How would you clone a gene that you have identified by a mutant phenotype in Drosophila?

How would you conduct a screen to identify recessive mutations in Drosophila that result in embryo lethality? How would you propagate the recessive mutant alleles?

In land plants, there is an alternation of generations between a haploid gametophyte generation and a diploid sporophytic generation. Both generations are typically multicellular and may be free-living. The male (pollen) and female (embryo sac) gametophytes are the haploid generation of flowering plants.

How would you conduct a screen to identify genes required for female gametophyte development in Arabidopsis?

In land plants, there is an alternation of generations between a haploid gametophyte generation and a diploid sporophytic generation. Both generations are typically multicellular and may be free-living. The male (pollen) and female (embryo sac) gametophytes are the haploid generation of flowering plants.

How would you conduct a screen to identify genes required for male gametophyte development?

Most organisms display a circadian rhythm, a cycling of biological processes that is roughly synchronized with day length (e.g., jet lag occurs in humans when rapid movement between time zones causes established circadian rhythms to be out of synch with daylight hours). In Drosophila, pupae eclose (emerge as adults after metamorphosis) at dawn.

Using this knowledge, how would you screen for Drosophila mutants that have an impaired circadian rhythm?

Most organisms display a circadian rhythm, a cycling of biological processes that is roughly synchronized with day length (e.g., jet lag occurs in humans when rapid movement between time zones causes established circadian rhythms to be out of synch with daylight hours). In Drosophila, pupae eclose (emerge as adults after metamorphosis) at dawn.

In most plants, such as Arabidopsis, genes whose encoded products have roles related to photosynthesis have expression patterns that vary in a circadian manner. Using this knowledge, how would you screen for Arabidopsis mutants that have an impaired circadian rhythm?

Most organisms display a circadian rhythm, a cycling of biological processes that is roughly synchronized with day length (e.g., jet lag occurs in humans when rapid movement between time zones causes established circadian rhythms to be out of synch with daylight hours). In Drosophila, pupae eclose (emerge as adults after metamorphosis) at dawn.

In each case, how would you clone the genes you identified by mutation?

Mutations in the Drosophila Ultrabithorax (Ubx) gene result in wings developing from two thoracic segments, rather than just one as in wild-type flies. In the mouse genome there are two Ubx orthologs . How would you determine whether the two mouse genes have distinct or redundant functions?

How would you edit a specific nucleotide in a genome?

Through a forward genetics screen in Arabidopsis you have identified a mutation that results in leaves curling upward, rather than being flat as in wild type. You have cloned the corresponding gene and note that it is a member of a small gene family composed of three additional members in Arabidopsis. How will you determine if the other three members of the gene family have similar or distinct functions as compared with the gene you first identified?

The CRISPR–Cas9 complex directs the Cas9 endonuclease to a specific genomic locus. If the endonuclease domain is inactivated and replaced with a transcriptional activator (or repressor) domain, what would be the functional consequence of directing such a complex to a specific chromosomal location?

How might you use CRISPR–Cas9 to create a large deletion?