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Building on and Integrating Previous Knowledge - by Ann Burgess
Week 13, April 13-17, 1998 Investigating the Cellular Functions of Human Breast Cancer Gene 1 (BRCA1): Controversial Beginnings.
Research Papers: Scully, R., Ganesan, S., Brown, M., DeCaprio, J.A., Cannistra, S.A., Feunteun, J., Schnitt, S. Livingston, D.M. (1996) Technical Comment: Location of BRCA1 in Human Breast and Ovarian Cancer Cells. Science 272:123-125. Chen, Y., Chen, P.-L., Riley, D.J., Lee, W.H., Allred, D.C., Osborne, C.K. (1996) Response: Location of BRCA1 in Human Breast and Ovarian Cancer Cells. Science 272:125-126.
Bishop, J.M. (1995) Cancer: The rise of the genetic paradigm. Genes and Development 9:1309-1315.
BRCA1 may encode a transcription factor. Describe: (1) the structural features of BRCA1 that led the authors to hypothesize that it is a DNA-binding protein, and (2) how a mutant gene for a transcription factor might cause the uncontrolled growth of breast epithelial cells. Describe how the authors characterized the cellular location of BRCA1 protein and explain why this is important. Explain the difference between monoclonal antibody and polyclonal antisera and tell how each is prepared. Compare and contrast the Chen group's findings with the Scully group's findings. Evaluate the differences. The ability to repeat reported results is a critical part of the scientific process. Be able to defend or refute the results reported in the Chen et al . (1995) research article. Study Guide The two review articles by Weinberg and Bishop give a synthesis of the converging themes in understanding cancer. Read these articles first. What is the mechanism common to all forms of cancer? Next read the Chen et al. research article and two related follow up notes. Be sure you understand the following terms and processes: Gene structure and function: transcription factor, exon, nuclear localization signal, intragenic mutation, zinc finger Cell and tumor biology: steroid hormone receptor, neoplasm, tissue, immunocytochemistry, phosphorylation/dephosphorylation, metabolic labeling using radioisotopes, lymphocyte, epitope.
Definitions Somatic and Germ Line Mutations - Read about the general properties of these mutations in the Principles of Genetics text. Loss of Heterozygosity (LOH) - This term refers to the loss of the usual heterozygous combination of maternal and paternal alleles of genes at any given region of a chromosome. During tumor formation, one allele may be lost by a deletion or inactivated by a point mutation (perhaps induced by an environmental carcinogen). The second allele may be inactivated in a similar way or lost by mitotic recombination. When this happens, the repair systems in the cell try to repair the loss (often along with flanking regions of the chromosome) by a copying the corresponding region of the homologous chromosome (which may itself be defective). Linkage analysis of kindreds - This type of analysis makes use of patterns of inheritance of markers throughout the genome. In linkage analysis, researchers determine whether any markers (already mapped genes, RFLPs, SSLPs) are consistently inherited along with the disease. If they do, they are said to cosegregate and the disease gene is assumed to lie somewhere in the area of the marker. (Note that this does not say the marker has anything to do with the disease.) It has been shown that BRCA1 is linked to more than half of all inherited breast cancers and 80% of families with both inherited breast and ovarian cancer. Polyclonal antibodies are prepared by injecting an animal with an antigen (such as a protein) and isolating the antibody fraction from the animal's serum several weeks later. These consist of a heterogeneous population of antibodies, each directed against a different part of the antigen and produced by different cells in the animal. Monoclonal antibodies, on the other hand, are homogeneous; each is directed against just one specific part of the antigen and is the product of a single clone of cells. Monoclonal antibodies are made by fusing an antibody-producing lymphocyte from an immunized animal with a tumor cell and then growing a large population of these hybrid cells ("hybridomas") in culture. Remember from Unit I that every antibody molecule consists of two sets of domains which have specific functions (See The World of the Cell and Biology texts for more details). Be sure you know the molecular weights of the polypeptide chains that make up an antibody molecule. Preimmune mouse serum is serum taken from a mouse prior to injecting it with antigen. Site-specific refers to tissues and could be reworded as tissue-specific.
Introduction (Note that this report has a brief introductory paragraph as opposed to a full Introduction Section.)
Results Figure 1
Two additional antibodies were used in the experiments found in Figure 1. In your opinion, was the use of these additional antibodies necessary?
Figure 2
What data is conspicuously missing from Figure 2A?
Discussion (Note that this report has a brief discussion consisting of only two paragraphs)
Be able to explain parallels between BRCA1 and steroid hormone receptor molecules.
Technical Comment and Response Regarding the Research Paper Scully, R., Ganesan, S., Brown, M., DeCaprio, J.A., Cannistra, S.A., Feunteun, J., Schnitt, S. Livingston, D.M. (1996) Technical Comment: Location of BRCA1 in Human Breast and Ovarian Cancer Cells. Science 272:123-125. Chen, Y., Chen, P.-L., Riley, D.J., Lee, W.H., Allred, D.C., Osborne, C.K. (1996) Response: Location of BRCA1 in Human Breast and Ovarian Cancer Cells. Science 272:125-126.
Why aren't these same heavily stained bands seen in Figure 1 of the Chen et al., report? How do the techniques used in these two figures differ from each other? In their reply to the technical comments of Sculy and coworkers, how did Chen et al.., directly address the problems associated with cross-reactivity of the antibody reagents and fixation procedures? What additional information was learned about BRCA1 location/mislocation in breast cancer cells as a result of the two technical comment articles?
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