PubMed Health⌕ Search

Biomedical subjects

J Köck

Publications and source records attributed to J Köck.

31 records · Page 2Linked to original sources
PubMed1993

Relevance of cysteine residues for biosynthesis and antigenicity of human hepatitis B virus e protein.

The mature form of the secretory core protein (HBe protein) of human hepatitis B virus contains four cysteines which are located at amino acid positions -7, 48, 61, and 107 relative to the HBc start methionine. In addition, there is a cysteine, Cys-183, located in the C-terminal domain of the HBe precursor, which is cleaved during HBe maturation. Here, the significance of these cysteines for biosynthesis and antigenicity of the HBe protein was examined. The cysteines at positions -7 and 61 were found to be crucial for HBe biosynthesis. As has already been described, if the Cys at position -7 is mutated, disulfide-linked HBe homodimers which have both HBe antigenicity and HBc antigenicity are expressed. Here we show that these dimers are due to Cys-61-Cys-61 disulfide bridges which are formed only if the Cys at position -7 is not present. In the wild-type protein, this dimerization appears to be inhibited by formation of intramolecular disulfide bridges between the Cys at -7 and one of the internal cysteines. Moreover, Cys-61 is important for HBe biosynthesis in general since mutation of this amino acid results in production of HBe proteins which are either only poorly secreted or possess a different antigenicity.

Antibodies, Monoclonal↗
PubMed1993

Analysis of the earliest steps of hepadnavirus replication: genome repair after infectious entry into hepatocytes does not depend on viral polymerase activity.

Hepadnaviruses contain a relaxed circular DNA genome (RC-DNA) with discontinuities in both strands. Upon infectious entry into a host cell, this genome is converted into a covalently closed superhelical form (CCC-DNA), which later serves as the template for transcription. Here we examined whether the viral polymerase activity is required for this repair reaction. Primary hepatocytes prepared from embryonated duck eggs were infected with the duck hepatitis B virus. Conversion of the RC-DNA into the CCC-DNA was then analyzed by a newly developed polymerase chain reaction technique. This method allows the efficient discrimination between the two DNA forms and is sensitive enough to monitor repair of the infecting viral DNA in the absence of replication and amplification. Thus, we were able to monitor this process in the presence of a potent inhibitor of the viral polymerase, the nucleoside analog 2',3'-dideoxyguanosine. The data show that inhibition of the viral polymerase activity has no influence on genome repair, suggesting that this enzymatic function is not required for conversion of the RC-DNA into the CCC-DNA. Consequently, antiviral drugs blocking the polymerase activity cannot prevent the infectious entry of the virus into a host cell.

Animals↗
PubMed1992

A cysteine and a hydrophobic sequence in the noncleaved portion of the pre-C leader peptide determine the biophysical properties of the secretory core protein (HBe protein) of human hepatitis B virus.

The molecular basis of the biophysical and antigenic differences between the cellular core protein (HBc protein) and the secreted core protein (HBe protein) of human hepatitis B virus was examined. The data show that the properties which distinguish the HBe protein from the HBc protein are due mostly to the 10-amino-acid portion of the HBe leader sequence which remains attached to the HBe protein after cleavage. A cysteine located within this region determines the quaternary structure and the antigenicity of the HBe protein. If this cysteine is lacking, the HBe protein, which is predominantly a monomer with only HBe antigenicity, is expressed as a disulfide-linked homodimer showing both HBe and HBc antigenicity. However, dimerization of the HBe protein was found to be neither sufficient nor required for particle formation. In fact, aggregation of the HBe protein was found to be inhibited by the strongly hydrophobic tripeptide Trp-Leu-Trp, which is also located in the noncleaved portion of the signal sequence. If this tripeptide was converted into either Asp-Asn-Asn or Ala-Asp-Leu, the HBe protein assembled into particles, independent of the presence of the cysteine.

Amino Acid Sequence↗
PubMed1991

Class I and class II ribonuclease H activities in Crithidia fasciculata (Protozoa).

The protozoan Crithidia fasciculata contains two different ribonuclease H activities. These enzymes display similar physical and biochemical characteristics to their homologues in higher eukaryotes, for instance calf thymus class I and class II ribonuclease H. Class I ribonuclease H of lower and higher eukaryotes can be activated by Mg2(+)- and Mn2(+)-ions. However, the presence of Mn2(+)-ions is inhibitory for the Mg2(+)-dependent class II ribonuclease H activity of Crithidia fasciculata and calf thymus. The protozoan class I-homologue enzyme appears to be serologically related to the class I ribonuclease H of calf thymus.

Animals↗
PubMed1991

Characterization of the RNA polymerases of Crithidia fasciculata.

We have characterized the crithidial RNA polymerases (RNAPs). The RNAPs from this organism were resolved by chromatography and could be classified into RNAP I, II and III because of their differential sensitivity to class-specific RNA polymerase inhibitors, such as alpha-amanitin (ama) and tagetitoxin. The three RNAP classes were subsequently characterized in nuclear run-ons using class-specific DNA templates and the inhibitory effect of ama on RNA chain elongation. These experiments showed that Crithidia fasciculata contains the normal set of eukaryotic RNAPs. However, RNAP II was found to be relatively resistant to ama, which seems to be a general feature of kinetoplastid parasites. Tagetitoxin, a potent inhibitor of purified crithidial RNAP III, does not inhibit RNAP III in nuclear run-on experiments, suggesting that the inhibitory effect of tagetitoxin depends on the enzyme conformation, i.e. 'naked' enzyme versus that in a transcription complex. Finally, the role of RNAP III in the transcription of the mini-exon genes, which provide the 5' end of each mRNA, is discussed.

Amanitins↗
PubMed1990

Phylogenetic analysis of the RNA polymerases of Trypanosoma brucei, with special reference to class-specific transcription.

We have sequenced the genes encoding to largest subunits of the three classes of DNA-dependent RNA polymerases of Trypanosoma brucei. The nucleotide and deduced amino acid sequences were compared and aligned with the corresponding sequences of other eukaryotes. Phylogenetic relationships were subsequently calculated with a distant matrix, a bootstrapped parsimony and a maximum-likelihood method. These independent calculations resulted in trees with very similar topologies. The analyses show that all the largest subunits of T. brucei are evolutionarily distant members within each of the three RNA polymerase classes. An early separation of the trypanosomal subunits from the eukaryotic lineage might form the fundamental basis for the unusual transcription process of this species. Finally, all dendrograms show a separate ramification for the largest subunit of RNA polymerase I, II and III. RNA polymerase II and/or III form a bifurcation with the archaebacterial lineage, RNA polymerase I, however, arises separately from the eubacterial beta' lineage. This suggests that the three eukaryotic RNA polymerase classes are not simply derived by two gene duplications of an ancestral gene with subsequent differentiation.

Amino Acid Sequence↗
PubMed1989

Trypanosoma brucei contains two RNA polymerase II largest subunit genes with an altered C-terminal domain.

We have identified and cloned four trypanosomal RNA polymerase largest subunit genes. Here, we present the molecular analysis of two genes, Trp4.8 and Trp5.9. The sequence of these genes shows that they are almost identical to each other and indicates that they encode the largest subunit of RNA polymerase II. Both genes contain a C-terminal extension that is clearly distinct from that of other eukaryotic RNA polymerase II genes, because it lacks the common tandemly repeated heptapeptide sequence and is rich in acidic amino acids. It shares many potential phosphorylation sites, however, with the C-terminal extension of other eukaryotic RNA polymerase II large subunits. The presence of two RNA polymerase II loci suggests that a fourth RNA polymerase could be formed. Interestingly, the fourth gene is only found in species exhibiting antigenic variation.

Amanitins↗
PubMed1988

Structure and sequence of the gene for the largest subunit of trypanosomal RNA polymerase III.

As the first step in the analysis of the transcription process in the African trypanosome, Trypanosoma brucei, we have started to characterise the trypanosomal RNA polymerases. We have previously described the gene encoding the largest subunit of RNA polymerase II and found that two almost identical RNA polymerase II genes are encoded within the genome of T. brucei. Here we present the identification, cloning and sequence analysis of the gene encoding the largest subunit of RNA polymerase III. This gene contains a single open reading frame encoding a polypeptide with a Mr of 170 kD. In total, eight encoding a polypeptide with a Mr of 170 kD. In total, eight highly conserved regions with significant homology to those previously reported in other eukaryotic RNA polymerase largest subunits were identified. Some of these domains contain functional sites, which are conserved among all eukaryotic largest subunit genes analysed thus far. Since these domains make up a large part of each polypeptide, independent of the RNA polymerase class, these data strongly support the hypothesis that these domains provide a major part of the transcription machinery of the RNA polymerase complex. The additional domains which are uniquely present in the largest subunit of RNA polymerase I and II, respectively, two large hydrophylic insertions and a C-terminal extension, might be a determining factor in specific transcription of the gene classes.

Amino Acid Sequence↗
PubMed1987

Primary structure of colicin M, an inhibitor of murein biosynthesis.

The DNA sequence of the colicin M activity gene cma was determined. A polypeptide consisting of 271 amino acids was deduced from the nucleotide sequence. The amino acid sequence agreed with the peptide sequences determined from the isolated colicin. The molecular weight of active colicin M was 29,453. The primary translation product was not processed. In the domain required for uptake into cells, colicin M contained the pentapeptide Glu-Thr-Leu-Thr-Val. A similar sequence was found in all colicins which are taken up by a TonB-dependent mechanism and in outer membrane receptor proteins which are constituents of TonB-dependent transport systems. The structure of colicin M in the carboxy-terminal activity domain had no resemblance to the pore-forming colicins or colicins with endonuclease activity. Instead, the activity domain contained a sequence which exhibited homology to the sequence around the serine residue in the active site of penicillin-binding proteins of Escherichia coli. The colicin M activity gene was regulated from an SOS box upstream of the adjacent colicin B activity gene on the natural plasmid pColBM-Cl139.

Amino Acid Sequence↗