Biomed research I and Oncogenesis

amplify gene or DNA of interest (DNA cloning)

-cell-based
-PCR

-DNA recombination (insert DNA of interest into cloning vector using restriction enzymes/sites)
-Transformation (put vector into host cell/E. coli)
-selective amplification (usually test for antibiotic resistance, gene usually inserted into vector with DNA of interest)
-isolation and sequencing of desired DNA

-plasmids
-lambda phage
-cosmid
-YAC

-double-stranded circular DNA
-can be found in bacteria and eukaryotes
-reproduce independently of cell cycle
-low transformation efficiency
-have engineered polylinker which can recognize various restriction enzymes
-replication origin for proliferation in host cell

-choose restriction enzyme that would recognize a restriction site in both the DNA of interest and vector
-cleave both DNA and vector with restriction site to insert DNA

-label ddNTPs with fluorescent probe
-use laser beam and run through detector to see different colors, which allows you to sequence DNA

-restriction fragment length polymorphisms
-restriction enzymes cut DNA at precise points making a collection of DNA fragments of precisely defined length
-create "DNA fingerprints"

-screen human DNA for disease genes
-criminal investigation using "DNA typing"

there must be a variation at a restriction site

-we all have the same important structural genes, but differ in the length of uncoded region
-compare evidence (DNA) to victim, suspects, and control
-probes will be complements to evidence

-cleave into fragments with restriction enzymes
-separate using gel electrophoresis
-denature and transfer to nitrocellulose membrane (better hybridization)
-add probe, and wash
-use x-ray or whatever method to visualize probes

-increase temp to denature DNA
-decrease temp to allow primers to anneal (must flank DNA region of interest)
-increase temp to allow DNA Pol to synthesize new DNA

-allows introduction of restriction sites
-add restriction sites to end of primer at 5' end
-as long as the 3' end of primer hybridizes to the DNA of interest, can still have polymerization

-uncontrolled cell growth
-invasion of other tissues (direct growth or implantation)

-carcinoma
-sarcoma
-leukemia

-adenomatous
-squamous
-myeloid
-lymphoid

-extent to which neoplastic (new abnormally grown) cells resemble normal cells

-cancer: mutated cell leaves origin and relocates to somewhere else (via blood)

-encapsulated growth
-no tissue differentiation so still looks like tissue of origin
-slow growth rate
-low recurrence risk (b/c still encapsulated)
-poor prognosis only if unable to remove
-slowly progressive

-uncontrolled slow or rapid rate of growth
-not encapsulated, infiltrated/metastasized to other regions
-tissue destruction is common
-usually progressive, fatal if untreated
-genetically unstable
-recurrence is common

high recurrence because not encapsulated, probably will grow in a different region of body

False: usually somatic mutations; small percentage is inherited as germ-line mutation

-environmental impact
-somatic mutations
-germ-line mutations (predisposition)
-infectious agents (viral or bacterial: eg. virus gets into cell, hijacks DNA and moves to different place)

-DNA damage repair
-apoptosis

-tumor suppressor genes
-oncogenes
-genetic stability genes

-regulates cell cycle (checkpoints)

-hereditary: multiple tumors, both eyes affected

False, usually no clear-cut pattern of inheritance

-nondisjunction
-nondisjunction and duplication (get 2 copies of mutant allele)
-mitotic recombination
-gene conversion
-deletion
-point mutation

cyclin-Cdk phosphorylates Rb which activates it and allows gene expression

p53

-transcription factor to activate p21 which inhibits cyclin-Cdk, which activates Rb, which activates gene transcription
-so p53 indirectly inhibits gene transription

mutated proto-oncogenes

-regulate cell growth, proliferation, and differentiation

-overactivity
-gene amplification (too much of normal protein), makes fusion protein with protein that is expressed a lot, moved closer to enhancer

Ras

-tyrosine kinase receptor
-nuclear transcription factor, MYC

-function is to maintain fidelity and stability of replication of genome so if these genes are messed up then will have bad replication--> mutations --> cancer (if mutations in the right genes)
-both genes must be mutated for cancerous growth

-genes involved in DNA repair (NER, BER, mismatch repair)
-genes involved in large chromosomal events (recombination, segregation)

-most cancers derive from a cells inability to control growth (lots of mutations), and all daughter cells will inherit these mutations and will not be able to control growth
-mutations increase cell proliferation, so each round of new cells occurs quicker so get selective growth advantage of cancerous cells

-cancerous cells are mutated so that they have uncontrolled cell proliferation
-each new round of cells will occur quicker than normal cells
-accumulation of mutation in cells makes them grow faster, overtaking normal cells

1) self-sufficient with growth signals
2) little response to growth-inhibitory signals
3) evasion of apoptosis
4) limitless replicative ability
5) sustained angiogenesis
6) tissue invasion and metastasis

-mitochondrion
-death receptor

-pro-apoptosis: caspase
-anti-apoptosis: Bcl-2

-telomerase; not expressed in most somatic cells, usually in stem and germ cells
-when telomeres shrink to a certain point, cell stops dividing (senesce), telomerase prevents shortening of telomeres

-Secrete VEGF
-stimulates vascular epithelial cell growth
-causes endothelial cells to release matrix metallothinoine proteases
-increases permeability of vessels, breaks down basal membrane

-reductionist's view
-heterotypic cell (interacts with other cells in microenvironment)

-need to detach from neighboring cells
-break through basement membrane
-evade immune system
-sitmulate angiogenesis

-sporadic

-tumor suppressor gene

Breast and ovarian (hormonally related)

Breast cancer in both women AND men and ovarian cancer

-Ashkenazi Jewish heritage
-family history of breast or ovarian cancer
-breast and ovarian cancer in same woman
-male breast cancer
-early onset (pre-menopause) breast cancer
-bilateral breast cancer

-estrogen receptor (ER)
-human epidermal growth receptor (HER)
-Progesterone receptor

tamoxilin

basaloid, triple negative

BRCA1, p53

-microarray
-test levels of gene mutations and various receptors

-cancer subtype (ER+, HER2+, basaloid)

recurrence risk of breast cancer by looking at gene expression

-what can kill one cell might not kill another