Chromosomes & Karyotyping

Question 1

Chromatin

Answer 1

• DNA, histones and other proteins = chromosome
• plays important role in DNA compaction, regulating replication, transcription, recombination and chromosome segregation

Question 2

Nucleosome

Answer 2

• 147 bp of DNA wrapped around octomer (two H2A-H2B dimers bound to an H3-H4 tetramer) and joined by linker DNA (34 bp)

Question 3

Nucleosome histone core

Answer 3

• 8 histones/ octamer
• two of: H2A, H2B, H3, H4
• nucleosome core DNA = 147 bp of DNA wrapped around octamer

Question 4

Nucleosome function

Answer 4

• Facilitates the compaction of ~200 bp of DNA
• Facilitates the compaction of the 30 nm chromatin fiber
• Template for chromatin enzymes which facilitate post-translational modifications = PTM facilitate higher levels of compaction

Question 5

Chromatosome

Answer 5

• consists of a nucleosome and a linker histone
• Histone protein H1 may bind to linker DNA = protect an additional 15 to 20 bp of DNA
• H1 bind at the DNA entering & exiting the nucleosome

Question 6

Histone tails

Answer 6

• Open for post-translation modification = changes chromosome structure
• Lysine and Arginine groups

Acetylation = less compact for transcription
Methylation = condensed

Question 7

Heterochromatin

Answer 7

Eukaryotic chromatin remains tightly compacted during interphase and is not transcribed

Question 8

Euchromatin

Answer 8

less condensed eukaryotic chromatin that is available for transcription

Question 9

Phase in Cell Cylce used for Karyotyping vs FISH

Answer 9

• Karyotyping = metaphase (most condensed)
• FISH = interphase (not as condensed for fluorescent probes to attach)

Question 10

Sister chromatids

Answer 10

In metaphase = one duplicated chromosome

Question 11

Homologous chromosomes

Answer 11

Pair of sister chromatids from each parent (maternal and paternal)

Question 12

Telomere vs Centromere

Answer 12

Telomere:
- end of a chromosome composed of repeated DNA sequences and associated proteins

Centromere:
- constriction in metaphase chromosomes.
- composed of repeated sequences, where chromosome that attaches to the mitotic spindle

Question 13

p arm vs q arm

Answer 13

p arm: petite arm

q arm: long arm

Question 14

Differentiate Metacentric, Sub metacentric, Acrocentric, Telocentric

Answer 14

Metacentric: centromere located centrally, the p and q arms are approximately the same length.

Sub metacentric: the centromere is located off center; the q arm is slightly longer than the p arm

Acrocentric: the centromere is located nearer one end of the chromosome; the q arm is significantly larger than the p arm

Telocentric: centromere is the distal end of the chromosome and there is no p arm; not normally found in humans

Question 15

G-banding

Answer 15

• karyotype used to ID chromosome abnormalities in genetic diseases and cancers
• differentiate by size, shape and banding patterns

Giemsa stain:
Light areas = Euchromatin
Dark = heterochromatin
Unstained centromeres

Question 16

WIP Resolution of G-banded chromosomes based on phase

Answer 16

• towards metaphase is easier to read
• towards prophase has better separation of bands

Question 17

How are bands on chromosomes numbered?

Answer 17

• Starts from the centromere and counts outwards

Chromosome> Arm > region > bands > subband
ie. 17q11.2

Question 18

Aneuploidies vs Structural abnormalities

Answer 18

Aneuploidies: abnormal number of chromosome, ie. trisomy

Structural: translocations, deletions, insertions, inversions, isochromosomes, ring chromosomes

Question 19

What is a balanced translocation ?

Answer 19

Two chromosomes that have a double-sided break
- no loss or gain of genetic material

Question 20

Isochromosomes

Answer 20

• two chromosomes become one = two p arms or two q arms separate together
• loss and gain of genetic material = unbalanced structural abnormality

Question 21

Unbalanced Translocation

Answer 21

• results from a balanced translocation carrier and normal chromosome
• gain or loss of genetic material

1/4 chance of:
- normal chromosomes

• balanced translocation carrier
• unbalanced duplication-deletion
• unbalanced duplication- deletion
  NOTE: miscarriage

Question 22

FISH

Answer 22

Fluorescence in situ Hybridization:

• DNA probes specific for nucleotide sequences
• probes made of DNA/ RNA fragments (100-1000bp)
• modified nucleotides in probes fluorescence under certain conditions

Question 23

FISH whole-chromosome probes

Answer 23

Collections of smaller probes, each of which binds to a different sequence along the length of a given chromosome

Question 24

FISH using SKY

Answer 24

Spectral karyotyping:
24 chromosome-specific painting probes are used in just one FISH experiment Very expensive (not used often)

Question 25

FISH: centromeric probes

Answer 25

- can be used on interphase and metaphase chromosomes - used to enumerate chromosomes

Question 26

FISH using Locus specific probes

Answer 26

- bind to a particular region of a chromosome - useful when scientists have isolated a small portion of a gene and want to determine on which chromosome the gene is located OR - how many copies of a gene exist within a particular genome

Question 27

FISH using CNV probes

Answer 27

Copy Number Variation probes (locus specific): - designed to hybridize to a precise gene location - used to identify gene deletions and amplifications

Question 28

FISH using Gene Fusion Probes

Answer 28

- locus specific - fusions occur when two normally separated genes are joined = show the signals less than one signal width apart - a normal cell will display the colors as separate signals

Question 29

FISH using Break-apart probes

Answer 29

- locus specific - single probe with 2 fluorophores - probe targets two areas of a specific gene sequence - Intact = probe appears yellow - Apart = red signal and a green signal are seen

Question 30

Name the nucleosome histones

Answer 30

Two of: H2A, H2B, H3, H4

Question 31

List the levels of compaction of chromatin

Answer 31

DNA > histones > nucleosome > chromatin > 30nm fibre > looped domains > metaphase chromosome

Question 32

Karyotype of a normal female vs male

Answer 32

46,XX = female 46, XY = male

Question 33

Karyotype of trisomy female

Answer 33

47,XX+21

Question 34

Purpose of Colcemid for Karyotyping

Answer 34

Arrests cells in metaphase

Question 35

Purpose of trypsin in Karyoptyping

Answer 35

Digests proteins (so stain can attach)* *NOTE: in Giemsa staining

Question 36

Stain for chromosomes

Answer 36

Giemsa stain