Molecular Structure of Genes and Chromosomes 2 (L9) Flashcards
chromatin
complex of DNA and protein that makes up the chromosome
nucleosome
147 bp of DNA wrapped 1 2/3 times around histones (2 each of H2A, H2B, H3, H4) - building block of chromatin
why do DNA and histones associate with each other?
histones have many basic aa’s (+) and DNA is (-)
linker DNA
b/w beaded nucleosome - 10 to 90 bps
how are nucleosomes stacked?
30 nm diameter double helix of nucleosomes stacked with H1 histone
what do all histones have?
flexible N-terminal tails (19-39 residues) on H3, H4
N and C-terminal tails on H2A, H2B
post-translational modifications of nucleosomes
phosphorylation, methylation, acetylation, ubiquitination
significance of acetylation
charge is neutralized -> DNA destabilized -> transcription activated
significance of methylation
charge is not changed -> DNA stabilized -> transcription inactivated
decondensed vs. condensed chromatin
decondensed: gene expression
condensed: no gene expression
regulation of acetylation
HATS (histone acetyltransferases) - add Ac onto Lys
HDACS (histone deacetylases) - remove Ac
where do acetylation and methylation occur?
on Lys side chain - epsilon amino group
what test can be used to determine the status of chromatin?
DNase 1 sensitivity assay of the nuclei DNA
number of times methylation can occur
three times on one amino group
regulation of methylation
HMTs (histone methyltransferases)
two types of chromatin in nucleus
euchromatin: active/open
heterochromatin: inactive/condensed
euchromatin
active/open - related to histone acetylation on H3 tails
what is the exception in euchromatin?
tri-methylation of Lys4 on H3 related to activation
heterochromatin
inactive/condensed - related to histone methylation - 2 H3 tails are tri-methylated
what is HP1
heterochromatin protein 1 - chromodomain and chromoshadow domain
what does HP1 recognize w/ chromodomain?
H3K9Me3 (trimethylated Lys 9 on H3)
what does HP1 recognize w/ chromoshadow domain?
allows for oligomerization of HP1 proteins -> aggregation -> forms heterochromatin
what else can HP1 also recruit?
HMTs -> spread area of methylation
function of boundary elements
isolate action of HP1 so there are regions of condensed and noncondensed DNA
TFIID
one of the first proteins bound to the promoter to promote transcription - recognizes acetylated Lys’s w/ bromodomain
how is animal cloning accomplished?
SCNT - somatic cell nuclear transfer
what is the epigenetic process that gives rise to variations in outcome of cloning?
random X chromosome inactivation (Barr body)
MeCP2
methyl CpG binding protein 2 - recognizes methylated C, recruits HDAC and HMT, changes post-translational modification to inactivated state of gene transcription
euchromatin vs. heterochromatin: what cell types have each?
stem cells: more euchromatin
differentiated cells: more heterochromatin
iPS cells (induced pluripotent stem cells)
pluripotency induced in already differentiated cells - done by transcription factors
what are iPS cells used for?
cloning; studying disease mechanisms in dish and also by correcting defect - create many fixed cells and put them back into patient (cell therapy)
ChIP - chromatin immunoprecipitation
only region that was originally decondensed will be bound to antibody - then secondary antibody for primary antibody -> use to get sequence info
what does the ChIP sequence tell?
get sequence info from pieces - see what sequences are associated w/ heavily Ac-proteins -> genes here in decondensed state -> most likely are expressed
what happens to chromatin during metaphase of mitosis?
condensation into chromosomes
cytogenetics
study of the structure and function of cell, especially the chromosomes
karyotyping
study of the number and appearance of chromosomes - each chromosome has unique banding pattern
what stain is used for karyotyping?
Giesma stain - shows G bands and R bands: G bands have more stain
what is one clinical application of karyotyping?
to detect chromosomal aberration
what causes CML?
transloation b/w chromosomes 9 and 22: Philadelphia chromosome - makes a new gene (oncogenic BCR-ABL tyrosine kinase) that is always on -> cancer
FISH - fluorescent in situ hybridization
like southern blot
SKY - special karyotyping
probes recognize a particular chromosome - use a different colored dye on each chromosome and see where translocations occur
ChIP steps
- isolate and shear chromatin mechanically
- add antibody-specific for acetylated N-terminal histone tail
- immunoprecipitate
- release immunoprecipitated DNA and assay by PCR
three elements for inheritance and replication of chromosomes
- autonomously replicating sequences (ARS) - “origin of rep”
- centromere - helps replicated plasmid to split into daughter cells
- telomere - allows linear plasmid to replicate
telomeres and DNA replication problem
lagging strand requires RNA primer to bind fragments -> so every time you replicate, the strand should get shorter… so you have telomeres
telomerase action
add TEL sequence to ends of chromosome by reverse transcription followed by translocation-hybridization
parts of telomerase-RNA complex
- ssDNA at terminus of telomere
- catalytic site for dNTP addition
- telomerase protein
- telomerase-associated RNA template
