Biomedical subjects
Publications and source records attributed to R Fries.
Genetically directed representational difference analysis (GDRDA) allows to isolate DNA fragments that differ between complex genomes. This approach was applied in the present work to isolate Y-specific bovine sequences. Of eight isolated and cloned fragments, two proved to be Y-specific.
The study comprised 236 pigs selected for resistance or susceptibility to oedema disease. The susceptibility to colonization of the small intestine by an Escherichia coli strain causing oedema disease was determined: (1) by monitoring faecal excretion of weaned pigs orally inoculated with E. coli strain O139:K12(B):H1:F18ab serotype; and (2) by an in vitro adhesion assay using an F18ab positive E. coli strain and small intestinal enterocyte preparations. Susceptibility to adhesion by these bacteria was shown to be controlled by a dominant (B) allele of the ECF18R locus and resistance by the alternative recessive allele (b). Pigs were typed for 14 blood group systems, 11 biochemical polymorphisms and the polymorphism at nucleotide 1843 of the RYR1 locus. Linkage was demonstrated between the locus for F18 E. coli receptors and the loci S, RYR1, GPI, EAH, A1BG and PGD (Z > 20). The most likely gene orders are: S-ECF18R-RYR1-GPI-PGD or GPI-RYR1-ECF18R-S-PGD. The recombination frequencies between ECF18R-S and ECF18R-RYR1 were estimated to be theta = 0.5% and 3.1%, respectively.
Between January and September 1995, 54 consecutive patients (male: 34, age: 66 +/- 10 years) with symptomatic chronic atrial fibrillation (median duration: 4.5 months) were referred for external electrical cardioversion to our hospital. Mean left atrial diameter was 49 +/- 9 mm, heart disease was apparent in 81%. All patients were under antiarrhythmic drugs (class III: 85%). In 49 patients (91%) sinus rhythm was achieved. In five patients atrial fibrillation persisted after delivery of 360 Joules. These five patients were characterized by a significantly higher body weight in comparison to patients with successful external cardioversion. All five patients underwent low energy internal cardioversion the following day: biphasic R-wave synchronous shocks were delivered through catheters positioned in the right atrium and the coronary sinus using stepwise increased energy levels. Internal cardioversion was successful in all patients resistant to external cardioversion: stable sinus rhythm was established at a mean energy level of 13 +/- 6.7 Joules. No complications were observed. During the follow-up, each patient revealed a relapse of symptomatic atrial fibrillation within 2 weeks after internal cardioversion despite antiarrhythmic therapy, whereas only 16 patients (33%) lost sinus rhythm during the same period of time after external cardioversion (p < 0.01). During a mean follow-up of 283 +/- 72 days 21 patients (43%) preserved stable sinus rhythm after external cardioversion. Internal low energy cardioversion seems to be effective and safe in conversion of chronic atrial fibrillation resistant to external cardioversion. The clinical value of this invasive, time- and material-consuming therapy seems to be limited in this setting because of the high early relapse-rate in the investigated patient population. Further clinical studies in a larger cohort of patients are necessary.
Chromosomal homologies between individual human chromosomes and the bovine karyotype have been established by using a new approach termed Zoo-FISH. Labeled DNA libraries from flow-sorted human chromosomes were used as probes for fluorescence in situ hybridization on cattle chromosomes. All human DNA libraries, except the Y chromosome library, hybridized to one or more cattle chromosomes, identifying and delineating 50 segments of homology, most of them corresponding to the regions of homology as identified by the previous mapping of individual conserved loci. However, Zoo-FISH refines the comparative maps constructed by molecular gene mapping of individual loci by providing information on the boundaries of conserved regions in the absence of obvious cytogenetic homologies of human and bovine chromosomes. It allows study of karyotypic evolution and opens new avenues for genomic analysis by facilitating the extrapolation of results from the human genome initiative.
N-Oct 3 is a human POU domain transcription factor that binds to the octamer sequence ATGCAAAT. The protein is expressed in the central nervous system during development and in adult brain. We have isolated and characterized genomic clones encoding the human N-Oct 3 gene (HGMW-approved symbol POUF3). Comparison of the structure of these clones with the N-Oct 3 cDNA revealed that POUF3 is an intronless gene. Sequencing of 650 bp of the promoter region showed 84% sequence identity of POUF3 with its murine homologue, the brain-2 (designated brn-2) gene. Whereas both POUF3 and brn-2 lack a TATA box, consensus sequences for AP-2, GCF, and SP1 transcription factors were identified within the highly conserved 5'-flanking region. These sequences may play a crucial role for the tissue-specific transcription activation of the POUF3 gene. Southern blotting and in situ hybridization localized the human POUF3 gene to chromosome 6q16.
Gene mapping in cattle has progressed rapidly in recent years largely owing to the introduction of powerful genetic markers, such as the microsatellites, and through advances in physical mapping techniques such as synteny mapping and fluorescence in situ hybridization (FISH). Microsatellite markers are often not physically mapped because they are generally isolated from small insert plasmid libraries, which makes their chromosomal localization inefficient. In this report we describe the FISH mapping of a large group of cosmid-derived bovine microsatellite markers, as our contribution to the European mapping initiative, BovMap. One objective of BovMap is to develop a set of anchored loci for the cattle genome map. Two cosmid libraries were screened with probes corresponding to the (AC)n microsatellite motif. Positive clones were mapped by FISH, and then a subset was further analyzed by sequencing the region flanking the microsatellite repeat. In total, 58 clones were hybridized with chromosomes and identified loci on 22 of the 31 different bovine chromosomes. Three clones contained satellite DNA. Two or more markers were placed on 12 chromosomes. Sequencing of the microsatellites and flanking regions was performed directly from 43 cosmids, as previously reported (Ferretti et al. Anim. Genet. 25, 209-214, 1994). Primers were developed for 39 markers and used to describe the polymorphism associated with the corresponding loci.
We have initiated a mapping strategy using cosmid clones to chromosomally anchor a high-resolution bovine genetic linkage map. Ten cosmids containing microsatellites were assigned to bovine chromosomes by fluorescence in situ suppression hybridization (FISH). Four cosmid clones, three of which contain an informative microsatellite, were assigned to autosomes 5, 13, 24, and 28. The assignment to autosome 13 anchors bovine syntenic group U11. Two additional cosmid clones, each containing informative microsatellites, are assigned to autosomes 9 and 29, anchoring bovine linkage groups U2 and U8, respectively. Four cosmid clones, three of which contain informative microsatellites, also provide the first assignment to autosome 25, anchoring bovine syntenic group U7 and orienting the corresponding linkage group relative to the centromere.
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Interleukin-4 (IL4) is involved in the immune response to certain parasites and possibly in the development of some atopic diseases since it triggers the T helper 2 lymphocyte response. Therefore, IL4 is a candidate gene, for example, for disease association studies and gene mapping. We isolated bovine IL4 cosmids and determined the genomic organization. Fragments carrying the exons as well as 725 base pairs (bp) from the 5' flanking and 190 bp from the 3' flanking region were cloned and sequenced. The first 481 base pairs of the 5' flanking region, including the putative promoter sequences, are surprisingly similar (92%) between cattle and human. In addition, we cloned and sequenced a mixed [(t/g)a]m(ca)n repeat located approximately 35 kilobases upstream from the IL4 gene. It showed seven repeat length alleles in a limited number of animals. The IL4 gene has been assigned to 7q15-q21 by fluorescence in situ hybridization in cattle. Evolutionary aspects are discussed on the basis of sequence data as well as interspecies chromosomal homologies.
As a new approach to parentage control we developed two multiplex coamplification polymerase chain reaction (PCR) systems containing a total of six different short tandem repeat (STR) loci; the microsatellite polymorphisms were visualized by automated fluorescence detection on the Applied Biosystems 373 DNA Sequencer with 672 Genescan Analysis software. Allele frequency data were determined from 238 animals. Thirty-five bovine parentage control cases not solvable by conventional blood typing could be solved.
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Swine-specific sequence tagged (microsatellite) sites, STS and STMS, were assigned chromosomally by polymerase chain reaction analysis of a somatic cell hybrid panel. This study confirms the localization from genetic mapping of seven anonymous microsatellites and the genes ANPEP, ATP2, CGA, DAGK, FSHB, IFNG, IGF1, IL1B and SPP1. New assignment for the gene BNP1 to chromosome 6 is reported. The confirmed and the new assignments extend the information of the previously established linkage maps and provide framework loci on which to order additional informative markers.
Eleven probes were assigned to bovine chromosomes 1 to 7 by fluorescence in situ hybridization (FISH). The identification of chromosomes was based on QFQ-banding prior to in situ hybridization and comparison with the Reading Conference (1976) and ISCNDA (1989) standards. The probes used for FISH can now be utilized as identification and discrimination features for bovine chromosomes 1 to 7 and particularly for chromosomes 4 and 6, which are difficult to distinguish. Comparison of our mapping data with previous assignments and of the standard chromosome banding patterns prompt us to propose a change in the ISCNDA nomenclature: ISCNDA chromosome 4 should be named chromosome 6 and vice versa. Chromosome 4 is marked by the ribosomal RNA cluster RNR3, and chromosome 6 is characterized by the casein gene cluster and an anonymous satellite (D6Z1).
This study describes the influence of bioptivet GB on minimum inhibitory concentrations (MIC) for oxytetracycline (OTC) of the intestinal E. coli flora of young broiler chickens after oral administration at a dosage equivalent to a prophylactic course of treatment. From day 6 until day 15 one group of 50 birds received a diet containing 124 ppm OTC, another group of 75 birds served as non medicated control. Investigated E. coli had been isolated from cloacal swabs and from caecal contents. MIC of 1581 E. coli strains were determined by agar dilution test procedures. MIC of OTC for the investigated strains were either > or = 128 micrograms/ml (resistant) or < or = 4 micrograms/ml (susceptible). Even from undosed birds resistant strains were isolated frequently, especially from samples of caecal contents. Administration of bioptivet GB resulted in a statistically significant increase in the average MIC. Statistically higher average MIC were recorded among isolates from cloacal swabs only during application of the drug. For strains from caecal contents this could be demonstrated until the end of the experiment.
The gene encoding the calcitonin receptor (CTR) was isolated from a porcine kidney epithelial cell line (LLC-PK1) genomic library and found to span approximately 70 kilobases. Analysis of the gene sequence revealed that the CTR mRNA encompasses 14 exons with 12 exons encoding the protein. Two splicing acceptor sites separated by 48 nucleotides were found in intron 7. The expression of two mRNA species in LLC-PK1 cells was subsequently confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and DNA sequencing. In LLC-PK1 cells the mRNA encoding the shorter CTR (CTR-1a) is approximately 1,000 times more abundant than the longer variant (CTR-1b), as estimated by the competitive RT-PCR. The transcription initiation site of the CTR gene was mapped by primer extension, S1 nuclease, and RT-PCR analysis. The proximal promoter region of 500 base pair is GC-rich (66%) and CpG-rich (CpG/GpC ratio 0.71). Transient transfection of CTR gene promoter-luciferase chimeras in LLC-PK1 cells led to the expression of luciferase activity. The CTR gene was mapped to chromosome band 9q11-q12.
Porcine lymphocytes and fibroblasts were fused with 3 different permanent rodent cell lines, and 21 stable somatic cell hybrid lines were established. These hybrid cell lines were characterized cytogenetically by sequential QFQ banding and chromosome painting using fluorescence in situ hybridization with porcine DNA. The lines were further characterized by PCR analysis with primer pairs derived from genes with confirmed mapping information. Using this panel, we assigned the locus encoding polyubiquitin (UBC) to chromosome 14, and the transition protein 2 locus (TNP2) and protamine loci (PRM1 and PRM2) to chromosome 3. Two chromosomal localizations have been further refined by radioactive in situ hybridization. UBC maps to chromosome 14q12-q15 and TNP2 to 3p11-p12.
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Five overlapping lambda EMBL-clones, containing the complete bovine lactoferrin gene (LTF), have been used to map this gene by fluorescence in situ hybridization to bovine Chromosome (Chr) band 22q24. Primers derived from promoter and exon I sequences were applied in polymerase chain reactions (PCRs) to DNA samples of a previously characterized panel of somatic cell hybrid lines, allowing the assignment of the bovine lactoferrin locus to syntenic group U12. These results permit the assignment of syntenic group U12 to bovine Chr 22.