Biomedical subjects

L Knoepfel

Publications and source records attributed to L Knoepfel.

6 recordsLinked to original sources

PubMed1996

Two versatile eukaryotic vectors permitting epitope tagging, radiolabelling and nuclear localisation of expressed proteins.

Two versatile eukaryotic expression vectors have been developed which permit the production of an epitope-tagged cDNA insert by transient transfection in mammalian cells or by in vitro transcription-translation. The first vector, pCATCH, can be used to clone cDNA inserts in three different frames via eight unique restriction sites in a multiple cloning site (MCS) located downstream from both the FLAG epitope and the specific heart muscle kinase phosphorylation site, conferring the possibility of in vitro radiolabelling. A specific protease cleavage site enables the removal of the FLAG epitope, simplifying affinity purification of recombinant CATCH proteins. pCATCH possesses stop codons in all three reading frames at the 3' terminal end of the MCS. A derivate of this vector, pCATCH-NLS, was constructed by incorporating an SV40 nuclear localisation signal upstream from the MCS, for directed localisation of the tagged proteins.

Amino Acid Sequence↗

PubMed1996

Cloning and characterization of the murine B-cell specific transcriptional coactivator Bob1.

From a murine B-cell cDNA-library we have cloned a cDNA encoding the murine B-cell specific coactivator mBob1. The protein is the murine homologue to the recently described human coactivator Bob1 (hBob1), also referred to as OBF-1 or OCA-B. We have also characterized the genomic mBob1 clone. Analysis of its intron-exon structure has allowed identification of a C-terminal splice variant. mBob1 is B-cell restricted, and is found in all B-cell lines representing different stages of B-cell differentiation. mBob1 interacts with the octamer transcription factors Oct-1 and Oct-2 and stimulates transcription mediated by these factors.

Amino Acid Sequence↗

PubMed1996

BZLF1 (ZEBRA, Zta) protein of Epstein-Barr virus selected in a yeast one-hybrid system by binding to a consensus site in the IgH intronic enhancer: a role in immunoglobulin expression?

We have used a yeast one hybrid screen to search for factors interacting with a subsegment of the immunoglobulin heavy chain (IgH) intronic enhancer. The 51 bp enhancer segment harbored a so-called E-box and an octamer site, known to bind helix-loop-helix transcription factors and Oct factors, respectively. Mammalian Oct-2A protein was also expressed in yeast, to select for transcription factors possibly cooperating with Oct-2. Six strongly interacting protein clones were selected from a peripheral blood lymphocyte library. These included a B cell-specific co-activator, termed Bob1, that directly binds to Oct-2 (Gstaiger et al., 1995, Nature 373, 360-362). Three further clones represent the helix-loop factors ITF-1 and ITF-2, another one the nucleolar protein nucleophosmin, or B23. Unexpectedly, the sixth clone with strong activity encoded the BZLF1 (= ZLF1, zta, ZEBRA, EB1) protein of Epstein-Barr virus (EBV). BZLF1 is a leucine zipper-related transcription factor and induces the switch from viral latency to lytic growth. We found that BZLF1 also activated transcription in transiently transfected mammalian cells via a consensus binding site located within the IgH intron enhancer. BZLF1 may thus influence immunoglobulin heavy chain expression in EBV-infected B lymphocytes.

Consensus Sequence↗

PubMed1995

A B-cell coactivator of octamer-binding transcription factors.

The octamer motif (ATGCAAAT) paradoxically plays a central role in mediating the activity of both B-cell specific and ubiquitous promoters. It has been widely assumed that the predominantly lymphoid-restricted octamer-binding factor Oct-2 mediates tissue-specific promoter activity, whereas the ubiquitously expressed Oct-1 mediates general promoter activity, but this view has been challenged. Here we use a modified yeast one-hybrid assay to isolate a B-cell factor, Bob1, which associates with either Oct-2 or Oct-1. In transfection experiments, this factor boosts Oct-1-mediated promoter activity and to a lesser extent, that of Oct-2. This coactivation is strictly dependent on the specific interaction with Oct-1 or Oct-2 because deletion of the octamer motif abolishes coactivation. We conclude that Bob1 could represent a new tissue-specific transcriptional coactivator which may convert a ubiquitously expressed transcription factor to a cell-type-specific activator.

Amino Acid Sequence↗

PubMed1994

Copper-dependent metabolism of Cu,Zn-superoxide dismutase in human K562 cells. Lack of specific transcriptional activation and accumulation of a partially inactivated enzyme.

The regulation of Cu,Zn-superoxide dismutase by copper was investigated in human K562 cells. Copper ions caused a dose- and time-dependent increase, up to 3-fold, of the steady-state level of Cu,Zu-superoxide dismutase mRNA. A comparable increase was also observed for actin and ribosomal protein L32 mRNAs, but not for metallothionein mRNA which was augmented more than 50-fold and showed a different induction pattern. The copper-induced mRNAs were actively translated as judged from their enhanced loading on polysomes, the concomitantly increased cellular protein levels and an augmented incorporation of [3H]lysine into acid-precipitable material. Cu,Zn-superoxide dismutase protein followed this general trend, as demonstrated by dose- and time-dependent increases in immunoreactive and enzymically active protein. However, a specific accumulation of Cu,Zn-superoxide dismutase was noticed in cells grown in the presence of copper, that was not detectable for other proteins. Purification of the enzyme demonstrated that Cu,Zn-superoxide dismutase was present as a reconstitutable, copper-deficient protein with high specific activity (kcat./Cu = 0.89 x 10(9) M-1.s-1) in untreated K562 cells and as a fully metallated protein with low specific activity (kcat./Cu = 0.54 x 10(9) M-1.s-1) in copper-treated cells. Pulse-chase experiments using [3H]lysine indicated that turnover rates of Cu,Zn-superoxide dismutase in K562 cells were not affected by growth in copper-enriched medium, whereas turnover of total protein was significantly enhanced as a function of metal supplementation. From these results we conclude that: (i) unlike in yeast [Carrì, Galiazzo, Ciriolo and Rotilio (1991) FEBS Lett. 278, 263-266] Cu,Zn-superoxide dismutase is not specifically regulated by copper at the transcriptional level in human K562 cells, suggesting that this type of regulation has not been conserved during the evolution of higher eukaryotes; (ii) copper ions cause an inactivation of the enzyme in intact K562 cells; and (iii) the metabolic stability of Cu,Zn-superoxide dismutase results in its relative accumulation under conditions that lead to increased protein turnover.

Animals↗

PubMed1994

Role of zinc-coordination and of the glutathione redox couple in the redox susceptibility of human transcription factor Sp1.

We show that thiol-groups confer redox-susceptibility to the zinc-finger transcription factor Sp1 and that this redox-susceptibility is prevented by DNA-binding and depends on zinc-coordination of the protein. Apo-Sp1 contained in metal depleted nuclear extracts of human K562 cells exhibited a markedly increased susceptibility towards oxidizing and alkylating agents, as compared to holo-Sp 1. Moreover, DNA binding of apo-Sp1, but not of the holo-protein, was dramatically decreased in the presence of GSH/GSSG ratios within the physiological range. We compared these results with the redox behaviour of two other transcription factors, OTF-1 and NF1, which was found to be different in several aspects from that of Sp1.

Apoproteins↗