Career Forum for the Biological Sciences

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Career Forum for the Biological
Sciences Wednesday March 12, 4-6 PM MCDB A2B70
A panel discussion with individuals from various
areas in Biology. A reception will follow the
presentations. Dr. Mark Winey will represent MCDB
Faculty and Research
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You cant make human CDC collections
This makes cloning genes by complementation
difficult
Cancer associated genes can be cloned
-their similarity to yeast CDC genes can be very
informative
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Linkage Between Physical and Genetic Markers
Genetic Marker
Physical Markers
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Identification of Cancer Genes by Chromosome
Walking
A
B
From the Human Genome Management Information
System Oak Ridge National Laboratory
http//www.ornl.gov/hgmis/publicat/primer/
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Sanger (dideoxy) DNA Sequencing
http//www.mri.sari.ac.uk/Genomics/wiDNASeq.htm
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Sanger (dideoxy) DNA Sequencing
Radioactive label
Radioactive label
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Sanger (dideoxy) DNA Sequencing
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Sanger (dideoxy) DNA Sequencing
Sequencing gels are awkward
0.1 cm
60 cm
20 cm
http//www.med.nus.edu.sg/life_sciences/images/DSC
00040.jpg
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Sanger (dideoxy) DNA Sequencing
Modern DNA Sequencing
Fluorescent labels (unique label for each base)
ddATP ddTTP ddCTP ddGTP
All reactions in 1 sample, loaded into capillary
Adapted from Alberts et al. Figure 8-36
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Capillary Electrophoresis
From http//www.yourgenome.org
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Automated Sanger DNA Sequencing
Applied Biosystems (ABI) 3700 DNA Sequencer
http//www.biosci.ohio-state.edu/pmgf/3700.htm
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Automated Sanger DNA Sequencing
Applied Biosystems (ABI) 3700 DNA Sequencer
http//www.biosci.ohio-state.edu/pmgf/3700.htm
http//gnn.tigr.org
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The Human Genome was Sequenced by Many Sequencers
http//abcnews.go.com/sections/science/DailyNews/c
hromosome991201.html
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Specific Genes can be Labeled on Chromosomes
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Cytogenetic Changes in Breast Cancer
From Jorma Isola, University of Tampere, FINLAND
www.uta.fi/imt/sgy/jorma/ Digital20Images.html
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Specific Cytogenetic Changes are Associated with
Some Cancers
Burkitts Lymphoma
Chronic Myelogenous Leukemia
From NCBI http//www.ncbi.nlm.nih.gov/cgi-bin/SCIE
NCE96/nph-gene?MYC
Alberts et al. Figure 23-5
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How do You Identify Genes that Cause Sporadic
(non-inherited) Cancers?
Some Sporadic Tumors Display Cytogenetic Changes
Gene Amplification Chromosomal Rearrangements etc.
These can be used to identify associated genes
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Not all Tumors Display Characteristic
Cytogenetic Changes
Many Cytogenetic Changes (no obvious correlation)
No Obvious Cytogenetic Changes
Chromosomes from Two Colon Cancers
Figure 23-40. Molecular Biology of the Cell, 4th
Edition
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Retroviruses and the Discovery of Oncogenes
Rertoviral Life Cycle
A Viral Oncogene (v-src)
A Viral Oncoprotein
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Transforming RSV Contains a Single Extra Gene
v-src viral sarcoma gene
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v-src is Derived From a Host Cell Gene, c-src
c-src is a proto-oncogene
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What is the src Oncogene?
src is a highly regulated tyrosine kinase
Goodsell (2001) The Oncologist 6474
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What is the src Oncogene?
src is a highly regulated tyrosine kinase
Phospho-Tyrosine Binding Domain
Poly-Proline Binding Domain
Phospho-Tyrosine
Poly-Proline Containing Domain
Tyrosine Kinase Domain
Goodsell (2001) The Oncologist 6474
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src is Autoregulated by its SH2 and SH3 Domains
src Activation is Driven by Dephosphorylation of
Inhibitory Tyrosine
Phospho-Tyrosine
De-phosphorylation of Tyrosine
Goodsell (2001) The Oncologist 6474
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src is Autoregulated by its SH2 and SH3 Domains
src Activation is Driven by Dephosphorylation of
Inhibitory Tyrosine
Phospho-Tyrosine
Phospho-Tyrosine Binding Domain
De-phosphorylation of Tyrosine
Goodsell (2001) The Oncologist 6474
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SH2 and SH3 Domains are Important in Biology
Src Homology 2 and Src Homology 3
Domains Homolgous to SH2 are found in many
proteins -they all bind P-Tyr
Domains Homolgous to SH3 are found in many
proteins -they all bind poly-Pro
(SH1)
Goodsell (2001) The Oncologist 6474
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SH2 and SH3 are Protein Interaction Domains
Proteins Interact with Growth Factor
Receptors through SH2 and SH3 Domains
An SH2 Domain
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SH2 and SH3 Domains in Signal Transduction
SH2 and SH3 Domains Participate in Scaffolding
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SH2 and SH3 Domains in Signal Transduction
SH2 and SH3 Domains Participate in Scaffolding
Src
www.biocarta.com
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Oncogenes Identified from Retroviruses
Acutely Transforming Retroviruses Carry v-oncs
v-onc
Virus
Host
src Rous sarcoma virus Chicken myc Avian
myelocytomatosis virus Chicken erb A, erb B
Avian erythroblastosis virus Chicken myb Avia
n myeloblastosis virus Chicken ets
Avian erythroblastosis
virus Chicken rel Avian reticuloendotheliosis
virus Turkey H-ras Harvey rat sarcoma virus
Rat K-ras Kirsten murine sarcoma
virus Mouse abl Abelson murine leukemia
virus Mouse raf Murine sarcoma
virus Mouse fos Mouse osteosarcoma
virus Mouse fms Feline sarcoma virus
Cat fes Feline sarcoma
virus Cat sis Simian sarcoma virus Monkey
From LSU Health Sciences Center
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v-oncs are Mutated Proto-oncogenes
c-oncs Regulate Cell Growth and Cell Cycle
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Retroviruses Lacking v-oncs can Activate c-oncs
Proto-oncogene
Host Chromosome
Promoter insertion
Viral Genome
Enhancer insertion
Retroviruses can Transform by Insertional
Mutagenesis -insertion of retroviruses into
proto-oncogene locus -LTR promoter or enhancers
activate c-onc expression
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Discovery of Oncogenes as Dominant Transforming
Genes by Shotgun Cloning
From www.orst.edu/instruction
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Discovery of Oncogenes as Dominant Transforming
Genes by Shotgun Cloning
From www.orst.edu/instruction
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What Kinds of Genes are Mutated in Cancer?
Oncogenes and Tumor Suppressors
Oncogenes are genes that positively regulate the
cell cycle - dominant proto-oncogene mutations
cause proliferation
Tumor Suppressors are genes that negatively
regulate the cell cycle - recessive tumor
suppressor mutations cause proliferation
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Oncogenes
Oncogenes are mutated proto-oncogenes (c-oncs)
c-oncs are positive regulators of the cell cycle
Oncogene Expression via Retroviral
Transformation -transforming retroviruses
express v-oncs, i.e. HSV v-src -virally
transduced versions of c-oncs -retroviruses
can also activate c-oncs by insertion
Oncogene Expression via Dominant Mutation of
c-oncs -dominant c-oncs identified
via -chromosomal rearrangement -transfection of
mouse cells with human tumor DNA
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What Classes of Genes can be Oncogenes?
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Receptor-Mediated Signal Transduction
SOS
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Receptor-Mediated Signal Transduction
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Receptor-Mediated Signal Transduction
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The ras pathway is a Major Oncogenic Target
-Growth factor receptors (i.e. EGF-R) can be
oncogenes v-erbB truncated EGF-R c-erbB
overexpressed in some breast cancers -activated
ras identified as a v-onc, and from bladder cancer
i.e. EGF-Receptor
src
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The ras pathway is a Major Oncogenic Target
v-erbB is a truncated EGF-R that signals in
absence of EGF
src
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v-erbB Expresses a Truncated EGF-Receptor
v-
http//www.wwnorton.com/cdly/cells/fig11_4.gif
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EGF-R Oncogenes and Breast Cancer Therapies
Overexpression of EGF-R (i.e. her2/neu) is
oncogenic
Herceptin
http//www.prous.com/journals/dnp/sample/html/dn13
0325/dn130325.html
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The ras pathway is a Major Oncogenic Target
mutations can activate ras in the absence of
hormone
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The ras pathway is a Major Oncogenic Target
-activated ras identified as a v-onc -activated
ras identified from bladder cancer
Adapted from http//peo.cshl.org/mangone/medbe
nch/last_ras.htm
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Activating ras Mutations
-Mutations that destroy GTPase activity, or
prevent GAP binding (residues 12, 13, 59,
61) -Mutations that weakly bind nucleotide or
cause high release rate (residues 116 and 119)
Adapted from http//peo.cshl.org/mangone/medbe
nch/last_ras.htm