Fourth Homework Assignment Drawing Restriction Maps Predicting Gel Electrophoresis Results Based on Maps Predicting and Interpreting Results of Southern Analyses Example: chicken genomic digest probed with chicken ovalbumin cDNA fragments Introduction The chicken ovalbumin gene was one of the very first genes in a higher eukaryote in which intervening sequences were found. Workers in the late 1970s and early 1980s used results from Southern blotting to formulate the hypothesis that the chicken ovalbumin gene contained intervening sequences. They designed additional Southern blotting experiments to further test the hypothesis. Finally, when both the cDNA and the genomic DNA had been sequenced, the investigators were able to confirm their results directly. These exercises include the Southern analyses the ovalbumin investigators might have done. Learning Objectives: Analytical skills *Using annotations provided by GenBank, you will be able to locate important features of a gene on the sequence. *Given restriction maps and/or cut site data, you will be able to draw expected agarose gel results. *Given a genomic DNA Southern blot of a predicted genomic digest, be able to show where on the blot you would expect to see signal with a full-length cDNA probe. *Given a genomic DNA Southern blot of a predicted genomic digest, be able to use selected probes from within a cDNA to help make an accurate genomic restriction map or to place the coding regions on a genomic map. GUIDED ACTIVITY I: Using the computer to map and locate the exons in the chicken ovalbumin gene. I.1. On a separate sheet of paper, draw a linear restriction endonuclease map to scale, using your results from NEBcutter for the chicken ovalbumin gene (not the cDNA). Use a scale of 1 cm = 500 bp. a. long. For the chicken ovalbumin mRNA, the line was: For the gene, the line will be: 1873/500 = ________ cm _____/ 500 = ________cm long. b. Label the 5’ end of the map “1” for the beginning nucleotide number and label the 3’ end with the appropriate ending nucleotide number. c. On the map, use your ruler to position the cut sites for each of the following enzymes, as appropriate according to your printouts. Bam HI, EcoRI, HhaI, Hind III, PstI, XbaI, NotI d. I.2. a. You should have five Eco RI sites on the genomic map, resulting in six fragments. Number those six fragments 1-6, from left to right. Remember to number the fragments, not the cut sites. Use your computer printout of information and sequence of the ovalbumin gene from the First Homework Assignment. i. On the printed sequence, highlight the exons. Note: the annotation has a historical error regarding the first two exons. Exon 1 runs from 1343-1390. Exon 2 runs from 2979 – 3163. The annotations for the remaining Exons are correct. You should wind up with 8 exons. ii. Draw a vertical slash through the sequence to show the transcription start site. (What nucleotide number is it?) iii. Circle the start codon. (What nucleotide numbers form the start codon?) iv. Put a square around the stop codon. (What nucleotide numbers form the stop codon?) b. Highlight the regions of exons on the map you drew in #1 above. Be sure to work to scale. c. Using the sequence information, the knowledge of the location of the exons in the gene, and your results from NEBcutter for both the cDNA and the gene, i. Locate the PstI site in the genomic sequence that corresponds to the PstI site in the cDNA. Draw a box around it in the genomic sequence. ii. Locate and draw a box around the Xba I site in the genomic sequence that corresponds to the Xba I site in the cDNA. d. Answer the questions below based on your work so far. i. Why are there no EcoRI sites in the cDNA sequence even though there are several EcoRI sites in the gene sequence? ii. Why are there no Hind III sites in the cDNA sequence? GUIDED ACTIVITY II: Using Southern analysis to demonstrate the existence of introns and to map genomic restriction fragments. You will not be able to answer this correctly if you have not understood and completed #I.1 and #I.2 above. You are going to reconstruct results that investigators in the late 1970s got from gel and blot hybridization analysis. They did not yet have sequence! II.1. Fill in all but the last column of the attached Table, using the map you drew in #1 above, the cDNA map you drew last time, and the information you have from your marked up computer printout of the ovalbumin gene sequence. Note that the table is about the ovalbumin gene, not the cDNA. II.2. In Lane 1 of the diagram below, sketch the positions in the gel at which you would expect EcoRI fragments 1-6 to be located if you had digested complete chicken genomic DNA. (Remember that if you were actually to run a gel of digested chicken genomic DNA, it would appear as a continuous smear because there would be so many other fragments in addition to the ones containing the sequence of the ovalbumin gene.) Remember that if you had actually digested chicken genomic DNA, you would not know the exact positions of Fragments 1 and 6, because you do not know the location of the next EcoRI site to either side of the mapped region. Put each of these two fragments in any logical place. Reproduce the diagram you made in Lane 1 in Lanes 2-4. At this point, Lanes 1-4 should look identical. In each lane, as specified below, circle the fragments that would be positive after hybridization with the cDNA probe mentioned. Use the table you filled out to help you. Lane 1 – the full-length cDNA Lane 2 – the cDNA fragment 5’ of the Pst I site Lane 3 – the cDNA fragment located between the PstI and Xba I sites Lane 4 – the cDNA fragment located 3’ of the Xba I site. 1 1 2 3 4 10,000 bp 7,0001 4,000 1,000 250 Analyses of Southern Data Imagine that you are one of the early investigators of the ovalbumin cDNA and gene. You have restriction enzyme and gel electrophoretic analysis of ovalbumin cDNA, and you have Southern blotting data of chicken genomic DNA probed with the ovalbumin cDNA and its fragments, but you have no sequence yet. In other words, you have only the experimental information from the gel electrophoresis of the cDNA from the third homework assignment and the Southern blotting and probing of the genomic DNA as you did it on the previous page. II.3. Fill in the last column of the table, numbering the fragments in the order in which you would expect them to appear in the gel. You will have one or more fragments for which you can’t be certain of the order. Note any such fragment with a “?”. II.4. Explain why the data shown in Lane 1 of the Southern blot and the data from restriction enzyme digests and gel electrophoretic analysis of the ovalbumin cDNA (last time’s work) would lead you to make the hypothesis that the ovalbumin gene must contain intervening sequences. II.5. Explain how the results in Lanes 2-4 of the Southern blot further support the hypothesis. II.6. Explain in words and with a diagram how your cDNA RE map and your Southern results would together allow you to partially determine the order of the genomic ovalbumin restriction digest fragments in the gene without knowing sequence. Note that the answer to this question requires absolutely no knowledge of sequence! Table. Analyzing EcoRI digestion and Southern blot analysis of the chicken ovalbumin gene Fragment number 1 2 3 4 5 6 Length of fragment in base pairs > 1731 458 486 2362 1806 > 2363 Is the 5’ end of the fragment in an intron? Is any exon sequence present in the fragment? (Yes/No) (Yes/No) Not applicable Southern blot hybridization analysis of total chicken DNA digested with EcoRI Try to number To which of the genomic fragment(s) will each cDNA the fragments probe hybridize? in the order of (+/-) their Full length Fragment 5’ of the Pst I site Fragment between the PstI and XbaI sites Fragment 3’ of the Xba I site appearance in the gel from top to bottom. Use “?” for unknowns.