PLNT3140 INTRODUCTORY CYTOGENETICS

Assignment 2

This assignment is worth 5% of the course grade.

Due Tue. Oct. 22.

1. (2 points) A map of a human gene is shown below. The naturally-occurring gene, in a, has Matrix Attachment Regions (MARs) on either side of the gene. When transformed into Drosophila, this gene inserts into the chromosomes and transcribes properly, as indicated by the fact that the mRNA is detected in qPCR*. Recombinant genes were created, in which an additional MAR was added. In b, a MAR was inserted into the intron, and in c, a MAR was inserted into an exon. These sequences were also transformed into Drosophila. However, no mRNA transcripts for the b or c construct were detectible. Explain this result. (P stands for promoter. PCR primers are shown in solid arrows.)

(Hint: The answer has nothing to do with RNA splicing (b) or interruption of the reading frame during translation. (c). )

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*qPCR - A method for detecting mRNA transcripts for a specific gene, using carefully-controlled PCR conditions that make it possible to quantify the relative amounts of the transcript in an RNA sample.
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2. (1 point) The following constructs were made, using either human MARs or Drosophila MARs:

The expression of these recombinant constructs in Drosophila is indicated at right. What do these experiments tell you?

3. (2 points) So far, we haven't said anything about whether the orientation of MARs is important. For example, suppose that a MAR in the arbitrarily-chosen R (right) orientation looked like this:

                          ------------->
           5'----------------GGCTCCAAT-------------------3' 
           3'----------------CCGAGGTTA-------------------5'

and in the L (left) orientation looked like:

           5'----------------ATTGGAGCC-------------------3' 
           3'----------------TAACCTCGG-------------------5' 
                           <------------

Draw recombinant constructs, similar to 1. a above, that would tell you whether the orientation of MARs is important. Be complete, ie. make sure you have all constructs necessary to test your hypothesis or hypotheses. You may ignore the trivial hypothesis that assumes that orientation is unimportant, which predicts expression with all constructs. If you assume that orientation does make a difference, which constructs will show expression of the gene, and which do not?

Submitting your assignment

Use the template report file as2_report_template.docx to answer the questions.
Make sure your report includes your name, student number and UM email address.
Save your assignment report as a PDF file, and upload it to the PLNT3140 UMLearn dropbox site in the Assignment 1 folder. Files in word processing formats (.doc, .docx, .rtf, .odt) are NOT acceptable.

Note on grading: In assigning a grade, some consideration may be given to how the answer communicates your ideas. Keep in mind the following:

use of precise and appropriate biological terms
organization of ideas in your answer, so that a clear chain of logic is apparent. Formatting tools such as s subheadings or bullet points help to show the reader the structure of your ideas
ambiguity - Try to read your answers from the viewpoint of your reader. Are there several possible ways to interpret what you have said?
Feel free to use figures or tables in cases where they will be useful get across your ideas.

Notes: If you have questions, it may help to send me a message at frist@cc.umanitoba.ca.