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PROBLEM 1. PERFORM AN ORF ANALYSIS OF THE LAC OPERON OF E. COLI.

The object will be to make a good guess as to the location of the four genes (lacI, lacZ, lacY, and lacA) of the E. coli lac operon in the operon sequence using the simple orf-scanning programs in DNA Strider and also using a Web site that searches for genes using a hidden Markov model of E. coli genes.

  1. Using DNA Strider, retrieve the lac genes from GenBank using the GenBank Accession no. J01636. If DNA Strider is not available, then answer the questions in steps 810.
    1. Start DNA Strider.
    2. Open a new DNA window.
    3. Open the E. coli lac operon sequence.
    4. Copy and paste the sequence from the browser window into the Strider window.
    5. Position the cursor at the first base in the sequence in the Strider window.
    6. Choose the "DNA" menu, choose "ORF Map," and then "6 frame" to show Met (1/2 bar) and Termination codons (full bar) in all six possible reading frames.
    7. Note where the longest ORFs are located on a piece of paper.
    8. If DNA Strider is not available, then examine Figure 9.1 and find the longest ORFs.
    9. Examine the GenBank features entry for J01636, looking for CDS (coding sequence).
    10. Compare the entries to your annotation.
  2. Genmark Web site
    1. Go to the Genmark Web site at Georgia Tech (http://opal.biology.gatech.edu/GeneMark/) or perform a Web search for GeneMark and paste in the lac operon sequence. If you have problems accessing the Genmark site, you can use the CDSB option on the Gene Finder location at the Baylor College of Medicine at http://searchlauncher.bcm.tmc.edu/seq-search/gene-search.html instead.
    2. Choose E. coli as the organism.
    3. Report the start and stop positions and lengths of the predicted ORFs and compare them to the rough locations found with DNA Strider. What differences (if any) exist between the Genmark output and the gene locations you predicted with DNA Strider?
  3. Note that the EMBOSS programs described in Chapters 3 and 12 may also be used to find the longest ORFs. The programs PLOTORF, GETORF, and SIXPACK may be used. However, this requires the instructor to have installed these programs and for students to have access to a UNIX or Linux server (see http://www.hgmp.mrc.ac.uk/Software/EMBOSS/Apps/index.html).




 

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