Chromosomes-I

Dr Imran Siddiqui

Human Genome Basics• Human cells are diploid, having 2 copies of each

chromosome, one from each parent.

• The gametes, sperm and egg, are haploid, with 1 copy of each chromosome.

• The X and Y chromosomes are sex chromosomes, and the other 22 chromosomes are called autosomes.

• Normal males are XY and normal females are XX. Thus, sperm cells either contain an X or a Y. Eggs always contain an X chromosome.

Cell Cycle and Chromosomes• A chromatid is a single DNA molecule along with supporting

proteins and RNA molecules.

• Chromosomes spend most of their time in the cell as single chromatids, the monad state. When a cell is getting ready to divide, the DNA in the chromosomes is replicated and the chromosomes become diads, two chromatids held together at the centromere.

– The diad state is what is typically seen in pictures of chromosomes, but it only occurs briefly in the life of the cell. During most of interphase

Cell Cycle• The 4 stages are: G1, S, G2, and M.• M = mitosis, where the cell divides into 2

daughter cells. The diad chromosomes go from the (2 chromatid) form to the monad (1 chromatid) form. That is, before mitosis there is 1 cell with diad chromosomes, and after mitosis there are 2 cells with monad chromosomes in each.

• S = DNA synthesis. Chromosomes go from monad to diad.

• G1 = “gap”. Nothing visible in the microscope, but this is where the cell spends most of its time, performing its tasks as a cell. Monad chromosomes

• G2 (also “gap”). Diad chromosomes, cell getting ready for mitosis.

• G1, S, and G2 are collectively called “interphase”, the time between mitoses

Chromosome Structure• DNA is long and thin and fragile:

needs to be packaged to avoid breaking.

• Lowest level is the nucleosome :150 bp of DNA wrapped 1 3/4 times around a core of 8 histone proteins (small and very conserved in evolution). A string of beads.

• The nucleosomes coil up into a 30 nm chromatin fiber. This level of packaging exists even during interphase.

• During cell division, chromatin fibers are attached in loops of variable size to a protein scaffold.

• The loops may be functional units: active vs. inactive in transcription.

• Further coiling gives the compact structures we see in metaphase.

Centromeres• Sometimes called the “primary

constriction” on a chromosome

• The centromere is the attachment point for the spindle.

• The centromere is a region of DNA on the chromosome. During cell division, a large protein structure, the kinetochore, that attaches to the centromere DNA sequences. The spindle proteins then get attached to the kinetochore.

• The centromere is a DNA many repeats of a about 170 bp element.

Telomeres

• Telomeres are the DNA sequences at the ends of chromosomes. • They keep the ends of the various chromosomes in the cell from

accidentally becoming attached to each other.• The telomeres of humans consist of as many as 2000 repeats of the

sequence: 5' TTAGGG 3'. • It is estimated that human telomeres lose about 100 base pairs

from their telomeric DNA during each mitosis (cell division). At this rate, after approximately 125 mitotic divisions, the telomeres would be completely gone, which is why normal cells will eventually die after healthy division.

• Chromosome shortening is prevented by telomerase, an RNA/protein hybrid enzyme.

• Telomerase is not active in most cells, but is active in stem cells, germ cells, hair follicles and most cancer cells.

Mitosis• Mitosis is ordinary cell division among the cells of the body. During

mitosis the chromosomes are divided evenly, so that each of the two daughter cells ends up with 1 copy of each chromosome.

• Prophase: --chromosomes condense --nuclear envelope disappears --centrioles move to opposite ends of the cell --spindle forms• Metaphase: --chromosomes are lined up on cell equator attached to the spindle at the centromeres• Anaphase: --centromeres divide. Now chromosomes are monads --the monad chromosomes are pulled to opposite poles by the spindle.• Telophase: --cytokinesis: cytoplasm divided into 2 separate cells --chromosomes de-condense --nuclear envelope re-forms --spindle vanishes

Meiosis• Meiosis is the special cell division that converts diploid body cells into the

haploid gametes. Only occurs in specialized cells.• Takes 2 cell divisions, M1 and M2, with no DNA synthesis between.• In humans, start with 46 chromosomes (23 pairs) in diad state. After M1,

there are 2 cells with 23 diad chromosomes each. After M2 there are 4 cells with 23 monad chromosomes each.

• Prophase of M1 is very long, with a number of sub-stages. • Main event in prophase of M1 is “crossing over”, also called

“recombination”. • In crossing over, homologous chromosomes pair up, and exchange

segments by breaking and rejoining at identical locations. • Several crossovers per chromosome, with random positions. This is the

basis for linkage mapping.• Chromosomes that don’t recombine seem to have a high rate of non-

disjunction (chromosome goes to the wrong pole). Maybe homologues are held together until anaphase by recombination proteins.

Chromosomes in the Microscope• Studied in metaphase cells, usually

white blood cells or skin cells.• Technique: arrest cell division at

metaphase with colchicine or colcemid (blocks spindle microtubules). Then, hypotonic treatment swells them and spreads out the chromosomes.

• Picture is a karyotype: chromosome pictures cut out and sorted by hand, or by computer.

• Length varies: longest is chromosome 1, shortest is 21).

• Bands seen with different stains, especially Giemsa stain, which produces G bands. R bands are “reverse Giemsa”, the light bands seen with Giemsa stain

Types of Chromosomes

There are four types of chromosomes based upon the position of the centromere.

1) Metacentric : Centromere occurs in the centre and all the four chromatids are of equal length.

2) Submetacentric : Centromere is a little away from the centre and therefore chromatids of one side are slightly longer than the other side.

3) Acrocentric : Centromere is located closer to one end of chromatid therefore the chromatids on opposite side are very long. A small round structure, attached by a very thin thread is observed on the side of shorter chromatid. The small round structure that is a part of the chromatid is termed as satellite.

4) Telocentric : Centromere is placed at one end of the chromatid and hence only one arm. Such telocentric chromosomes are not seen in human cells.

FISH• Fluorescence in situ hybridization.• Hybridize a DNA probe labeled

with a fluorescent marker to chromosomes, then visualize in fluorescence microscope.

• See location of the gene: often can see sister chromatids even.

• Chromosome painting: use many probes from a single chromosome (there is lots of unique DNA on each chromosome). Good for seeing rearrangements.

• Picture is translocational Down syndrome. Two copies