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1. Explain why molecular biologists think it is important to sequence the complete genome of an organism.

2. Explain why genome sequencing projects have focused on a few already well studied model organisms.

3. Describe how gene-trap reporter genes, which are only evident when the "engineered transposon" inserts itself in an expressed gene, are used to identify a cell specific gene?

4. Define the term "microarray" and describe how they are used as a biotechnology tool.

5. List and discuss the potential significance of some of the ethical issues facing us as we learn more about genetic diseases.

6. Answer the following question: If your family had a long history of members dying of a genetic disease for which there is no current cure, but a genetic test is available that can tell you if you have the mutant gene that is responsible for the disease, would you want to be tested? Why or why not?

genome
genome sequencing
model organism
homologous recombinations
marker gene
bacterial artificial chromosome (BAC)
knock-out
transposable elements (transposons)
gene trap
reporter gene
microarrays (DNA chips)
human genome project
gene therapy

Web resources:

• From Maps to Medicine: About the Human Genome Research Project

• Principles Of Biotechnology North Central Regional Extension Publication Iowa State University - University Extension

• The WASHINGTON BIOTECHNOLOGY ACTION COUNCIL: KEEPING AN EYE ON THE GENE - IES This organization provides an alternative view of the potential benefits of biotechnology

• Microarray Project (ľAP): Laboratory of Cancer Genetics The National Human Genome Research Project.

• The Brown Lab Microarrayer equipment.

• GENE CHIP BREAKTHROUGH an economic perspective from Fortune Magazine

• GeneChip Probe Array Synthesis from Affymetrix technology

• !!!!!! Don't Miss this Gene Chip Movie from Affymetrix!!!!!

• Knock-Out Mice from Access Excellence

Projects designed to learn about the evolution and function of organisms by determining the chromosome location and nucleotide sequence of all the genes of a group of model organisms representing the three life domains:

• Archaea
  • Methanococcus janaschii
• Bacteria
  • Escherichia coli
  • Helicobacter pylori
• Eukarya
  • yeast Saccharomyces cerevisiae
  • Plants
    • Arabidopsis thaliana (about 20% complete)
    • rice (the staple food of about 60% of the human population)
  • Animals
    • Nematode Worm (Caenorhabditis elegans) see Lewis text, pg 213-215
    • Fruit Fly (Drosophila melanogaster) see Lewis text pg 215-216
    • Homo sapiens

Homologous Recombination (Knock-Out Genes): Determining the Function of Specific Genes in a cell or organism

A specific gene in the chromosomes of thousands of cells is "mutated" by the specific replacement with a "bogus" bit of DNA containing an antibiotic resistance gene and a visible marker that color codes the cell in which it is expressed.

The ends of the "knock out gene" include sections of DNA that are homologous (same sequence of nucleotides) to a specific location in the genome.

Homologous Recombination

• Some of the genes are successfully "Knocked-out" and some are not.

• The cells are treated with antibiotics that will kill all the cells in which the gene was not "knocked out".

• The surviving cells are studied to see how they function or fail to function with the gene knocked out.

• If the organism is multicellular the cells are grown into complete organism.

• If the gene is actually expressed, the visible marker gene will produce a visible pigment those cells.

Plant Genomics: Arabidopsis thaliana

Characteristics that make Arabidopsis a good model organism:

• Short life span (seed to seed in 5 weeks)
• Small plant (thousands can be grown in a small area)
• Small genome (120 megabases of DNA - about 1/30 of the human genome)
• About 20,000 genes
  • 40% is expected to be of unknown function
  • 20% of the functional genes have been sequenced
  • Expected to complete sequencing by 2000

How to Sequence the Whole Plant Genome

An enzyme is used to break the genome (all the DNA from all the chromosomes - about 120 million base pairs) into overlapping smaller segments about 100,000 base pairs in length.

• Each short segment of DNA is placed into bacterial artificial chromosomes (BACs).

• The BACs are placed into bacteria which produce thousands of copies of the BACs as they grow and reproduce.

• The DNA sequence of the ends of each short segment of DNA is sequenced.

• A contiguous map of the segments is created by using a computer data base to match the overlapping ends of the short sequences of DNA.

• Once the sequence of segments is known the entire genome is sequenced.

How to determine the function of Arabidopsis genes

Gene Traps using Transposable Elements (Transposons) to Tag Single, Specific Genes

• A method to use Homologous Recombination has not yet been developed to "knock-out" specific genes on plant chromosomes.

• Transposons, also known as "jumping genes" are segments of DNA that are capable of moving from chromosome to chromosome in a random fashion.

• Wherever a transposon "jumps into" a gene it usually mutates it and destroyes its function.

• This effectively inactivates genes, similar to homologous recombination, but transposon insertion is entirely random in where the transposon is inserted in the genome.

• Using genetic engineering to incorporate a color producing reporter gene and a selection antibiotic resistance gene in the transposon allows the researcher to tell when and where the gene is expressed and eliminate unsuccessful attempts by killing those cells with antibiotics.

• Arabidopsis plants containing engineered transposons that are expressed have pigmented cells because a specific gene, that is expressed only in those cells, has been tagged by the transposon. This tagging effectively "Traps the Gene" in action.

• By producing thousands of Arabidopsis plants, each with one random transposon insertion, you can produce a collection of plants with "knock outs" for virtually every one of the 20,000 genes in the genome.

• Studying the effects of these gene knockouts helps us understand the function of each of those genes.

!!!!!!!Be sure to see this movie explaining microarrays!!!!!!!

• Samples of DNA from part of the genome or even each and every known gene sequence are applied to the surface of a glas slide in a series of microscopic dots - an array.
  
  
• When a gene is activated it produces messenger RNA (mRNA) in the process of constructing the protein encoded in the gene.

• Messenger RNA can be specifically extracted from cells in certain tissues and/or at different times of development.

• The question to be answered is which genes are involved in which biological activities.

• The goal of microarray analysis is to match each type of mRNA extracted from a cell with the DNA from the gene that produced it.

• An enzyme (reverse transcriptase) is used to produce complimentary DNA molecules (cDNA) using the mRNA extracted from the cells as templates.

• The cDNA is labeled with a fluorescent dye.

• The cDNA produced from the cell sample is unwound by heating and literally washed over the microarray of DNA samples on the chip.

• Wherever DNA molecules from the sample match up with complimentary DNA molecules on the chip they bind.

• The chip is then examined under a fluorescence microscope with laser light and a photograph is taken to record the fluorescence of dots on the chip that have hybridized with cDNA prepared from the cell sample.

• The location of each and every segment of DNA on the chip is known so determining which genes were active in the cell is just a matter of comparing the pattern of fluorescing dots to the gene map of the chip.

• 3 billion base pairs (~30X larger than the Arabidopsis genome)

• Projected to be complete by 2005

• Why do it?
  • To determine a basic understanding of the function of genes

  • There are ~4000 known genetic diseases caused by mutations that alter the expression of a gene or change the protein gene product that leads to a physiological problem.

  • The ability to development of a treatment for a genetic disease is based on three important questions:
    1. Which gene is altered?
    2. What protein does the gene normally make?
    3. Can the altered gene or protein be fixed or replaced?

• Genome research will generate ethical, legal, and social implications and/or problems.