KH8 Flashcards
what does the genome of each species of eukaryote consist of
a characteristic number of independent linear DNA molecules
each linear DNA molecules is a
chromosome
what do chromosomes never exist as
naked DNA
what do chromosome always exist as
DNA/protein complex termed chromatin
describe a key feature of chromatin organization
condensation or compaction
DNA molecule in average human chromosome is 5 cm long if stretched out = ~5000 times longer than width of typical cell nucleus
So DNA molecule is highly folded/packed/coiled even in the interphase nucleus
when are chromosomes more tightly folded/packed/coiled
mitotic metaphase (more packed than interphase)
why are chromosomes more tightly packed during mitotic metaphase
to facilitate equal distribution between the 2 daughter cells
describe metaphase
highly condensed for transmission to daughter cells
no DNA replication or transcription
only concerned with getting chromosomes to right place
describe interphase
real functional chromosome
undergoing replication and transcription
where action is
chromatin fiber of chromosome unwinds to a degree (from metaphase to interphase) - never to DNA/simplest chromosome structure
dynamic and controlled
what is chromatin
eukaryotic DNA and associated proteins
how many base pairs of DNA in each chromosome for humans
~50-250 million base pairs
what are topological domains
folded domains with definite boundaries
interact among themselves
what is nucleosome
DNA wrapped around histone octamer
where are giant interphase chromosomes from
polytene chromosomes of the fly (drosophila) salivary galdn
describe polytene chromosomes - why they happen
cellular gigantism driven by DNA ~10 cycles of DNA replication without cell division
duplicates but chromosome does not separate
describe polytene chromosomes - physically
all ~1024 daughter chromatids are in perfect alignment making a giant chromosome with regional differences in chromatin condensation
many parallel chromatids
describe regional differences in chromatin condensation
dark bands = condensed chromatin (topological domains)
light bands = boundary elements
describe interphase chromatin organization
dynamic
polytene chromosome puffs show how chromatin decondensation with transcriptional activation
describe puffs of polytene chromosome
puff up = active transcription regions, open and close
associated with active form of RNA polymerase 2 = active transcription
describe puffs of polytene chromosome - colours
red = pol 2 phosphoCTD = active
green = non phosphorylated = inactive
name parts of metaphase chromosome
sister chromatids
centromere
telomere
chromatid
describe metaphase sister chromatids
identical products of the previous semiconservative replication of a single chromosomal DNA molecule
what do metaphase chromosomes show
karyotype (morphology)
chromosomal complement of the species
what characteristics of chromosomes vary
number
shape
size
species specific and sometimes sex specific
describe ex of karyotype
human female karyotype revealed by fluorescence in situ hybridization (FISH) with a panel of probes representing sequences differentially distributed among the chromosomes
“chromosome painting” - identification
what are chromosome rearrangements
breaks/translocation
describe chromosome rearrangements
chromosomes can break and rejoin giving translocations
when can translocations happen
mutations can happen during somatic cell division cycle in life and can cause disease like cancer
also can occur in germ line
describe ex of chromosome rearrangements (cancer)
chronic myelogenous leukemia
results of chromosome breakage and fusion in blood cells
chromosome fusion generates chimeric gene encoding an oncogenic fusion protein
creates protein that deregulated cell growth in blood cells with mutation = cancer
called philadelphia chromosome
describe chromosome rearrangements in germ line
gives gametes - eggs or sperm - with variant chromosomes
offspring often have reduced fertility
usually a dead end
this is why karyotype is so consistent across a species
can happen that it is successful and passed on = rare and karyotype can evolve over time
describe evolution of human karyotype
from primate ancestral karyotype
unchanged Chr 11 (=13)
breakage Chr 14, 15 (=5)
reciprocal translocation Chr 12, 22 (=14, 21) end-to-end fusion Chr 2 (=9 + 11)
what are the elements required for replication and stable inheritance of linear chromosomes - 3
origin of replication
centromere
2 telomeres - ends
how do we learn about protein structure and function
by building artificial chromosomes
how did we discover elements required for chromosome function
Experimental discovery in a simple eukaryote: yeast
describe what yeast cells need
yeast leu- cells have LEU gene inactivated by a mutation and need exogenous leucine for growth
what does LEU gene code for
makes leucine
describe initial experiment done with yeast
wild type LEU gene cloned into circular bacterial plasmid
introduce to yeast cells and ask if it rescues leucine independent growth
aka does LEU plasmid replicate as cell grows
describe initial experiment done with yeast - results
LEU plasmid replicates well in bacteria BUT INCAPABLE OF REPLICATION IN YEAST
since bacterial origins of replication do not work in eukaryotes
how do we fix plasmid not working in yeast
insert into plasmid a random piece of yeast DNA that happened to contain a yeast origin of replication
now plasmid can support growth of leu - yeast in absence of leucine
describe origin of replication (yeast experiment)
ARS = autonomously replicating sequenced = yeast origin of DNA replication
REQUIRED FOR REPLICATION
but cells not portioned well between daughter cells - mitotic segregation is faulty
how to improve mitotic segregation
add centromere
CEN = DNA sequence from yeast chromosome centromere
drives good mitotic segregation
what is kinetochore
attached to centromere
spindle microtubules attach here
then 2 chromatids separate and walk in opposite directions
the centromere…
link to spindle microtubules
describe yeast CEN
sequences common to various yeast centromere
present on nucleosome that includes a centromere specific histone variant CENP-A (centromeric protein A)
what does CENP-A do
recruits CBF3 complex which in turn recruits Ndc80 complex which attaches to microtubules
initiates cascade of assembly
describe image of CENP-A
different colour since its a different histone
tags it so it can recruit kinetochore apparatus - lateral attachment then to end on conversion
Kinetochores attached here and migrates and microtubule dissolves behind it as it moves
drags whole chromosome with it - this is how chromosome moves apart
what shape are yeast chromosomes
linear DNA molecules
not circular
Previously we used a plasmid in yeast experiment which is circular
how to convert circular plasmids to linear DNA molecules
cutting a single site with restriction endonucleases
Does a plasmid with ARS and CEN that works well as an experimental circular chromosome in yeast also work well as a linear chromosome?
nOOOOOOOOOO
lose everything - linear does not work
so add telomeres
what are telomeres
special sequences at ends of eukaryotic chromosomes
cloning a bit of telomere DNA at end and it works - needed for linear molecules to survive
describe conclusions of yeast experiment - telomeres
cut plasmid without telomeres = unstable
linear plasmids containing ARS and CEN act normal if genomic fragment telomeres added to both ends
name the functions of telomeres - 3
protect from exonuclease
prevent end-to-end fusion
solve a replication problem faced by linear DNA
what is telomere problem
because lagging strand synthesis cannot be completed - chromosomes shorten at ends in each replication
unsustainable since at some point you will lose an essential gene
15-20 bases of RNA and no way to make it DNA
what is solution to telomere problem
telomerase
DNA polymerase that can extend telomeres
restores chromosome length to overcome lagging strand end shortening
what do telomeres contain
simple repeat DNA sequences
ciliate protist tetrahymena (TTGGGG)
human (TTAGGG)
describe telomerase
reverse transcriptase that carries its own template RNA complementary to the telomeric DNA repeat
by extending template strand telomerase gives primase more template DNA to prime on
what is a reverse transcriptase
DNA polymerase that uses RNA as template
describe how telomerase works informally
binds to telomere
complementary to telomere
so reverse transcriptase can extend it
slips so can extend next primer - adds telomere repeats
where is telomerase active
germ cells and stem cells not in somatic cells since divide only a few times (so existing telomeric repeats are long enough)
when is telomerase often reactivated
in cancer cells - a target for cancer therapy
describe mice lacking telomerase gene
ok for 3 generations then fertility decline
has enough repeats so it can go through 3 generations before ends are shortened too much and a key gene is not replicated (it is lost)
describe ciliated protist tetrahymena
unusual gene expression mechanism
does not express genes from its “main” genome. maintained in a non-transcribed form in the micronucleus
transcription occurs in the macronucleus from millions of gene-sized DNA pieces.
presence of so many DNA ends made it possible for researchers to identify the telomere sequence and to isolate telomerase
