Panel discussion

Risk assessment is part of the risk analysis process as it is used in veterinary medicine to estimate risks related to international trade and food safety. Data from monitoring and surveillance systems (MO&;SS) are used throughout the risk assessment process for hazard identification, release assessment, exposure assessment and consequence assessment. As the quality of risk assessments depends to a large extent on the availability and quality of input data, there is a close relationship between MO&;SS and risk assessment. In order to improve the quality of risk assessments, MO&;SS should be designed according to minimum quality standards. Second, recent scientific developments on state-of-the-art design and analysis of surveys need to be translated into field applications and legislation. Finally, knowledge about the risk assessment process among MO&;SS planners and managers should be promoted in order to assure high-quality data.

     

Genetic structure and origin of t haplotypes of mice, analyzed with H-2 cDNA probes

We investigated the genetic organization and evolutionary origin of t chromosomes of mice by examining the restriction fragment patterns of DNA from t haplotypes and normal chromosomes with cDNA probes to H-2 class I genes. On genomic DNA blots, the restriction fragments containing H-2-related sequences were highly variable among different inbred strains of mice, whereas they were very similar among different t haplotypes even when the t haplotypes carried serologically different H-2 haplotypes. These observations suggest that all t haplotypes have a common origin and are not products of independent mutational events. We also mapped the position of several restriction fragments characteristic of t DNA by using a battery of recombinant t haplotypes, defined with respect to their t-lethal factors and H-2 haplotypes. We thus show that restriction fragments containing H-2-related sequences map to the left of the H-2 class I genes in t chromosomes, a region in which the tw32 b-lethal factor also maps. The cloning of these fragments can be expected to provide an entry for the structural analysis of t DNA.     

Panel discussion on submammalian systems

A test of X-ray-induced recessive lethal mutations in mouse spermatogonia (500 rad) was carried out. The test was based on familial analysis, which allowed division on the P pairs into those with lethal heterozygous members and in others assumed to be lethal-free. The F1 males from the latter group, in back-crosses to their daughters, gave an excessive rate of intra-uterine death in comparison with lethal-free males. The excessive death is assumed to reflect the rate of new (induced + spontaneous) recessive lethals or rather lethal equivalents.Three ways of estimating the rate of new recessive lethal mutations gave a mean of 5.5% per genome. From previous tests we can assume that 1% are spontaneous mutations. Thus the data indicate that the mutation rate per rad per gamete is 9 × 10^?5. This value is identical with a previous estimate.The results are discussed in relation to population tests performed in the early 60'2. It is concluded that the lack of observable deterioration in the populations after several consecutive generations of exposure is in accord with the estimates in the present analysis which are more than an order of magnitude lower than assumed at the start of the population tests.It is also stressed that species with different DNA contents show similarities in point estimates of doubling dosages.     

Yak lactate dehydgogenase A4: purification, properties, and cDNA cloning

Lactate dehydrogenase A4 (LDH-A4) was purified for yak skeletal muscle. Michaelis constant (Km) analysis showed that yak LDH-A4 for pyruvate was significantly higher than that of cattle. cDNA cloning of LDH-A revealed two amino acid substitutions between yak and cattle. We suggest that the higher Km of yak LDH-A4 might be a result of molecular adaptation to a hypoxic environment.     

Pea choline kinase: purification, properties and isolation of a cDNA

Choline kinase has been partially purified from pea seedlings and its properties studied. Using sequence information from soya bean and other choline kinases, we have also isolated a cDNA encoding the enzyme. It encodes a protein of 343 amino acids (calculated molecular mass of 39785 Da), which shows 82% homology with the soya bean choline kinase. The protein has been expressed in Escherichia coli with very good activity and high expression levels.     

Human guanylin: cDNA isolation, structure, and activity.

Guanylin is a mammalian peptide homologue of heat-stable enterotoxins that acts on intestinal guanylate cyclase to elicit an increase in cyclic GMP. We have isolated a cDNA encoding an apparent precursor of guanylin from a human intestinal cDNA library. The mRNA is expressed at high levels in human ileum and colon. Human guanylin stimulated increases in T84 cell cyclic GMP levels, displaced 125I-labelled heat-stable enterotoxin (STa) binding to this cell line, and stimulated increases in short-circuit current (Isc) of isolated rat proximal colonic mucosa. This peptide may play a role in regulating fluid and electrolyte absorption in human intestines.     

cDNA cloning, purification and properties of Paralithodes camtschatica metalloprotease

Hepatopancreas of king crab Paralithodes camtschatica produces a metalloprotease, which belongs to the astacin family, as cDNA cloning and sequencing showed. The metalloprotease has been purified chromatographically to apparent homogeneity. The purification factor was 16 and activity recovery was 20%. pH and temperature optimum have been determined. In its properties (molecular weight, pI, metal content) the metalloprotease is close to crayfish astacin. However, analysis of the enzyme sequences revealed differences, which account for differences in substrate specificities and imply a different activation mechanism.     

Genetic diversity in cocoa revealed by cDNA probes

The variability of the cocoa (Theobroma cacao) nuclear genome was investigated. A total of 203 cocoa clones was surveyed for restriction fragment length polymorphisms (RFLPs) using four restriction endonuclease and 31 seed cDNA probes. A high level of polymorphism has been found. This study points to a structuring of the species that fits with the distinction between the Criollo and Forastero populations. These results combined with previously obtained nuclear rDNA and mtDNA data allow us to propose new hypotheses on the origin and evolution of the different cocoa populations.     

Circular dichrotic probes for aspects of chromatin structure: aromatic polycationic ionen probes

Two ionens (II and X) formed complexes with DNA and chromatin with extrinsic CD bands and reduced intrinsic bands. The salt and urea sensitive, AT-specific probe (II) gave Δε > 100 L-(residue II)^?1-cm^?1 with DNA and Δε=0–14 with chromatin; II reduced the intrinsic bands from Δε≈0.7 to Δε≈0.5. Ionen X gave Δε₃₄₅=30 with DNA, and Δε₃₄₅=15–20 with chromatin. X reduced the intrinsic band to ?1.6. X show less base specificity. Extrinsic Δε_λ of X increased linearly to r (residue/phosphate) = 0.5 for DNA and only 0.3 in chromatin. DNA in chromatin may have ～10% of the II and 50–60% of the X binding sites and those in an altered conformation.     

Simplified purification and testing of colloidal gold probes

Summary A novel efficient method for purifying and testing colloidal gold probes has been developed. The method consists of concentrating colloidal gold particles conjugated to IgG or protein A in dialysis bags over silica gel and purifying them by gel chromatography on small columns of Sephacryl S-400. Fractions collected are tested by paper immunocytochemical models. Comparisons to gold probes purified by conventional ultracentrifugation documents that ultrastructural staining intensities and total yield of gold probes is the same, but that the chromatographically purified gold probes are less prone to aggregation or clumping. The method has been extensively used for preparing conjugates of 5, 10 or 15 nm gold particles with antirabbit immunoglobulins but has also been exploited for preparing streptavidin-gold conjugates, protein A-gold conjugates and antirabbit immunoglobulin-silver conjugates.     

Simplified purification and testing of colloidal gold probes

A novel efficient method for purifying and testing colloidal gold probes has been developed. The method consists of concentrating colloidal gold particles conjugated to IgG or protein A in dialysis bags over silica gel and purifying them by gel chromatography on small columns of Sephacryl S-400. Fractions collected are tested by paper immunocytochemical models. Comparisons to gold probes purified by conventional ultracentrifugation documents that ultrastructural staining intensities and total yield of gold probes is the same, but that the chromatographically purified gold probes are less prone to aggregation or clumping. The method has been extensively used for preparing conjugates of 5, 10 or 15 nm gold particles with antirabbit immunoglobulins but has also been exploited for preparing streptavidin-gold conjugates, protein A-gold conjugates and antirabbit immunoglobulin-silver conjugates.     

Nucleoside diphosphate kinase from pea chloroplasts: purification,cDNA cloning and import into chloroplasts

Nucleoside diphosphate kinase (NDPK; EC 2.7.4.6) was enriched 1900-fold from purified pea (Pisum sativum L. cv. Golf.) chloroplasts. The active enzyme preparation contained two polypeptides of apparent molecular weight 18.5 kDa and 17.4kDa. Both proteins were enzymatically active and were recognized by an antiserum raised against NDPK from spinach chloroplasts, suggesting the existence of two isoforms in pea chloroplasts. The N-terminal protein sequence data were obtained for both polypeptides and compared with the nucleotide sequence of a cDNA clone isolated from a pea cDNA library. The analysis revealed that the two NDPK forms are encoded for by one mRNA, indicating that the lower-molecular-weight form could represent a proteolytic breakdown product of the 18.5-kDa NDPK. The pea chloroplastic NDPK is made as a larger precursor protein which is imported into chloroplasts. The NDPK precursor is then processed by the stromal processing peptidase to yield the 18.5-kDa form.Abbreviations NDPK nucleoside diphosphate kinase - preNDPK precursor NDPK - ps-NDPK cDNA coding for Pisum sativum NDPK II We thank Dr. Schmidt, University Göttingen, Germany, for doing the protein sequencing. This work was supported in part by grants from the Deutsche Forschungsgemeinschaft.     

Cytokinin oxidase from Zea mays: purification, cDNA cloning and expression in moss protoplasts

Cytokinins are degraded by cytokinin oxidases (CKOs) which catalyse cleavage of the N6-(isopent-2-enyl)-side chain resulting in formation of adenine-type compounds. CKO activity has been recorded in many plants and is thought to play a key role in controlling cytokinin levels in plants. Several partially purified CKOs have been characterised but no genes have been isolated yet. CKO activity is known to be inhibited by phenylureas, cytokinin agonists. We used 1-(2-azido-6-chloropyrid-4-yl)-3-(4-[3H])phenylurea ([3H]-azidoCPPU) to photolabel a glycosylated CKO from maize kernels. This enabled us to purify the enzyme. Peptide sequences were determined and the corresponding cDNA was cloned. The deduced amino acid sequence shares homology domains with FAD-dependent oxidases. An original assay based on transient expression of the enzyme in moss protoplasts allowed the functionality of the recombinant enzyme to be demonstrated.