MCB Exam 3 02 10 2014 Lecture 20 Structure of the Nucleus aqueous compartment inside the envelope nucleoplasm chromatin fibers nucleolus outer membrane of nuclear envelope inter membrane of inter envelope in between perinuclear space nuclear pore complexes nuclear lamina attachment for heterochromatin Function domains 1885 each chromosome occupies a distinct territory Carl Rabl 1984 Rabl model was proven true by detailed studies of polytene replicated but don t get separated chromosomes in Drosophila Visualizing Nuclear Domains fluorescence can visualize and identify chromosomes FISH fluorescence in situ hybridization 1 Chromosome preparation immobilization denaturation 2 Probe labeling 3 Probe denaturation 4 Mix probe with chromosome prep allow to hybridize 5 Activate probe and detect fluorescence 6 Visualize in fluorescence microscope chromosome painting can help us visualize chromosomal domains extension of FISH chromosome painting on an interphase nucleus organized domains each chromosome occupies a distinct territory within the nucleus and is arranged in an organized fashion nuclei are divided into discrete functional domains that play an important role in regulating gene expression and in replication Lecture 21 Nuclear Domains provide localized regions of function for the activities that occur in the nucleus DNA replication finds the origins and unwind incorporate fluorescent dNTPs Fluorescence detection of replication foci each spot is a focus of replication each replication focus is 200 300 ori replicons replicons number of oris probably 100 foci 300x100 30 000 oris in the human genome eukaryotic cell cycle S synthesis synthesizes DNA DNA replication pattern incorporate one fluorescent dye during early S phase and a different dye during late S phase BLUE early replication high gene density RED late replication low gene density Early replication high gene density Late replication low gene density Transcription chromatin more dna brighter strain speckles clusters of splicing factors actively transcribing genes are at the periphery of the territory interchromosomal domain is highly organized GREEN chromatin RED splicing factors Nontranscribing in periphery on nucleus Fluorescence microscopy brighter more Electron microscopy darker more Splicing factor clusters are in areas where there isn t dna A Chromosomes occupy distinct territories within the nucleus and extend regions of euchromatin out into the interchromosomal territorities for transcription because that is where the transcriptional machinery is located The red regions within the chromatin loop in the inset indicate transcriptionally active genes B Not only does the chromosome occupy its own space but the arms of the chromosome occupy their own territories and do not overlap each other The green and red indicate chromosome arms while the gold indicates the centromeric region of that chromosome C The darker the color the more compact the chromatin So the lightest yellow indicates euchromatin found predominantly at the periphery of the chromosomal territory where transcription and or replication are occurring while the deepest orange red indicates heterochromatin buried in the interior of the territory D Foci of replication Green color represents regions replicating early during S phase when the chromatin is already more unwound and represents regions of high gene density Red regions represent areas of low gene density tightly bound into heterochromatin which will not be replicated until late in S phase E Regions of chromatin with transcriptionally active genes white dots will be on the periphery of a chromosomal territory while transcriptionally inactive or silent genes black circles will be in the interior of a territory Gold circles represent Speckles or clusters of transcription replication or repair machinery F Speckles are recruited to regions of activity from much larger collections of machinery N nucleolus Lecture 22 The Nucleolus Mutations Part I Nucleolus ribosome production facility highly organized no membrane site for ribosomal RNA rRNA synthesis rRNA processing assembly of ribosomal subunits Eukaryotic ribosomes 4 types of ribosomal RNA 75 different proteins actively growing mammalian cells have 5 10 mil ribosomes that must be synthesized each time the cell divides to meet this demand all cells have multiple copies of their rRNA genes human cells have 280 copies of 5 8 18 and 28S and 2000 copies of the 5S rRNA genes tandem arrays of genes for 5 8S 18S and 28S rRNAs are transcribed by RNA polymerase I into primary transcripts or pre rRNAs snorps cut up 45S ribosomal genes are found in tandem arrays 45S gene arrays 5 8S 18S 28S are found on 5 different human chromosomes 13 14 15 21 22 5S rRNA genes found in huge array on chromosome 1 transcribed by a different RNA polymerase III nucleolus forms around the arrays of 45S rRNA genes which are therefore called nuclear organizing regions or NOR if the cell is not actively transcribing these rRNA genes there will not be a nucleolus transcription of the rRNA genes is very active and very efficiently initiated when needed under electron microscopy 3 distinct regions of the nucleolus can be distinguished fibrillar region or center contains DNA dense fibrillar region surrounds fibrillar region contains newly transcribed rRNA bound to ribosomal proteins pre RNA granular zone contains RNA bound to ribosomal proteins that are beginning to assemble into ribosomes pre ribosomal particles transcription processing assembly in 3 regions rDNA in fibrillar center pre rRNA in dense fibrillar region pre ribosomal particles in granular zone replication transcription and translation all depend on accurate complementary base pairing but none are perfect DNA replication is most reliable DNA needs to stay intact mistakes in replication lead to alterations in the nucleotide sequence in DNA these alterations are passed on to daughter cells when the cell divides Mutations heritable changes in the DNA in single cell organisms all daughter cells have the mutation in multi cell organisms mutations can be somatic or germ line somatic non sex cell passed to daughter cells in area germ line sex cell passed to new organism Why are mistakes in transcription or translation not as critical Many copies of RNA produced RNAS are not heritable over multiple generations Gene gene lacI is wild type lacI is mutated form of the gene DNA sequences can be changed by many factors uncorrected mistakes in