To begin to examine the function of genes that control early development in the hindbrain, we have screened an embryonic zebrafish cDNA library with a murine krox-20 gene probe that contained the conserved zinc finger regions. We have isolated two overlapping cDNAs, zf187 and zf201 which are homologues of the murine krox-20 gene. The N-terminal of the longest cDNA (zf201) contains two acidic regions identical to those of the murine krox-20. This indicates that the functional organisation of these proteins is probably conserved. Northern Blot analysis identified a single transcript of 2.0 kb. Wholemount in situ hybridisation established that expression of the zebrafish gene (krx-20) first appears at 100% epiboly as a single anterior domain of the prospective neuroepithelium, followed very soon after by a second more posterior domain. The alternating pattern of expression of this gene in rhombomeres(r) r3 and r5 is apparent by 12 hr post-fertilisation, that is prior to the morphological appearance of the rhombomeres. Around 14 hr neural crest migration begins from the dorsal surface of r5, moving caudally into r6 and then ventrally towards the pharyngeal arches. Crest migration is not apparent at or after 16 hr. No neural crest migration was observed from r3. Expression of krx-20 is down regulated firstly in r3 around 26 hr and later in r5 around 30 hr.

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The nucleotide sequence and derived amino acid sequence of two different beta-glucosidase cDNA clones were determined. One clone (TRE104) was identified as the cyanogenic beta-glucosidase by homology with the N-terminal and internal peptide amino acid sequence of the purified enzyme. The biological function of the other beta-glycosidase (TRE361) is not known. Co-segregation of genomic restriction fragments uniquely identified by each cDNA clone shows that these two genes are linked in the white clover genome. Both TRE104 and TRE361 fragments co-segregate with cyanogenic beta-glucosidase activity. Extensive homology was found between the white clover beta-glucosidase sequences and a group of prokaryote and mammalian beta-glycosidases. This group of sequences has no homology with a separate set of beta-glucosidase genes isolated from fungi and the thermophilic bacterium Clostridium thermocellum.

The Li locus in white clover controls the presence of cyanogenic beta-glucosidase (linamarase) activity in leaf tissue, such that plants homozygous for the 'null' allele (li) have no linamarase activity in this tissue. The isolation of a cDNA clone from linamarase mRNA is described. The cDNA clone is used to further characterise alleles of the Li locus. Northern blot analysis shows that plants homozygous for the 'null' allele (li li) produce very reduced levels of mRNA which hybridises to the cDNA. Heterozygous plants (Li li), which have intermediate levels of enzyme activity, produce intermediate levels of mRNA. Southern blot analysis of Hind III digested genomic DNA shows that the white clover genome contains three genes with homology to the linamarase cDNA and that at least two of these genes segregate independently. Analysis of the cosegregation of linamarase activity and the presence of genomic restriction fragments identifies the genomic sequence specifying linamarase structure and indicates either a structural or cis acting control function of the Li locus.

Mammalian cytochrome P450s provide our first line of defence against the toxic effects of environmental chemicals. Ironically these enzymes also convert some compounds to their ultimate toxic or mutagenic species. Our knowledge of these mammalian enzymes and the role they play in chemical toxicity and mutagenesis has stemmed mostly from in vitro studies. In order to establish the role of specific enzymes in the toxicological response in vivo we have generated transgenic Drosophila which express mammalian cytochrome CYP2B1, which is a member of a large gene family encoding several important drug metabolising enzymes. The gene was fused to a Drosophila promoter which confers expression in the larval fat body. Using the Somatic Mutation And Recombination Test (SMART) we have demonstrated that transgenic larvae expressing the P450 are hypersensitive to the anticancer drug cyclophosphamide, a procarcinogenic substrate which is activated by the enzyme. This work demonstrates the potential of such transgenic Drosophila strains as an in vivo model for studying the role of specific mammalian drug metabolising enzymes in the pathways and metabolic cascades associated with the action of cytotoxic and carcinogenic chemicals, and also the chemical properties of specific classes of mutagen to be determined.