Lecture 16 - Chromosomes Flashcards
How is DNA compacted
- nucleosome cores are joined by linker DNA (34 bps)
- nucleosomes consist of a histone octomer with 147 bp wrapped around it
What is a histone core composed of
Eight histones, two copies of each: H2A, H2B, H3, H4
What is a chromatosome
a nucleosome and a linker histone (H1)
What are histone tales
made of lys and arg, modification of them changes the structure
What is heterochromatin
tightly compacted chromatin
What is euchromatin
less condensed chromatin that is available for transcription
what is a telomere
the end of a chromosome composed of repeated DNA sequences
What is the p arm
the short arm
what is the q arm
the long arm
what is a centromere
appears as a constriction in metaphase chromosomes, composed of repeated sequences in the area of chromosome that attaches to the mitotic spindle
what is a satellite
part of the end of the chromosome that is separated from the rest of the chromosome by a secondary constriction
When do we usually karyotype
during metaphase
What is metacentric
the centromere is located centrally
what is submetacentric
the centromere is located off center
what is acrocentric
the centromere is located nearer one end of the chromosome
What is telocentric
the centromere is at the distal end of the chromosome (no p arm)
How are lymphocutes cultured for karyotyping
- peripheral blood is collected in heparin
- it is centrifuges at 100g
- plasma is removed and the buffy coat is transfered to media
- cell count is done
- 4x10^7 cells are added to 10ml media
- it is uncubated at 37C with 5% CO2 and 97% humidity
- growth is checked for daily and media is changed every 3 days
- colcemid is added when cells are 60-70% confluent
- incubated for 24 hours
- media is transfered to a 15mL tube
- the plask is washed with PBS and transfered to tube
- trypsin is added to the flask and incubated for 1-5 min
- transfer to tube
- centrifuge for 10min 100g
- remove all but 1mL of the supernatant
- suspend the cells in the supernatant
How to harvest the chromosomes
- transfer the cells to a hypotonic solution
- incubate 37C for 20 minutes
- layer carnoys fixative on top
- incubate
- centrifuge 100g
- remove supernatant
- add to fixative
- incubate
- centrifuge 100g
- remove supernatant
- repeat steps 7 to 10
How to view chromosomes
using giesma staining to look at G-banding
How to read G banding
- light areas contian euchromatin
- dark areas contain heterochromatin
- centromeres do not stain
What abnormalities can be seen in karyotypes
aneuploidies and structural changes
What is an unbalanced translocation
a translocation resulting in the gain or loss of genetic material
What is a balanced translocation
a 1 for 1 translocation that results in no visual change
what is isochromosome
two p arms or two q arms separate together, resulting in a loss and gain of genetic material
What is FISH used for
it deploys DNA probes specific for each chromosome to view changes
What are CNV probes
copy number variation probes designed to hybridize to a precise gene location and are used to identify gene deletions and amplifications
What are gene fusion probes
identify when two normally separated genes are joined (BCR-ABL)
What are break-apart probes
a single probe with two fluorophores that show a yellow signal when intact and red/green signals when separated
