Biomedical subjects

Rob D Chapman

Publications and source records attributed to Rob D Chapman.

4 recordsLinked to original sources

PubMed2005

Stringent doxycycline-dependent control of gene activities using an episomal one-vector system.

Conditional expression systems are of pivotal importance for the dissection of complex biological phenomena. Here, we describe a novel EBV-derived episomally replicating plasmid (pRTS-1) that carries all the elements for conditional expression of a gene of interest via Tet regulation. The vector is characterized by (i) low background activity, (ii) high inducibility in the presence of doxycycline (Dox) and (iii) graded response to increasing concentrations of the inducer. The chicken beta actin promoter and an element of the murine immunoglobin heavy chain intron enhancer drive constitutive expression of a bicistronic expression cassette that encodes the highly Dox-sensitive reverse tetracycline controlled transactivator rtTA2(S)-M2 and a Tet repressor-KRAB fusion protein (tTS(KRAB)) (silencer) placed downstream of an internal ribosomal entry site. The gene of interest is expressed from the bidirectional promoter P(tet)bi-1 that allows simultaneous expression of two genes, of which one may be used as surrogate marker for the expression of the gene of interest. Tight down regulation is achieved through binding of the silencer tTS(KRAB) to P(tet)bi-1 in the absence of Dox. Addition of Dox releases repression and via binding of rtTA2(S)-M2 activates P(tet)bi-1.

Animals↗

PubMed2005

Role of the mammalian RNA polymerase II C-terminal domain (CTD) nonconsensus repeats in CTD stability and cell proliferation.

The C-terminal domain (CTD) of mammalian RNA polymerase II (Pol II) consists of 52 repeats of the consensus heptapeptide YSPTSPS and links transcription to the processing of pre-mRNA. The length of the CTD and the number of repeats diverging from the consensus sequence have increased through evolution, but their functional importance remains unknown. Here, we show that the deletion of repeats 1 to 3 or 52 leads to cleavage and degradation of the CTD from Pol II in vivo. Including these repeats, however, allowed the construction of stable, synthetic CTDs. To our surprise, polymerases consisting of just consensus repeats could support normal growth and viability of cells. We conclude that all other nonconsensus CTD repeats are dispensable for the transcription and pre-mRNA processing of genes essential for proliferation.

Amino Acid Sequence↗

PubMed2004

The last CTD repeat of the mammalian RNA polymerase II large subunit is important for its stability.

The phosphorylation of the RNA polymerase II (Pol II) C-terminal domain (CTD) has been shown to affect the initiation, and transition to elongation of the Pol II complex. The differential phosphorylation of serines within this domain coincides with the recruitment of factors important for pre-mRNA processing and transcriptional elongation. A role for tyrosine and threonine phosphorylation has yet to be described. The discovery of kinases that express a preference for specific residues within this sequence suggests a mechanism for the controlled recruitment and displacement of CTD-interacting partners during the transcription cycle. The last CTD repeat (CTD52) contains unique interaction sites for the only known CTD tyrosine kinases, Abl1/c-Abl and Abl2/Arg, and the serine/threonine kinase casein kinase II (CKII). Here, we show that removal or severe disruption of the last CTD repeat, but not point mutation of its CKII sites, results in its proteolytic degradation to the Pol IIb form in vivo, but does not appear to affect the specific transcription of genes. These results suggest a possible mechanism of transcription control through the proteolytic removal of the Pol II CTD.

Amino Acid Sequence↗

PubMedDate not supplied

Targeted gene expression using a 1.1 kilobase promoter fragment of the tumour-associated antigen EpCAM.

The epithelial cell adhesion molecule EpCAM is over- or de novo expressed during carcinogenesis. EpCAM expression correlates with increased proliferation and dedifferentiation. Recently, we reported the cloning of a 1.1 kilobase fragment upstream of the epcam gene and demonstrated its specific transcriptional activity. Here, we analyzed the potential of this fragment for targeted gene expression. The fragment was used to regulate the expression of the green fluorescent protein (GFP) and HSV-1 thymidine kinase (HSV-TK), as a model therapeutic gene. Transfection of the pEpProm-control or pEpProm-GFP plasmids resulted in the expression of functional GFP and HSV-TK proteins specifically in EpCAM-positive cells. Expression levels of both proteins correlated with the amount of EpCAM. Additionally, the targeted expression of HSV1-TK transferred a marked sensitivity to ganciclovir treatment in EpCAM-positive HEK293-EBNA1 and SkBr3 carcinoma cells. The EpCAM promoter fragment is, thus, a novel tool to allow for the transcription of therapeutic genes, specifically, in EpCAM-positive carcinomas.

Antigens, Neoplasm↗