Download smokers - West High School

DNA Chip Scanning
•DNA Chips are scanned with a
laser to excite the fluorescein
dye that is attached to the
target cDNA.
•Only those probe spots where
target cDNA hybridized will
fluoresce.
GENE EXPRESSION CENTER
Scanner - DNA chip goes in here
Image on
screen as
chip is
scanned
Smoker vs. Former Smoker
Smoker vs. Non-Smoker
Probe DNA
(each grid)
1
2
3
4
5
6
7
8
9
10
11
DNA CHIP before hybridization with target DNA
(after spotting Probe DNA)
Sometimes the SDS used in the wash
solutions will fluoresce.
This is all background signal.
A Real DNA Chip
Each spot is a different gene
Example:
Green
Smoker
Red
Non-smoker
Yellow
Both tissues
express the
gene
Bioinformatics - It’s a BLAST
BLAST: “Basic Local Alignment Search Tool”
Sequences at each spot on the
chip are provided on kit CD
Probe Gene Sequences - Cancer / Smoking Chip
GENE 1 - CTCATCAGTAATGGTCAGAGCAT
GENE 2 – GAACAGCTGTTCAGTCTGCAAGT
GENE 3 – CTGGGGTTAGTGCTGGCGATCCT
GENE 4 – CCAGGAGCCCCAGTTACCGGGAG
GENE 5 – GGAGAAGGTAGATTTCAATACGT
GENE 6 – CATGATAAGGCTCTTACCCCCTT
GENE 7 – GTCATGGCGACTGTCCAGCTTTG
GENE 8 – TTGTTTTTGAGACAGAACCTTGC
GENE 9 – ATCCTCCAGGTGCACAAGCTCCA
GENE 10 – ACCATGGATGATGATATCGCCGC
GENE 11 – TGAACGCATTCATCGTGTGGTCT
Gene Ontology
Describes three features about a gene:
Where its protein product is located in the cell
(cellular compartment)
What process its protein product is part of
(cellular process)
The function of that protein product
(molecular function)
What do these Genes Do?
The genes on this chip are not intended
to give a complete model of lung cancer,
but they can be used to illustrate
several important biological principles.
http://www.madison.k12.wi.us/west/science/biotech/at-genes.htm
Biological Principles:
Gene Expression - Tumor suppressors; oncogenes
Developmental stage-dependent genes
Genetic Polymorphism – Aldehyde dehydrogenase has
several different versions (alleles)
among human populations
Biochemical reaction catalysis – Aldehyde dehydrogenase,
Glutathione Peroxidase, Cytochrome
P450 metabolize a variety of
substances
Evolutionary Conservation of Protein Structure – Actin
and Slit homolog code for similar
proteins in several other species
Smoking increases expression (Up-regulates) these genes:
Gene 1 - CYP1A1 cytochrome P450 superfamily of enzymes
• involved in drug metabolism
• located in the endoplasmic reticulum.
• induced in the lung up to 100-fold because of tobacco smoking.
Gene 4 - ALDH3A1 - aldehyde dehydrogenase
• detoxification
• located in the cytosol.
• 16 different alleles exists, one predisposes cancer
Gene 5 - GPX2 - Glutathione peroxidase
• detoxification and antioxidant functions.
• located in the cytoplasm.
• highly expressed in squamous carcinomas of the lung
Quitting smoking is worth it!
Off in smokers; On in non- and former smokers
Gene 3 - SLIT2 (Slit Homologue)
* Involved in cell adhesion
* silenced in many cancers (including lung)
* may be a tumor suppressor
Gene 7 TP53 - p53 tumor suppressor
* guardian of the genome: central role in cell cycle
* loss of p53 function allows pre-malignant cells to continue
proliferating
* found in very low levels in normal cells
* located in the mitochondrion and in the nucleolus
It’s better to NEVER START smoking!
UP-Regulated (ON) in smokers and former smokers:
Gene 6 - CEACAM6 Human Carcinoembryonic Antigen
• an oncogene
• involved in adhesion between cells
• extracellular matrix
• Over-expression inhibits cell differentiation and disrupts how
cells are organized in lung tissue.
DOWN - Regulated (OFF) in smokers and former smokers:
Gene 9 - CX3CL1 - chemokine ligand
• tumor suppressor
• involved in cell adhesion and in inflammation.
• located on the cell surface, membrane & extracellular
compartment.
UP-Regulated (ON) in smokers and former smokers:
Gene 2 - GPC3 Glypican
• controls cellular response to damage from external substances
• may control cell growth and cell death (apoptosis)
• Decreased expression may represent an early step in cigarette
smoke–associated lung carcinogenesis
• located in the plasma membrane and extracellular matrix.
On in smokers and former smokers, OFF in non smokers
Gene 8 - CLDN10 – claudin
• Involved in cell adhesion and inflammation
• located in the plasma membrane