Centre Education Program: Rural Joules Project

Gene Analysis Project

1. Investigate an Arabidopsis gene from TIGR.

Use the locus name query with the locus name At5g10400 or At3g45930

  • Investigate where these genes are found on the Arabidopsis chromosomes, enter the locus name into the query window.

2. Using the TIGR site collect the CDS sequence of the gene

Copy the DNA sequence and translate it to a protein sequence using in silico translation tools such as:

  • Bioblundel
  • In-silico (choose the frame for the translation which produces the longest protein sequence)
  • copy translated sequence to use in further applications

3. Blast the protein sequence to find similar sequences.

  • Use Basic local alignment sequence tool (BLAST) at NCBI, choose protein-protein Blast.
  • Enter the protein sequence for your histone into the search window
  • click on BLAST!
  • when new window opens click on Format! and wait for results.

4. Are there similar sequences in the genomes of other organisms?

  • Investigate your blast output looking for hits from other plant and animal genomes.
    • e.g. Triticum aestivum = wheat
    • Oryza sativa = rice
    • Zea mays = maize
    • Chlamydomonas reinhardtii = green algae
    • Glycine max = soybean
    • Mus musculus = mouse
    • Homo sapiens = human
    • Drosophila melanogaster = fruit fly
    • Caenorhabditis elegans = worm
  • Investigate the hits by clicking the active link. To extract protein sequences from the NCBI window that opens choose FASTA in the 'display' window and then copy the protein sequence.

5. How similar are the protein sequences from different species that you found using the blast search?

  • In a new web window open the multiple sequence alignment tool ClustalW, choose protein alignment.
  • Paste in the original Arabidopsis protein sequence (the sequence you translated). Note: It is important that you paste in the sequences in FASTA format for this to work, your entry should look like:
    >Arabidopsis gene naming information
    MARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRFRPGTVALREIRKYQKSTE
    LLIRKLPFQRLVREIAQDFKTDLRFQSSAVAALQEAAEAYLVGLFEDTNLCAIHAKRVTI
  • Add sequences from other organisms that you identified using the Blast search by coping the sequences and pasting each sequence (just choose a couple from a range of species).
  • You will need to keep the Blast window open and open each new sequence in a new window.
  • If you copy the sequences in FASTA format (described in pt 4) you should be able to paste each straight from the Blast window in the correct format.
  • Excute multiple sequence alignment. Examine the multiple sequence alignment output.
  • How similar is the Arabidopsis sequence to the human sequence?

6. What do these protein encode?

Investigate the information on the TIGR and TAIR websites.

7. Where and when is your gene expressed in the Arabidopsis plant?

  • Explore the wealth of microarray data available on line via Genevestigator (you will need to enter an email address).
  • Explore the mRNA expression associated with the genomic loci around these genes using the SALK tiling array database.
  • Investigate if mutant lines are available with these genes interrupted by a T-DNA insertion using the SIGnAL T-DNA gene mapping tool.

 


The DNA is tightly wound around the histone complexes

Storing Information: Histones

To fit into the cell each chromosome is subjected to several levels of packaging. In chromatin DNA is folded in a hierarchical fashion.

On the first level roughly 200 bp DNA are associated with a globular octameric aggregate of 4 histone proteins (2 molecules each of the four core histones H2A, H2B, H3 and H4) - called the nucleosome.

Histones act as spools around which DNA winds. This enables the compaction necessary to fit the large genomes of eukaryotes inside cell nuclei: the compacted molecule is 50,000 times shorter than an unpacked molecule.

Histones also act in epigenetic gene regulation. Histones undergo posttranslational modifications which alter how tightly they bind to wrapped DNA. In particular, methylation causes tighter binding, which down-regulates gene transcription; acetylation loosens binding to encourage transcription and translation. Sourced from Wikipedia.