Answers, problem set A

1) Transcription factors - regulate transcription of target genes, and thereby determine what genes a cell expresses and therefore what proteins it has.

Promoters of genes - have binding sites for transcription factors, and therefore determine whether a particular gene will be expressed in a cell in which the corresponding transcription factors are active.

Cytoskeleton – required for cell movements that sculpt embryo during gastrulation, and for localizations of molecules that may confer polarity (asymmetry) on cells.

Cell surface proteins - include receptors that perceive extracellular signals, and adhesive molecules that keep cells in correct place.

2) a) probe b) substrate

i) Southern blot labelled DNA DNA on blot

ii) Northern blot labeled DNA RNA on blot

iii) Western blot antibody protein on blot

iv) in situ hybridization labeled RNA mRNA in tissue

v) microarray labeled cDNA arrayed DNA molecules

3) Mammal egg cells are arrested in metaphase of meiosis II before fertilization

4) Put the following events associated with fertilization in the correct sequence:

meiosis

binding of galactosyltransferase to the ZP3 protein

the acrosomal reaction

fusion of egg and sperm cell membranes

release of cortical granules to the zona pellucida

(meiosis II finishes after fertilization in many animals)

mitosis

5) a) when a sperm encounters the zona pellucida of an egg - acrosomal reaction

b) once a sperm encounters the egg plasma membrane - sperm/egg fusion

c) upon fertilization of the egg by a sperm - cortical reaction

6) a) Ca⁺⁺ ions will induce the cortical reaction and prevent sperm entry

b) EGTA will counteract the effect of Ca⁺⁺ ions and thereby allow sperm entry

c) IP₃ will cause Ca⁺⁺ release and thereby prevent sperm entry as Ca⁺⁺ does

7) i) A frog skin cell normally makes only more skin.

ii) The blastomere may give rise to one of several tissue types (ectodermal, endodermal, etc.), depending on what part of the embryo it was taken from. The specification map indicates which parts of the blastula will give rise to which sorts of tissues.

iii) An oocyte with a nucleus from a skin cell will develop into a blastula but no further (unless the nucleus is serially transferred to another oocyte after several cell divisions).

iv) An oocyte with a nucleus from a blastomere can develop into a tadpole or frog.

v) A skin cell with a nucleus from a fertilized egg will probably still only make skin.

b) It appears that skin cells can only make skin, although their nuclei can be reprogrammed to other fates if placed in the context of an oocyte. However, complete reprogramming of skin cell nuclei (allowing them to direct development of the whole organism) apparently requires serial transfer. Particular blastula cells also have limited potential (although not as limited as skin cells). Blastula nuclei can be reprogrammed to produce all cell types by placing them in an oocyte.

c) Correct development requires both the correct oocyte cytoplasm as well as a competent nucleus from a cell that is not too differentiated (i.e. from a blastomere but not from a skin cell).

8) a) Dolly resembles the parent that contributed the nucleus.

b) To clone a male sheep, obtain the donor nucleus from a ram.

9) Take a cell from the horse you want to clone, and fuse it with a horse oocyte that has had its own nucleus removed. Activate the “zygote” with an electric pulse. Implant the activated zygote into the uterus of a foster mother horse, and wait for a birth. The cloned horse will share genes with the nuclear donor, but will only run as fast if it is trained properly, i.e. has the appropriate environmental influences.

10) Neurons should express a different set of genes from skin cells (as well as some of the same genes. Some gene expression differences should be present before the cells look different. To distinguish these, you would need to use a detection method for expression of a diagnostic gene or genes, for example an RNA hybridization (northern blot) using a neuron-specific gene as a probe.