BIOL 200 Final Review - The exam will be cumula.ve-\u00ad\u2010 70 material from RR 30 material from GB The exam is worth 65 marks It will consist of 50

BIOL 200 Final Review - The exam will be...

This preview shows page 1 out of 216 pages.

Unformatted text preview: The exam will be cumula.ve-­‐ 70% material from RR 30% material from GB The exam is worth 65 marks It will consist of 50 mul.ple choice ques.ons -­‐ 35 marks ROY -­‐ 15 marks BROWN There will be 15 short answer ques.ons -­‐10 marks ROY -­‐ 5 marks BROWN READ ALL THE QUESTIONS CAREFULLY! Email for post-­‐review ques.ons [email protected] STUDENT SUGGESTIONS FOR BIOL 200 CRASH COURSE “I would like to go over molecular geneFc techniques, such as cloning and gene targeFng.” “Im wondering if you guys can go over possible short answer ques.ons.” “Can we go over lecture 29 more in depth, chromaFc modificaFons, kind of lost” “An area that I would like to be covered is miRNA and RNA interference.” “Im having difficulty with the last part (funcFonal genomics) so I was just a bit confused with that part.” “ Some topics that would be helpful to cover: 1) different methods of protein analysis (Northern, Southern, Western Blots) 2) electrophoreFc mobility shiS assays” “ Ques.on: can you go over lecture 18 and explain PCR genotyping and how diseased copy of allele can be cut by enzyme and lead higher risk in developing a disease?” Q: In lecture 20, can you re-­‐explain how to use secondary anFbody in SDS Polyacrylamide Gel Electrophoresis ? I'm confused on how that works. Q: can you clarify the linker scanning LS mutaFon in lecture 21 where we learn to iden.fy DNA sequences involved in ac.va.on of transcrip.on? I would like all of the data collec.ng processes gone over, if possible. e.g. northern, southern blots ADDITIONAL REQUESTS: -­‐Mapping enhancers -­‐Nuclear Import/Import of NLS containing cargo proteins. -­‐Domains and their funcFon. Please include topics on: 1) T7 RNA pol gene and Lac operon in a plasmid 2. electrophoreFc mobility shiS assays Can we go over transposable elements and BLAST from Dr. Bureau's sec.on, please? r RNA processing (chapter 8) and more specifically the phosphorylaFon, Carboxy terminal domain and splicing with the trans-­‐esterificaFon reacFons. EcoRI (and other enzymes like it) cuts DNA at a specific six nucleo.de, palindromic sequence (GAATTC) that occurs rela.vely infrequently throughout the genome The reac.on bi-­‐products have overhanging ends that are fully complementary and are thus “cohesive” Specialized plasmids called vectors were engineered to carry out specific func.ons based on cuing out undesirable, non-­‐essen.al regions of the plasmid, followed by liga.ng in useful bits of DNA using restric.on enzyme sites Polylinkers (or mul.ple cloning sites) are regions with many restric.on enzyme sites to facilitate the introduc.on of DNA fragments. Permanent collec.ons of genes can be obtained and maintained in DNA libraries. Different libraries are used cut Vector w/ BamHI depending on the informa.on (plasmid or phage) you want to study. Genomic libraries contain copies of the DNA present in the genomic/chromosomal context (intergenic regions, introns, exons, repe..ve sequence) cDNA libraries represent mRNA (.ssue specificity, abundance) mRNA is converted to complementary DNA or cDNA by priming the poly A tail with a single-­‐ stranded poly T oligonucleo.de. RT uses this primer to ini.ate single-­‐strand DNA synthesis that is fully complementary to the mRNA template. RNA is then removed and a poly dG adapter is annealed to the 3’ end. A poly dC primer is used to ini.ate synthesis of the second DNA strand. E. coli DNA polymerase I progresses through any remaining hybrid regions and extends the second strand Remove RNA -­‐alkali or -­‐RNaseH Transient transfec.on: only some cells express the transgene Stable transfec.on: all the cells express the transgene A complex mixture of macromolecules Binding to a solid phase support Nitrocellulose or Nylon Probe specific for target recogni.on ( ) Remove non-­‐specific Target Detec.on Known sequence that corresponds to gene or gene product of interest Lys Glu Ala Phe Thr His His Gly Synthesize an oligonucleo.de that has the complementary sequence to the specific region of the gene (cDNA) of interest-­‐this may be a “degenerate” pool. 5’-­‐GCC ATG ATG GGT AAA TGC CTC CTT-­‐3’ A G G A G G T T T T A C C C Polynucleo.de Kinase (PNK) (yes, yet another enzyme from a the lowly bacteriophage…) will phosphorylate nucleo.des by transferring the γ phosphate of ATP to the free hydroxyl at the 5’ end of the oligonucleo.de 5’-­‐AAG GAG GCA TTT ACC CAT CAT GGC-­‐3’ P 5’-­‐GCC ATG ATG GGT AAA TGC CTC CTT-­‐3’ A G G A G G T T T T A C C C By incorpora.ng deoxyribo-­‐ nucleo.des that carry a radiolabel on the α-­‐phosphate into PCR amplified DNA, specific DNA molecules can be very efficiently labelled. Unincorporated radioac.ve substrates are subsequently removed and the radiolabelled DNA can then be used as a probe. + dGTP, dTTP, dATP, dCTP (α-32P dCTP) It must be rendered single stranded prior to use GGATTCCA CTTCTGCGTTA……….CGGAGTAGAATTCCGGAAT CCTAAGGTGAAGACGCAAT……….GCCTCATCT TAAGGCCTTA Both DNA and RNA can be separated according to size using an agarose gel. DNA is cut with a restriction enzyme and then run through an agarose gel-a diagnostic signature that reflects the DNA sequence mRNAs of different sizes will correspond to the various genes that encode them. Both of these analytical techniques require that the molecules be transferred to a solid state support and be denatured (single stranded) for subsequent analysis. DNA-> nylon, nitrocellulose RNA->nitrocellulose. The nucleic acids are then bound covalently to the support This permanently records the levels (abundance) and the position (size) of the molecules following separation on the gel. The support or “blot” can then be “hybridized” with probes to any sequence that may be of interest. Washes remove non-specific signal and only complementary sequences will be detectable on the blot following autoradiography. Nucleic acids of complementary sequences can base pair to form double stranded hybrids, ie… Molecular idenFficaFon of RFLPs -a gene’s DNA with a gene’s DNA (Southern Blot) Allele 2   An example: A specific allele of the Breast Cancer Related 1 (BRCA1) gene can be detected by the presence of a parFcular restricFon enzyme site-­‐restricFon fragment length polymorphisms (RFLP) can be detected. isolate genomic DNA from individual’s blood cells amplify individual’s BRCA1 alleles with specific primers digest PCR product with indicative restriction enzyme and run on gel (RFLP) DNA of wild type allele cannot be cut Breast cancer probability: individual has low risk can be cut DNA of mutant allele individual has high risk (loss of heterozygosity) individual will develop breast cancer a gene’s DNA with a gene’s DNA (Southern Blot) a gene’s RNA with a gene’s DNA (Northern Blot) a gene’s RNA with a gene’s cDNA (Northern Blot) From Sasaki et al. Biochemical J. 2002  Text •  The combina.on of powerful compu.ng tools and biology gave rise to efficient algorithms for gene predic.on •  Genes are predicted from open reading frames (ORFs) that span more than 100 codons without a stop. Their expression can be confirmed by Expressed Sequence Tags (EST) •  Intron-­‐Exon boundaries can be predicted from highly conserved 5’ splice sequence and the resump.on of ORFs •  May provide evidence of genome modifica.on ie… duplica.on of genes or transposons •  Syntenic comparisons in non-­‐coding and intergenic regions can be made with related species REGULATORY FUNCTION •  Proteins that share a conserved func.on oqen exhibit similari.es in amino acid sequence ie…DNA binding, kinase, GTP binding or GTPase, helicase Yellow is iden.cal Pink is similar •  By comparing the primary amino acid sequence of a query protein with known proteins such similari.es can uncover key func.onal insights •  Key amino acids that may be involved in an important domain/mo.f need not be iden.cal. Conserva.on of the biochemical nature of the residue can be sufficient ie… hydrophobic, charged, neutral Polo-­‐like Kinase (PLK4) •  Iden.fied protein sequences can be queried using programs such as BLAST (Basic Local Alignment Search Tool) that will compare amino acid sequence with all the available data compiled from the various organism databases Important informa.on about func.on can be inferred from mo.fs or shared domains with known proteins. •  Query results are then ranked according to p-­‐value: – -­‐the probability that two proteins would have the same sequence based on chance •  Lower p-­‐values indicate greater shared similarity •  (p<10-­‐3 proteins can be considered to share some common ancestry) Put your protein sequence here! •  Protein sequence informa.on indicates substan.al HOMOLOGY or “shared sequence iden.ty” between the various proteins (tubulin) of organisms. •  An ancient sole family member likely carried out all tubulin func.ons •  Tubulin genes might have duplicated and thereaqer gave rise to PARALOGUES •  If the organism then underwent specia.on the tubulin genes present in each new species would be the tubulin ORTHOLOGUES in the related species. •  The basic cellular toolkit shared by each organism is strikingly conserved •  Genes required for cellular metabolism make up a large propor.on of the total number of genes •  Transcrip.on and transla.on related genes are also present in significant number •  The vast majority of the genes iden.fied are considered to be of unknown func.on •  The basic cellular toolkit shared by each organism is strikingly conserved •  Genes required for cellular metabolism make up a large propor.on of the total number of genes •  Transcrip.on and transla.on related genes are also present in significant number •  The vast majority of the genes iden.fied are considered to be of unknown func.on Microarrays are produced by synthesizing specific oligonucleo.des on limited areas of a glass slides. Since the oligonucleo.de synthesis is directed we know exactly which oligonucleo.de (gene product) is located at which posi.on on the slide! The oligonucleo.des are designed in a way that they can only hybridize with one specific cDNA molecule! •  Cells can be subjected to different condi.ons •  mRNA is extracted from the treated and untreated cells •  Each mRNA is used to make cDNA just like in a cDNA library, but the two different pools of cDNA (treated vs untreated) are differen.ally tagged with two different fluorescent tags •  Each is used to hybridize a full genome microarray independently and the differences are recorded Aqer hybridiza.on, the microarray can be scanned by a special instrument ( r e a d e r ) t h a t c a n d e t e c t t h e fluorescence emiued by the labeled cDNAs Since the oligonucleo.des are in excess, the fluorescence is a measure, albeit not accurate, for the abundance of a mRNA in the original sample -­‐> the more mRNA -­‐> the more labelled cDNA -­‐> the more cDNA hybridized to the oligonucleo.de -­‐> the stronger the emission detected signal from the cDNA derived from the mRNA of a particular gene A) represents a cluster of genes involved in cholesterol biosynthesis Fibroblasts arrest in G0 in the absence of serum. mRNA is collected over A .me course and global gene ac.vity is determined over .me B) Corresponds to cell cycle genes Green indicates decrease C) Immediate early genes D) Angiogenesis E) Wound healing Red indicates increase Overview of the RNA-seq Procedure •  Informa.on about how the loss of gene func.on affects phenotype is informa.ve…doing this at the genome scale is extremely valuable-­‐ FUNCTIONAL GENOMICS •  In yeast, individual genes can be disrupted using a gene targe.ng/replacement strategy based on homologous recombina.on •  A disrup.on construct (also referred to as a “gene targe.ng” or “targe.ng construct/vector”) is made using PCR primers that have sequence homology to the regions flanking the target sequence (20-­‐40bp on each end). This sequence iden.ty will direct the recombina.on event. •  The disrup.on construct is transformed into diploid yeast cells to replace the appropriate region. •  Presence of the dominant selectable marker confers drug resistance (G-­‐418) so the cells can grow on drug. •  When allowed to sporulate the haploid progeny (spores) will either have a wild type chromosome or a recombinant chromosome. •  The effects of the gene replacement can then be assessed ie…viability or growth rate C. elegans researchers “knocked-­‐down” every predicted transcrip.on unit on chromosome 1 by using feeding RNAi… …of the analysed genes, 339 were assigned some func.on as determined by the visible RNAi phenotype Later they did it for each predicted gene in the C. elegans genome (~19,000 genes) A variety of genes were found to give rise to the various visible phenotypes While the genes that cause embryonic lethality and sterility were oqen involved in basal cellular func.on… …genes that affected postembryonic traits were mostly novel or very specialised! Taken from Nature, 2000 dsRNA injected into the C. elegans gonad causes systemic loss of func.on phenotypes that correspond to the injected gene… white RNA-­‐hairpin wt Taken from Nature, 2002. The effect is highly conserved and is not unique to worms… •  It works post-­‐transcrip.onally since there is no effect on transcrip.onal ini.a.on, while the mRNA rapidly disappears •  En.re families of genes can be silenced depending on the region of dsRNA chosen •  In C. elegans the effect is systemic: although the dsRNA is injected into the gonad or taken up through feeding it can effect all the cells of the en.re animal •  The effect can be obtained through injec.on of dsRNA into the gonad or by simply soaking the worms or feeding them bacteria that expresses dsRNA •  The effect is cataly.c in most organisms. A minute quan.ty of dsRNA can eliminate highly abundant gene products (RNA) ie…ac.n +1 Promoter linker Target sequence 5’ 3’ Expression vector 3’ Target sequence 5’ 5’ T7 Promoter 3’ +1 Target sequence Target sequence +1 3’ 5’ 3’ 3’ 5’ Expression vector T7 Promoter 5’ •  Does not require the target mRNA •  Target mRNA is also cleaved into 21-­‐23nt fragments •  The whole process is ATP-­‐ dependent •  The enzyme acFvity was referred to as “DICER” Zamore et al., Cell, 2000. •  Dicer is an RNAse III like enzyme •  It acts as a dimer to cleave dsRNA into 22nt fragments that are then bound by the RNA-­‐induced silencing complex (RISC). •  ATP hydrolysis drives the unwinding (helicase) ac.vity of RISC which then uses the ssRNA product as a guide to target the complex to complementary cellular RNAs, finally associa.ng through Watson-­‐Crick base pairing. •  The resul.ng cleaved transcripts are then degraded by cytoplasmic ribonucleases •  In the nematode C. elegans lin-­‐4 mutants (lin stands for lineage abnormal) have an opposite phenotype to those of lin-­‐14 mutants. •  lin-­‐4 encodes a small RNA that has considerable homology (an.sense) to regions of the lin-­‐14 3’ UTR. •  lin-­‐4 RNA is synthesized as a longer precursor then it has to be processed, aqer which it binds its target sites and affects transla.on of the lin-­‐14 mRNA. •  Argonaute, which is a component of RISC and Dicer are both involved in this pathway. •  microRNAs (miRNAs) have been predicted through bioinforma.c analyses to target numerous RNAs •  Stringent biochemical and molecular approaches have so far iden.fied mul.ple cases where they play an important regulatory role…the list con.nues to grow •  Metabolism, .ssue growth, neural development, developmental .ming, stem cell biology/pluripotency and also in cancer. Taken from Nature Reviews/ Genetics, 2004. •  dsRNA •  Dicer/siRNA •  RISC •  Target cleavage •  microRNAs (miRNA) are transcribed as longer precursor mRNAs that are processed through the ac.vity of Dicer or a Dicer-­‐like enzyme. •  The resul.ng miRNA is recognised/bound by the RISC complex, but its target substrates are oqen not fully complementary. •  This difference seems to spare the targeted mRNA from degrada.on by the RNAi pathway, but does however block its transla.onal efficiency. For lin-­‐14 this occurs at the level of transla.onal elonga.on. Both pathways are triggered by a dsRNA molecule microRNA-­‐like (lin-­‐4) siRNA •  What appears to be true for silencing the centromeres in S. pombe is likely true for higher eukaryotes like us… •  Recent data suggest that the microsatellite regions of repe..ve DNA around the centromeres in vertebrate chromosomes are silenced through an analogous dsRNA-­‐mediated mechanism. . taken from Science, 2002 . taken from Nature Cell Biology, 2004 •  Overexpression of gene products can give some informa.on but can also lead to artefacts •  Func.on is best addressed through removal of gene ac.vity and analysis of the resul.ng phenotype •  Abnormal phenotypes indicate specific processes have been disrupted that rely of the ac.vity of the affected gene •  Removal of gene ac.vity: –  –  -­‐forward gene.c analysis -­‐reverse gene.c analysis –  –  RNAi GENE TARGETING •  Homologous recombina.on (HR) requires a minimum shared homology of flanking regions between the targe.ng vector and the host gene •  Introduce a dominant selectable marker flanked by homologous regions to facilitate homologous recombina.on and gene replacement •  Transforma.on of diploid yeast cells with the targe.ng construct will incorporate the fragment and permit HR to occur •  Strong selec.on based on a dominant selec.ve marker will permit iden.fica.on of transformants •  Following sporula.on the yeast cells that are homozygous for the modified locus can be isolated through selec.on •  If the gene is essen.al the spores will not be able to grow •  Owen Smithies and Mario Capecchi originally showed that homologous recombina.on could also be performed in pluripotent embryonic mouse cells (ES cells). •  The combined strength of this kind of gene.c manipula.on with the, at that .me, newly characterized ES cell lines isolated from mouse embryos made this strategy extremely powerful •  Provided a means of manipula.ng gene ac.vity in a temporally-­‐ controlled, even in a desired .ssue-­‐ type •  G-­‐418 will select for all recombina.on events… •  Ganciclovir is toxic in the presence of the herpes virus tk gene, so it will nega.vely select against all non-­‐homologous recombina.on events •  Only ES cells that have undergone Homologous Recombina.on (HR) can survive the two selec.on steps. •  ES cells are then used to populate and blastocyst of an acceptor mouse. This mouse strain has to be another coat colour that is recessive. •  The blastocyst is transferred to a surrogate mouse mother •  The progeny will be a mixture of both genotypes if the cells were viable and the process worked properly. Brown coat colour is dominant!! •  The popula.on of to.potent cells is therefore heterogeneous in the blastocyst •  Homogeneous host embryos will give rise to a black mouse •  Embryos that are heterogeneous (have cells from the host and the targeted ES cell genotypes) will be CHIMERIC ie… coat colour will be spoued/patched) •  This means that the implanted cells contribute to various .ssues. The hope is that one of the .ssues they contribute to is the germ line! •  Transgenes integrate randomly into the genome so posi.onal effects may affect gene expression •  Transgenic reporter genes are very important for understanding expression pauerns of genes •  Can “turn on” genes at a specific .me in development or using a .ssue-­‐ specific promoter to assess what a given gene ac.vity may have on the cells/.ssue/organism. •  Heavy metals •  Tetracyclin •  Hormones  Text •  Matrix contains pores or indents of a specific size (Agarose, Sephadex) •  Protein frac.on is loaded onto the column and individual proteins or complexes will flow through the channels between the beads and/or through the pores. •  Small proteins make it through the pores while big proteins are confined to space between the beads. •  Very large proteins simply flow through the column while smaller proteins take the long way! •  Longer column lengths provide greater separa.on efficiency Affinity chromatography is the most efficient method of purifyin...
View Full Document

  • Left Quote Icon

    Student Picture

  • Left Quote Icon

    Student Picture

  • Left Quote Icon

    Student Picture