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The institute of Veterinary Medicine is the oldest veterinary school of Germany and nowadays it is part of the agricultural faculty of the Georg-August-University of Goettingen. Because of shifts in the emphasis of research in veterinary science in the area of animal production and hygiene several structural changes have been made over the last few years. In this brief introduction latest developments at the institute of Veterinary Medicine are reviewed.

The ryanodine receptors (RYR) are a family of intracellular Ca2+ release channels that were first identified in the terminal cistenae of the sarcoplasmic reticulum of the skeletal and cardiac muscle. Mutations within the skeletal muscle isoform were shown to cause malignant hyperthermia in swine and man. We have analysed the genomic structure of the porcine skeletal muscle ryanodine receptor and its expression using chimeric reporter gene constructs consisting of the RYR1 gene promoter and the chloramphenicol acetyltransferase gene after transfection in muscle and non-muscle cells.

We have cloned a porcine gene, designated TBP1O, that belongs to the Tat-binding protein/26S protease subunit family. The genomic structure of the porcine TBP1O gene was analyzed after isolation of three overlapping genomic phage lambda clones. The TBP10 gene harbors 12 exons spanning 4.5 kb of chromosomal DNA. The TBP1O gene was assigned to Chromosome (Chr) 12 by fluorescence in situ hybridization (FISH) on metaphase chromosomes. The chromosomal location was confirmed by PCR analysis of a porcine-rodent hybrid cell panel. The TBP1O protein is encoded by a 1221 nucleotide cDNA and has a molecular mass of 45.6 kDa. The predicted amino acid sequence has highest similarity to the human and bovine p45 subunit of the 26S protease and the human transcription factor TRIP1. Further similarities were detected to the slime mold protein DdTBP1O and the Schizosaccharomyces pombe and Saccharomyces cerevisiae protein SUG1. Like DdTBP1O and other members of the protein family, the porcine TBP1O harbors a leucine zipper motif in the N-terminal region and a domain characteristics of ATP-dependent proteases in the C-terminal region.

The ryanodine receptors (RYR) are a family of calcium release channels that are expressed in a variety of tissues. Three genes, i. e. ryr1, ryr2, and ryr3, have been identified coding for a skeletal muscle, cardiac muscle, and brain isoform, respectively. Although, the skeletal muscle isoform (RYR1) was shown to be expressed predominantly in skeletal muscle, expression was also detected in the esophagus and brain. To analyze the transcriptional regulation of the RYR1 gene, we have constructed chimeric genes composed of the upstream region of the RYR1 gene and the bacterial chloramphenicol acetyltransferase (CAT) gene and transiently transfected them into primary cultured porcine myoblasts, myotubes, and fibroblasts. A 443-base pair region upstream from the transcription start site was sufficient to direct CAT activity without tissue specificity. Deletion of a 61-base pair fragment from the 5'-end of the promoter resulted in a marked reduction of CAT activity in all three tissue types. A similar reduction of expression was observed when using a construct with the first intron in antisense orientation upstream from the promoter. In contrast, the first intron in sense orientation enhanced expression only in myotubes, while expression was repressed in fibroblasts and myoblasts. Gel retardation analyses showed DNA binding activity in nuclear extracts for two upstream DNA sequence elements. Our data suggest that (i) RYR1 gene expression is regulated by at least two novel transcription factors (designated RYREF-1 and RYREF-2), and (ii) tissue specificity results from a transcriptional repression in nonmuscle cells mediated by the first intron.

We have mapped two genes from human chromosome 1, urate oxidase (UOX) and transforming growth factor beta 2 (TGFB2), by fluorescence in situ hybridization (FISH) in the pig genome. Porcine-specific polymerase chain reaction (PCR) primers for both genes were designed from the porcine cDNA sequence. With the help of these primers yeast artificial chromosome (YAC) clones for UOX and TGFB2 were isolated from a pig YAC library. These DNA probes were used for FISH analysis. TGFB2 was localized to SSC 10p16. With the YAC probe for UOX two porcine chromosome regions 6q26 and 6q32, revealed specific signals. These results, help to refine the comparative mapping data between human and pig.

We have mapped in the pig (Sus scrofa) the genes for zona pellucida glycoprotein 3 (ZP3), erythropoietin (EPO), and malate dehydrogenase 2 (MDH2) by somatic cell hybrid analysis in the pig genome. Previously, the gene for the T-cell receptor beta cluster (TCRB) was assigned to SSC 18 and that for interleukin 6 (IL6) to SSC 9. However, statistical analysis of mapping data for ZP3, EPO, and MDH2 did not discriminate between SSC 9 and SSC 3. Porcine-specific PCR primers for ZP3 and IL6 were used to isolate probes from porcine YAC libraries. These two genes were mapped by FISH to porcine chromosome regions 3pter --> p15 (ZP3) and 9q14 --> q15 (IL6). Comparative mapping considerations permit the tentative assignment of porcine EPO and MDH2 to 3pter --> p15.

Large-scale physical mapping of the porcine genome has been limited because up to now no suitable genomic libraries for this purpose have been available. Therefore, we have constructed a yeast artificial chromosome (YAC) library from porcine lymphocytes. The library was cloned in the amplifiable vector pCGS966. A total of 10080 YAC clones was obtained and has been ordered into 105 96-well microtiter plates. An average insert size of 300 kb was calculated from the analysis of 78 randomly selected clones, giving a one-fold coverage of the porcine genome. To analyze the complexity, we have screened the library for five different genes and isolated four different clones containing parts of three of these genes. One YAC clone harboring parts of the porcine cardiac muscle ryanodine receptor (RYR2) gene allowed us to assign this locus to Chromosome (Chr) 14q22-q23. The data were confirmed by PCR analysis of a rodent-porcine hybrid cell panel.

The rapid development of modern biotechnology has introduced a new dimension to veterinary medicine and animal breeding. Genome analysis of important breeding species will ultimately allow to detect and consequently to eliminate any possible genetic disorder. Moreover, infectious diseases may also be detected easier and more reliable. The production of transgenic livestock opens the possibility to generate animals with improved health and productivity without time consuming breeding programmes. In the following review I will focus on the description of recent developments in the application of molecular biological techniques in veterinary medicine and animal production. Special emphasis will be given to the physiological consequences of transgene expression rather than the techniques for the production of transgenic animals.

Excitation-contraction coupling in skeletal muscle is mediated by two calcium channels located in the membranes of the transverse tubule and the sarcoplasmic reticulum. Calcium is released from the terminal cisternae of the sarcoplasmic reticulum via the ryanodine receptor. Abnormal increases in myoplasmic free calcium caused by a defect in the ryanodine receptor have been reported in malignant hyperthermia. Malignant hyperthermia is a life-threatening pharmacogenetic disorder in a variety of species and is triggered by volatile anesthetics and depolarizing muscle relaxants. To study the genomic organization of the porcine skeletal muscle ryanodine receptor gene, we have isolated six genomic fragments spanning approximately 80 kb of chromosomal DNA. In this report, we describe the genomic organization of a 15.5-kb genomic fragment comprising 18 exons coding for region 4624 to 7929 of the porcine skeletal muscle ryanodine receptor gene.

An important aspect of gene transfer into farm animals is the improvement of disease resistance. The mouse Mx1 protein is known to be sufficient to confer resistance to influenza viruses. Gene constructs containing the mouse Mx1 cDNA controlled by the human metallothionein IIA promoter (hMTIIA::Mx), the SV40 early enhancer/promoter region (SV40::Mx) and the mouse Mx1 promoter (mMx::Mx) were transferred into pigs. The results of the gene transfer experiments with the hMTIIA::Mx and the SV40::Mx constructs indicate that the permanent high-level synthesis of Mx1 might be deleterious to the organism: the gene transfer efficiency was surprisingly low, and all transgenic piglets born had rearrangements in their transgene copies that abolished protein synthesis. The use of the interferon (IFN)- and virus-inducible mMx::Mx construct resulted in normal gene transfer efficiency. Two transgenic pig lines could be established which expressed IFN-inducible mouse Mx1 mRNA. Extensive protein analysis did not detect mouse Mx1 in IFN-treated transgenic animals.

In this study we describe the isolation of genomic clones of the 5' region of the porcine ryanodine receptor gene, a candidate for malignant hyperthermia in pigs and humans. The recombinants were isolated from a porcine liver, genomic DNA library in phage EMBL3A after screening with PCR amplified DNA fragments. The exon/intron structure of the ryanodine receptor gene was determined by DNA sequencing. Based on the sequence data it was possible to develop a simple test for the detection of malignant hyperthermia susceptible and normal pigs.

Pronuclear microinjection was used to produce transgenic mice harboring gene constructs, in which 110 base pairs (WAP1) or 2.4 kilobases (WAP2) of the 5' flanking sequences of the whey acidic protein (WAP) gene were fused to human growth hormone (hGH)-coding sequences. Female WAP-hGH transgenic mice expressed the transgenes in the mammary gland, the expression of the WAP2-hGH transgene mirroring that of the endogenous WAP gene. When other organs were examined, high level expression of hGH was observed in the brains of WAP2-hGH transgenic mice. Using in situ hybridization and immunohistochemistry, hGH expression from the transgene was seen to occur specifically in Bergmann glia cells. While normally neither WAP nor hGH is expressed in this type of cell, it appears that a combination of the regulatory region of the WAP gene and the hGH structural gene results in a novel tissue specificity in Bergmann glia cells.

Mapping of complex genomes has been influenced substantially by the isolation of locus-specific, but repetitive DNA elements known as VNTRs. Since a high GC-content is characteristic of most of these elements one might expect them to be clustered at least partially in CpG-islands. To address this question we have constructed a porcine liver DNA NotI-linkage library in pUC18 using isolated HTF-islands. HpaII tiny fragments ranging from less than 100 bp to 1 kb in length were randomly selected and analysed by sequencing. As expected from the source of DNA the clones were rich in CpG-, C- and G-content. Several clones exhibiting a repetitive (GGC)n-motif and sequences originally present in human VNTR-markers were shown to detect DNA fingerprints in different species (Homo sapiens, Sus scrofa domestica, Gallus domesticus, Glycine max, Saccharomyces cerevisiae). A subset of clones was used for hybridization experiments with porcine DNA and were shown to detect single-copy linkage fragments.

The structure of eukaryotic transcription units and already known DNA elements which influence gene expression and eukaryotic gene regulation are described. In recent years molecular genetics approaches have been used to identify and characterize the molecular anatomy of protein-coding eukaryotic genes. Within these genes there are modular sequences, consisting of arrays of short, highly conserved recognition elements that are essential for the initiation of transcription, translation, and tissue-specificity of gene expression. The skillful combination of promoters, enhancers and other regulatory sequences allows the expression of every gene in every organ or tissue of a transgenic animal.

Transgenic mice harbouring growth hormone gene constructs have been produced by DNA microinjection into pronuclei of fertilized oocytes. We examined transgenic mice carrying a mouse metallothionein I-human growth hormone (mMT I-hGH) fusion gene. Here, we present our results concerning gene integration, gene expression, and phenotypical, clinical and pathomorphological alterations found in mice expressing the hGH transgene. Body and organ growth was significantly increased in transgenic mice, whereas fertility was found to be reduced. The life-span was markedly shortened indicating detrimental side-effects of the high levels of circulating hGH. Lesions of kidneys, liver and heart were the predominant pathological findings. Our own results are compared with those obtained by other authors who have investigated mice carrying rat, bovine or ovine growth hormone fusion genes. GH-transgenic mice may serve as a model system to investigate ectopic expression of hormone genes thus circumventing endogenous feedback control mechanisms in complex hormonal cascades.