Question

PROBLEM 1) You are a scientist in a lab that studies how cell fates are specified during development. In particular, you are curious about muscle development. Muscle cells are derived from the mesoderm (one of the three primary germ layers) and are specified rather early in development. You are using the fruit fly, Drosophila melanogaster, as a model system to study the phenomenon of muscle fate specification. You have isolated a line of flies carrying a loss of function mutation in a gene called “whimpy” (wpy). Embryos homozygous for the wpy mutation die during mid-embryonic development. Analysis of the mutant embryos suggests that muscle cells are not made. This suggests that the wildtype (normal) form of the wpy gene is required (necessary) for normal muscle specification during development (this is a HYPOTHESIS).

a) Suppose you have cloned the wild type (normal) and mutant form of the wpy gene. There is a single nucleotide difference between the wild type and mutant forms of the gene. List at least two possible explanations for how a single NT difference could result in the mutant phenotype.

b) Briefly, how would you obtain information about the kind of protein encoded by the wpy gene (and thus make hypotheses about its function)?

c) How might you go about determining when and where the wpy gene is expressed during development? Explain your answer briefly.

d) How would you test the hypothesis that wpy is sufficient to specify muscle cell fate? Explain your answer.

Answer 1

a) The single nucleotide difference in a gene, known as point mutation affects gene function. This single base pair change may lead to missense mutation in which an aminoacid of the encoded protein changes. Another possibility could be nonsense mutation, in which a nucleotide base change leads to the formation of a stop codon, leading to premature termination of translation, hence producing a truncated protein. This leads to mutant phenotype

b) DNA sequencing can be carried out and the DNA sequence of the gene can then be matched to amino acid sequence of the corresponding protein of the gene from the database on NCBI. This will indicate the kind of protein encoded by the wpy gene, whether it is mutated or not.

c) The gene expression can be measured using RNA expression analysis (qRT-PCR, microarray, SAGE etc) by obtaining samples of the organism from different stages of development. In addition, proteins can be isolated from the same samples for western blot to measure the expression at protein level.

d) The hypothesis that wpy is sufficient to specify muscle cell fate can be tested by comparing the gene expression of wild type and mutant gene.