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H Domdey

Publications and source records attributed to H Domdey.

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

Assignment of the human organic anion transporting polypeptide (OATP) gene to chromosome 12p12 by fluorescence in situ hybridization.

BACKGROUND/AIMS: The organic anion transporting polypeptide (OATP) of human liver mediates the basolateral hepatocellular uptake of numerous cholephilic anions and steroidal compounds. The aim of this study was to clone the human OATP gene and to map its chromosomal localization by fluorescence in situ hybridization. METHODS: A polymerase chain reaction-amplified fragment of the human OATP gene was used to isolate a genomic OATP clone from a P1-derived artificial chromosome human genomic library. Human metaphase chromosomes were hybridized with digoxigenin-labeled DNA from the genomic OATP clone and incubated in fluoresceinated antidigoxigenin antibodies for in situ detection of specific hybridization signals. RESULTS: Sequence analysis revealed that the isolated P1-derived artificial chromosome clone contained a large portion of the human OATP gene. Fluorescence in situ hybridization of human chromosomes with the genomic OATP clone resulted in the specific labeling of the OATP gene on the short arm of chromosome 12 at band 12p12. CONCLUSIONS: Mapping of a genomic OATP clone to chromosome 12p12 represents a first step towards the molecular characterization of the human OATP gene. While no liver disease has so far been associated with cytogenetic abnormalities of the short arm of chromosome 12, the genomic OATP sequence provides the basis for studies on gene structure and on the tissue-specific regulation of OATP gene expression.

Anion Transport Proteins↗
PubMed1996

Cloning and characterization of the ALG3 gene of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae alg3-1 mutant is described as defective in the biosynthesis of dolichol-linked oligosaccharides (Huffaker and Robbins, Proc. Natl. Acad. Sci. USA, 80, 7466-7470, 1983). Man5GlcNAc2-PP-Dol accumulates in alg3 cells and Endo H resistant carbohydrates are transferred to protein by the oligosaccharyltransferase complex. In this study, we describe the cloning of the ALG3 locus by complementation of the temperature sensitive growth defect of the alg3 stt3 double mutant. The isolated ALG3 gene complements both the defect in the biosynthesis of lipid-linked oligosaccharides of the alg3-mutant and the under-glycosylation of secretory proteins. The inactivation of the nonessential ALG3 gene results in the accumulation of lipid-linked Man5GlcNac2 and protein-bound carbohydrates which are completely Endo H resistant. The ALG3 locus encodes a potential ER-transmembrane protein of 458 amino acids (53 kDa) with a C-terminal KKXX-retrieval sequence.

Amino Acid Sequence↗
PubMed1996

Pre-mRNA topology is important for 3'-end formation in Saccharomyces cerevisiae and mammals.

Various signal motifs that are required for efficient pre-mRNA 3'-end formation in the yeast Saccharomyces cerevisiae have been reported. None of these known signal sequences appears to be of the same general importance as is the mammalian AAUAAA motif. To establish the importance of yeast pre-mRNA termini in 3'-end formation, the ends of a pre-mRNA transcript synthesized in vitro were ligated before incubation in a yeast whole-cell extract. Such covalently closed circular RNAs were not cleaved at their poly(A) sites. Interestingly, pseudocircular RNAs with complementary 3'- and 5'-terminal sequences allowing the formation of panhandle structures were also resistant to cleavage. However, 3'-end processing was impeded neither by terminal hairpins at either or at both ends nor by RNA oligonucleotides complementary to either or both ends of a linear pre-mRNA. Intriguingly mammalian pseudocircular pre-mRNAs also were not cleaved at their poly(A) sites when incubated in a HeLa cell nuclear extract. These results provide evidence for the general importance of RNA topology in the formation of an active 3'-end processing complex in S. cerevisiae and higher eukaryotes. The possibility of a torus-shaped factor involved in 3'-end formation is discussed.

Alcohol Dehydrogenase↗
PubMed1995

Sequence analysis of a 78.6 kb segment of the left end of Saccharomyces cerevisiae chromosome II.

We report the sequence analysis of a 78,601 bp DNA segment on the left arm of chromosome II of Saccharomyces cerevisiae. This 78.6 kb segment spans the region from the start of a subtelomeric Y' element up to the ILS1 gene. It contains 49 open reading frames (ORFs) with more than 100 amino acids length including 14 internal and five overlapping ORFs. The gene density, excluding the internal ORFs, was calculated as one ORF per 2.2 kb. Eight ORFs (PKC1, TyA, TyB, ATP1, ROX3, RPL17a, PET112 and ILS1) correspond to previously characterized genes. ORF YBL0718 was identified as CDC27; YBL0706 as TEL1. Four other ORFs show strong similarities to already known genes. The gene product of YBL0838 is 60% identical to the ribosomal protein RPL32 from rat, mouse and man. YBL0701 encodes a protein with significant similarity to the initiation factor eIF2 associated p67 glycoprotein from rat. Eight ORFs were disrupted and the resulting yeast strains analysed with respect to their phenotype.

Amino Acid Sequence↗
PubMed1995

PTF1 encodes an essential protein in Saccharomyces cerevisiae, which shows strong homology with a new putative family of PPIases.

Complementation of a temperature sensitive mutant of the yeast Saccharomyces cerevisiae resulted in the isolation of PTF1 (processing/termination factor 1), an essential gene encoding a putative 3'-end processing or transcription termination factor of pre-mRNAs. Ptf1p shows significant homology to a newly discovered family of PPIases. This family is characterized by its insensitivity to immunosuppressive drugs and the lack of homology with cyclophilins and FK-506 binding proteins [Rahfeld et al. (1994) FEBS Lett. 352, 180-184]. Should Ptf1p display PPIase activity, it would be the first characterized, eukaryotic member of this putative family, which is essential for growth.

Amino Acid Isomerases↗
PubMed1995

Splicing of a circular yeast pre-mRNA in vitro.

We have tested the fate of a circularized synthetic pre-mRNA transcript in a whole cell splicing extract of Saccharomyces cerevisiae. Our results demonstrate that this circular precursor RNA is able to induce spliceosome formation in vitro and that the products of the following splicing reaction are the lariat-shaped intron, and a mature circular mRNA. Thus, it would appear that free 5' and/or 3' ends are not obligatory for a splicing reaction to occur, although we find its efficiency to be strongly influenced by the presence or lack of free ends. To our knowledge, this is the first demonstration that a circular pre-mRNA molecule is recognized as a suitable substrate by an eukaryotic mRNA splicing apparatus.

Base Sequence↗
PubMed1995

TH1 cells trigger tumor necrosis factor alpha-mediated hypersensitivity to Pseudomonas aeruginosa after adoptive transfer into SCID mice.

Recent experiments have shown that gamma interferon (IFN-gamma), either administered or induced in vivo, e.g., by certain bacteria, is a key mediator in inducing hypersensitivity to bacterial lipopolysaccharides. The source of endogenous IFN-gamma in this context (natural killer versus TH1 cells) has not been investigated yet. In order to investigate the role of antigen-specific, IFN-gamma-producing TH1 cells in murine Pseudomonas aeruginosa infection, a murine TH1 cell line was propagated in vitro by using recombinant P. aeruginosa outer membrane protein I. Adoptive transfer experiments were performed by intravenous injection of various amounts of TH1 cells into P. aeruginosa-challenged SCID mice. Adoptive transfer of 5 x 10(6) T cells into SCID mice followed by an intraperitoneal challenge with 1.4 x 10(6) CFU of live P. aeruginosa resulted in the rapid death of the animals within 12 h postchallenge, whereas transfer of lower T-cell doses and saline as a control did not cause any detrimental effects. After challenge with 2.8 x 10(6) CFU of P. aeruginosa, similar results were obtained 18 h postchallenge; however, at the end of the 72-h observation period, no significant differences in survival rates were obtained between the groups treated with different amounts of T cells. The rapid death of mice treated with 5 x 10(6) T cells was reflected by 860-fold-elevated levels of tumor necrosis factor alpha (TNF-alpha) present in serum 2 h postchallenge, whereas no significant differences in TNF-alpha serum levels were detectable in mice treated with lower doses of T cells or with saline. Pretreatment of T-cell-reconstituted SCID mice with neutralizing anti-IFN-gamma monoclonal antibodies completely protected mice from bacterial challenge and reduced TNF-alpha levels in serum. We conclude that under the experimental conditions described here, IFN-gamma- and interleukin-2-producing TH1 cells represent an important trigger mechanism inducing TNF-alpha-mediated hypersensitivity to bacterial endotoxin.

Animals↗
PubMed1995

Protection of immunocompromised mice against lethal infection with Pseudomonas aeruginosa by active or passive immunization with recombinant P. aeruginosa outer membrane protein F and outer membrane protein I fusion proteins.

Recombinant outer membrane proteins (Oprs) of Pseudomonas aeruginosa were expressed in Escherichia coli as glutathione S-transferase (GST)-linked fusion proteins. GST-linked Oprs F and I (GST-OprF190-350 [GST linked to OprF spanning amino acids 190 to 350] and GST-OprI21-83, respectively) and recombinant hybrid Oprs (GST-OprF190-342-OprI21-83 and GST-OprI21-83-OprF190-350) were isolated and tested for their efficacy as vaccines in immunodeficient mice. GST-OprF-OprI protected the mice against a 975-fold 50% lethal dose of P. aeruginosa. Expression of GST-unfused OprF-OprI failed in E. coli, although this hybrid protein has been expressed without a fusion part in Saccharomyces cerevisiae and used for immunizing rabbits. The immune rabbit sera protected severe combined deficient (SCID) mice against a 1,000-fold 50% lethal dose of P. aeruginosa. Evidence is provided to show that the most C-terminal part of OprF (i.e., amino acids 332 to 350) carries an important protective epitope. Opr-based hybrid proteins may have implications for a clinical vaccine against P. aeruginosa.

Animals↗
PubMed1994

Complete DNA sequence of yeast chromosome II.

In the framework of the EU genome-sequencing programmes, the complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome II (807 188 bp) has been determined. At present, this is the largest eukaryotic chromosome entirely sequenced. A total of 410 open reading frames (ORFs) were identified, covering 72% of the sequence. Similarity searches revealed that 124 ORFs (30%) correspond to genes of known function, 51 ORFs (12.5%) appear to be homologues of genes whose functions are known, 52 others (12.5%) have homologues the functions of which are not well defined and another 33 of the novel putative genes (8%) exhibit a degree of similarity which is insufficient to confidently assign function. Of the genes on chromosome II, 37-45% are thus of unpredicted function. Among the novel putative genes, we found several that are related to genes that perform differentiated functions in multicellular organisms of are involved in malignancy. In addition to a compact arrangement of potential protein coding sequences, the analysis of this chromosome confirmed general chromosome patterns but also revealed particular novel features of chromosomal organization. Alternating regional variations in average base composition correlate with variations in local gene density along chromosome II, as observed in chromosomes XI and III. We propose that functional ARS elements are preferably located in the AT-rich regions that have a spacing of approximately 110 kb. Similarly, the 13 tRNA genes and the three Ty elements of chromosome II are found in AT-rich regions. In chromosome II, the distribution of coding sequences between the two strands is biased, with a ratio of 1.3:1. An interesting aspect regarding the evolution of the eukaryotic genome is the finding that chromosome II has a high degree of internal genetic redundancy, amounting to 16% of the coding capacity.

Base Composition↗
PubMed1994

Complete DNA sequence of yeast chromosome XI.

The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome XI has been determined. In addition to a compact arrangement of potential protein coding sequences, the 666,448-base-pair sequence has revealed general chromosome patterns; in particular, alternating regional variations in average base composition correlate with variations in local gene density along the chromosome. Significant discrepancies with the previously published genetic map demonstrate the need for using independent physical mapping criteria.

Base Sequence↗
PubMed1994

The beta-tubulin genes of Drosophila auraria are arranged in a cluster.

When the beta 1-, beta 2- and beta 3-tubulin-specific DNAs from Drosophila melanogaster were used as probes to recognize tubulin-specific sequences in the chromosomes of Drosophila auraria, they were found to hybridize to the same polytene band in region 32C of the 2L polytene chromosome. Three overlapping clones were isolated from a lambda EMBL3 genomic library of D. auraria, and they all contain beta-tubulin-specific sequences based on hybridization and partial-sequencing experiments of subcloned fragments. These clones hybridize in situ to the same polytene chromosome band in region 32C and they represent an approximately 35-kb fragment of genomic DNA.

Amino Acid Sequence↗
PubMed1994

Protection of immunosuppressed mice against translocation of Pseudomonas aeruginosa from the gut by oral immunization with recombinant Pseudomonas aeruginosa outer membrane protein I expressing Salmonella dublin.

AroA Salmonella dublin was used as recipient for a plasmid coding for the outer membrane protein I (OprI) of Pseudomonas aeruginosa. Oral immunization of Balb/c mice with recombinant S. dublin induced serum IgG and IgA antibodies against P. aeruginosa. In spleen and Peyer's patches anti-P. aeruginosa IgG- and IgA-secreting cells could be measured by the ELISPOT technique. In an oral challenge of immunosuppressed mice with P. aeruginosa the orally immunized animals had a 58-fold higher LD50 than control animals.

Administration, Oral↗
PubMed1994

Flexibility and interchangeability of polyadenylation signals in Saccharomyces cerevisiae.

Various signal motifs have been reported to be essential for proper mRNA 3'-end formation in the yeast Saccharomyces cerevisiae. However, none of these motifs has been shown to be sufficient to direct 3'-end processing and/or transcription termination. Therefore, several structural motifs have to act in concert for efficient 3'-end formation. In the region upstream of the three polyadenylation sites of the yeast gene for alcohol dehydrogenase I (ADH1), we have identified a hitherto unknown signal sequence contained within the octamer AAAAAAAA. This motif, located 11 nucleotides upstream of the first ADH1 polyadenylation site, is responsible for the utilization of this site in vitro and in vivo, since mutational alteration drastically reduced 3'-end formation at this position. Insertion of 38 ADH1-derived nucleotides encompassing the (A)8 motif into the 3'-end formation-deficient cyc1-512 deletion mutant restored full processing capacity in vitro. Insertion of the octamer alone did not restore 3'-end formation, although mutation of the (A)8 motif in the functional construct had abolished 3'-end processing activity almost completely. This demonstrates that the sequence AAAAAAAA is a necessary, although not sufficient, signal for efficient mRNA 3'-end formation in S. cerevisiae.

Alcohol Dehydrogenase↗
PubMed1994

Outer membrane proteins of Pseudomonas aeruginosa as vaccine candidates.

We tested the ability of recombinant outer membrane proteins of Pseudomonas aeruginosa to serve as a protective vaccine against this gram negative pathogen under two main pathophysiological events leading to P. aeruginosa sepsis. i) systemic infection during immunosuppression, and ii) bacterial translocation. A hybrid vaccine was cloned combining protective epitopes of outer membrane protein F (OprF) and outer membrane protein I (OprI). This vaccine proved to be highly protective against an intraperitoneal challenge with P. aeruginosa in immunosuppressed mice. Oral immunization of mice, with recombinant Salmonella dublin expressing OprI induced s-IgA antibodies in the gut mucosa against OprI and provided protection against translocation of P. aeruginosa in an immunosuppressed mouse model. To test whether OprI is safe for use in humans, recombinant OprI was purified and used for immunization of volunteers. Vaccination was well tolerated and no major side effects were observed. The induction of serum antibodies against OprI was found to be dose-dependent and was observed in total in 65% of the volunteers.

Animals↗
PubMed1992

Rapid sequencing of the Sendai virus 6.8 kb large (L) gene through primer walking with an automated DNA sequencer.

The determination of the complete DNA sequence of the large (L) polymerase gene of Sendai virus strain Fushimi was used to explore the potential and feasibility of primer walking with fluorescent dye-labelled dideoxynucleotide terminators on an automated ABI DNA sequencer. The rapid identification of the complete sequence demonstrated that this approach is a time- and cost-saving alternative to classical sequencing techniques. Analysis of the data revealed that the L gene of Sendai virus strain Fushimi consists of exactly 6800 nucleotides and that the deduced amino acid sequence identifies a single open reading frame encoding a protein of 252.876 kDa. In contrast to Sendai virus strain Enders, the L mRNA of strain Fushimi is monocistronic. The comparison of the deduced amino acid sequences of the L genes of three different Sendai virus strains confirmed the existence of conserved as well as variable regions in the L protein and revealed a high grade of conservation in the carboxyterminal third. Furthermore, functional amino acid sequence motifs, like elements of RNA-dependent RNA polymerases and ATP-binding sites as postulated previously, were identified.

Base Sequence↗
PubMed1992

Antibody production in silkworm cells and silkworm larvae infected with a dual recombinant Bombyx mori nuclear polyhedrosis virus.

We have examined the efficiency of coexpression of two heterologous genes from a recombinant Bombyx mori nuclear polyhedrosis virus for the production of antibodies in silkworm larvae. The cDNAs encoding the light and the heavy chains of a murine immunoglobulin, directed against lipoprotein I of Pseudomonas aeruginosa, were brought under the control of two separate copies of the viral polyhedrin promotor. Infection of silkworm cells with the recombinant baculovirus yielded a maximum of 6.4 micrograms/ml IgG2A in the culture supernatant 72 hours post infection, while 800 micrograms/ml IgG2A was found in the hemolymph of infected fifth instar silkworm larvae seven days after infection with the same construct. The recombinant antibody exhibited a similar antigen specificity and avidity to that of the monoclonal antibody derived from ascites fluid.

Animals↗
PubMed1992

Identification of pre-mRNA polyadenylation sites in Saccharomyces cerevisiae.

In contrast to higher eukaryotes, little is known about the nature of the sequences which direct 3'-end formation of pre-mRNAs in the yeast Saccharomyces cerevisiae. The hexanucleotide AAUAAA, which is highly conserved and crucial in mammals, does not seem to have any functional importance for 3'-end formation in yeast cells. Instead, other elements have been proposed to serve as signal sequences. We performed a detailed investigation of the yeast ACT1, ADH1, CYC1, and YPT1 cDNAs, which showed that the polyadenylation sites used in vivo can be scattered over a region spanning up to 200 nucleotides. It therefore seems very unlikely that a single signal sequence is responsible for the selection of all these polyadenylation sites. Our study also showed that in the large majority of mRNAs, polyadenylation starts directly before or after an adenosine residue and that 3'-end formation of ADH1 transcripts occurs preferentially at the sequence PyAAA. Site-directed mutagenesis of these sites in the ADH1 gene suggested that this PyAAA sequence is essential for polyadenylation site selection both in vitro and in vivo. Furthermore, the 3'-terminal regions of the yeast genes investigated here are characterized by their capacity to act as signals for 3'-end formation in vivo in either orientation.

Alcohol Dehydrogenase↗