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Biomedical subjects

H Schaller

Publications and source records attributed to H Schaller.

At least 127 records · Page 7Linked to original sources
PubMed1982

Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5.

The nucleotide sequence of 1200 bp from the unique region of transposon Tn5 containing the neomycin phosphotransferase gene (neo) was determined, and the location of the neo gene was identified by deletion mutants in a translational reading frame of 792 bp. The derived gene product, an aminoglycoside 3'-phosphotransferase (APH) II, consists of 264 amino acid residues and has a calculated Mr of 29053. Its amino acid sequence shows sequence homologies to the APH type I enzyme coded for by transposon Tn903 (Oka et al., 1981).

Amino Acids↗
PubMed1982

Segment-specific mutagenesis: extensive mutagenesis of a lac promoter/operator element.

A method for highly efficient segment-specific mutagenesis is described. The method uses as target for sodium bisulfite mutagenesis the DNA single strands of a DNA restriction fragment that had been separated by cloning into base-complementary regions of a pair of phage fd vectors. After repair synthesis in vitro, the mutagenized DNA fragment is recovered by cloning into a nonmutated plasmid vector and analyzed for sequence and by functional tests. By using this method, the nucleotide sequence of a 109-base pair restriction fragment containing the lac promoter/operator from Escherichia coli was extensively modified. More than 90% of the 235 isolates obtained showed a change in phenotype; all of 22 analyzed for their nucleotide sequence were found to carry multiple C leads to T point mutations in up to 60% of the possible target positions. Nevertheless, few isolates showed major changes in promoter activity relative to the nonmutated promoter element, which indicates a high degree of flexibility in the promoter sequence.

Base Sequence↗
PubMed1982

Export of a protein into the outer membrane of Escherichia coli K12. Stable incorporation of the OmpA protein requires less than 193 amino-terminal amino-acid residues.

The cloned ompA gene encoding the major outer membrane protein OmpA of Escherichia coli has been shortened in vitro by exonuclease digestion from the end corresponding to the CO2H terminus of the protein. Nine derivatives were identified which still possessed substantial parts of the ompA gene and one was constructed which had suffered a small deletion early in the gene. Gene fragments encoding NH2-terminal OmpA sequences of 45, 133, 193, and 227 residues of the 325 amino acids of OmpA were examined in detail at the DNA level and for OmpA protein fragments synthesized. The latter two fragments were incorporated into the outer membrane and all known functions of the OmpA protein were expressed whereas the fragment with 133 OmpA-specific residues was not stably incorporated into this membrane. In all cases where OmpA functions were observed, an OmpA-specific polypeptide of Mr 24 000 was found in cell envelopes, regardless of the size of the residual ompA sequences and of the fused coding sequences in the vector DNA. Pulse-label experiments revealed larger initial translation products, most of which were degraded to the protein of Mr 24000. The 133-residue OmpA fragment was also detected but proved to be entirely unstable. It is argued that the OmpA protein consists of two domains and that the NH2-terminal moiety from residues 1 to about 180 represents the membrane domain of the polypeptide. Therefore, the loss of about 50, possibly less, CO2H-terminal residues from this domain suffices to interfere with stable incorporation into the outer membrane.

Amino Acid Sequence↗
PubMed1982

Structure and function of the hepatitis B virus genome.

A preliminary map of the hepatitis B virus genome has been derived from the nucleotide sequence of cloned human hepatitis virus (HBV) DNA. The genes for two viral antigens were identified and their expression was studied using SV40 vector systems. HBV DNA cloned and amplified in bacteria induces hepatitis in chimpanzees demonstrating that the cloned HBV DNA is functionally intact.

Base Sequence↗
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↗
PubMed1981

Nucleotide sequence and corresponding amino acid sequence of the gene for the major antigen of foot and mouth disease virus.

A segment of 1160 nucleotides of the FMDV genome has been sequenced using three overlapping fragments of cloned cDNA from FMDV strain O1K. This sequence contains the coding sequence for the viral capsid protein VP1 as shown by its homology to known and newly determined amino acid sequences from this man antigenic polypeptide of the FMDV virion. The structural gene for VP1 comprises 639 nucleotides which specify a sequence of 213 amino acids for the VP1 protein. The coding sequence is not flanked by start and stop codons which is consistent with the mode of biosynthesis of VP1 by post-translational processing of a polyprotein precursor.

Amino Acid Sequence↗
PubMed1981

Cloning of cDNA of major antigen of foot and mouth disease virus and expression in E. coli.

Double-stranded DNA copies of the single-stranded genomic RNA of foot and mouth disease virus have been cloned into the Escherichia coli plasmid pBR322. A restriction map of the viral genome was established and aligned with the biochemical map of foot and mouth disease virus. The coding sequence for structural protein VP1, the major antigen of the virus, was identified and inserted into a plasmid vector where the expression of this sequence is under control of the phage lambda PL promoter. In an appropriate host the synthesis of antigenic polypeptide can be demonstrated by radioimmunoassay.

Antigens, Viral↗
PubMed1981

Production of immunologically active surface antigens of hepatitis B virus by Escherichia coli.

Several plasmids have been constructed which direct the synthesis of hepatitis B virus surface antigens in Escherichia coli either as the native polypeptide or fused to other plasmid encoded polypeptides. When injected into rabbits, extracts from bacteria carrying some of these plasmids induced the synthesis of antibodies to the antigens even though the extracts did not give satisfactory positive results in radioimmunoassay for them. Either the NH2-terminal segment or the COOH-terminal segment of the surface antigens alone was sufficient to elicit the immune response, but antibodies against the two segments showed different specificities. The results emphasize the value of an in vivo assay for the presence of antigens in crude cell extracts and illustrate the feasibility of this type of screening with laboratory animals.

Antibody Specificity↗
PubMed1981

Structural analysis of Tn5.

Nucleotide sequences have been determined for the 1.5-kb inverted repeats of Tn5 and for their junctions with the central unique region and with host DNA. The primary findings stemming from this analysis are: 1. Integration of Tn5 is accompanied by the duplication of 9 bp of host DNA. 2. Loss of Tn5 occurs by crossover between short, homologous nucleotide sequences near the junction between Tn5 and host DNA. 3. The IR sequences contain long, open translational reading frames that may code for transposase proteins. 4. The two IR sequences differ by a single-base change. This alteration accounts for the two functional differences observed between IRL and IRR: It shortens the reading frame for the transposase gene in IRL, and it improves the efficiency of a promoter for the nearby Km-resistance gene. 5. The NH2 terminus of the structural gene for the Km-resistance gene maps at the very left border of the unique region, i.e., very close to the end of IRL. These results support the view that the inverted repeats of Tn5 stem from two identical copies of an originally independently moving DNA element. In the transposon, one of these, IRL, seems to have evolved toward a close physical and functional linkage with the antibiotic-resistance gene.

Base Sequence↗
PubMed1980

Conversion of bacteriophage fd into an efficient single-stranded DNA vector system.

Single-stranded DNA vectors were constructed in vitro by insertion of various DNA fragments into the Intergenic Region of the single-stranded DNA phage fd. These inserts introduce into the phage genome unique cleavage sites for restriction nucleases which are suited for sticky joining in cloning experiments. Since these sites are usually located within genes coding for antibiotic resistance, inactivation of a resistance gene by insertion can be used as a marker for the successful cloning of a DNA fragment. Resistance genes also allow to select for recombinant DNA phages and to minimize the loss of DNA inserts which otherwise becomes significant above an insert size of about one kb. Cloning of several DNA fragments is described and strand separation of double-stranded DNA fragments by means of cloning into fd DNA is given as an example for application of single-stranded DNA vectors.

Bacteriophages↗
PubMed1979

Hepatitis B virus genes and their expression in E. coli.

A composite DNA sequence of regions of hepatitis B virus, determined from a series of recombinant plasmids, reveals the genes for the surface antigen and the core antigen of the virus. The sequence of the core antigen shows it to be a DNA binding protein. The core antigen gene is expressed in Escherichia coli and when injected into rabbits the bacterial product induces antibodies which react with core antigen isolated from human sources.

Amino Acids↗
PubMed1979

Nucleotide sequence of the recognition site of the B-specific restriction modification system in E. coli.

Two sB mutations in the genome of bacteriophage fd were located by sequence analysis in the fd sequence at positions 971 and 6341. Base changes at or close to these positions in phage M13 and in phage fl am 124 also correlate with a loss of sensitivity to B restriction. From the sequence homology between the sequences at the two sB sites the recognition signal for the E. coli B restriction/modification enzzyme is predicted to be: 5' TGA---8N---TGCT 3' 3' ACT---8N---ACGA 5'.

Base Sequence↗
PubMed1979

Nucleotide sequence of the origin of replication of the Escherichia coli K-12 chromosome.

The origin of replication, oriC, of the Escherichia coli chromosome was mapped within a DNA segment of 422 base pairs. The nucleotide sequence of this segment was determined. The source of DNA for the sequence analysis was a minichromosome constructed in vivo, consisting exclusively of chromosomal DNA and a minichromosome constructed by cloning in vitro. The nucleotide sequence of the replication origin is characterized by a high degree of repetitiveness due to both inverted and direct repeats. Sequence homologies were found between portions of the replication origins of E. coli and phages lambda and G4. This suggests similarities in some steps in the initiation of replication of the different replicons.

Base Sequence↗
PubMed1978

Transposition of a DNA sequence determining kanamycin resistance into the single-stranded genome of bacteriophage fd.

Derivatives of bacteriophages fd which transduce kanamycin resistance were selected after growth of the phage in an E. coli strain that carried transpoon 5 (Tn5). Different clones of transducing phage and their DNAs were characterized by gel electrophoresis, electron microscopy, and by their ability to multiply in the absence of helper phage. Integration of the intact transposon into the full size phage genome was correlated with an increase in size of the phage particle from 0.95 mu to 1.7 mu, and with the appearance in the phage DNA of the stem loop structure characteristic for single-stranded Tn5 DNA. In non-defective phages the site of insertion was mapped by heteroduplex analysis within the intergenic region of the phage genome. Defective transducing phages were characterized as an insertion of Tn5 into a phage gene, and/or as a partial deletion or duplication of phage and transposon DNA. The size of the transducing phage from different defective clones varied from 0.6 mu to 3.0 mu and was directly proportional to the DNA content. These results demonstrate that filamentous bacteriophage are highly capable to replicate and package very different amounts of foreign DNA.

Coliphages↗
PubMed1978

Structure of the orgin of DNA replication of bacteriophage fd.

An RNA-polymerase-protected DNA fragment of 125 nucleotides from the origin of single-strand to double-strand replication of bacteriophage fd (ori-DNA) was located on the physical map of the phage genome. A stretch of 187 base pairs of DNA including the ori-DNA was sequenced. This DNA segment contains regions with a highly asymmetric pyrimidine/purine distribution next to regions with 2-fold symmetry that form stable hairpin structures in the viral DNA strand.

Base Sequence↗