Lecture 16 - Chromosomes

Question 1

How is DNA compacted

Answer 1

• nucleosome cores are joined by linker DNA (34 bps)
• nucleosomes consist of a histone octomer with 147 bp wrapped around it

Question 2

What is a histone core composed of

Answer 2

Eight histones, two copies of each: H2A, H2B, H3, H4

Question 3

What is a chromatosome

Answer 3

a nucleosome and a linker histone (H1)

Question 4

What are histone tales

Answer 4

made of lys and arg, modification of them changes the structure

Question 5

What is heterochromatin

Answer 5

tightly compacted chromatin

Question 6

What is euchromatin

Answer 6

less condensed chromatin that is available for transcription

Question 7

what is a telomere

Answer 7

the end of a chromosome composed of repeated DNA sequences

Question 8

What is the p arm

Answer 8

the short arm

Question 9

what is the q arm

Answer 9

the long arm

Question 10

what is a centromere

Answer 10

appears as a constriction in metaphase chromosomes, composed of repeated sequences in the area of chromosome that attaches to the mitotic spindle

Question 11

what is a satellite

Answer 11

part of the end of the chromosome that is separated from the rest of the chromosome by a secondary constriction

Question 12

When do we usually karyotype

Answer 12

during metaphase

Question 13

What is metacentric

Answer 13

the centromere is located centrally

Question 14

what is submetacentric

Answer 14

the centromere is located off center

Question 15

what is acrocentric

Answer 15

the centromere is located nearer one end of the chromosome

Question 16

What is telocentric

Answer 16

the centromere is at the distal end of the chromosome (no p arm)

Question 17

How are lymphocutes cultured for karyotyping

Answer 17

1. peripheral blood is collected in heparin
2. it is centrifuges at 100g
3. plasma is removed and the buffy coat is transfered to media
4. cell count is done
5. 4x10^7 cells are added to 10ml media
6. it is uncubated at 37C with 5% CO2 and 97% humidity
7. growth is checked for daily and media is changed every 3 days
8. colcemid is added when cells are 60-70% confluent
9. incubated for 24 hours
10. media is transfered to a 15mL tube
11. the plask is washed with PBS and transfered to tube
12. trypsin is added to the flask and incubated for 1-5 min
13. transfer to tube
14. centrifuge for 10min 100g
15. remove all but 1mL of the supernatant
16. suspend the cells in the supernatant

Question 18

How to harvest the chromosomes

Answer 18

1. transfer the cells to a hypotonic solution
2. incubate 37C for 20 minutes
3. layer carnoys fixative on top
4. incubate
5. centrifuge 100g
6. remove supernatant
7. add to fixative
8. incubate
9. centrifuge 100g
10. remove supernatant
11. repeat steps 7 to 10

Question 19

How to view chromosomes

Answer 19

using giesma staining to look at G-banding

Question 20

How to read G banding

Answer 20

• light areas contian euchromatin
• dark areas contain heterochromatin
• centromeres do not stain

Question 21

What abnormalities can be seen in karyotypes

Answer 21

aneuploidies and structural changes

Question 22

What is an unbalanced translocation

Answer 22

a translocation resulting in the gain or loss of genetic material

Question 23

What is a balanced translocation

Answer 23

a 1 for 1 translocation that results in no visual change

Question 24

what is isochromosome

Answer 24

two p arms or two q arms separate together, resulting in a loss and gain of genetic material

Question 25

What is FISH used for

Answer 25

it deploys DNA probes specific for each chromosome to view changes

Question 26

What are CNV probes

Answer 26

copy number variation probes designed to hybridize to a precise gene location and are used to identify gene deletions and amplifications

Question 27

What are gene fusion probes

Answer 27

identify when two normally separated genes are joined (BCR-ABL)

Question 28

What are break-apart probes

Answer 28

a single probe with two fluorophores that show a yellow signal when intact and red/green signals when separated