BIOL2005 WORKSHEET 2008 Answer all 6 questions in the space provided using additional sheets where necessary. Hand your completed answers in to the Biology office by 3 p.m. Friday 8th February. 1. Your lab performs an F1 screen for recessive developmental mutations in zebrafish. If you spot a potentially interesting developmental defects in haploid embryos what can you do to (i) 2 marks (ii) 1 mark Generate homozygous mutant diploid embryos for further phenotypic analysis. Maintain a library of your mutants without having to keep large numbers of live fish 2. Which of the following phenotypes would it be possible to identify in an F1 screen of haploid zebrafish embryos:- i) Dorso-ventral patterning defects ii) Defective mesoderm formation iii) Defects in swimming ability iv) Hearing defects Give your reasoning in each case 2 marks
3. No tail (ntl) is a recessive, homozygous lethal mutation. Heterzygotes are completely wild type in phenotype. Homozygous embryos show tail defects and die between 2-3 days after fertilisation. The phenotype is very similar to that of the mouse T mutant. The mouse T gene has been cloned and a gene producing a very similar (homologous) protein sequence (Zf-T) identified in zebrafish. The ntl mutation is maintained by breeding from proven heterozygous carriers of the mutation ie males and females that when crossed together produce 25% mutant embryos. DNA extracted from carriers (ntl/+) digested with an appropriate restiction enzyme and subjected to Southern analysis gives a single 8.4kb band when probed with the Zf-T sequence. DNA extracted from an unrelated wild type fish stock (+ ind ) and subjected to the same procedure gives a 5.7kb band. This shows that there is an RFLP for Zf-T that has different forms in the ntl stock and the + ind stock. Describe a simple genetic test you could perform to determine whether ntl and Zf-T were closely linked. Include diagrams showing the crosses you would perform noting the genotypes and phenotypes of the parents and their possible offspring and the genotypes of the gametes produced by any given parent. Note that ntl/ntl fish cannot be used as parents since fish with this genotype die as embryos. They can, however, be scored when generated as offspring of a cross. It has now been shown that the Zf-T and ntl genes are the same. How do you reconcile this with the fact that heterozygous carriers of ntl show only one form of the Zf-T RFLP? 1 mark
4. Describe a breeding programme you could use when screening for new dominant mutations affecting swimming ability in the offspring of mutagenised zebrafish. Include the crosses you would do to confirm that any defects observed were genetic in origin.
5. The mouse Pdgfra gene is expressed in glial cell precursors in the embryonic mouse spinal cord. It is not expressed in the dorsal root ganglia. Transgenic mice were made by microinjection of two different constructs. Both constructs carry lacz reporter gene inserted into exon 2 of the Pdgfra gene so that it forms an in frame fusion protein which has full β-galactosidase activity. Construct A includes the Pdgfra promoter region, the entire coding sequence, 150kb of the genomic sequence immediately downstream of the Pdgfra coding region. Construct B includes 250 kb of downstream sequence but is otherwise identical to construct A. A B Five independent transgenic mouse lines were made with construct A and three with construct. Β-galactosidase activity was assessed in the spinal cord of transgenic embryos from each of these lines. Embryos from all 5 construct A lines showed β-galactosidase activity in the dorsal root ganglia but not in glial precursors. Embryos from all 3 construct B lines showed β-galactosidase activity in glial precursors but not in the dorsal root ganglia. What do these results suggest about the location of regulatory elements that enhance or suppress Pdgfra expression (i) in glial precursors (ii) in the dorsal root ganglia Draw a map showing the regions downstream of Pdgfra that might include these regulatory elements. Pdgfra lacz
6. In mice, as in other mammals, sex determination is regulated by a genetic pathway that includes the Y linked gene Sry, and two autosomal loci Sf1 and Wnt4. In wild type mice the presence of the Y chromosome determines sex (XY mice are male XX mice are female). In molecular terms, Sf1 is a transcription factor, which activates the expression of testosterone and other hormones that promote testes and male development. Female development is the default pathway that takes place when these hormones are absent. Wnt4 activity represses Sf1 expression and Sry activity represses Wnt4 expression. Thus:- Sry Wnt4 Sf1 Knockout mice with mutations in Sry, Wnt4 and Sf1 show different sex reversal phenotypes:- XY mice in which the Sry gene has been knocked out develop as females (male to female sex reversal). XX mice which are homozygous mutant for the knockout allele of Wnt4 develop as males (female to male sex reversal). XY mice which are homozygous mutant for the knockout allele of Sf1 develop as females (male to female sex reversal). Given this information can you predict the phenotypic sex of the following double mutant combinations:- (i) XX mice which are homozygous mutant for both Sf1 and Wnt4 (ii) XY mice which are mutant for Sry and homozygous mutant for Wnt4 (iii) XY mice which are homozygous mutant for both Sf1 and Wnt4 (iv) XY mice which are mutant for Sry and homozygous mutant for Sf1 Show your reasoning in each case 4 marks