#9. 9.12.96, Dr.Pherson. How is DNA organized within the nucleus, how does this affect functionality of DNA? Mammalian organisms all have large amts of DNA so they can produce the many different cell types. DNA contents of several genomes #pair #chromosomes length E.coli 4x10 to the 6th 1 1.4 mm yeast 1.4x10 to the 7th 16 4.6mm fruit fly 1.7x10 to the 8th 4 56mm human 3.9x 10 to the 9th 23 990mm DNA not found as single molecule. It is organized into distinct chromosomes which are DNA plus proteins. Histones: 5 major classes (see chart in notes) Major structural protein associated w/DNA, present in amount nearly equal to amt of DNA. they are rich in lysine and/or argenine. At neutral pH in the cell, those AAs are + charged, while DNA is - charged, so these molecules are ionically attracted to eachother. This is good, because the histones are responsible for "packaging' the DNA in the cell. Histone proteins interact w/one another to form a "core" consisting of 8 subunits (see diagram) which the DNA will interact with. The negatively charged DNA strand wraps around the histone core protein complex. this is called a nucleosome, and is repeated along the DNA at about 200 base pair (146 bp) intervals. There are millions of nucleosomes in each cell of the body. The 200 # reflects # of base pairs w/in one core region and one linker region. see diagram for how the nucleosomes are linked by "linker" dna, which is not fixed in length. Many nucleases preferentially CUT the linker DNA, producing DNA fragments that are multiples of the nucleosome repeat length, which is usually around 200 bp. This was one of the first clues that this structure existed. [slide- trinucleosomes, dinucleosomes, mononucleosomes on a pherisis plate of some kind.] When extnended by low ionic strength, the nucleosome chain resembles "beads on a string", but normally the nucleosomes are folded into more condensed structures such as the 30 nm fiber. Eg, the nucleosomes coil up -see photocopy. The arrangement of nucleosomes on the 30 nm fiber isn't known. The DNA strand in the 30 nm fiber has a packing ratio of about 40:1 (?) What causes the nucleosomes to coil up? who knows. [slide- cartoon of how nucleosomes might be folded, with crisscrossing linker DNA, which zigzags through middle of fiber, with nucleosomes arranging themselves on the outside of the fiber. This would accomodate any length of linker DNA, and would be extremely flexible.] Posttranslational modifications of histones are associated w/transcription and replication. It is believed that these modifications are involved in opening up the chromatin to facilitate access to the DNA. These modifications include phosphorylation of histone H1 and acetylation of core histones. DNA LOOPS Experiment was done. Chromosome in metaphase was taken and treated with 2 molar sodium chloride solution which removed the histone proteins and caused the dna to unfold and fan out. Chromatin is arranged in large loops which extend from a chromosomal "scaffold" and this can be shown by removing the histones from a metaphase chromasome. [see diagram]. Note the ENORMOUS amount of DNA released when the histones are removed. Most of this DNA is organized in loops about 20 to 100 kilobases in length. These loops are of course folded into 30 nm fibers, and usually emerge and return to the scaffold at nearly the same spot. One of the proteins which attaches the DNA to the scaffold is topoisomerase II. This enzyme catalyses changes in the topology of DNA by making a double stranded cut in the DNA and passing a DNA strand through that cut. Topological stress created by the passage of a polymerase through a segment of DNA can be relieved by topoisomerases. So, it can just kind of untie the DNA knots, let things in, and seal it back up again, or it can relax a strained area of DNA which is supercoiled. why do we need these enzymes? If you have a fixed DNA strand, and you have to unravel it with polymerase to transcribe it...so, you need toposiomerase II to untangle the tangling caused by this process. Yeast cells containing a mutation in topoisomerase II can complete one round of DNA replication but can't segregate their chromosomes properly. It appears that DNA strands get tied into a knot at the completion of replication. The tangled chromosomes can't be separated w/o topoisomerase II. Topoisomerases are targets for several drugs. Some are anticancer drugs, since they selectively kill dividing (replicating) cells. [see diagram-supercoiling is introduced by forward motion of polymerase, and it must be relieved by topoisomerase in order to allow polymerase to proceed forward.] lack of topoisomerase can cause cell to not be able to separate off replicated chromosomes. They get stuck together as the daughter DNA peels off and you reach the end but can't break the chain between old and new. cartoon summary of chromosome structure p 5 of handout. _____________________ ___________ |___________________ \/ ___________| ____________________| |__________ |____________________/ \____________| | | telomere (end) centromere: center [temporarily skipped this part] X chromosome inactivation: The Y chromosome is kind of puny, so males are deficient in genetic information according to Dr.Pherson. Hmm. Females have XX in mammals, males have XY, so are haploid for genes that are only on X chromosomes, so males are more susceptible to x-linked genetic dz. In females, one of the two X chromosomes is inactive, present in a highly condensed state, often seen as a Barr body. This condensation prevents expression of most of the genes. The inactivation occurs very early in development. It also occurs RANDOMLY. It is clonally propagated to later stages. So, adult female mammals will have x-linked traits expressed in heterogeneous patches - note fur color of calico cat. Mechanism of x inactivation is not known. Condensation of regions of chromatin that aren't being used is an important aspect of gene regulation in other regions of the chromatin. Sperm chromatin: The DNA in mammalian sperm is not complexed with histones. Instead - small argenine rich proteins called protamines are present. The histones are removed late in the developement of the sperm and replaced w/the protamines. THey form a VERY compact DNA, so the nucleus can be MUCH more reduced in size, and is completely inactivated. This highly condensed state facilitates sperm mobility. Erythrocytes: Birds and amphibians have nucleated erythrocytes (nRBCs). Their chromatin is in a highly condensed and inactive state. This is accomplished partly by presence of a histone called H5 - which is structurally related to histone H1. H5 is found only in nRBCs.