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MOLECULAR SWITCHES IN THE LIVER

We have seen how RNA polymerase and general transcription factors assemble on a promoter to transcribe a mRNA.  In this section we will explore how this process is regulated by the DNA itself and other transcription factors and signalling molecules.

 

Role of DNA and chromatin structure

One question that puzzled many scientists in the 1980s was how proteins binding to one region of DNA could regulate transcription of genes 100s-1000s of bases away.  This paper elegantly addresses this question.  (Note:  only portions of the paper are shown)

In vivo DNA loops in araCBAD: Size limits and helical repeat.  Dong-Hee Lee and Robert F. Schleif.  PNAS 86, 476-80 (1989).

1.  What questions are the authors trying to answer about size limits and helical repeats?  What is the general design of their experiment?

2.  How do the authors increase the distance between the two AraC binding sites (araI and araO₂)?  What is the purpose of the enzyme BstEII?  The E. coli DNA fragments?  The short oligonucleotides?

3.  How did the authors decrease the distance between the two AraC binding sites (araI and araO₂)?  What was the reason for using BAL-31 exonuclease?

4.  When AraC binds to both the araI and araO₂ sites what affect does this have on the araCBAD promoter (P_BAD)?

4.  What is the average number of bp per turn in normal DNA in solution?  What periodicity do the authors find in vivo? 

5.  What interesting observation do the authors make in figure 3?  Using this data, suggest a mechanism by which proteins binding to one region of DNA could regulate transcription of genes 100s-1000s of bases away.  (Hint:  look at figure 1).

Answer

Proteins regulating transcription

One way to study the role of a gene in an organism is to delete the entire gene in a mouse and examine the phenotype of the animals.  Such animals are call transgenic animals, or gene knock-outs.  We will go into how this is done later, for now assume that when they say genotype +/+ they mean a mouse with both copies of the indicated gene, +/- is a heterozygote and -/- is a homozygous mouse which has lost both copies of the indicated gene.  Null ES cells refer to cells from these mice.

Article:  Regulation of a Transcription Factor Network Required for Differentiation and Metabolism Stephen A. Duncan, * M. Angeles Navas, Daniel Dufort, Janet Rossant, Markus Stoffel. Science, 281:5377, 692-5, (1998)

1.  RT-PCR is reverse transcriptase PCR.  What will the authors be detecting using this technique? 

What type of genes are being knocked out? 

What is a hepatocyte?

2.  In figure 2 which gene has been knocked-out?  Which genes appear to be regulated by this protein?  Does it appear to be a positive or negative regulator?

3.  In figure 3 which gene has been knocked-out?  Which genes appear to be regulated by this protein?  Does it appear to be a positive or negative regulator?

4.  Comparing the HNF-3a and HNF-3b bands in both figures 2 and 3, explain the presence or absence of each band in the (-/-) mice.

5.  Draw a diagram of how HNF-3a and HNF-3b may regulate the expression of genes in a cell.