Human Genome Basics Most 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. Humans have a haploid number n of 23, meaning that there are 23 chromosomes in a complete haploid set. Thus, gametes have 23 chromosomes and diploid cells have 46. The pairs of chromosome in a diploid cell are called homologues. 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 dyads, two chromatids held together at the centromere. The dyad 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, chromosomes are in the monad state.
Cell Cycle The cell cycle is a theoretical concept that defines the state of the cell relative to cell division. The 4 stages are: G1, S, G2, and M. M = mitosis, where the cell divides into 2 daughter cells. The chromosomes go from the dyad (2 chromatid) form to the monad (1 chromatid) form. That is, before mitosis there is 1 cell with dyad chromosomes, and after mitosis there are 2 cells with monad chromosomes in each. S = DNA synthesis. Chromosomes go from monad to dyad. 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). Dyad 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 DNA probably attaches at specific AT-rich areas called scaffold attachment regions. 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, based on microscopic appearance. The centromere is the attachment point for the spindle. Acentric chromosomes, which dont have a centromere, dont attach to the spindle and dont end up in either nucleus after mitosis. 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 many repeats of a about 170 bp element (very difficult to clone in humans but well known in yeast). Called alpha-satellite DNA. Centromere regions also contain large amounts of repeated sequence DNA and transposable elements (more on this later).
Origins of Replication DNA replication starts at specific spots on the chromosome, the origins of replication. In yeast, these sequences are called autonomously replicating sequences (ARS) and are short well defined sequences. In humans, it seems that origins of replication can vary a bit between cells and under different conditions. May be many of these on each chromosome.
Telomeres Telomeres are the DNA sequences at the ends of chromosomes. Chromosomes that lose their telomeres often fuse with other chromosomes or become degraded. There are telomere-binding proteins that protect the chromosome ends. Telomeres are also needed to ensure complete replication of the DNA: the end-replication problem DNA polymerase must have a double stranded primer region with a free 3 OH to build on. The primer is made of RNA, synthesized by primase. At the 3 end of the chromosome, the RNA primer gets degraded, leaving a single stranded region of DNA. In the next round of replication, one DNA molecule will be shorter than the other. Process repeats, gradually shortening the chromosomes. Thought to be a cause of cell mortality.
More Telomeres Chromosome shortening is prevented by telomerase, an RNA/protein hybrid enzyme. Telomerase has a short RNA that is used as a template for a reverse transcriptase: binds to 3 end of chromosome, then synthesizes DNA extension. This extension acts as a template for regular DNA polymerase, keeping chromosome length intact. Telomere sequences are multiple repeats of a highly conserved 7 base sequence.
Euchromatin and Heterochromatin Euchromatin is the location of active genes (although many genes in euchromatin are not active: depends on cell type). During interphase euchromatin is extended and spread out throughout the cell. Heterochromatin is darkly staining, condensed, and late replicating. Genes in heterochromatin are usually inactive. Some heterochromatin is constitutive : always heterochromatin: especially around centromeres. Composed mostly of repeat sequence DNA. Other heterochromatin is facultative: can be heterochromatin or euchromatin: e.g. inactive X chromosome in females., the Barr body.
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 dyad state. After M1, there are 2 cells with 23 dyad 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 dont recombine seem to have a high rate of nondisjunction (chromosome goes to the wrong pole). Maybe homologues are held together until anaphase by recombination proteins.
More Meiosis Metaphase of meiosis 1 is very different from metaphase in mitosis (or M2). In metaphase of M1, pairs of homologous chromosomes line up together. In mitosis and M2, chromosomes line up as single individuals. Anaphase of M1: the spindle pulls the two homologues to opposite poles. However, the centromeres dont divide, and the chromosomes remain dyads. Telophase of M1: cytoplasm divided into 2 cells, each of which has 1 haploid set of dyad chromosomes Meiosis 2 is just like mitosis. In prophase, the chromosomes condense and the spindle forms. Metaphase of M2: dyad chromosomes line up singly on the cell equator. Anaphase of M2: centromeres divide, chromosomes are now monads which get pulled to opposite poles. Telophase: cytoplasm divided into 2 cells. After M2: total of 4 cells from the original cell. Each contains one haploid set of monad chromosomes
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 (should be 22, but mistakes were made early on). Centromere position: centromere index: length of short arm divided by total length. Used to define metacentric, sub-metacentric, acrocentric. (No human telocentrics) Bands seen with different stains, especially Giemsa stain, which produces G bands. R bands are reverse Giemsa, the light bands seen with Giemsa stain. Bands seem to derive from large scale variations in GC content.
Nomenclature Short arm is p (petite) and long arm is q. cen is centromere, ter is terminus (telomere): pter and qter. proximal means closer to the centromere, and distal means father away from the centromere Regions divided at major bands: p1, p2, p3, etc. Then each region is divided into lesser bands; p11, p12, etc (pone-one, not p-eleven). Even smaller bands too: p12.1, etc.
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
Chromosome Abnormalities Three basic types: polyploidy: having more than 2 sets of chromosomes. aneuploidy: hav