Chromosomes & Karyotyping Flashcards
Chromatin
- DNA, histones and other proteins = chromosome
- plays important role in DNA compaction, regulating replication, transcription, recombination and chromosome segregation
Nucleosome
- 147 bp of DNA wrapped around octomer (two H2A-H2B dimers bound to an H3-H4 tetramer) and joined by linker DNA (34 bp)
Nucleosome histone core
- 8 histones/ octamer
- two of: H2A, H2B, H3, H4
- nucleosome core DNA = 147 bp of DNA wrapped around octamer
Nucleosome function
- 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
Chromatosome
- 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
Histone tails
- Open for post-translation modification = changes chromosome structure
- Lysine and Arginine groups
Acetylation = less compact for transcription
Methylation = condensed
Heterochromatin
Eukaryotic chromatin remains tightly compacted during interphase and is not transcribed
Euchromatin
less condensed eukaryotic chromatin that is available for transcription
Phase in Cell Cylce used for Karyotyping vs FISH
- Karyotyping = metaphase (most condensed)
- FISH = interphase (not as condensed for fluorescent probes to attach)
Sister chromatids
In metaphase = one duplicated chromosome
Homologous chromosomes
Pair of sister chromatids from each parent (maternal and paternal)
Telomere vs Centromere
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
p arm vs q arm
p arm: petite arm
q arm: long arm
Differentiate Metacentric, Sub metacentric, Acrocentric, Telocentric
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
G-banding
- 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
WIP Resolution of G-banded chromosomes based on phase
- towards metaphase is easier to read
- towards prophase has better separation of bands
How are bands on chromosomes numbered?
- Starts from the centromere and counts outwards
Chromosome> Arm > region > bands > subband
ie. 17q11.2
Aneuploidies vs Structural abnormalities
Aneuploidies: abnormal number of chromosome, ie. trisomy
Structural: translocations, deletions, insertions, inversions, isochromosomes, ring chromosomes
What is a balanced translocation ?
Two chromosomes that have a double-sided break
- no loss or gain of genetic material
Isochromosomes
- two chromosomes become one = two p arms or two q arms separate together
- loss and gain of genetic material = unbalanced structural abnormality
Unbalanced Translocation
- 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
FISH
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
FISH whole-chromosome probes
Collections of smaller probes, each of which binds to a different sequence along the length of a given chromosome
FISH using SKY
Spectral karyotyping:
24 chromosome-specific painting probes are used in just one FISH experiment Very expensive (not used often)
FISH: centromeric probes
- can be used on interphase and metaphase chromosomes
- used to enumerate chromosomes
FISH using Locus specific probes
- 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
FISH using CNV probes
Copy Number Variation probes (locus specific):
- designed to hybridize to a precise gene location
- used to identify gene deletions and amplifications
FISH using Gene Fusion Probes
- 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
FISH using Break-apart probes
- 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
Name the nucleosome histones
Two of: H2A, H2B, H3, H4
List the levels of compaction of chromatin
DNA > histones > nucleosome > chromatin > 30nm fibre > looped domains > metaphase chromosome
Karyotype of a normal female vs male
46,XX = female
46, XY = male
Karyotype of trisomy female
47,XX+21
Purpose of Colcemid for Karyotyping
Arrests cells in metaphase
Purpose of trypsin in Karyoptyping
Digests proteins (so stain can attach)*
*NOTE: in Giemsa staining
Stain for chromosomes
Giemsa stain
