Class notes Functional Nuclear Domains Nucleus allows for organization more efficient processes Nuclear domains provide localized regions for the activities that occur in the nucleus DNA replication Florescent detection of replication foci flores ori if the whole thing was pinkish then not organized if there are red spots organized each spot is a focus of replication each replication focus is 200 300 ori replicons probably 100 foci of replication in the human nucleus LCQ 30 000 origins or replication in the human genome sidebar G1 space between M phase and DNA replication S synthesizing DNA 8 hours G2 end of S and G2 important because there is a finite time you can do S phase also S takes several hours DNA replication pattern incorporate one florescent dye during early S phase DNA Synthesis and a different dye during late S phase do all ori start at beginning all blue or all red experiment a whole bunch of blue and some red early replication high gene density how many genes do i have per unit length late replication low gene density when you don t need it pack it up highly organized transcription DNA that needs to be transcribed at the periphery of stain where chromatin is chromatin spread out but space space is where macheriy is inter chromosomal territory speckles spots of machinery microscopic tech florescence microscopy brighter more electron microscopy darker more True or False When an interphase eukaryotic cell is stained for chromatin and viewed under fluorescence microscopy the nucleolus appears dark because no DNA is present in that area of the nucleus false The nucleolus mutations part 1 nucleolus only a functional domain no membrane separation nucleolus is the site for ribosomal RNA rRNA synthesis rRNA processing cut out what we want and use it and throw out rest Assembly of ribosomal subunits Eukaryotic ribosomes consist of 4 types of ribosomal RNA designated 5S 5 85S Actively growing mammalian cells have 5 10 million ribosomes that must be synthesized time the cell divides amplify signal make multiple mRNA and have them be transcribed by many ribosomes 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 tandem arrays of genes for 5 8 S 18S and 28 rRNAs are transcribed by RNA Polymerase 1 into primary transcripts or pre rRNAs Ribosomal genes are found in tandem arrays 45s gene arrays Will be processed into 5 8S 18S and 28S rRNA are found on 5 different human chromosomes 13 14 15 21 22 two copies of each so ten chromosomes you need 280 28 on each 5S rRNA genes found in huge array on chromosome 1 rna poly 3 the nucleolus forms around the arrays of 45S rRNA genes which are therefore called Nucleolar Organizing Regions or NOR part of chromosome you push into middle so if the cell in not actively transcribing these rRNA genes there will not be a nucleolus M phase transcription of rRNA genes is very active and very efficiently initiated when needed christmas tree trunk rna polymerase ornaments snorps shortened length snorps cutting through why are ornaments not ribosomes no translation of rRNA this is in nucleus and translation occurs in cytoplasm ribosomes can t find cap on them strongest promoters consensus regions highly conserved under electron microscopy three distinct regions of the nucleolus can be distinguished fibrillar region or center DNA dense fibrillar region so many proteins and enzymes the ornaments granular zone unwound DNA fibrillar region transcription ribosomal proteinsdense fibrillar region processing granular zone pre ribosomal assembly 5S rRNA comes from elsewhere Which of the following molecules would you expect to find functioning or being worked on in the nucleolus Choose all that apply A snoRNPs D snRNPs B RNA polymerase I C TFIID E ribosomal proteins Replication transcription and translation all depend on accurate complementary base pairing but none are perfect mistakes in replication leads to alteration in the nucleotide sequence in the DNA these alterations are passed onto daughter cells when cell divides Heritable changes in the DNA are called mutations in the single cell organisms all daughter cells have the mutation in multi cell organisms mutations can be somatic or germ line somatic passed to daughter cells in area germ line passed to new organism Why are mistakes in transcription or translation not as critical many copies of RNA produced RNA are not heritable over multiple generation Important terminolgy gene gene lacI wild type lacI is mutated DNA sequences can be changed by many factors uncorrected mistakes in replication chemical mutagens high intensity radiation x rays UV etc Among these incorrect mistakes in replication account for the most mutations this is when millions to billions of new are formed how often do errors occur in DNA replication frequency of mutations that end up in ecoli is about 04 mutations cell division can also be 4 mutations for 100 cell divisions average in all organisms is 1 mistake per billion nucleotides in vitro e coli DNA polymerase make 400 mistakes cell division 10 000X the observed number how does E coli reduce errors it makes by 10 4 Mutations part 2 Proofreading a DNA polymerase s backspace key an example of 3 5 exonuclease activity If exonuclease activity is gone error frequency is 100 times higher accounts for half of 10 4 reduction in error frequency not as efficient in RNA polymerase they don t use 3 5 exonuclease activity If proofreading doesn t catch the mistake mismatch repair system in E coli scans recently synthesized DNA looking for mismatches and hemimethylated DNA New DNA gets methylated at adenine residues within the sequence 5 GATC 3 10 minutes after replication biological palindrome one strand read 5 to 3 is the same as when the other strand is read 5 to 3 mistake is in the new strand mismatch repair enzymes fix problem in unmethylated new strand Methyl directed mismatch repair MMR in ecoli MutS scans DNA for issues MutH looks for hemimethylated sequences endonuclease activity MutL link together MutS and MutH MutH cuts nonmethylated strand An exonuclease removes bases just beyond mismatch DNA polymerase III fills in the gap Ligase seals the nick If mismatch repair enzymes are missing error frequency is 100 times higher one type of colon cancer can be traced to a mutation in the human mismatch repair system Proofreading and mismatch repair decrease error frequency by 100 x 100 x 100 10 000 which