Chromosome and chromatin basics
What DNA sequences do you need to make a chromosome?
- Centromere
o Region to which the spindle fibers attach during mitosis
- Telomeres
o The ends of chromosomes
o Very special, usually linear ends of DNA molecules are not stable in the cell
- Origin of replication
o Where DNA replication begins
- Artificial chromosomes
o First eukaryotic artificial chromosome was a YAC
o Need those three DNA elements plus a minimum size
§ 11,000bp chromosome has segregation errors in 50% of mitoses
§ 55,000bp has segregation error in 1.5%
§ 100,000bp has segregation error in .3%
What is a chromosome?
- one piece of DNA with the three elements above plus all the genes (1/3 of chromosome)
- lots of proteins
o histones (1/3 of chromosome)
§ H1, H2A, H2B, H3, H4
§ Lots of “basic” amino acids lysine and arginine
· Positively charged at neutral pH (because accept H+, makes these basic)
· Allows them to bind negatively charged phosphodiester backbone
§ Involved in packaging DNA so it fits in nucleus
§ Also intimately involved in transcriptional regulation
o Great conservation of amino acid sequence between different organisms
§ Pea to bovine H4, only 2 differences in 102 amino acids
§ Suggests any mutations are deleterious
o nonhistone chromosomal proteins (1/3 of chromosome)
§ a whole grab-bag of different proteins
§ RNAP, DNAP
§ Transcriptional regulatory proteins
§ Other structural proteins that help package the DNA
The nucleosome and higher-order chromatin structure
- nucleosome
o octamer of histones, two each of 2A, 2B, 3, 4
o arranged very specifically
o DNA wrapped around
§ 147bp DNA
§ DNA wrapped around slightly less than twice
o linker DNA between nucleosomes (Fig. 13.3)
o how are nucleosomes formed?
§ Not clear, assembled shortly after replication
- 30nm (300A) fiber,
o solenoid of nucleosomes, H1 may help form this
o condensed chromatin (Fig. 13.4a)
o hard for proteins to have access (like RNAP) so less txn (????)
- Scaffold
o a protein structure to which the DNA/nucleosomes may be hooked up
o gives chromosomes characteristic shape and this scaffold can be seen even when DNA is digested away
o many proteins thought to be involved
o a bit nebulous (Fig. 13.4b)
o scaffold-attachment regions – these are DNA sequences that are thought to associate with the scaffold (probably via DNA binding proteins)
o have done fluorescence studies – fluorescently labeled probes from regions far apart on the chromosome end up close together in the cell
Karyotypes and banding
- number and size of chromosomes in an organism is its karyotype
- get from cells in metaphase of mitosis, when chromosomes are condensed
- often do banding
o G-banding with Geimsa reagent
o Leads to light and dark bands
§ 2000 identifiable bands in human karyotype (Fig.13.6)
o generally dark bands have condensed chromatin, light bands less condensed
o Characteristic pattern for each chromosome. Every time they are G-stained they give the same pattern of bands
o Can ID chromosomes this way
o Can report location of a gene this way (Fig. 13.6c)
§ P arm (short arm)
· 1p3.5
§ Q arm (long arm)
o Can diagnose chromosomal abnormalities with this
§ Down’s
§ others
- Spectral karyotype (chromosome painting)
o Make probe DNA specific for each chromosome
o Label with dye
§ Two dyes that fluoresce at different wavelengths
§ Can do each with different proportion of dyes
§ Hybridize probe to metaphase chromosomes
§ So each chromosome has different proportion of two dyes
§ Computer can ID each chromosome based on dye proportion and make false-color image