Oct 13, KH8, Chromosomes

Question 1

what does the genome consist of?

Answer 1

characteristic number of independent linear molecules

Question 2

what is each of these linear molecules called?

Answer 2

a chromosome

Question 3

what do chromosomes exist as?

Answer 3

never as naked DNA
DNA/protein complex called chromatin

Question 4

what is a key feature of chromatin organisation?

Answer 4

condensation/compaction
DNA molecule of average chromosome is 5cm long, 5000 times larger than typical nucleus
tightly folded/packed/coiled

Question 5

when are the chromosomes even more compactly folded than during interphase? and why?

Answer 5

during mitotic metaphase
facilitates equal distribution between the two daughter cells

Question 6

what happens in metaphase?

Answer 6

highly condensed for transmission to daughter cells
no DNA replication, no transcription

Question 7

what happens in interphase?

Answer 7

the real functional chromosome
replication, transcription and all the action

Question 8

what happens in the transition from metaphase to interphase?

Answer 8

the chromatin fiber of the chromosome unwinds to a degree

Question 9

what is a chromatin?

Answer 9

eukaryotic DNA and associated proteins

Question 10

what is a nucleosome?

Answer 10

DNA wrapped around a histone octamer

Question 11

how many base pairs of DNA in each chromosome in humans?

Answer 11

~50-250 million

Question 12

what are polytene chromosomes?

Answer 12

giant interphase chromosomes
have thousands of DNA strands
many parallel identical chromatids

Question 13

what do the dark bands represent?

Answer 13

condensed chromatin (topological domains)

Question 14

what are metaphase sister chromatids?

Answer 14

identical products of the previous semi conservative replication of a single chromosomal DNA molecule

Question 15

what are the ends of the sister chromatids called?

Answer 15

telomeres

Question 16

what do metaphase chromosomes show?

Answer 16

the karyotype

Question 17

what is the karyotype?

Answer 17

the chromosomal complement of the species

Question 18

what characteristics are species specific? (and sometimes sex specific)

Answer 18

number, shape and size of chromosomes

Question 19

how can chromosomes rearrange, when does it happen and what does this do?

Answer 19

chromosomes can break and rejoin, which gives translocations
these mutations can happen during a somatic cell division cycle in the life of an organism
can cause disease (ie cancer)

Question 19

what is the germ line?

Answer 19

refers to the sex cells (eggs and sperm) that sexually reproducing organisms use to pass on their genomes from one generation to the next (parents to offspring)

Question 20

what happens when chromosome rearrangement occurs in the germ line?

Answer 20

this gives gametes (egg or sperm) with variant chromosomes
offspring arising from such variant gametes often have reduced fertility
usually germ line chromosome rearrangements are a dead end
this is why the karyotype is so consistent across a species

Question 21

what happens when a chromosomal rearrangement variant is successfully passed on from one generation to the next?

Answer 21

the karyotype can evolve over evolutionary time

Question 22

what are the elements required for the replication and stable inheritance of linear chromosomes?

Answer 22

1. origin of replication
2. centromere
3. 2 telomeres (ends)

Question 23

how does yeast demonstrate the elements required for chromosome function?

Answer 23

yeast leu- cells have the LEU gene inactivated by a mutation, and therefore require exogenous leucine for growth
wild type yeast LEU gene is cloned into a circular bacterial plasmid
introduce LEU plasmid into leu- yeast cells and ask if this “rescues” leucine-independent growth
such rescue would require the LEU plasmid to replicated as the cells grow
the LEU plasmid replicates well in bacteria, but incapable of replication in yeast because bacterial origins of DNA replications do not work in eukaryotes
this plasmid cannot support leu- yeast growth in absence of leucine
BUT suppose the plasmid is inserted in a random piece of yeast DNA that happened to contain a yeast origin of replication
now the plasmid can support leu- yeast growth in the absence of leucine

Question 24

what is that yeast origin of DNA replication called?

Answer 24

ARS = autonomously replicating sequence it is required for plasmid replication

Question 25

how does mitotic segregation compare from when the plasmid has LEU and when it has LEU +ARS?

Answer 25

mitotic segregation does not happen when only LEU is present because there is no growth of that plasmid in presence of ARS, plasmid replication occurs but mitotic segregation is faulty (5-20% of cells have the plasmid)

Question 26

what is the CEN?

Answer 26

the DNA sequence from a yeast chromosome centromere

Question 27

what does the CEN do when added to the plasmid?

Answer 27

increases mitotic segregation (>90% of cells have plasmid)

Question 28

what is the kinetochore?

Answer 28

a complex of proteins associated with the centromere of a chromosome during cell division, to which the microtubules of the spindle attach.

Question 29

where do the spindle microtubules attach?

Answer 29

the kinetochore at the centromere

Question 30

explain how the centromere is the link to spindle microtubules

Answer 30

there are sequences common to the various yeast centromeres present on a nucleosome that incudes a centromere specific histone variant CENP-A (centromeric protein A) CENP-A recruits the CBF3 complex which in turn recruits the Ndc80 complex, which attaches the microtubules

Question 31

how can circular plasmids be converted to linear DNA molecules? and what's the issue with that?

Answer 31

by cutting at a single site with a restriction endonuclease BUT these do not work well as linear chromosomes (no growth without leucine and no mitotic segregation)

Question 32

how can the issue of circular plasmids not working well as linear DNA molecules be fixed?

Answer 32

add genomic fragment TEL (telomeres) to both ends there will be growth without leucine and there will be good mitotic segregation

Question 33

what do telomeres do?

Answer 33

protect from exonuclease prevent end to end fusion solve a replication problem face by linear DNA

Question 34

what is the telomere problem and what is the solution?

Answer 34

because the lagging strand cannot be completed, chromosomes should shorten at the ends of each replication unsustainable because at some point you will lose an essential gene telomerase is the solution a DNA polymerase that can extend telomeres, restoring chromosome length to overcome lagging strand end shortening

Question 35

what do telomeres contain

Answer 35

simple repeat DNA sequences

Question 36

where is telomerase active and why?

Answer 36

only active in germ cells and stem cells somatic cells divide only a few times, so existing telomeric repeats are already long enough

Question 37

where is telomerase often re-activated?

Answer 37

in cancer cells --> divide too much telomerase is a target for cancer therapy

Question 38

what are topological domains?

Answer 38

folded domains that have somewhat of a definite structure but have definite boundaries these regions have regulatory DNA sequences and interact within each other

Question 39

how is the DNA arranged around the histone proteins, and how are they positioned relative to each other?

Answer 39

around 100 base pairs wrapped twice around histone protein (octomer) the nucleosomes forms "beads on a string" shape

Question 40

how are polytene chromosomes created?

Answer 40

DNA over-replication happens when the DNA replicated but chromosomes do not separate

Question 41

how are regional differences in chromatin condensation seen under a microscope?

Answer 41

dark bands = topological domains light bands = boundary elements

Question 42

what are boundary elements?

Answer 42

region between each topological domain separate neighboring genes to ensure their independent regulation

Question 43

what does it mean exactly "replication but chromosomes do not separate"?

Answer 43

the replication fork doesn't go all the way to the end another round start, the replication fork once again doesn't go to the end, stops before where the previous replication fork had stopped replication without separation (see slide 6)

Question 44

what happens in interphase chromatin organisation?

Answer 44

it is dynamic polytene chromosome puffs show chromatin decondensation when transcription occurs (puff out)

Question 45

how can those puffs be shown?

Answer 45

puffs associated with active form of RNA polymerase II are undergoing active transcription red puffs= polll phosphoCTD = active green = non phosphorylated = inactive

Question 46

what is chromosome paiting?

Answer 46

fluorescent in situ hybridisation (FISH) with a panel of probes which represent sequences differently distributed among the chromosomes color in the repeating DNA sequences

Question 47

explain how a cancer (name it) is caused by chromosome rearrangement

Answer 47

chronic myelogenous leukemia in blood cells there is a break on chromosome 22 and on chromosome 9 the broken off end of chromosome 22 joins to 9 and the broken off of chromosome 9 goes on 9 (the ends swap) creates a chimeric gene which codes for a protein that deregulates cell growth (cancer)

Question 48

what are some changes that happened in the karyotype of ancestral primates to bring that of humans?

Answer 48

there are several changes which lead to the karyotype that we see in humans now those changes were preserved through evolution some chromosomes were unchanged some chromosomes (14 and 15) came from the same ancestral chromosomes (5) --> there was a break there is reciprocal translocation (switching) chromosome 12,22 (=14,21) and end to end fusion chromosome 2 = chr 9+11

Question 49

what DNA does the centromere contain?

Answer 49

contains DNA that can drive mitotic segregation

Question 50

how do the microtubules join at the centromeres initially and then finally?

Answer 50

first there is a lateral joining which turns into a end to end joining

Question 51

explain the structure of telomerase and how it works

Answer 51

telomerase is a reverse transcriptase which carries its own template RNA complementary to the telomeric DNA repeat it is made up of part protein and part RNA the complementary RNA template binds to the telomeric DNA end slips/shifts over to match somewhere else from there reverse transcriptase can extend that and and make a complete telomere repeat