Phase 1

Each group of students will begin working with one pair of genes, first identifying T-DNA insertion mutants (if they exist), ordering seeds and designing PCR primers.  The three gene families will will work with this semester are nitrate reductase, alpha-amylase and beta-amylase.

Group 1 - Nitrate reductase (At1g77760, At1g37130)

Group 2 - alpha-amylase (At1g69830, At1g76130)

Group 3 - beta-amylase (At4g00490, At5g45300)
 

A.  Identify a knockout mutant

Go to the SIGnAL Arabidopsis Gene Mapping Tool on the Arabidopsis links page and scroll down below the map labeled Chromosome 1, type a gene name (e.g. At1g77760) into the box labeled "Gene Name" then press "submit".  When the screen refreshes look for your gene name in red above the blue chromosome bar.  Other genes are indicated by green arrows.  Notice that the arrows indicate the direction of transcription and the intron/exon structure of the genes.  Below the blue bar and drawn to scale will be a number of mapped entries for T-DNA insertions and sequenced cDNAs from various sources.  Look to the left to see what the colors represent.  Pink labels showing "Salk_#" are the mapped sequences adjacent to T-DNA-insertions from the Salk Institute.  The arrow above the number represents the sequence in the direction away from the point of insertion.  Notice where the base of the arrow is located with respect to the gene map above the bar.  Which insertions are more likely to result in effective knockouts?  Now click on one of the Salk T-DNA links to pull up information on it.  If no insertions have been mapped to the gene, then you are out of luck for now.

Study the information in the resulting pop-up window to glean any useful information.  Print this page for later reference.  What is the identity of the gene (TITL)?  Is the insertion in an exon (LOCA)?  Now click on the "[sec]" link to see the sequence adjacent to this insertion.  Save this sequence for later analysis.

B.  Pick Primers

Return to the SIGnAL Arabidopsis Gene Mapping Tool on the Arabidopsis links page and click on the link:  "5. To Design Verification Primers".  Read this page carefully to learn about verification of T-DNA inserts using PCR.  Now type your Salk_#s into the box and press "submit.  Print the resulting primer information and discuss ordering them with your instructor.
 

C.  Learn about your genes

Find the entries for both of your genes on each of the Arabidopsis genome annotation sites from the Arabidopsis links page.  Print the gene annotation pages and study them carefully.  How are the genes similar?  How are they different?  Has the cellular location of the protein they encode been predicted?  If not, use TargetP or  PSORT to look for targeting sequences.  How many other related genes are there in the Arabidopsis genome?
 

Proceed to Phase 2

1/22/03 Copyright (C) 2003, Jonathan Monroe, monroejd@jmu.edu. All rights reserved.
URL: http://csm.jmu.edu/biology/courses/bio455_555/phase1.html