Techniques in Molecular Biology

VTMC 831



VTMC 831
Introduction Exercises Prerequisites Schedule Guest Lectures Useful Web sites References

  1. Module 1: “Molecular boot camp” – basic laboratory and research skills:
    • Best laboratory practices
    • Searching the literature
    • Units and conversions
    • Measurement of nucleic acids
    • Buffers and pH
    • Basic statistics and experimental design
  2. Module 2: Recovery of coding sequences for a gene from several species (human, horse, dog, rat, little brown bat),
    • Determining DNA sequence and comparison and analysis of sequences
    • Purification of protein, RNA and DNA from cultured cells (to be used in modules 3 and 4 as well)
    • Design of primers to extract sequences by PCR
    • Polymerase chain reaction (PCR)
    • Purification, cloning and sequencing of PCR products
    • Determining sequence of PCR products and cloned inserts (using NRC service)
    • Searching amino acid sequence of protein for structural and functional domains
    • Comparison of sequences by CLUSTAL and interpretation of results
    • Cloning coding sequences in a mammalian expression vector and detection by immunofluorescence of protein produced .
  3. Module 3: To determine if a treatment increases the expression of a gene (transcripts)
    • Determining relative amount of transcripts of genes by quantitative real-time PCR (qRT-PCR)
    • qRT-PCR – applications, limitations and pitfalls
  4. Module 4: To determine if a treatment increases the level of a protein
    • Separation of polypeptides by SDS-PAGE and detection of specific proteins by immunoblotting
  5. Module 5: To determine if the putative nuclear factor binding site (NFBS) of the protein Zhangfei is required for its effect on estrogen-induced growth of breast cancer cells.
    • Establish that Zhangfei has an effect on estrogen-induced growth of the cells
    • Change the putative NFBS in Zhangfei by site-directed mutagenesis
    • Assess effect of mutation on ability of ZF to suppress transcriptional activation by Zhangfei
  6. Module 6: Detection of DNA sequences in complex samples using bead-based hybridization.

Introduction to Modules 2-5:

Several years ago (7) our laboratory discovered a human neuronal protein that appears to regulate the ability of herpes simplex viruses (viruses that cause "cold sores", among other problems) to become latent in neurons. We called this protein Zhangfei. Zhangfei (ZF) belongs to the basic leucine-zipper class of proteins and it can activate (4) or suppress the transcription of other genes (5,6). Recently we also discovered that ZF may play a role in neuronal differentiation (2,3) and may induce tumour cells derived from neuronal precursors to differentiate or commit apoptosis (1). Your adventure in VTMC 831 will follow from these observations. 

1. The effect of Zhangfei on the unfolded protein response and growth of cells derived from canine and human osteosarcomas. Bergeron, T., Zhang, R., Elliot, K., Rapin, N., MacDonald, V. Linn, K., Simko, E. and Micra, V. Vet Comp Oncol. 2012 Jan 15 doi:10.1111/J.1476-5829.2011.00310x

2. Zhangfei induces the expression of the nerve growth factor receptor, trkA, in medulloblastoma cells and causes their differentiation or apoptosis.

Valderrama X, Rapin N, Verge VM, Misra V., J Neurooncol. 2009 Jan;91(1):7-17.

3. Zhangfei, a novel regulator of the human nerve growth factor receptor, trkA. Valderrama X, Rapin N, Misra V. J Neurovirol. 2008 Oct;14(5):425-36.

4. Novel Brn3a cis-acting sequences mediate transcription of human trkA in neurons. Valderrama X, Misra V. J Neurochem. 2008 Apr;105(2):425-35.

5. Cooperative interaction of Zhangfei and ATF4 in transactivation of the cyclic AMP response element. Hogan MR, Cockram GP, Lu R. FEBS Lett. 2006 Jan 9;580(1):58-62.

6. The neuronal host cell factor-binding protein Zhangfei inhibits herpes simplex virus replication. Akhova O, Bainbridge M, Misra V. J Virol. 2005 Dec;79(23):14708-18.

7. Zhangfei is a potent and specific inhibitor of the host cell factor-binding transcription factor Luman. Misra V, Rapin N, Akhova O, Bainbridge M, Korchinski P. J Biol Chem. 2005 Apr 15;280(15):15257-66.

8. Zhangfei: a second cellular protein interacts with herpes simplex virus accessory factor HCF in a manner similar to Luman and VP16. Lu R, Misra V. Nucleic Acids Res. 2000 Jun 15;28(12):2446-54.

Papers on Zhangfei/CREBZF/SMILE from other labs:

Curcumin differentially regulates endoplasmic reticulum stress through transcriptional corepressor SMILE (small heterodimer partner-interacting leucine zipper protein)-mediated inhibition of CREBH (cAMP responsive element-binding protein H).
Misra J, Chanda D, Kim DK, Li T, Koo SH, Back SH, Chiang JY, Choi HS.
J Biol Chem. 2011 Dec 9;286(49):41972-84. Epub 2011 Oct 12.

Amino acid deprivation induces CREBZF/Zhangfei expression via an AARE-like element in the promoter.
Zhang Y, Jin Y, Williams TA, Burtenshaw SM, Martyn AC, Lu R.
Biochem Biophys Res Commun. 2010 Jan 15;391(3):1352-7. Epub 2009 Dec 22.

Transcriptional corepressor SMILE recruits SIRT1 to inhibit nuclear receptor estrogen receptor-related receptor gamma transactivation.
Xie YB, Park JH, Kim DK, Hwang JH, Oh S, Park SB, Shong M, Lee IK, Choi HS.
J Biol Chem. 2009 Oct 16;284(42):28762-74. Epub 2009 Aug 18.

Molecular characterization of SMILE as a novel corepressor of nuclear receptors.
Xie YB, Nedumaran B, Choi HS.
Nucleic Acids Res. 2009 Jul;37(12):4100-15. Epub 2009 May 8.

SMILE, a new orphan nuclear receptor SHP-interacting protein, regulates SHP-repressed estrogen receptor transactivation.
Xie YB, Lee OH, Nedumaran B, Seong HA, Lee KM, Ha H, Lee IK, Yun Y, Choi HS.
Biochem J. 2008 Dec 15;416(3):463-73.

Identification and characterization of the DNA-binding properties of a Zhangfei homologue in Japanese pufferfish, Takifugu rubripes.
Cockram GP, Hogan MR, Burnett HF, Lu R.
Biochem Biophys Res Commun. 2006 Jan 27;339(4):1238-45. Epub 2005 Dec 6.

Cooperative interaction of Zhangfei and ATF4 in transactivation of the cyclic AMP response element.
Hogan MR, Cockram GP, Lu R.
FEBS Lett. 2006 Jan 9;580(1):58-62. Epub 2005 Dec 6.

April 30, 2012

Vikram Misra Dept. Veterinary Microbiology Western College of Veterinary Medicine University of Saskatchewan