Search PubMed⌕ Search

Biomedical subjects

G Hobom

Publications and source records attributed to G Hobom.

At least 37 records · Page 2Linked to original sources
PubMed1996

Structural deviations in a bovine low expression lysozyme-encoding gene active in tissues other than stomach.

Lysozyme-encoding genes (Lys) constitute a gene-family in ruminants. While several of these genes are highly expressed in stomach (sLys), few other copies are weakly expressed in other tissues, notably in polymorphnuclear granulocytes and macrophages (mLys). Searching an understanding for these grossly different levels of expression, we isolated the bovine variant of the gene being expressed in granulocytes and characterized it by sequencing, together with its promoter. Spanning about 9 kb of genomic DNA, the gene is found to be segmented into four exons, in common with all other Lys, as known from vertebrates. Sequence homologies between all bovine sLys-variants exceeds 70% over much of the entire coding sequence and promoter region. This indicates (i) that bovine lysozymes expressed either in stomach or granulocyte originate from a common ancestral gene and (ii) also excludes the possibility that the observed weak expression of the mLys gene is due to major structural rearrangements within the promoter segment. However, primer extension analysis based on RNA isolated from kidney locates the transcription startpoint (tsp of that gene) 44 nt further upstream than observed in both, bovine stomach lysozyme RNA or any of the homologous genes in mice and man. The observed weak expression of this bovine mLys gene appears to be a consequence of both the presence of an extra ATG codon in the extended 5'-UTR, and a severe down mutation of the ancestral TATA-box which is only partially compensated for by the presence of another mutation further upstream resulting in a weak substitute promoter sequence.

Animals↗
PubMed1996

Promoter elements in the influenza vRNA terminal structure.

The role of the partially double-stranded influenza vRNA terminal structure and its constitutive elements as a promoter signal was studied in vivo in a series of nucleotide substitution and insertion derivatives. A series of single and complementary double exchanges restoring intrastrand base pairing shows that a distal promoter element consists of a six-base pair double-stranded RNA rod in long-range complementary interaction. Within the distal element, all base pair positions are freely exchangeable, and hence no nucleotide-specific recognition could be identified. The proximal promoter element consists of nine partially complementary nucleotides at the vRNA 5' and 3' end. The nine plus six base pair panhandle rod of protein-free vRNA is interrupted by a central third element, a single unpaired nucleotide: adenosine 10 or various substitute residues, which appears to cause a bulged conformation in the overall structure. Mutagenization studies in the promoter proximal element indicate that, upon binding to polymerase, nucleotides at positions 2 and 3 interact with positions 9 and 8 within each branch (5' or 3') in short-range base pairing. In this conformation, the intermediate positions 4-7 are exposed as a single-stranded tetra-loop, which includes invariant guanosine residue 5 in the top conformational position of the 5' segment loop. Altogether, the three base paired segments in angular conjunction to each other adopt a conformation that is described in a "corkscrew model" for an activated stage of vRNA/polymerase interaction.

Adenosine↗
PubMed1995

Brugia spp. and Litomosoides carinii: identification of a covalently cross-linked microfilarial sheath matrix protein (shp2).

A microfilarial sheath protein gene (shp2) coding for the major constituent of the insoluble, cross-linked sheath remnant (SR) from Brugia malayi, Brugia pahangi and Litomosoides carinii has been cloned and sequenced, based on peptide partial amino-acid sequences. All three closely related single-copy shp2 genes in the two genera carry a single intron in identical position; shp2 mRNAs are post-transcriptionally modified by both cis-splicing and trans-splicing. In accordance with their extracellular destinations the encoded proteins include signal peptide sequences; molecular masses of approx. 23 kDa are hence predicted for the mature secreted polypeptides. In their structures sheath matrix proteins shp2 may be regarded as extreme cases of a modular constitution, since these proteins largely consist of two different segments of multiple sequence repetitions, PAA and QYPQAP (or QYPQ), separated by elements of unique sequence. Extreme insolubility and cross-linking are likely to originate from these repetitive sequences within shp2, and to constitute the basic properties of a microfilarial matrix largely consisting of an shp2 network.

Amino Acid Sequence↗
PubMed1995

Early and late pre-mRNA processing of budgerigar fledgling disease virus 1: identification of viral RNA 5' and 3' ends and internal splice junctions.

Budgerigar fledgling disease virus 1 (BFDV-1) is the first avian polyomavirus to be identified, and it possesses uncommon structural and biological properties. Here we present an analysis of the processed viral RNAs in infected chicken embryo fibroblast cells. Two early and 18 late BFDV-1 mRNAs were defined according to their 5' ends and internal splice patterns. In the early region of the genome an incomplete splice reaction covering 195 nt is responsible for creating two mRNAs that could encode small t and large T antigens, which would be initiated from a hypothetical early promoter, PE. The late mRNA 5' ends define two putative promoter regions (PL1 and PL2), 111 nt apart in the BFDV-1 genome non-coding region. The overall splicing pattern of the late mRNAs is further complicated by an alternative splice reaction of intron 2 (deletion of either 64 nt in intron 2a or of 256 nt in intron 2b) and a splice removing intron 3 (870 nt), resulting in deletion of most of the VP2-VP3 coding region. The positions of the late mRNA 5' ends and the splicing pattern indicate the existence of two open reading frames, putatively encoding two pairs of agnoproteins, in the 5' region of several late mRNAs. These mRNAs appear to be bicistronic and to encode one of the agnoproteins together with one of the viral coat proteins.

Animals↗
PubMed1995

Mutational analysis of influenza virus promoter elements in vivo.

RNA polymerase I transcription in vivo in transiently DNA-transfected cells has been used to express influenza virus vRNA molecules coding for chloramphenicol acetyltransferase (CAT) in an antisense orientation. Influenza virus superinfection provided viral RNA polymerase and other proteins required for transcriptional conversion of minus-strand vRNA into plus-strand viral mRNA molecules expressing CAT activity. This system has been used for analysis of the vRNA sequences which cooperatively constitute the vRNA promoter structure via nucleotide exchanges as well as deletions and insertions of both terminal segments. Several mutants caused greatly enhanced expression over wild-type levels, which was transmitted during serial passage of progeny virus. The data obtained for the mutations in various promoter elements support a model implicating double-stranded vRNA promoter structures in binding of viral polymerase, and in consecutive steps during initiation of RNA synthesis.

3T3 Cells↗
PubMed1995

OmpA fusion proteins for presentation of foreign antigens on the bacterial outer membrane.

The ompA genes of Escherichia coli and Shigella dysenteriae have been used to construct a group of enterobacterial surface expression vectors for foreign genes. Linker oligonucleotides were inserted into the sequence corresponding to the third or fourth outer domain to allow in-frame sandwich fusion of foreign genes or epitopes into ompA. Influenza haemagglutinin was inserted without its leader peptide and anchor sequences and shown to be transferred as an ompA fusion protein to the bacterial surface in large amounts. The stability of this system depends on the stem structure (i.e. the bottom part) of the haemagglutinin unit which apparently initiates the folding process that extends into the ompA segment. This fusion construct can be used as a vector system and has been used to transfer to the bacterial surface several other proteins inserted into it, including beta-galactosidase, foot-and-mouth disease virus (FMDV) and malaria antigens. All are exported from the cytoplasm across both the inner and outer membranes to become exposed on the bacterial surface. Very hydrophobic segments or inserts with distinct secondary structures, such as the capsid protein, VP1 of FMDV, will, however, block this process.

Animals↗
PubMed1994

A single point mutation results in A allele-specific exon skipping in the bovine alpha s1-casein mRNA.

Bovine alpha s1-casein (alpha s1-CN) allele A is found in low allelic frequencies among different cattle breeds and is known to be characterized by the deletion of amino-acid residues 14 to 26 of the mature protein (as defined via the most common allele B), and a corresponding deletion of 39 bp from its cDNA. Based upon the genomic sequence of bovine alpha s1-CN [Koczan et al., Nucleic Acids Res. 19 (1991) 5591-5596], this allelic deviation can be interpreted as an absence of exon 4 from the A allele mRNA and protein product. We demonstrate that this allelic aberration is not caused by a genomic deletion across the exon-4 DNA, but is correlated with a single point mutation at position +6 in the splice donor sequence distal of exon 4, which results in upstream exon skipping during the serial splice reactions of the A allele alpha s1-CN pre-mRNA. The A-allele-specific mutation at position +6 is able to interrupt the perfect complementarity of the intron-4 splice donor signal (positions one to eight) with U1-snRNA, which may then no longer be able to compensate for a rather weak exon-4 upstream splice acceptor sequence in facilitating the initial binding of U2 auxiliary factor/65-kDa (U2AF65) to that polypyrimidine tract. This interpretation of the exon skipping mechanism in alpha s1-CN allele A is in agreement with similar results obtained [Hoffmann and Grabowski, Genes Dev. 6 (1992) 2554-2568] in an analysis of the rat preprotachykinin-encoding gene and in vitro experiments.

Alleles↗
PubMed1994

Structure of the bovine lactoferrin-encoding gene and its promoter.

Lactoferrin (Lf), a ferric ion (Fe3+)-binding glycoprotein, is found most notably in milk, probably to mediate protection against microbial infection of the mammary gland. Based on an initial isolation and sequencing of a complete cDNA of the bovine Lf gene (bLf), the complete gene was obtained from genomic libraries on five overlapping phage lambda EMBL3 clones. A detailed restriction map and the complete exon/intron structure of the gene are presented, together with 1 kb of sequence data of the promoter upstream from the proximal exon. The coding sequence is dispersed over 17 exons spanning 34.5 kb of genomic DNA. While the exons are of similar size, as in other members of the transferrin gene family (Tf), some of the intron sizes are very different. Evolutionary conservation of both exon sizes and their contribution to the various domains of the protein molecule add to the evidence that Lf originated via an internal sequence duplication. The promoter sequence lacks some of the sequence motifs for transcriptional enhancers found in the promoters of human and mouse Lf, suggesting a potential reason for the relatively weak expression of bLf.

Animals↗
PubMed1994

Ribozyme mediated destruction of influenza A virus in vitro and in vivo.

Short catalytic RNAs with inherent, specific endoribonuclease activity, called ribozymes, have recently been shown to exist in nature. According to the structural models artificial ribozymes have been designed that can potentially hydrolyse any chosen target RNA sequence in trans at a specific site. We have constructed and characterized in vitro hammerhead and hairpin ribozymes designed to cleave viral RNA segment 5 of influenza A virus. Both ribozymes were functional under optimal in vitro conditions, but quantitative measurements indicate that the hammerhead ribozyme is considerably more efficient at this target site than the hairpin ribozyme. Mg2+ dependent hammerhead ribozyme-mediated cleavage reactions were enhanced at higher temperature and in presence of spermidine, but catalytic activities were retained also in cellular extract S-100 or nuclear extracts at physiological temperatures. Recombinant plasmids derived from transfection vector pSV2-neo were engineered to allow the expression of specific ribozymes under the control of SV40 early promoter or SV40 early+ late promoters. These plasmids were introduced by transfection into COS cells, and their expression and enzymatic activities were analyzed in stable cell lines after selection of neomycin-resistance. Several permanent ribozyme-expressing clones were established and characterized: ribozyme coding DNA sequences and synthesis of ribozyme RNA molecules in the transfected cells were determined and monitored by polymerase chain reactions. It was found that the highest levels (up to 70-80%) of resistance to influenza A virus strain X-31 super-infection was observed in COS cells transfected with plasmids containing SV40 early or SV40 early+late promoters coinciding with relatively high and constitutive rates of ribozyme expression. These results suggest the feasibility of developing ribozymes designed against influenza virus to achieve therapeutic value.

Animals↗
PubMed1994

RNA polymerase I-mediated expression of influenza viral RNA molecules.

RNA polymerase I transcription has been used for expression of influenza vRNA molecules, with influenza hemagglutinin or other cDNAs precisely inserted between mouse rDNA promoter and terminator sequences. In in vitro studies generation of HA vRNA transcripts in high rates and correct formation of their 5' ends as well as their 3' ends has been achieved for such hybrid DNA templates. For in vivo expression studies, the HA coding region was replaced by chloramphenicol acetyltransferase (CAT), also in vRNA antisense orientation, with both influenza terminal sequences beyond start and stop codons being retained on the resulting transcript. Following transfection with precisely constructed hybrid DNA templates and depending on infection with influenza virus, CAT activity could be demonstrated. Templates resulting in 3' extended vRNA molecules did not give this result. vRNA-CAT molecules were not only recognized by influenza viral RNA polymerase for synthesis of plus strand mRNAs, but also were packaged into progeny virus particles, as shown by CAT activity in infected cells after passaging of virus containing supernatants.

3T3 Cells↗
PubMed1994

Litomosoides carinii microfilarial sheaths: partial amino acid sequences of several major polypeptide constituents.

Isolated sheaths from Litomosoides carinii microfilariae were disintegrated by reduction with dithiothreitol and were 14C-carboxymethylated. Five major sheath proteins thus solubilized were purified by size exclusion chromatography and reversed-phase HPLC (rpHPLC). Proteolytic fragments of complete sheaths and of the single sheath proteins were isolated by rpHPLC and were N-terminally sequenced. A library of 27 partial sheath polypeptide sequences was thus established, 21 of which could be assigned to three L. carinii sheath structural genes (shp1,2, and 3/3a) isolated on the basis of this and of previous amino acid sequence information. The remaining peptides document the presence of at least one additional major sheath constituent.

Amino Acid Sequence↗
PubMed1994

OmpA-FMDV VP1 fusion proteins: production, cell surface exposure and immune responses to the major antigenic domain of foot-and-mouth disease virus.

Exposure at the bacterial outer surface of the major antigenic epitope of the foot-and-mouth disease (FMDV) viral protein VP1 was studied using protein fusion with outer membrane protein A (OmpA) of Shigella dysenteriae for production and transport of the foreign polypeptide to the outer membrane of Escherichia coli. Fusion constructs with VP1 peptide insertions of up to 56 amino acids in the third outer domain of OmpA could be demonstrated on the bacterial surface by indirect immunofluorescence and immunogold labelling. OmpA fusion proteins with large insertions from sequences of the FMDV protein VP1 were shown to elicit virus-specific immune responses in rabbits.

Amino Acid Sequence↗
PubMed1994

Expression and DNA binding of budgerigar fledgling disease virus large T antigen.

Budgerigar fledgling disease virus (BFDV) represents the first non-mammalian member of the polyomavirus genus and possesses uncommon structural and biological properties. Recombinant baculoviruses were constructed to express BFDV small t antigen, large T antigens, as well as a large T deletion mutant Td and beta-galactosidase-Td fusion proteins to high levels in infected insect cells. A recombinant virus containing a genomic copy of the BFDV early region was used for small t antigen expression, and corresponding intron-deleted cDNAs for production of large T antigen derivatives. Recombinant T as well as authentic T antigen proteins from infected chicken embryo fibroblasts were purified using both immunoaffinity and DNA affinity column chromatography. We present evidence that the large T antigen interacts specifically with DNA sequences present in the non-coding region of BFDV; by indirect DNA immunoprecipitation mapping and DNase I footprinting, four regions including 12 DNA-binding sites have been determined that cover most of the BFDV non-coding region. The T antigen binding pattern observed suggests a protein-DNA interaction system considerably different from those of simian virus 40 and other polyomaviruses.

Animals↗
PubMed1994

Host restriction in the productive cycle of avian polyomavirus budgerigar fledgling disease virus type 3 depends on a single amino acid change in the common region of structural proteins VP2/VP3.

The three avian polyomaviruses budgerigar fledgling disease virus types 1 to 3 (BFDV-1 to -3) contain genomes of identical size, 4981 bp. With differences of up to only 15 bp between the three genomes, these viruses show distinct tropism for cultured cells of various avian species: infection of chicken embryo (CE) cells with BFDV-1 and -2 results in virus propagation, whereas BFDV-3 is not replicated; all three viruses replicate, with different efficiencies, in infected Muscovy duck cells. Transfection of CE cells with BFDV-3 DNA results in a single productive cycle. As shown by construction of hybrid virus genomes and site-directed mutagenesis, a single amino acid difference (glycine instead of valine or alanine) within the common region of the minor structural proteins VP2/VP3 is responsible for this type of abortive infection of CE cells. Further experiments indicate a defect in one of the early steps during infection, at or prior to uncoating.

Animals↗
PubMed1994

Histidine residues near the N terminus of staphylococcal alpha-toxin as reporters of regions that are critical for oligomerization and pore formation.

Chemical modification of histidine residues in staphylococcal alpha-toxin leads to loss of functional activity. Site-directed mutants of the toxin in which each of the four histidine residues was replaced by several amino acids were therefore produced. The mutant proteins were purified and characterized. Exchange of H-259 or H-144 was sometimes tolerated without reduction in hemolytic activity. These histidine residues are thus not essential for toxin function. Exchange of H-35 and H-48, however, had marked effects. H-35 mutant toxins bound with high affinity to rabbit erythrocytes but displayed faulty oligomerization and were unable to form pores. H-48 mutant toxins also had severely impaired hemolytic activity due probably to faulty hexamerization. We interpret these results to indicate that the N-terminal domain of alpha-toxin in the region of H-35 and H-48 is involved in protomer-protomer interactions that underlie the hexamerization and pore-forming process.

Animals↗
PubMed1993

RNA polymerase I catalysed transcription of insert viral cDNA.

RNA polymerase I has been used for transcription of influenza hemagglutinin (HA) cDNA precisely linked in the anti-sense configuration to both mouse rDNA promoter and terminator segments. In transcription reactions based on Ehrlich ascites cell nuclear extracts, specific uniform RNA products are synthesized in high rates that are comparable to original rDNA template transcriptions. Primer extension reactions show the 5' ends of these RNA transcripts to be located exactly at position +1, corresponding to the 5' end of negative strand HA viral RNA. RNA 3' ends in a first series of constructs were found extended beyond the accepted location of pre-rRNA 3' ends, in using both hybrid cDNA and original rDNA templates. But upon deletion of six basepairs from the rDNA termination region RNA polymerase I transcription has been adapted to yield correctly terminated influenza viral RNA in vitro. This result has been confirmed in an in vivo experiment via synthesis of an anti-sense viral RNA molecule containing the chloramphenicol acetyltransferase (CAT) gene, which in turn is recognized at its terminal sequence by viral RNA dependent RNA polymerase for plus strand mRNA synthesis and expression of CAT activity.

Animals↗
PubMed1993

The gene coding for the major sheath protein of Litomosoides carinii microfilariae, gp22, is transcribed in oocytes and embryonic cells.

The transcription and translation of the gene encoding gp22, a major constituent of the microfilarial sheath of the filarial parasite Litomosoides carinii were studied by in situ hybridisation and immunohistology. Transcription of the gp22 gene is confined to oocytes and embryos in the reproductive organs of adult female worms. It starts in oocytes in the rhachis zone, is maximal in multicellular embryos and decreases slowly as the microfilariae develop. Blood microfilariae lack the gp22 transcript. The gp22 gene product is first detectable in parasites recovered on day 32 post infection. Expression of gp22 begins in multicellular embryos in the uteri of mature female worms and can be detected in all further developed intrauterine stages. The gp22 gene product appears to be exported by the embryonic cells and becomes integrated into the sheath where it may contribute to the flexibility of the latter structure.

Amino Acid Sequence↗