genetics

Protocol 2: Details on Your Gene of Interest

Purpose: work with data from a few sources to collect and organize some of the details about the isoforms of your gene of interest.

1. Collect the specifics about the differentially expressed and spliced isoforms from the Planells paper, from which you chose your gene.

a. Open the accompanying excel file (posted in D2L) called “Redacted_additional_file_7.xlsx”. This file is a n edited version of the “Additional File 7” that was prepared by the authors and available by a link to the online paper. Note that this is becoming more common for papers that analyze a lot of data, which would be too much to embed within the test of the publication. Your instructor has trimmed the file to just what is needed for this project. You are focusing on genes that are differentially express and alternatively spliced in the genital ridge at day E11 of mouse embryonic development, right before differentiation of that tissue into testes or ovary.

b. Once you open the file, you will see that there are many genes in the file. You will have to search for the information for your gene. The information in this file will give you the information for portions of the tables at the end of step 1. (labeled green)

c. To search for your data, you will need to “enable editing” if editing is disabled (this will allow you to copy and paste from the table cells, rather than try to re-type the information).

d. Click in box A1 so that you will search the entire table. Open a “find” box by clicking the “Find and Select” in the tool bar or use control^F. Type your gene name into the box and select “find next.” If you get a message that it could not be found, confirm that your cursor starts in box A1. You will now be in the first row where the information for your first isoform is located. Capture the necessary data for the table, and repeat until you get the information for all isoforms. (Most people will have only 2 isoforms, one “male up” and one ”female up”)

Table: Details on _____________________(your gene of interest)

Feature	Mouse	Human
Gene name
Gene stable ID
Chromosome
Refseq mRNA ID
Description

Table: Features of _______________ gene expression in the genital ridge of Mus musculus at day E11

Feature	Increased expression in Males	Increased expression in Females
Transcript name
Ensembl Transcript ID
Transcript start
Transcript end
Transcript length
Number of exons
Number of amino acids

2. Now that you have the green components of the tables filled in, calculate the transcript length for each isoform and fill that in the table (yellow boxes)

3. In order to find the number of exons and the number of amino acids, you will need to go to http://www.ensembl.org/Mus_musculus/Info/Index and copy/paste your Ensembl transcript ID into the search box.

a. When you get your results, choose the first one that has your search term included and highlighted.

b. You will get a lot of information here.

First, snip and save the exon-intron diagram provided (circled in red). Save it in your notebook with the specific transcript name, so you can find it again later for your poster. Copy the number of exons into your table for this transcript (blue box)

c. Next, Click on the box called “Show transcript table” (circled in blue).

From this table you can complete the blue boxes on the table above.

Notice that from this table, you can directly go to your other transcript isoform -- or you can go back to the search bar at the beginning of step 3 and repeat this. Be sure to capture the exon-intron structure of the second isoform also.

In this table you can explore a lot of interesting elements. For example, I can directly see that both of my isoforms (202 and 203) have the same number of amino acids, but different number of bp. Also, if you click on the “UniProt” link, you will see a wealth of information about your gene/isoform, stored in another database.

d. After you have completed the second table, there is still more information to be gathered at this site. Hide the transcript table and go to the menu at the left side. Click on “Protein” under the heading “sequence”

Here you should copy and paste the sequence into a word document or click on “download sequence”

NB: Save the file with a name that is specific for the isoform, and place a copy in your online notebook (dropbox). Also, when you want to open the file again, you will have to open Word first, then open the file from inside word. After that, you can save it as a word doc.

e. Alignment and regulation: Click on the Gene:(name) tab circled in red above the protein sequence figure. From here, scroll down and look over the alignment diagram. This is similar to the alignments that we did in part 1 of the course. Notice at the bottom, below the alignments are boxes indicating regulatory elements. Are any of these in regions that are differentially spliced in your isoforms? If so, record your observation and use it in your final discussion on your poster.

f. Finally, on the menu at the left, click on “Gene expression” Here you can see a summary of gene expression experiments in adult tissues. How does this compare to what you know about your gene’s expression at day E11 (embryonic day 11)? Toggle the information shown between adult male and female by clicking the symbols at the left. Is the gene still expressed in sexually differentiated tissues? Note your observations and use it in your final discussion.

Save a copy of your tables so far in your online notebook. We will get the human data in another protocol.

So- from this lab, you should have at least 3 more types of items in your notebook, likely 4:

1- exon-intron snipped/screen-shott figures 2- protein sequence files 3- tables with data 4- (hopefully) written notes of your observations for use in your results/conclusions.