gene regulation vehicles
transcription intiation, rna processing, stability of rna molecules, protein modifications/transport, protein degradation
What is in-situ (in tissue) hybridization?
mrna chased with probe (dna, complimentary to target mrna) that has been fluorescently labelled to bind/ hybridize to mrna to detect it
evaluate spatial differences in body of embryo
evaluate results at different stages of embryonic development
detects a few mrna at one time
why is mrna indicative of gene regulation/expression levels
mrna products of a gene are only transcribed when the gene is active
compare normal breast epithelial and breast carcinoma cells if a sample of each is observed in a dish in culture
normal: don”t pile up, stop growing when they touch eachother
Cancerous: jagged edges, varying nucleus sizes, pile on top of each other, can outgrow their space
difference caused by epigenomes: what genes are or are not expressed and to what degree in either cell
microarray
look at all mrna
a kit will blow up cells and seperate contents to isolate mrna
make complimentary fluorescent dna / oligonucleotides different colours
tiny wells in glass slide, each well represents gene corresponding to mrna product
well that shows red: only cancer cells have gene on
Green: only normal cells have gene on
Yellow: both normal and cancerous cells equally express this gene
blanks: control, genes that shouldn’t be expressed in either cell type, if they fluoresce a mistake has been made
summarize transcriptional gene regulation
methylation: methyl groups attached to cytosines in CpG islands block alpha helices of transcription factors from binding to the promoter. HDAC further inhibits by winding up dna by removing SOMETHING groups of histone tails
summarize rna interfering machinery
miRNA forms hair pin loops from base pairing with mrna and halts translation then RISC (RNA induced silencing complex) complex inhibits translation
siRNA (part of RISC complex) forms hair pin loops then chops up mrna into segments
selective degradation
:proteins have shelf life, can be targeted for destruction when not needed
proteasomes (large protein complexes) break down peptide bonds into small fragments of a few nucleotides which seperate to individual aas to be recycled for another translation
Small ubiquitin proteins tag proteins ( process is ATP dependant). ubiquitin joins to target protein substrate. polyubiquitin chain attaches to substrate target protein, allowing proteasome to degrade protein. degrades transcription factors, cell cycle regulators, etc.
