Biomedical subjects

E Dehlin

Publications and source records attributed to E Dehlin.

6 recordsLinked to original sources

PubMed2000

Cap-dependent deadenylation of mRNA.

Poly(A) tail removal is often the initial and rate-limiting step in mRNA decay and is also responsible for translational silencing of maternal mRNAs during oocyte maturation and early development. Here we report that deadenylation in HeLa cell extracts and by a purified mammalian poly(A)-specific exoribonuclease, PARN (previously designated deadenylating nuclease, DAN), is stimulated by the presence of an m(7)-guanosine cap on substrate RNAs. Known cap-binding proteins, such as eIF4E and the nuclear cap-binding complex, are not detectable in the enzyme preparation, and PARN itself binds to m(7)GTP-Sepharose and is eluted specifically with the cap analog m(7)GTP. Xenopus PARN is known to catalyze mRNA deadenylation during oocyte maturation. The enzyme is depleted from oocyte extract with m(7)GTP-Sepharose, can be photocross-linked to the m(7)GpppG cap and deadenylates m(7)GpppG-capped RNAs more efficiently than ApppG-capped RNAs both in vitro and in vivo. These data provide additional evidence that PARN is responsible for deadenylation during oocyte maturation and suggest that interactions between 5' cap and 3' poly(A) tail may integrate translational efficiency with mRNA stability.

Adenosine Monophosphate↗

PubMed1998

The deadenylating nuclease (DAN) is involved in poly(A) tail removal during the meiotic maturation of Xenopus oocytes.

Exonucleolytic degradation of the poly(A) tail is often the first step in the decay of eukaryotic mRNAs and is also used to silence certain maternal mRNAs translationally during oocyte maturation and early embryonic development. We previously described the purification of a poly(A)-specific 3'-exoribonuclease (deadenylating nuclease, DAN) from mammalian tissue. Here, the isolation and functional characterization of cDNA clones encoding human DAN is reported. Recombinant DAN overexpressed in Escherichia coli has properties similar to those of the authentic protein. The amino acid sequence of DAN shows homology to the RNase D family of 3'-exonucleases. DAN appears to be localized in both the nucleus and the cytoplasm. It is not stably associated with polysomes or ribosomal subunits. Xenopus oocytes contain nuclear and cytoplasmic DAN isoforms, both of which are closely related to the human DAN. Anti-DAN antibody microinjected into oocytes inhibits default deadenylation during progesterone-induced maturation. Ectopic expression of human DAN in enucleated oocytes rescues maturation-specific deadenylation, indicating that amphibian and mammalian DANs are functionally equivalent.

Amino Acid Sequence↗

PubMed1997

Bacterial ghosts as multifunctional vaccine particles.

Expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts have been produced from a variety of bacteria including Escherichia coli. Salmonella typhimurium, Salmonella enteritidis, Vibrio cholerae, Klebsiella pneumoniae, Actinobacillus pleuropneumoniae, Haemophilus influenzae, Pasteurella haemolytica, Pasteurella multocida, and Helicobacter pylori. Such ghosts are used as non-living candidate vaccines and represent an alternative to heat or chemically inactivated bacteria. In recombinant ghosts, foreign proteins can be inserted into the inner membrane prior to E-mediated lysis via specific N-, or C-, or N- and C-terminal anchor sequences. The export of proteins into the periplasmic space or the expression of recombinant S-layer proteins vastly extents the capacity of ghosts or recombinant ghosts as carriers of foreign epitopes or proteins. Oral, aerogenic or parenteral applications of (recombinant) ghosts in experimental animals induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of ghosts and recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in the production of ghosts used as vaccines or as carriers of relevant antigens. The inserted target antigens into the inner membrane or into S-layer proteins are not limited in size.

Adjuvants, Immunologic↗

PubMed1996

Repression of beta interferon gene expression in virus-infected cells is correlated with a poly(A) tail elongation.

Expression of beta interferon (IFN-beta) is transiently induced when Namalwa B cells (Burkitt lymphoma cell line) are infected by Sendai virus. In this study, we found that an elongation of the IFN-beta mRNA could be detected in virus-infected cells and that such a modification was not observed when the IFN-beta transcript was induced by a nonviral agent, poly(I-C). Treatment of the cells with a transcriptional inhibitor (actinomycin D or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) resulted in further elongation of the transcript. Characterization of the elongated IFN-beta transcript by primer extension and RNase H treatment showed that the modification was a result of an elongated poly(A) tail of up to 400 nucleotides. We conclude that the poly(A) tail elongation of the IFN-beta transcript is associated with the viral infection. Furthermore, the presence of the elongated IFN-beta transcript correlated with a decrease of IFN-beta protein in the medium and in cell extracts. Sucrose gradient analysis of cytoplasmic extracts showed that IFN-beta transcripts with elongated poly(A) tails were found in the nonpolysomal fractions, whereas the shorter transcripts could be detected in both polysomal and nonpolysomal fractions. A longer form of the IFN-beta mRNA was also found in the nonpolysomal fractions of cells not treated with transcriptional inhibitors. Thus, the observed regulation of IFN-beta mRNA is not entirely dependent on the inhibition of transcription. To our knowledge, this study provides the first example of a poly(A) tail elongation in somatic cells that negatively influences gene expression in vivo.

Base Sequence↗

PubMed1990

The importance of the 5'-region in regulating the stability of sdh mRNA in Bacillus subtilis.

The decay of the polycistronic Bacillus subtilis sdh mRNA was analysed using probes specific for each of the component cistrons, sdhC, sdhA and sdhB. In exponentially growing cells, the entire sdh mRNA seems to decay with an 'all or nothing' mechanism and with a uniform half-life of 2-3 min for all cistrons. In stationary-phase cells, the half-life of the 5'-part had dropped to about 0.6 min whereas that of the 3'-part was about 1.2 min. Decay of sdh mRNA was also measured in exponentially growing cells containing a 'down-mutation' in the ribosomal binding site preceding sdhC which decreases the expression of sdhC by about 90%. The mutation has a moderate effect on expression of the downstream cistron sdhA. In this mutant, the half-life of the 5'-part of sdh mRNA was about 0.5 min (i.e. the same as in stationary phase wild-type cells) and the half-life of the 3'-part about 1.3 min. Also, analysis of the decay of an sdh-cat fusion transcript revealed that the sdh (5') part decayed more rapidly than the cat part and this difference was more pronounced in stationary-phase cells compared to exponentially growing cells. The results of these experiments demonstrate the importance of the 5'-segment of sdh mRNA in controlling the stability of the transcript under different growth conditions.

Bacillus subtilis↗

PubMed1982

The binding of [3H]-5-hydroxytryptamine to homogenates of human brain.

The binding of [3H]-5-hydroxytryptamine ([3H]-5-HT) to homogenates of human brain has been studied. The specific binding is saturable, with a Kd (frontal cortex) of 12 /+- 2 nM, and is inhibited by non-radioactive 5-HT (IC50=26 nM) and D-Lysergic acid diethylamide (IC50=20 nM). Specific, but not non-specific binding of [3H]-5-TH was inhibited by incubation of the homogenates at 50 degrees C. The binding of [3H]-5-HT across the human brain was not uniform, the highest binding being found in the substantia nigra and hippocampus, and the lowest in the thalamus and pons. The Kd of the binding sites towards 5-HT did, however, appear to be similar for the different brain regions.

Animals↗