Current Level

 

 

 Unit 1

 Unit 2

 Unit 3

 Unit 4

 Temp

 Genetics Ex

 

 

 

Previous Level

 

 

 

 BioWeb Home

 Modeling

 Seq Anal

 Theory

 Bioinformatics

 Mo Bio Lecture

 Mo Bio Lab

 

 

 Genetics Ex

 

 

Bioinformatics Exercise--- Genetics (BIO306)

 

 

The purpose of this lab exercise is to acquaint you with some of the publicly available biological databases and genome analysis tools.  Some of these databases and tools you have used in previous laboratory exercises (e.g. NCBI Blast, OMIM).  Herein, you will be presented with a whirlwind tour of the interface between biology and computers.

You are conducting research on the model plant, Arabidopsis thaliana, and have isolated a mutant defective in leaf development.  The leaves become severely lobed (Figure A, the wild-type leaf is on the left) and form stem-like structures on the leaf surface (Figure C), as shown below. 

knat1 

You have mapped your mutation to chromosome four in Arabidopsis, and you wish to isolate and characterize the affected gene.

1. Open two new browser windows to this page.

2. Go to The Arabidopsis Information Resource site (TAIR). You will need to open Map Viewer under the Analysis Tools heading. Use the Display box to view the Arabidopsis chromosomes individually.

How many chromosomes are there in Arabidopsis?

3. Change the Display box to Chromosome 4 to display a map of the chromosome. 

How large is chromosome 4 (cM or base pairs)?

Why do you suppose the recombination-based maps (listed in cM) are not all the same size?

For the purposes of this lab, you will only need to refer to the AGI (Arabidopsis Genome Initiative) map. Note the AGI map is green, other maps have different colors.

You know your mutation maps between the markers mi167 and nga8.  In the Search window at the top of the page, type in mi167 and search. To make things easier to view, zoom to 20X (on the left, under the AGI map). The green bar depicts the chromosome, and the red box shows the part of the chromosome displayed. To the right, the markers are listed under the base pair scale. Beneath the markers in the section labeled AnnotUnits (don’t ask me why it’s called AnnotUnits) you will find all the BAC (Bacterial Artificial Chromosome) clones used to determine the DNA sequence of Arabidopsis.  

Where is mi167 located (in Mb) on the chromosome?

Scroll back up and repeat the search, but this time use nga8. 

Where is nga8 located (in Mb) on the chromosome?

4. Using the black arrows above the base pair scale to navigate the chromosome, you can view the clones that make up the contig spanning the region between the two markers. 

What BAC clones make up the contig between markers nga8 and mi167?

 

5.  The planets align properly and finer mapping experiments indicate that your mutation is likely on BAC F9M13.  Unfortunately, BAC F9M13 is 101,644 bp long and likely contains many genes. 

How can you determine which DNA bases on the BAC correspond to your mutant gene?  How do you know where the promoters, ORFs, introns, etc. are in the BAC DNA sequence?  Is two hours long enough to find out if you do this by hand?  

 

 

6. Double click on the F9M13 tag and you will get information on the clone.  Other organisms that have extensive genomic sequencing projects have similar web sites (e.g. yeast, mouse, humans).  In the "sequence" area for this clone click on the genomic link under the Genbank Accession Sequence.  This takes you to the sequence file for BAC F9M13. Copy the DNA sequence from the sequence file to the clipboard (Yes, all 101,644 bp). 

7. Thankfully, there is a program that will predict (not always accurately!) the locations of all the genes (and ORFs) in the BAC (on both strands of DNA).  The program is called GENSCAN (http://genes.mit.edu/GENSCAN.html).  When you get to the GENSCAN site, make sure you change the organism from "vertebrate" to "Arabidopsis".  Paste you DNA sequence into the appropriate window and scroll down to click "Run GENSCAN". Be patient, this may take a minute!

How many putative (predicted) genes are on BAC F9M13?

How many genes are encoded on each strand of DNA (+ versus -)?

Which gene has the largest number of introns?

Which gene covers the longest stretch of DNA?

8.  Mutations that change the fate of cells are called homeotic mutations.  For example, the Antennapedia mutation in fruit flies causes legs to grow where the antennae should be.  Because of the change from leaf tissue to stem tissue you suspect that your mutation may be in a homeotic gene.  Such genes are kind of master control genes; they regulate the transcription of other genes by binding to specific DNA sequences, called homeoboxes.  

How could you determine which predicted peptides (from GENSCAN) might be homeobox or homeobox-like genes?

9. Your instructor will assign you some of the predicted peptides to run through BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) in order to determine the possible functions of the predicted peptides.  Keep in mind you will be doing protein-protein comparisons rather than DNA sequence comparisons.  Simply copy the amino acid sequence from GENSCAN into the BLAST sequence box.

Which predicted protein is most likely altered in the mutant Arabidopsis plant?

What is the name of this protein?

Does the GENSCAN predicted amino acid sequence exactly match the amino acid sequence predicted from the cDNA clone?  Why not (take a guess)?

Have similar proteins been cloned in other organisms?  Which ones?

How many homeobox genes are in Arabidopsis? (HINT: Go to Entrez Genomes at NCBI and search the Arabidopsis genome.)

10. The knat1 gene you have mutated is similar to knotted 1. To see if other organisms (like people!) have proteins similar to knotted-1 you return to the NCBI home page (http://www.ncbi.nlm.nih.gov/) and go into the "Entrez Home" program.  Use "knotted 1" as your query. Once you have run the search, click the Gene link.

How many loci were found?  In what organisms?

Choose the GeneID 5316 gene and click on the link.

What does the protein do?

 

 

11. You want to know under what conditions the human PKNOX1 gene is highly expressed and if its expression is suppressed under other conditions.  You would prefer not to have to do all the work yourself, so you check out the Stanford Microarray Database (SMD) at the following URL:  http://genome-www5.stanford.edu/MicroArray/SMD/.  You will be doing a Public Login.  As there are well over 15,000 total experiments, select human as your experimental organism before Displaying Data. 

How many experiments are there with human tissue?

Find experiment 2151 dealing with breast cancer.  By examining the Data from this experiment (under Options), you can see how different genes are expressed in breast tumor cells relative to normal breast tissue.  Currently, you are interested in the PKNOX1 gene.   Sort by "Log(base2) of R/G Normalized Ratio (Mean)"  in "Ascending" order and be sure to display "LocusID".  Because you are looking for LocusID number 5316, to save time in finding it, you should Display Rows 5001-5200.

12. Find PKNOX1 (5316) in the list and click "zoom".  This allows you to look at that spot in the microarray.  Not very interesting, is it?  Click on the spot to see if the array is just lousy.  The array will have your spot of interest outlined with a yellow square.  Note that there are other green and reddish-brown spots, so the array looks okay.  Click "Back" to get back to your spot, and click "view expression history".  This will show you the R/G ratio for every array in the database that has measured expression of this same PKNOX1 gene.  If expression varies by a lot in any array, the expression history will tell us.  Ignore the yellow bars, as the hybridization levels were too low to be reliable in these experiments.  Look at the green bars.

Does the expression of the PKNOX1 gene vary significantly (greater than fourfold) in any of the experiments? (Remember that the ratios are in Log(base2)). 

In which experiment(s) did gene expression increase significantly? 

In which experiment(s) did the expression decrease significantly?

SUMMARY:  Today you have become acquainted with some of the resources available to save you time and money when investigating particular phenotypes in any organism, including humans.  As more and more data becomes available, the demand for people who can exploit (interpret) the information in the databases will increase. You have also seen (hopefully) that proteins involved in cell patterning or differentiation can be conserved across species, and even kingdoms.  They may have different roles in each organism, but the basic protein domains are used over and over to provide great diversity in form and function of flora and fauna. (It’s all about the alliteration baby!)

 

 

 

 

 

uwsa_l5

 

  2002 The Board of Regents of the University of Wisconsin System.

Click here to email comments to Scott Cooper regarding this site or its links.