BioSci 203 lecture 21 page 1 © copyright Bruce Blumberg All rights reserved Bio Sci 203 Lecture 21 - cDNA library screening &sequence characterization Bruce Blumberg –office Bio Sci II – –lab 5427 (x46873), 5305 (x43116) –office hours Wednesday 1-2. This week –Protein protein binding assays –Characterization of Selected DNA Sequences DNA sequence analysis –mRNA Analysis Techniques to detect and quantitate mRNA –Northern –RNase protection –RT-PCR –in-situ hybridization –Transcript mapping

BioSci 203 lecture 21 page 2 © copyright Bruce Blumberg All rights reserved How to identify your gene of interest (contd) Two hybrid screening –originally used in yeast, now other systems possible –prepare bait - target protein fused to DBD (GAL4) usual stable cell line is commonly used –prepare library as fusion proteins with a known activation domain

BioSci 203 lecture 21 page 3 © copyright Bruce Blumberg All rights reserved How to identify your gene of interest (contd) Two hybrid screening (contd) –approach transfect library into cells and either select for survival or activation of reporter gene purify and characterize positive clones –advantages seems simple and inexpensive on its face –in materials functional assay –disadvantages fusion proteins bias the screen against full-length cDNAs. Binding parameters not manipulable bait must not have activation function Difficult or impossible to detect interactions between proteins and complexes. Doesn’t work for secreted proteins Many months to screen –savings in materials are eaten up by salaries –avg grad student costs $30k/year –avg postdoc or tech costs $40k/year MANY false positives

BioSci 203 lecture 21 page 4 © copyright Bruce Blumberg All rights reserved How to identify your gene of interest (contd) In vitro interaction screening –based on in vitro expression cloning (IVEC) transcribe and translate cDNA libraries in vitro into small pools of proteins (~100) test these proteins for their ability to interact with your protein of interest –EMSA –co-ip –FRET –SPA –advantages functional approach smaller pools increase sensitivity automated variant allows diversity of targets –proteins, protein complexes, nucleic acids, protein/nucleic acid complexes, small molecule drugs –very fast –disadvantages can’t detect heterodimers unless 1 partner known expensive consumables (but cheap salaries) –typical screen will cost $10-15K expense of automation

BioSci 203 lecture 21 page 5 © copyright Bruce Blumberg All rights reserved Current generation binding assays scintillation proximity assay –Target is bound to solid phase - bead or plate –radioactive protein or ligand is added and allowed to reach equilibrium 35 S, 125 I, 3 H work best –radioactive decay is quenched in solution, only detected when in “proximity” of the solid phase, e.g. when bound to target –applications ligand-receptor binding with 3 H small molecules protein:protein interaction protein:DNA

BioSci 203 lecture 21 page 6 © copyright Bruce Blumberg All rights reserved Current generation binding assays SPA (contd –advantages homogeneous, equilibrium assay –washing is not necessary! Can detect weak interactions, insensitive to high off rate large dynamic range –disadvantages radioactive equipment in vitro only

BioSci 203 lecture 21 page 7 © copyright Bruce Blumberg All rights reserved Current generation binding assays (contd) FRET - fluorescent resonance energy transfer –based on the transfer of energy from one fluor to another that is not normally excited at that wavelength –Many types of fluorescent moieties possible rare earth metals europium cryptate fluorescent proteins –GFP and variants –allophycocyanin Tryptophan residues in proteins –application very commonly used for protein:protein interaction screening in industry FRET microscopy can be used to prove interactions between proteins within single cells –Roger Tsien

BioSci 203 lecture 21 page 8 © copyright Bruce Blumberg All rights reserved Current generation binding assays (contd) FRET (contd) –advantages can be very sensitive may be inexpensive or not depending on materials non-radioactive equilibrium assay single cell protein:protein interactions possible time resolved assays possible –disadvantage poor dynamic range fold difference full scale must prepare labeled proteins or ligands tunable fluorometer required (we have one here)

BioSci 203 lecture 21 page 9 © copyright Bruce Blumberg All rights reserved Current generation binding assays (contd) AlphaScreen - amplified luminescent resonance proximity –Bind proteins to two types of beads donor bead accepts laser light at 680 nm and emits singlet oxygen acceptor bead receives singlet oxygen and emits light at –principle is that singlet oxygen can only diffuse one bead diameter before decaying only closely association between donor and acceptor gives a signal –applications receptor:ligand binding protein:protein binding discovery of peptide ligands for proteins

BioSci 203 lecture 21 page 10 © copyright Bruce Blumberg All rights reserved Current generation binding assays (contd) AlphaScreen (contd) –advantages very sensitive, equilibrium assay very fast –disadvantages requires instrument ~$100K cost of beads must bind proteins to beads single source for beads and instrument completely in vitro assay

BioSci 203 lecture 21 page 11 © copyright Bruce Blumberg All rights reserved Current generation binding assays (contd) BRET2 (Bioluminescence Resonance Energy Transfer) –based completely on bioluminescent reaction from Renilla reniformis can make lots of money by copying nature! –Renilla luciferase emits blue light in presence of its substrate coelenterazine –If GFP is nearby, it accepts this blue light and emits green light make two fusion proteins, one to rluc the other to GFP mix everything together if proteins interact then green light is detected if not, only blue light

BioSci 203 lecture 21 page 12 © copyright Bruce Blumberg All rights reserved Current generation binding assays (contd) BRET2 (contd) –applications protein protein interactions in solution protein:protein interactions within a single cell –advantages fairly sensitive no laser required to excite –minimal equipment required works in living cells (substrate is permeable) –disadvantage need to make fusion proteins single source for reagents

BioSci 203 lecture 21 page 13 © copyright Bruce Blumberg All rights reserved Current generation binding assays (contd) Eletrochemiluminescent assays –Origen system IGEN –based on a molecule that emits light when stimulated in an electrical field ruthenium derivative –capture a molecule with magnetic beads, if binding occurs, you can elicit light –Applications becoming widely used in clinical diagnostics as a radioimmunoassay (RIA) or enzyme linked immunosorbent assay (ELISA) substitute some applicability to protein:protein and protein:DNA binding

BioSci 203 lecture 21 page 14 © copyright Bruce Blumberg All rights reserved Current generation binding assays (contd) Eletrochemiluminescence (contd) –advantages rapid and sensitive compared with RIA or ELISA good quantitation –disadvantages requires instrument proprietary reagents completely in vitro assay

BioSci 203 lecture 21 page 15 © copyright Bruce Blumberg All rights reserved Current generation binding assays (contd) Biacore (surface plasmon resonance) –surface plasmon waves are excited at a metal/liquid interface –Target bound to a thin metal foil and test sample flowed across it –Foil is blasted by a laser from behind SPR alters reflected light intensity at a specific angle and wavelength Binding to target alters refractive index which is detected as change in SPR Change is proportional to change in mass and independent of composition of binding agent

BioSci 203 lecture 21 page 16 © copyright Bruce Blumberg All rights reserved Current generation binding assays (contd) Biacore (contd) –Advantages Can use any target Biological extracts possible Measure kinetics Small changes detectable with correct instrument –360 d ligand binding to 150 kd antibody Can use as purification and identification system –Disadvantages Machine is expensive (we have two) “high throughput” very expensive Not trivial to optimize

BioSci 203 lecture 21 page 17 © copyright Bruce Blumberg All rights reserved Analysis of genes and cDNAs Characterization of cloned DNA –what things do we want to know about a new gene? Complete DNA sequence –cDNA sequence –genomic sequence? –Restriction enzyme maps? Where are introns and exons? –Particularly if knockouts are coming where is the promoter(s)? –Alternative promoter use? –Mapping transcription start(s) where and when is mRNA expressed? –How abundantly is it expressed in each place? –Is there any association between expression levels and putative function? What is the function of this gene? –Loss-of-function analysis decisive »knockout »antisense »mutant mRNA e.g. dominant negative –gain of function may be helpful »transgenic »mutant mRNA - constitutively active transcription factor

BioSci 203 lecture 21 page 18 © copyright Bruce Blumberg All rights reserved Analysis of genes and cDNAs (contd) Landmarks in DNA sequencing –Sanger, Nicklen and Coulson. Sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. 74, (1977). –Sanger, F. et al. The nucleotide sequence of bacteriophage ΦX174. J Mol Biol 125, (1978). –Sutcliffe, J. G. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol 43, (1979). –Sanger et al., Nucleotide sequence of bacteriophage lambda DNA. J Mol Biol 162, (1982). –Messing, J., Crea, R. & Seeburg, P. H. A system for shotgun DNA sequencing. Nucl.Acids Res 9, (1981). –Anderson, S. et al. Sequence and organization of the human mitochondrial genome. Nature 290, (1981). –Deininger, P. L. Random subcloning of sonicated DNA: application to shotgun DNA sequence analysis. Anal Biochem 129, (1983). –Baer et al. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature 310, (1984). (189 kb) –Innis et al. DNA sequencing with Taq DNA polymerase and direct sequencing of PCR-amplified DNA Proc. Natl. Acad. Sci. 85, (1988)

BioSci 203 lecture 21 page 19 © copyright Bruce Blumberg All rights reserved Analysis of genes and cDNAs (contd) Landmarks in DNA sequencing (contd). – Haemophilus influenzae (1.83 Mb) – Mycoplasma genitalium (0.58 Mb) – Saccharomyces cerevisiae genome (13 Mb) – Methanococcus jannaschii (1.66 Mb) – Escherichia coli (4.6 Mb) – Bacillus subtilis (4.2 Mb) – Borrelia burgdorferi (1.44 Mb) – Archaeoglobus fulgidus (2.18 Mb) – Helicobacter pylori (1.66 Mb) – Treponema pallidum (1.14 Mb) – Caenorhabditis elegans genome (97 Mb) – Deinococcus radiodurans (3.28 Mb) – Drosophila melanogaster (120 Mb) – Arabidopsis thaliana (115 Mb) – Escherichia coli O157:H7 (4.1 Mb) – Human “genome”

BioSci 203 lecture 21 page 20 © copyright Bruce Blumberg All rights reserved Analysis of genes and cDNAs (contd) – Haemophilus influenzae (1.83 Mb) first bacterium sequenced, human pathogen – Mycoplasma genitalium (0.58 Mb) smallest free living organism – Saccharomyces cerevisiae genome (13 Mb) – Methanococcus jannaschii (1.66 Mb) first Archaebacteria – Escherichia coli (4.6 Mb) – Bacillus subtilis (4.2 Mb) – Borrelia burgdorferi (1.44 Mb) Lyme disease – Archaeoglobus fulgidus (2.18 Mb) first sulfur metabolizing bacterium – Helicobacter pylori (1.66 Mb) first bacteria to cause cancer – Treponema pallidum (1.14 Mb) syphillus – Caenorhabditis elegans genome (97 Mb) – Deinococcus radiodurans resistant to radiation, starvation, ox stress – Drosophila melanogaster (120 Mb) – Arabidopsis thaliana (115 Mb) (plant) – Escherichia coli O157:H7 (4.1 Mb) – Human “genome”

BioSci 203 lecture 21 page 21 © copyright Bruce Blumberg All rights reserved The human genome In Feb , Celera and Human Genome project published “draft” human genome sequencs –Celera -> –Ensembl -> –Consensus from all sources ~30K Number of genes –C. elegans – 19,000 –Arabidopsis 25,000 Predictions had been from k human genes –What’s up with that? –Are we only slightly more complicated than a weed? –How can we possibly get a human with less than 2x the number of genes as C. elegans –Implications? UNRAVELING THE DNA MYTH: The spurious foundation of genetic engineering, Barry Commoner, Harpers Magazine Feb, 2002

BioSci 203 lecture 21 page 22 © copyright Bruce Blumberg All rights reserved The human genome The answer – Sloppy science –Gene sets don’t overlap completely –Floor is 42K and likely total 50K or more 96k UniGene clusters from ESTs = 42113

BioSci 203 lecture 21 page 23 © copyright Bruce Blumberg All rights reserved DNA Sequence analysis Complete DNA sequence –complete sequence is desirable but takes time how long depends on size and strategy employed –which strategy to use depends on various factors how large is the clone? –cDNA –genomic How fast is sequence required? sequencing strategies –primer walking –cloning and sequencing of restriction fragments –progressive deletions bidirectional unidirectional –Shotgun sequencing whole genome with mapping –map first (C. elegans) –map as you go (many)

BioSci 203 lecture 21 page 24 © copyright Bruce Blumberg All rights reserved DNA Sequence analysis (contd) Primer walking - walk from the ends with oligonucleotides –sequence, back up ~50 nt from end, make a primer and continue

BioSci 203 lecture 21 page 25 © copyright Bruce Blumberg All rights reserved DNA Sequence analysis (contd) Primer walking (contd) –advantages very simple no possibility to lose bits of DNA –restriction mapping –deletion methods no restriction map needed best choice for short DNA –disadvantages slowest method –about a week between sequencing runs oligos are not free (and not reusable) not feasible for large sequences –applications cDNA sequencing when time is not critical targeted sequencing –verification –closing gaps in sequences

BioSci 203 lecture 21 page 26 © copyright Bruce Blumberg All rights reserved DNA Sequence analysis (contd) Cloning and sequencing of restriction fragments –once the most popular method make a restriction map subclone fragments sequence –advantages straightforward directed approach can go quickly cloned fragments often useful otherwise –RNase protection –nuclease mapping –in situ hybridization –disadvantages possible to lose small fragments –must run high quality analytical gels depends on quality of restriction map –mistaken mapping -> wrong sequence restriction site availability –applications sequencing small cDNAs isolating regions to close gaps

BioSci 203 lecture 21 page 27 © copyright Bruce Blumberg All rights reserved DNA Sequence analysis (contd) nested deletion strategies - make sequential deletions from one end of the clone –cut, close and sequence make restriction map digest with enzymes that cut in polylinker and insert religate and sequence from end with restriction site repeat until sequence is finished, filling in gaps with oligos

BioSci 203 lecture 21 page 28 © copyright Bruce Blumberg All rights reserved DNA Sequence analysis (contd) nested deletion strategies (contd) –cut, close and sequence advantages –fast –simple –efficient disadvantages –limited by restriction site availability in vector and insert –need to take the time to make a restriction map –BAL31 mediated deletions (archaic) digest insert from both ends with BAL31 repair, subclone and sequence advantages –was once the only way to make progressive deletions disadvantages –bidirectional –can’t protect -> must reclone applications –no longer used –superseded by ExoIII-mediated deletion cloning

BioSci 203 lecture 21 page 29 © copyright Bruce Blumberg All rights reserved DNA Sequence analysis (contd) nested deletion strategies (contd) –Exonuclease III-mediated deletion -

BioSci 203 lecture 21 page 30 © copyright Bruce Blumberg All rights reserved DNA Sequence analysis (contd) –Exonuclease III-mediated deletion (contd) cut with polylinker enzyme –protect ends - »3’ overhang »phosphorothioate cut with enzyme between first cut and the insert –can’t leave 3’ overhang timed digestions with Exonuclease III stop reactions, blunt ends ligate and size select recombinants sequence advantages –unidirectional –processivity of enzyme gives nested deletions

BioSci 203 lecture 21 page 31 © copyright Bruce Blumberg All rights reserved DNA Sequence analysis (contd) Nested deletion strategies –Exonuclease III-mediated deletion (contd) disadvantages –need two unique restriction sites flanking insert on each side –best used successively to get > 10kb total deletions –may not get complete overlaps of sequences »fill in with restriction fragments or oligos applications –method of choice for moderate size sequencing projects »cDNAs »genomic clones –good for closing larger gaps