April 27, 1992 TIME: 8:00 - 10:00 A M



(20) 1. Discuss the properties of the basic helix-loop-helix (bHLH) group of proteins, focussing on the prototypical bHLH protein, MyoD. Include in your answer how MyoD was discovered, its biochemical and functional properties, and its putative role in myogenesis, both in tissue culture cells and in the embryo.

(5 ) 2. What is Id?

3. We are now going to consider the possible role of Id in myeloid differentiation. Myeloid cells are erythrocytes, platelets, granulocytes, and monocytes; they are produced in the bone marrow and spleen of the adult. Where are erythroid cells produced in the embryonic and fetal human?


These experiments are going to utilize 32DC13(G) cells, which are hematopoietic precursors of granulocytes. 32DC13(G) cells are dependent upon interleukin-3 (IL-3) for the in vitro maintenance. They will differentiate into granulocytes after treatment with granulocyte stimulating factor (G-CSF). The concentration of Id mRNA was measured by RNA blot analysis. In the figure shown below, basal refers to RNA isolated from cells growing exponentially in the presence of IL-3. The numbers above the lanes refer to the number of hours of treatment with G-CSF. Given below the lanes are the relative intensities of the Id signals. Describe the technique, the controls used, and interpret the results obtained.

(Figure inserted here)


Basic HLH proteins bind to a consensus sequence known as:


An oligo corresponding to this sequence was synthesized and incubated with nuclear extracts of 32DC13(G) cells growing in IL-3 (basal) or incubated with G-CSF for the number of hours indicated above the lanes and a mobility shift assay was conducted. Describe the technique and interpret the results (the unbound oligo is not shown here). What is the sequence of the oligo? What controls would you use to examine the specificity of these results?

(Figure inserted here)


32DC13(G) cells were transformed with an Id gene under the control of a constitutive promoter. When nuclear extracts of these cells were used in mobility shift assays, the following results were obtained. Interpret the results.

(Figure inserted here)


When the differentiation properties of the transformed cells were examined, the following results were obtained (expressed as percentage of cells). From these results, propose a role for Id in differentiation of granulocytes.

(20 )

Cells in IL-3 Cells in G-CSF
Cell Line Undifferentiated Granulocytes Undifferentiated Granulocytes
Control 100 0 0 100
Id transformed 100 0 100 0

(5 ) 4. Discuss the experiments that have led to the discovery of the genetic basis for testis determination in mammals.