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Biomedical subjects

R Sprengel

Publications and source records attributed to R Sprengel.

84 records · Page 5Linked to original sources
PubMed1987

Homologous recombination between hepadnaviral genomes following in vivo DNA transfection: implications for studies of viral infectivity.

The cloned DNA of duck hepatitis B virus (DHBV) can initiate a productive infection of susceptible ducklings when presented by direct intrahepatic injection. We have examined the effects of the structure of the incoming DNA upon the outcome of this in vivo transfection. Plasmid-linked DHBV genomes of greater than unit length regularly give rise to infectious virus irrespective of the location of the plasmid insert on the transcriptional map. When mutant DHBV genomes are coinjected with subgenomic viral DNA fragments spanning the mutation, wild-type recombinants arise. These results indicate that, as in transfection of cultured cells, in vivo transfection of hepatocytes is regularly followed by homologous recombination involving incoming DNA molecules. These frequent recombination events will complicate efforts to use in vivo transfection for certain genetic analyses of hepadnaviruses.

DNA, Viral↗
PubMed1987

Human hepatic triglyceride lipase: cDNA cloning, amino acid sequence and expression in a cultured cell line.

By immunoscreening of a human cDNA expression library and hybridization of colonies, four partially overlapping cDNA clones of human hepatic triglyceride lipase (HTGL) mRNA were isolated. The clones included the complete coding sequence, the 3'- and at least part of the 5'-untranslated region. The length of the composite HTGL cDNA segment (1.7 kb) was consistent with the size of the mRNA identified in an established human hepatoma cell line. DNA-sequence analysis of cDNAs of partially unspliced mRNAs, and of cloned genomic DNA indicated that the HTGL coding sequence comprises at least six exons. As predicted from the cDNA, the unprocessed HTGL protein has a molecular weight of 56, three potential glycosylation sites, and a signal peptide of 23 amino acids. Sequence comparison with cDNA of other lipases, including rat hepatic lipase, revealed 30%-75% protein-sequence homology. The data establish that HTGL is a secretory protein produced in the hepatocyte, and that its synthesis can be continued in permanent cell lines of hepatoma origin. Our studies also showed that HTGL is another member of a lipase gene family which has interfacial binding sites and possibly other functional domains in common.

Amino Acid Sequence↗
PubMed1987

Expression of the precore region of an avian hepatitis B virus is not required for viral replication.

The core-antigen-coding region of all hepadnaviruses is preceded by a short, in-phase open reading frame termed precore whose expression can give rise to core-antigen-related polypeptides. To explore the functional significance of precore expression in vivo, we introduced a frameshift mutation into this region of the duck hepatitis B virus (DHBV) genome and examined the phenotype of this mutant DNA by intrahepatic inoculation into newborn ducklings. Animals receiving mutant DNA developed DHBV infection, as judged by the presence in hepatocytes of characteristic viral replicative intermediates; molecular cloning and DNA sequencing confirmed that the original mutation was present in the progeny genomes. Infection could be efficiently transmitted to susceptible ducklings by percutaneous inoculation with serum from mutant-infected animals, indicating that infectious progeny virus was generated. These findings indicate that expression of the precore region of DHBV is not essential for genomic replication, core particle morphogenesis, or intrahepatic viral spread.

Animals↗
PubMed1987

Replication strategy of human hepatitis B virus.

To study the replication strategy of the human hepatitis B virus, the 5' end of the RNA pregenome and the initiation sites of DNA plus and minus strands have been mapped. The RNA pregenome was found to be terminally redundant by 120 nucleotides; it is initiated within the pre-C region and may also function as mRNA for synthesis of the major core protein and the hepatitis B virus reverse transcriptase. The hepatitis B virus DNA minus strand is initiated within the direct repeat sequence DR1, it contains a terminal redundancy of up to eight nucleotides, and its synthesis does not require any template switch. The DNA plus strand is primed by a short oligoribonucleotide probably derived from the 5' end of the RNA pregenome, and its synthesis is initiated close to the direct repeat sequence DR2. For its elongation to pass the discontinuity in the DNA minus strand an intramolecular template switch occurs using the terminal redundancy of this template. Thus, the route of reverse transcription and DNA replication of hepatitis B viruses is fundamentally different from that of retroviruses.

Animals↗
PubMed1985

Translationally coupled initiation of protein synthesis in Bacillus subtilis.

The neomycin phosphotransferase gene (neo) from Transposon Tn5 is active in Gram-negative bacteria but silent in B. subtilis since it lacks an appropriate ribosome binding site for Gram-positive bacteria. Neo translation could be reactivated by coupling its initiation to the translational termination of the highly expressed beta-lactamase gene (penP) from B. licheniformis. This initiation occurred at the authentic neo start codon. Its efficiency was independent of the nucleotide sequence 5 to the neo gene, but strongly affected by the distance between the termination and initiation codon. It was the highest if both codons overlapped in the sequence ATGA. In B. licheniformis, a translationally coupled neo gene was inducible expressed as the penP gene demonstrating the potential of the technique to monitor the activity of expression units for which no direct assays exists.

Bacillus subtilis↗
PubMed1985

Comparative sequence analysis of duck and human hepatitis B virus genomes.

We have cloned and sequenced an infectious, functionally active genome of a duck hepatitis B virus (DHBV). It is 3,021 base pairs (bp) in length and shows little DNA sequence homology to the genome of human hepatitis B virus (HBV). However, the amino acid sequences of predicted viral gene products are similar between DHBV and HBV, and the genome organization present in DHBV reflects that of HBV. As in the mammalian virus the long minus strand of the DHBV genome encodes three long overlapping reading frames designated as P, S, and C. The fourth open reading frame, termed X, is absent in DHBV. A comparison with a sequence of a second DHBV isolate [Mandart et al, Journal of Virology 49:782-792, 1984] revealed a nucleotide sequence variation of 5.6% and confirmed the presented overall gene organization of DHBV.

Animals↗
PubMed1984

Protein fusions with the kanamycin resistance gene from transposon Tn5.

The gene for the neomycin phosphotransferase II (NPT II) from transposon Tn5 was fused at the amino or carboxy terminus to foreign DNA sequences coding for 3-300 amino acids and the properties of the fused proteins were investigated. All amino-terminal fusions examined conferred kanamycin resistance to their host cell, but profound differences in their enzymatic activity and stability were detected. Short additions to the amino terminus of the NPT II resulted in highly enzymatically active fusion proteins whereas long amino-terminal fusions often had to be proteolytically degraded to release active proteins. Fusions at the carboxy-terminal end of the NPT II protein did not always induce kanamycin resistance and their enzymatic activity depended more stringently on the nature of the junction sequence.

Base Sequence↗
PubMed1984

A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell extracts.

A general method is described for the detection and quantification of low amounts of neomycin phosphotransferase in crude cell extracts. The assay is based on the electrophoretic separation of the enzyme from other interfering proteins and detection of its enzymatic activity by in situ phosphorylation of the antibiotic kanamycin. Both kanamycin and [gamma-32P]ATP acting as substrates are embedded in an agarose gel placed on the polyacrylamide gel containing the separated proteins. After the enzymatic reaction, the phosphorylated kanamycin is transferred to P81 phosphocellulose ion exchange paper and the radiolabeled kanamycin is visualised by autoradiography. With this method 1 ng of active enzyme can easily be detected. Both prokaryotic and eukaryotic cell extracts can be examined, and changes in the size of enzymatically active proteins can be determined.

Animals↗
PubMed1984

Cloned duck hepatitis B virus DNA is infectious in Pekin ducks.

Approximately 10% of German-bred Pekin ducks were found to be chronically infected with duck hepatitis B virus (DHBV). The genomes of three German DHBV isolates analyzed were closely related but showed substantial restriction site polymorphism compared with U.S. isolates. We tested the infectivity of three sequence variants of cloned DHBV DNA by injecting them into the liver of virus-free ducklings. Most of these animals injected with double-stranded closed-circular or plasmid-integrated dimer DHBV DNA developed viremia, demonstrating the infectivity of all three cloned DHBV DNA variants. The cloned viruses produced were indistinguishable from those from naturally infected animals, implying that our experimental approach can be used to perform a functional analysis of the DHBV genome.

Animals↗
PubMed1981

Penicillinase from Bacillus licheniformis: nucleotide sequence of the gene and implications for the biosynthesis of a secretory protein in a Gram-positive bacterium.

The gene for the penicillinase from B. licheniformis has been cloned in a functional stat on a 1.5 kb DNA fragment and its nucleotide sequence has been determined. A sequence of 307 amino acid residues is infered for the penicillinase precursor. Of these 34 amino acids precede the sequence of the secreted form of the enzyme. This peptide extension shows the features of a signal for secretion and also provides the hydrophobic anchor for the membrane-bound form of the enzyme.

Bacillus↗