Answers, problem set C

22) Xenopus - large oocyte that develops outside the mother

zebrafish - similar to Xenopus, but with the added benefit of convenient genetics

mouse - a mammal and therefore more similar to humans, but harder to observe developing embryo; can also do reverse genetics

elephant - large and expensive to maintain; might be good to study development of the trunk, which is unique to elephants, or to study persistence of memory in an animal that never forgets!

23) one

24) inner cell mass, epiblast

25) a) The mouse can be entirely red, entirely green, or have red and green patches

b) Entirely red or entirely green

26) First engineer a piece of DNA that has i) a mutation in the gene to be replaced, flanked by regions of homology with the genomic copy of the gene to be mutated; ii) a positive selectable marker gene (such as that encoding neomycin resistance), between the regions of homology; and iii) a counter-selectable gene (such as that encoding thymidine kinase) outside the region of homology. Introduce this construct into embryonic stem cells and select those cells in which a homologous recombination event has occurred using the two selectable marker genes. Culture the resulting cells, and introduce them into the blastocoel of a several developing embryos. Introduce these into the uterus of a foster mother, and find those mice that have incorporated transgenic cells into the germline. Breed those mice to get progeny homozygous for the engineered mutation.

27) a) gridlock - aorta doesn't develop properly, resulting in poor blood flow. The wild-type Gridlock protein is involved in correct development of arteries.

b) lim1 – lacks head. The wild-type LIM1 protein is a transcription factor that is expressed in the organizer (Hensen’s node), and presumably is required to activate genes involved in inducing differentiation of anterior dorsal structures.

28) Stem cells are self-renewing cells that give rise to more of themselves as well as to other more differentiated cell types. They are the sources of new cells and tissues, both during development and during maintenance of a living organism. Interest in stem cells arises from the possibility that they may be cultured in vitro and then introduced into patients needing replenishment of particular cell types.

29) i) Make a knockout mouse lacking the gene for the signal (using method outlined in question 26 above). If the mutant fails to devlop mesoderm, then the molecule was necessary.

ii) Add the molecule to undifferentiated cells and see whether mesoderm forms. One could also make a transgenic mouse that expresses the gene for the signal ectopically, and see whether extra mesoderm forms.

30) i) To test whether the molecule is necessary, one could inject mRNA encoding a dominant negative version of the receptor for the signal and see whether this prevented mesoderm formation.

ii) To test whether the molecule is sufficient, one could inject it into an embryo and see whether it induces mesoderm, or one could add it to cultured animal cap cells.

iii) The molecule should also be present in cells that induce mesoderm (vegetal cells of blastula), which could be tested by in situ hybridization (to detect the mRNA) or immunolocalization (to detect the protein).

31) Several mutations may be needed before the cells actually become cancerous, and it may take some time for each of the needed mutations to accumulate in the same clone of cells.

32) a) Extra cell divisions.

b) Loss of contact with other cells and with basal lamina, ability to migrate and colonize other tissues. Cells break through the basal lamina and spread into the muscle layer.

c) Mutations

33) People with hereditary non-polyposis colon cancer (HNPCC) have a predisposition to develop cancer.

a) Are these people born with cancer? No

b) What biochemical deficiency do these people have? They have mutations in genes encoding proteins that are used to repair DNA mismatches.

c) Why do they develop cancer of the colon rather than of other tissues? Colon cells may be exposed more than other cells are to mutagens that cause base pair changes requiring repair by the mismatch repair system. They may also have different regulatory makeup from other cells that makes them especially susceptible to such damage. For example, they may be less good at halting the cell cycle before damage is repaired than other cells are. Finally, they may divide more than other cells, which would provide more opportunities for mutations to be passed on to daughter cells.

34) Early in development, mutant mouse embryos homozygous for a recessive mutation in the Rb gene develop lens cells that fail to differentiate properly, and instead proliferate (divide) too much. Many apoptotic (dying) cells are also seen in the vicinity of these proliferating cells. If the same mice are also mutant in the p53 gene, there is still excess cell division but apoptotic cells are not present.

a) Rb is a tumor suppressor gene. The product acts as a brake on the progress of cell replication. In its unphosphorylated state, it binds to the E2F transcription factor and prevents it from activating genes required for DNA replication.

b) In response to DNA damage, p53 prevents cell replication (by activating expression of the gene encoding the p21 CDK inhibitor protein), and it also induces cell death (apoptosis). In the rb mutant background, p53 apparently induces many of the lens cells to undergo cell death.

c) In a mouse lacking p53, immune cells will survive despite the DNA damage induced by X-rays because the p53 is not present to induce apoptosis.