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169 Cards in this Set
- Front
- Back
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Introns discovered in 1977 by...
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Philip Sharp (adenovirus), Alec Jeffries (hemoglobin)
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Sharp
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He isolated the RNA that was made from an adenovirus, when he tried to hybridize it to genomic DNA he found segments and loops.
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Hemoglobin
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2000bp, 3 exons, 2 introns
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Jeffries
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showed that the introns had repetitive DNA...used it work out the method of DNA finger printing
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Human factor 8 gene
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responsible for blood clotting, 200k bp, gene is so big it cant even be in a plasmid clone....however can clone the cDNA
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Prokaryotes vs. eukaryotes
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in prokaryotes the genes are closely spaced and frequently cotranscribed (polycistronic message). In eukaryotes genes are never cotreanscribed, always a single gene per RNA, exons have to be united and introns are taken out.
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Getting rid of introns is important because
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its usually ridded with stop codons
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The only exon that has to have the AUG is
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the first one
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You have to have a stop in the...
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in the last exon
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RNA processing in Eukaryotes
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5' cap, 3' polyA addition, splicing
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5' cap and polyA tail
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very important for translation initiation, transport, and stability
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In eukaryotic translation the 5' cap is important for
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binding factors
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In eukaryotic translation
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one open reading frame, cant make a polycistronic rna because there is no way for the ribosomes to recognize internal AUGs
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RNA processing: Early experiments indicated that RNA was synthesized as a ____________.
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longer precursor
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This larger class of RNAs was referred to as __________.
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heteronuclear RNA or hnRNA (many different sizes) not found in the cytoplasm
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The mRNA found associated with polyribozomes was on average much _______ than hnRNA. They quickly migrated to the __________.
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smaller, cytoplasm
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Pulse-Chase Experiment
Pulse: |
Label RNAs with 32P for 30 mins, separate by rate zonal centrifucation.
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Chase:
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Stop transcription with actinomycin D, allow cells to grow for 3 hours, separate by rate zonal centrifugation.
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OD-
Radioactivity- |
OD-total RNA (old and new), Radioactivity-Newly synthesized RNA
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Other experiments
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total RNA and isolated RNAs from polyribosomes are separated by rate zonal sedimentation through a sucrose gradient distribution of masses are compared.
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Conclusions
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mRNA is derived from hnRNA, this process results in an overall decrease in size of RNA
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Other experiments showed-
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the size difference between hnRNA and mRNA primarily due to removal of introns
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The process of removing the introns is called ________.
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splicing
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The splicing reaction is catalyzed by enzymes composed of a complex of RNA and protein, reffered to as ______________.
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snRNPs, small nuclear ribonucleoprotein particles
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Each of the snRNPs has a single RNA associated with it is rich in __________ and they are referred to as ___
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uradine and are referred to as U1, U2, U4/U6, and U5
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Each snRNA is composed of
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one RNA and about 10 or more proteins
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When 5 snRNPs (U1, U2, U4, U5, U6) form together on an RNA is called a __________.
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spliceosome
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Once assembled there are 2 ____________ ____.
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transesterification steps.
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2 transesterfication steps
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branch-5' splice site (formation of a lariat), 5' splice site-3'splice site. this releases the intron as a lariat
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The first step in spliceosome formation is...
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the recognition of the juction between the exons and introns.
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This recognition involves _______________ assisted by __________.
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RNA-RNA base pairing, proteins
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There are four conserved sequences that have been found to be necessary:
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5'splice site, branch point, pyrimidine track, 3'splice site
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5'splice site is next to the __________.
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5'exon
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3'splice site is next to the _______.
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3'exon
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Branch site A
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is important for the first tranesterification step.
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5'splice site
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donor site, the 5'exon ends in AG, and the intron begins with G,U, (A/G), A, G, U
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3' splice site
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acceptor site, introns ends with pyrimidine track, A, G, then 3' exon begins with G
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Formation of the Spliceosome. 1st step-
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Binding of the U1 snRNP to 5' splice site, BBP to branch point and U2AF to polyPy
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2nd Step
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Release of BBP and binding of U2 to branch point
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3rd step
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Binding of U4/U6, U5, and release of U2AF
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Recognition involveds
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U1 RNA base pairing to 5'splice site and U2 RNA base pairing to branch point
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The A at the branch point doesnt base _________ with ___, instead it ________.
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pair, U2, bulges out (important for strating the splicing reaction)
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U4 and U6 come in as a __________.
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Complex
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The RNA found in U4 and U4...
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base pair with eachother (in assembly process)
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In order for splicing to occur...
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a major rearrangement in the spiceosome proceeds or is concurrent with the transesterification reactions
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This rearragement...
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breaks the U4 and U6 apart and places U6 in position to catalyze the reactions
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The U6 then...
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base pairs with the 5'splice site and U2 which is base paired with the branch region
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Rearrangement releases...
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U1 from 5'splice site and U4 and U6
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U6 then....
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catalyzes first transestrification reaction
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U5
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assists in the second transesterification reaction
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The introns is then released as a _______ and degraded. The spliceosome ___________ and the mRNA is ready to be transported out of the nucleus.
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lariat, dissociates
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Average human gene: exons-_____bp, introns-______bp, __________introns
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150, 3000, 8 or 9
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Introns are usually much _____ than exons
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bigger
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How does the spiceosome find its sites?
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Co-transcriptional spicing, Exonic splicing enhancers (ESE)
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Co-transcriptional splicing
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Splicing factors are loaded on P-CTD of RNA polymerase, Factors bind to 1st 5' splice site, Next 3'splice site is used thus lowering the chance of exon skipping.
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Exonic splicing enhancers (ESE)
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Bind SR proteins (serine arginine rich), SR proteins attract U2AF to nearby 3'splice sites and U1 snRNA to 5" splice sites.
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Sometimes splicing variants are purposely made
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alternative splicing
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alternative splicing can be _______ or ________.
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Constitutive, Regulated
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Constitutive
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two or more splice variants are always made
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Regulated
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splice variants are made in only certain cell types or certain times of development.
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Alternative splicing is a reason we have fewer ____.
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genes, because we can make varients of them.
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Tissue specific alternative splicing
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you make various versions of proteins by mixing exons, theory is exons form their own domains
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Cell surface protein has ________ splice varients.
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38016. cell adhesion protein that are required for the formation of neuronal connections, variations of exons lead to high number
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White blood cells have different DNA...
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because genes for anti bodies are mixed and matched to form different DNA.
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ESE play important role in regulating __________.
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Splicing
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ESE play importnat rolde in regulating splicing. Presence of absences of specific...
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SR protein in a particular cell type or stage of development can determine if a splice site is used.
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Exonic Splicing silencers can also inhibit use of specific splice site
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hnRNPs bind silencers but lack the ability to recruit splicing machinery, sometimes silencers overlap enhancers, other times they overlap the splice sites
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What is the advantage of having introns that need to be eliminated before the mRNA can be translated?
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Allowed evolution to proceed at an increased pace, alternative splicing allows a variety of related proteins to be synthesized from a single gene, exons frequently encode different domains of a protein which can be combined via DNA rearrangements to generate new proteins relatively quickly (exon shuffling)
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Exon shuffling
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allows recombination of exons between genes...exons bring splice sites with it.
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Epigenetics
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Changes in gene expression that are stably inherited, but are not due to changes in DNA sequence.
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___________ modifications can stably change gene expression.
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chromatin
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Sometimes __________ modifications are sufficient. ex. ____________
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histone, polycomb regulated genes involving H3K27 methylation
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Other times both ________ and _________ modifications are required. ex.________
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DNA, Protein, ex. cytosine methylation, H3K9 methylation
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Example of Polycomb regulation
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Antennapedia
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Antennapedia
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1970s mutations were isolated that transformed one body part into another.
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Called these __________ mutations.
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homeotic mutations, ex. flys leg grow instread of antennae
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WHen they looked at the genes responsible for these mutations they had a sequence which they put a box around and called it the _______.
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homeobox (hox)
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The homeobox contains __________.
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homeodomain DNA binding motif
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Each of the genes turned out to be a _______________.
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Transcription factors which the same DNA binding motif known as the homeodomain
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The hox genes are regulated by_______ _______.
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polycomb proteins
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Hox genes are expressed in...
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order and correlate with location in body of fly.
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Conserved in __________ as well.
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mammals
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Hox genes are regulated permanitely by ________ __________.
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histone modifications
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Polycomb regulation
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named after another Drosophila mutant, complex of proteins that act through polycomb response element(PRE), represses transcription permanently (maintained through cell divisions):silencing
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PRE
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polycomb response element
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________ is a complex of proteins which methylates __________.
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PRC2, H3K27
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PRC2 acts as a __________.
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methyl transferase
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Once methylated, __________ complex binds to the methylated K27 and inhibits ____________.
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PRC1, transcription
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Gene silencing through DNA methylation and H3K9 methylation. Found commonly in__________.
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chromatin (silence transposable elements)
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Examples of genes silencing though DNA methylation and H3K9 methylation.
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Imprinted genes, Inactive X, Miscellaneous other genes.
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Imprinted genes
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genes that are expressed from only one chromosome (either mothers or father's chromosome)
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Inactive X
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in mammals, females have randomly one of their X chromosomes turned off.
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DNA methylation occurs primarily at __________.
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cytosines, increases mutation rate
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5-Aza-cytosine
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cannot be methylated
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hemimethylated
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one strand methylated, one strand not
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Maintenence methylation
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use hemimethylated to methylate other strand
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DNA methylation can be detected using methylation sensitive ______________.
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restriction enzymes.
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Hpa II
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cleaves CCGG, but doesnt cleave C(m)CGG, CC(m)GG, C(m)C(m)GG
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Msp I
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cleaves CCGG, CC(m)GG, but doesnt cleave C(m)CGG, C(m)C(m)GG
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________________ _____ is used to detect methylation.
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southern blot. Probe is used after treating with restriction enzymes. Fragments are analyzed to determine methylation.
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____________ sequencing can also detect DNA methylation.
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bisulfite
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Bisulfite
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turns cytosine into uracil befor you sequence it.
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This change from C to U is useful because
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because cytosine methylation blocks this first step, so you know methylated Cs will stay the same and unmethylated Cs will become Us
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______ and _____ ________ allows one to determine exactly which bases are methylated
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PCR and DNA sequencing
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Agouti mouse: ______ ____ fed to obese mother gave rise to normal progeny.
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Folic acid, Vitamin B12
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Methyl-tetrahydrofolate and Vitamin B12 are involved in generating _______________.
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S-adenosyl-methionine (methyl donor in DNA and histone methylation)
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Mice fed Vit B12 and folic acid turned off _______ gene thorugh ______ and _____ ______.
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agouti, DNA and histone methylation.
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The silencing resulted in return of normal _________.
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appetite
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DNA methylation is not found in all organisms
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many model organisms have no DNA methylation. such as C. elegans, D. melanogaster, Yeast.
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Mammals have mostly methylated _____.
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CGs, elimination of all methylation is lethal.
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Plants have methylated ___ in all sequences
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C, elimination of all methylation is not lethal but causes phenotypes
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Bacteria have methylated __ or methylated __________.
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A or CC(A/T)GG
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Example of Genomic Imprinting
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Parent of origin dependent expression. ex. IGF2
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IGF2
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insulin-like growth factor 2,
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IGF2 expressed only from the ________chromosome
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paternal
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H19
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non coding RNA (regulatory RNA)
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H19 is only expressed from the __________chromosome
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maternal
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These two genes are separated by an ______________.
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imprinting control region (ICR), acts as an activator for H19 and an insulator for IGF2.
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ICR is bound by a protein known as ________.
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CTCF
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CTCF
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is a zinc finger protein (11 Zn fingers) which binds to a CG rich sequence (ICR)
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CTCF binds __________ and doesnt bind ____________.
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unmethylated ICR, methylated ICR. this affects gene expression.
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Monoallelic expression at the imprinted Igf2/H19 locus occurs by at least two disting mechanisms:
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paternal H19 promoter is not activated, Igf2 promoters insulated from enhancer, both mechanisms are dependent on the ICR.
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Imprinting: CTCF binding to unmethylated ICR results in...
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insulation of IGF2 from the H19 enhancer
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H19 transcription is activated on the maternal chromosome, ________ is not transcribed
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IGF2
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Deletion underited in mother results in lack of expression of H19 and results in
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Angelmans Syndrome (happy puppets)
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Imprinting: CTCF does not bind to methylated ICR resulting in...
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no insulation and IGF2 is transcribed
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H19 is not transcribed when...
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CTCF is not bound
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Deletion inherited from father results in loss of ________.
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IGF2, Prader-Willie syndrome (obesity phenotype)
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Together, each gene is expressed from just one chromosome
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(monoallelic)
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In humans loss of imprinting at this locus is associated with ______ and developmental disorders such as __________________ and ___.
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tumors, Prader-Willi syndrome, Angelman syndrome
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Prader Willi syndrome: no expression of _______ from paternal chromosome.
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IGF2
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Angelman syndrome: no expression of ___ from maternal chromosome.
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H19
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Transposable elements
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movable elements that can disrupt genes or alter expression.
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They are ______ to the cell if left unchecked.
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harmful
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_______ and _________ found on silenced transposable elements
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DNA methylation and Histone methylation
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Silencing is considered __________ ________ ________.
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genome immune system.
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FLower: Spreading of methylation from the TE into the promoter _________ gene responsible for blue pigment
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turned off.
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DNA methylation is generally correlated with _______methylation
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H3K9, reinforces eachother
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Epigenetic marks are stably inherited
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through development, genes that are turned off or turned on remain that way, during life of organism, epigenetic changes can occur in response to environment...can lead to cancer, autism?
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Genomics
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is thr study of all the genes of a cell, or tissue, at the DNA (genome), mRNA (transcriptome, or protein (proteome) levels
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The first genome sequenced what a ________________ in 1977 by Frederick sanger (invented DNA sequencing)
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bacteophage phi x174
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It was about ________bp, and had no ________, simple analysis revealed all ______ ________ ______.
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5375, introns, open reading frames. start with ATG and end with one stop codon, genes packed into small genome.
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From the DNA sequence of the genome, all...
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protein sequences can be deduced
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Surprising find:
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overlapping genes
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What have we learned from sequencing genomes?
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much of DNA in higher organisms does not encode proteins, large duplication and rearrangements have occured in addition to singlebase changes, humans have the same number of genes as many other eukaryotes.
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The DNA that doesnt encode proteins are...
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reptitive DNA, transpoable elements, etc.
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Introns larger than exons, gene prediction best by comparing
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mRNA (cDNA clone) to genomic sequence.
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In the human, there are an estimated _____________ genes predicted which is about ____ of human genome.
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25000, 2%, means our complexity is not due to the number of genes but how they are regulated.
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How are genes identified amongst the millions of base pairs?
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computer annotation
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Computer annotation-
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search for start and stop codons, promoters sequences, exon intron junctions, homology to other known genes
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Comparative analysis: Sequence comparisons with non-mammalian genomes provide information on the evolution of:
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anatomy, physiology, development, behavior
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Comparison of human and chimpanzee genomes show...
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35 thousand single nucleotide substitutions, 5 million small insertions, deletions and local arrangements, one chromosomal fusion
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We are ___ percent identical to chimpanzee in terms of genome.
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99
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Only ____ the number of differences as between any two humans
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10X
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Sequences are considered ________ if they share a common evolutionary ancestor
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homologous
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2 types
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othologous and paragolous
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_________ genes are homologs in different species
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orthogolous, ex. B-hemoglobin in man or mouse
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_________ genes are homologs in same species
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paralogous, ex. alpha-hemoglobin and B-hemoglobin in man
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What are the function of each paralogs? SU(VAR)3-9
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redundant
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Analysis of sequences within a species can identify when...
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groups separated physically through migration
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Lab involved analyzing mitochondrial DNA to determine...
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maternal heritage (we looked at the highly variable region which is not as accurate)
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High-throughput analysis of gene function
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activities of thoughsands of genes and their products studied at the same time
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They can do this using...
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DNA microarrays, protein arrays
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Microarray
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each spot has a different sequence and gene,corresponding DNA will hybridize to array and can be visualized using tags
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Expression Analysis
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using microarray chips-isolate cDNA and add a flourescent tag, when they hybridize you can visualize it
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Each spot contains...
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partial sequence of one gene.
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Protein arrays can measure
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binding to small molecules, DNA, RNA, etc.
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Protein array: steps (analytical)
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label proteins with fluorescent tags, incubate with ligand array, strength of signals corresponds to strength of interaction.
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2 Proteins arrays:
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analytical and functional
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