weber uiuc edu 12 October 2007 MCB150 Lecture 21 Lecture 21 12 October 2007 Announcements More broken hand notes Next week is the exam and there will be discussion section questions posted online o Review them Regular lecture on Monday Monday night at 5 7PM in 228 NHB Review Review on Wednesday too Chromsomal Domains Territories How do you experimentally determine where the DNA resides during interphase FISH Fluorescense In Situ Hybridization o Label specific regions on the chromosomes o In the example can see specifically labeled area o Can also label entire chromosomes paternal or maternal Called chromosome painting Do Interphase Chromsomes Mitotic Chromosomes Occupy Territories Chromosomal painting can help visualize the locations of entire chromosomes o Can actually see where one begins and another ends o Use this to tell if there are different locations for each DIAGRAM Chicken chromosomes o One is stained normally o The other image shows staining using painting Each chromosome has a different fluorescent dye attached What would we see if there were different colors used o Can see the different chromosomes are in relation to one another o Again the picture is of a mitotic cell We still do not know for interphase chromosomes What if we do this during interphase o Won t see such tight X chromosomes o If they don t occupy particular regions will not see distinct colors o If they do we should see regions of distinct color The result is there are clear regions They are not compact because they are in interphase Beautiful illustration due to painting o Does not appear to infringe on neighbor s territory Nuclei are divided up into distinct regions of function o Functional domains organized regions of chromosomal activity o Put each process into its own domain o Typically obey the rule of bringing the DNA to the machinery instead of the opposite Chromosomal Domains Territories DIAGRAM Region of red includes all of the machinery for working with the DNA Page 1 of 6 weber uiuc edu 12 October 2007 MCB150 Lecture 21 These are called interchromsomal domains o Any space in the nucleus where there isn t a chromosome There is a lot there and a lot going on in that space It is not empty space Localization of Regions Domains proide localized regions of function o E g DNA replication Use fluorescently labeled dNTPs to find out how the DNA is organized DIAGRAM of eukaryotic DNA More than one ori present Now incorporating dNTPs in both directions away from the ori on both strands using the fluorescent dNTPs Simply look for the fluorescence What are we looking for We want to see if the DNA is organized into a particular region If we know there are 30 000 oris then we will see either a smear of color or one location of red Smears mean no organization One region of color means that all replication is happening in one location a bunch of oris are being worked on in one region Fluorescence Detection All we want to see is fluorescence At one point we see a spot A close look at that spot reveals that it is not all of the oris nor just a single ori Instead probably it is a region of the chromosome with a cluster of oris o May be some spacing between them We only need to loop out the ori regions to the machinery for replication o The nucleus will cluster together oris o Makes it easier to keep the machinery all in one location Each spot is considered a focus of replication o There will be many foci of replication in the nucleus Within human chromosomes there are 200 300 replicons o Replicons are the replication complexes containing an ori Probably 100 or more foci of replication in the human nucleus Thus 200 300 oris all operating in 100 or more foci of replication o Doing the math we see that 300 oris 100 foci 30 000 replications happening simultaneously which is what we expect to happen The Eukaryotic Cell Cycle M phase the phase when a cell splits off o M phase is very short because it is condensing and rearranging only No protein synthesis happens while the DNA is condensed and being separated G1 phase the gap phase Period between M phase and S phase o Will take some time to prepare for replication o Accumulate protein etc Lots of things happening o We do not stop growing and protein synthesis throughout interphase S phase synthesizing of DNA o The finite period of time during interphase when DNA is replicated G2 phase the second gap phase Page 2 of 6 weber uiuc edu 12 October 2007 MCB150 Lecture 21 o Check for mistakes and check for preparedness for M phase o Shorter time period than others except M phase We have to break up interphase into three new interphases Order of cell cycle M G1 S G2 The most important thing for DNA to do is to accurately replicate itself and separate itself into daughter cells DNA Replication Pattern Incorporation of fluorescent dye during S phase and a different dye later in Sphase o Use two different markers o Used to check if all ori operate simultaneously Or does some DNA get replicated later in S phase than others Will need to move things out of the way when not needed the chromatin should be packed Is all DNA able to be replicated at the same time o No Some DNA is more tightly packed so it is inaccessible o Cannot replicate that until later in S phase Use red and blue color to get the image in the notes o Each spot is a focus of replication o Throughout all of S phase some is later than the others We can start making guesses about what was replicated early o Early replication means easy early access Which means it was being used probably transcribed Thus euchromatin is likely to be replicated earlier o Heterochromatin will tend to be replicated later because those genes are more tightly packed and are not being used Do not confuse gene density the number genes in a sequence of base pairs with heterochromatin density which is the type of packing o Regions of high gene density are loosely packed because they are being used euchromatin o Regions of low gene density are tightly packed because they are infrequently or never used heterochromatin Statistically speaking euchromatin DNA is more likely to have higher gene density o There will be some regions of low gene density o However unwound genes are more likely to be higher density Can extend this to say that regions of euchromatin have higher gene density The late replication red is more tightly packed and it will take more time to get to it It isn t used at the time so these are regions of lower gene density In other