Problem-solving test: E2F: A family of S phase regulating transcription factors

Authors


Abstract

Terms to be familiar with before you start to solve the test: transcription factors, gene induction, DNA synthesis, S phase, retinoblastoma, immunoprecipitation, radioactive labeling, oligonucleotide, enhancer element, polyacrylamide gel electrophoresis, autoradiography, gel retardation assay © 2013 by The International Union of Biochemistry and Molecular Biology, 42(1):88–90, 2014

Abbrevations
Rb

retinoblastoma

MCQ

multiple-choice question

The Experiment

Members of the E2F transcription factor family (E2F-1 to E2F-5) induce genes coding for proteins required to initiate DNA synthesis (these genes are often called S phase genes). In resting cells, E2F proteins are complexed with and inactivated by the retinoblastoma (Rb) protein. In cells entering the S phase E2F transcription factors are released from these complexes.

In the experiment described in this test, two members of the E2F family, E2F-1 and E2F-4, were studied in cultured cells. Nuclear extracts were prepared from cells in G0 phase and were immunoprecipitated with an anti-Rb antibody. The immunoprecipitate was incubated with a 32P-labeled double-stranded oligonucleotide containing the E2F enhancer region. The sample was then divided into three aliquots: no antibody was added to sample 2, whereas anti-E2F-1 and anti-E2F-4 antibodies were added to samples 3 and 4, respectively. Sample 1 served as a control that did not contain protein. Samples were then electrophoresed in nondenaturing polyacrylamide gels and autoradiography was performed (Fig. 1). (The technique used in this experiment, gel retardation assay, was described in an earlier test of this series [1].)

Figure 1.

Gel retardation assay of samples containing a radioactively labeled E2F enhancer oligonucleotide (the vertical arrow indicates the direction of electrophoresis; for details see the text).

Five-Choice Completion

This type of question consists of a question or incomplete statement followed by five suggested answers or completions. Select the one best answer.

  1. ____What was the aim of using a nondenaturing gel for electrophoresis?

    1. To prevent dissociation of oligonucleotide-protein complexes

    2. To prevent dissociation of E2F protein-antibody complexes
    3. To induce separation of the two strands of the oligonucleotide
    4. A and B
    5. A, B, and C

Four-Choice Association

(In this type of question a set of lettered headings is followed by a list of numbered words or phrases

Select:

  1. if the word or phrase is associated with A only;
  2. if the word or phrase is associated with B only;
  3. if the word or phrase is associated with A and B;
  4. if the word or phrase is associated with neither A nor B.)
  1. E2F-1 protein
  2. E2F-4 protein
  3. Both of them
  4. Neither of them
  1. ____It is present in band a.
  2. ____It is present in band b.
  3. ____It is present in band c.
  4. ____It is present in band d.

Figure Analysis

(The following statements are related to the information presented above. Based on the information given

Select:

  1. if the statement is supported by the information given;
  2. if the statement is contradicted by the information given;
  3. if the statement is neither supported nor contradicted by the information given.)
  1. ____All bands in Fig. 1 contain the E2F enhancer oligonucleotide.
  2. ____Binding of anti-E2F antibodies prevented the binding of the transcription factors to their enhancer elements.
  3. ____The cells contain an E2F transcription factor that is neither E2F-1, nor E2F-4.

Five-Choice Completion

(This type of question consists of a question or incomplete statement followed by five suggested answers or completions. Select the one best answer.)

  1. ____What conclusions can be drawn from comparing samples 1 and 2? At the time when the protein extract was prepared from them, G0 cells

    1. contained Rb protein

    2. contained E2F protein
    3. contained Rb/E2F complexes
    4. B and C
    5. A, B and C
  2. ____Which of the following statements could explain the identical image of samples 2 and 3? The cells

    1. did not contain E2F-1 transcription factor

    2. contained E2F-1, but it did not bind to Rb
    3. did not contain the Rb protein
    4. Both A or B can provide an explanation
    5. A, B or C can give an explanation
  3. ____Which conclusion can be drawn from comparing samples 2 and 4?

    1. About half of E2F-1 molecules was complexed to Rb protein molecules in the G0 cells

    2. All E2F-1 molecules were complexed to Rb protein molecules in the G0 cells
    3. About half of E2F-4 molecules was complexed to Rb protein molecules in the G0 cells
    4. All E2F-4 molecules were complexed to Rb protein molecules in the G0 cells
    5. Figure 1 does not give an answer to this question

Correct Answers

  1. D
  2. B
  3. B
  4. D
  5. D
  6. A
  7. B
  8. A
  9. E
  10. D
  11. E

Explanations

Gel retardation assay is a technique used to analyze protein-DNA complexes formed in vitro: radioactively labeled oligonucleotides containing specific protein-binding sites are incubated with protein extracts, complexes are resolved by electrophoresis and free (band d in Fig. 1; MCQ 5: D) and protein-bound oligonucleotides (bands a to c) are detected in the gel by autoradiography (MCQ 6: A). To preserve protein-DNA and protein-protein complexes, a low-salt buffer is used for electrophoresis (MCQ 1: D).

Protein-bound oligonucleotides are slowed down during electrophoresis (a phenomenon called electrophoretic mobility shift). The complex is even more retarded if a specific antibody binds to it (band a in Fig. 1; this phenomenon is called supershift). Band b was supershifted by anti-E2F-4 to band a (MCQ 2: B; MCQ 3: C), E2F-4 is thus present and is complexed to Rb in G0 cells (MCQ 7: B; MCQ 9: E). (It is, however, not possible to tell from the results whether all E2F-4 molecules were bound to the Rb protein at the time of immunoprecipitation/MCQ 11: E/.) In contrast, the anti-E2F-1 antibody did not cause supershift, the cells therefore did not contain E2F-1/Rb complexes (MCQ 10: D). These observations also indicate that band c is the result of the binding of an E2F protein, other than E2F-1 and E2F-4 to the oligonucleotide (MCQ 4: D; MCQ 8: A). (This test is based on a figure in the 2006 Catalog of Santa Cruz Biotechnology/Santa Cruz, CA, USA.)

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