Molecular Structure of Genes and Chromosomes 2 (L9)

Question 1

chromatin

Answer 1

complex of DNA and protein that makes up the chromosome

Question 2

nucleosome

Answer 2

147 bp of DNA wrapped 1 2/3 times around histones (2 each of H2A, H2B, H3, H4) - building block of chromatin

Question 3

why do DNA and histones associate with each other?

Answer 3

histones have many basic aa’s (+) and DNA is (-)

Question 4

linker DNA

Answer 4

b/w beaded nucleosome - 10 to 90 bps

Question 5

how are nucleosomes stacked?

Answer 5

30 nm diameter double helix of nucleosomes stacked with H1 histone

Question 6

what do all histones have?

Answer 6

flexible N-terminal tails (19-39 residues) on H3, H4

N and C-terminal tails on H2A, H2B

Question 7

post-translational modifications of nucleosomes

Answer 7

phosphorylation, methylation, acetylation, ubiquitination

Question 8

significance of acetylation

Answer 8

charge is neutralized -> DNA destabilized -> transcription activated

Question 9

significance of methylation

Answer 9

charge is not changed -> DNA stabilized -> transcription inactivated

Question 10

decondensed vs. condensed chromatin

Answer 10

decondensed: gene expression
condensed: no gene expression

Question 11

regulation of acetylation

Answer 11

HATS (histone acetyltransferases) - add Ac onto Lys

HDACS (histone deacetylases) - remove Ac

Question 12

where do acetylation and methylation occur?

Answer 12

on Lys side chain - epsilon amino group

Question 13

what test can be used to determine the status of chromatin?

Answer 13

DNase 1 sensitivity assay of the nuclei DNA

Question 14

number of times methylation can occur

Answer 14

three times on one amino group

Question 15

regulation of methylation

Answer 15

HMTs (histone methyltransferases)

Question 16

two types of chromatin in nucleus

Answer 16

euchromatin: active/open
heterochromatin: inactive/condensed

Question 17

euchromatin

Answer 17

active/open - related to histone acetylation on H3 tails

Question 18

what is the exception in euchromatin?

Answer 18

tri-methylation of Lys4 on H3 related to activation

Question 19

heterochromatin

Answer 19

inactive/condensed - related to histone methylation - 2 H3 tails are tri-methylated

Question 20

what is HP1

Answer 20

heterochromatin protein 1 - chromodomain and chromoshadow domain

Question 21

what does HP1 recognize w/ chromodomain?

Answer 21

H3K9Me3 (trimethylated Lys 9 on H3)

Question 22

what does HP1 recognize w/ chromoshadow domain?

Answer 22

allows for oligomerization of HP1 proteins -> aggregation -> forms heterochromatin

Question 23

what else can HP1 also recruit?

Answer 23

HMTs -> spread area of methylation

Question 24

function of boundary elements

Answer 24

isolate action of HP1 so there are regions of condensed and noncondensed DNA

Question 25

TFIID

Answer 25

one of the first proteins bound to the promoter to promote transcription - recognizes acetylated Lys's w/ bromodomain

Question 26

how is animal cloning accomplished?

Answer 26

SCNT - somatic cell nuclear transfer

Question 27

what is the epigenetic process that gives rise to variations in outcome of cloning?

Answer 27

random X chromosome inactivation (Barr body)

Question 28

MeCP2

Answer 28

methyl CpG binding protein 2 - recognizes methylated C, recruits HDAC and HMT, changes post-translational modification to inactivated state of gene transcription

Question 29

euchromatin vs. heterochromatin: what cell types have each?

Answer 29

stem cells: more euchromatin | differentiated cells: more heterochromatin

Question 30

iPS cells (induced pluripotent stem cells)

Answer 30

pluripotency induced in already differentiated cells - done by transcription factors

Question 31

what are iPS cells used for?

Answer 31

cloning; studying disease mechanisms in dish and also by correcting defect - create many fixed cells and put them back into patient (cell therapy)

Question 32

ChIP - chromatin immunoprecipitation

Answer 32

only region that was originally decondensed will be bound to antibody - then secondary antibody for primary antibody -> use to get sequence info

Question 33

what does the ChIP sequence tell?

Answer 33

get sequence info from pieces - see what sequences are associated w/ heavily Ac-proteins -> genes here in decondensed state -> most likely are expressed

Question 34

what happens to chromatin during metaphase of mitosis?

Answer 34

condensation into chromosomes

Question 35

cytogenetics

Answer 35

study of the structure and function of cell, especially the chromosomes

Question 36

karyotyping

Answer 36

study of the number and appearance of chromosomes - each chromosome has unique banding pattern

Question 37

what stain is used for karyotyping?

Answer 37

Giesma stain - shows G bands and R bands: G bands have more stain

Question 38

what is one clinical application of karyotyping?

Answer 38

to detect chromosomal aberration

Question 39

what causes CML?

Answer 39

transloation b/w chromosomes 9 and 22: Philadelphia chromosome - makes a new gene (oncogenic BCR-ABL tyrosine kinase) that is always on -> cancer

Question 40

FISH - fluorescent in situ hybridization

Answer 40

like southern blot

Question 41

SKY - special karyotyping

Answer 41

probes recognize a particular chromosome - use a different colored dye on each chromosome and see where translocations occur

Question 42

ChIP steps

Answer 42

1. isolate and shear chromatin mechanically 2. add antibody-specific for acetylated N-terminal histone tail 3. immunoprecipitate 4. release immunoprecipitated DNA and assay by PCR

Question 43

three elements for inheritance and replication of chromosomes

Answer 43

1. autonomously replicating sequences (ARS) - "origin of rep" 2. centromere - helps replicated plasmid to split into daughter cells 3. telomere - allows linear plasmid to replicate

Question 44

telomeres and DNA replication problem

Answer 44

lagging strand requires RNA primer to bind fragments -> so every time you replicate, the strand should get shorter... so you have telomeres

Question 45

telomerase action

Answer 45

add TEL sequence to ends of chromosome by reverse transcription followed by translocation-hybridization

Question 46

parts of telomerase-RNA complex

Answer 46

- ssDNA at terminus of telomere - catalytic site for dNTP addition - telomerase protein - telomerase-associated RNA template