds to the C-terminal portion of p300/CBP within amino acids 2045 to 2191. The same p300 region interacts with the p160 coactivators and with the adenovirus E1A protein. Accordingly, E1A competed for binding to p300 in vitro. Coexpression of E1A or of small fragments of p300 containing the Vpr binding site resulted in inhibition of Vpr's transcriptional effects. The C-terminal part of p300 containing the transactivating region is required for Vpr transactivation, whereas the histone acetyltransferase enzymatic region is dispensable. Vpr mutants that bind p300 but not the GR did not activate expression of the MMTV promoter and had dominant-negative effects. These results indicate that Vpr activates transcription by acting as an adapter linking transcription components and coactivators

Vpr Protein of Human Immunodeficiency Virus Type 1 Binds to 14-3-3 Proteins and Facilitates Complex Formation with Cdc25C: Implications for Cell Cycle Arrest

Vpr and selected mutants were used in a Saccharomyces cerevisiae two-hybrid screen to identify cellular interactors. We found Vpr interacted with 14-3-3 proteins, a family regulating a multitude of proteins in the cell. Vpr mutant R80A, which is inactive in cell cycle arrest, did not interact with 14-3-3. 14-3-3 proteins regulate the G(2)/M transition by inactivating Cdc25C phosphatase via binding to the phosphorylated serine residue at position 216 of Cdc25C. 14-3-3 overexpression in human cells synergized with Vpr in the arrest of cell cycle. Vpr did not arrest efficiently cells not expressing 14-3-3σ. This indicated that a full complement of 14-3-3 proteins is necessary for optimal Vpr function on the cell cycle. Mutational analysis showed that the C-terminal portion of Vpr, known to harbor its cell cycle-arresting activity, bound directly to the C-terminal part of 14-3-3, outside of its phosphopeptide-binding pocket. Vpr expression shifted localization of the mutant Cdc25C S216A to the cytoplasm, indicating that Vpr promotes the association of 14-3-3 and Cdc25C, independently of the presence of serine 216. Immunoprecipitations of cell extracts indicated the presence of triple complexes (Vpr/14-3-3/Cdc25C). These results indicate that Vpr promotes cell cycle arrest at the G(2)/M phase by facilitating association of 14-3-3 and Cdc25C independently of the latter's phosphorylation status