MOLECULAR BIOLOGY – Basic Genetics EXAMS – SAVE THE DATE! KMB 758 MCQ KMB 758 MCQ The exam format will be a multiple choice question (MCQ) test with 40 questions. There will be two possible exam dates. Please mark them in your diaries. Should you fail or are not satisfied with your grade you can have an aural retake exam with me.

Potential revision Class next time!! Alexander Bruce Ph.D. Aleksandar Mihajlovic M.Sc. Vasanth Thamodaran M.Sc. USUAL TIME & PLACE 10 am in lecture theatre

MOLECULAR BIOLOGY TECHNIQUES III. analysis of gene expression MOLECULAR BIOLOGY – Gene expression analysis MOLECULAR BIOLOGY TECHNIQUES III. analysis of gene expression

MOLECULAR BIOLOGY – Gene expression analysis HYBRIDIZATION RNA detection Y ANTIBODY protein detection

MOLECULAR BIOLOGY – Gene expression analysis Where in organism is the gene expressed? Detection of RNA by in situ hybridization: Stage 5 Stage 10 Stage 11 Stage 13

Green Fluorescent Protein MOLECULAR BIOLOGY – Gene expression analysis REPORTER DNA Gen X Green Fluorescent Protein Other reporter genes e.g. LacZ http://www.youtube.com/watch?v=MMLVqGEOD58 Drosophila – GFP and embryonic development:

MOLECULAR BIOLOGY – Gene expression analysis Mouse embryo histone H2B-GFP reporter

Green Fluorescent Protein MOLECULAR BIOLOGY – Gene expression analysis REPORTER DNA Gen X Green Fluorescent Protein Other reporter genes e.g. LacZ How? Enhancer Trap, Knock-in …. http://www.youtube.com/watch?v=MMLVqGEOD58 Drosophila – GFP and embryonic development:

MOLECULAR BIOLOGY – Gene expression analysis Various reporter gene vectors for random integration into cell genomes (e.g. ES cells) leading to production of transgenic animals Reporter has minimal promoter that can be activated by a functioning proximal enhancer Viral intergration elemets Transposon elements random integration Antibiotic resistence allows selection of successfully integrated transgenes Reporter activity if integration site is proximal to an active gene enhnacer i.e. cell marked/ highlighted Enhancer-, gene- and promoter-trap vectors, which all contain a lacZ reporter gene and a NEOMYCIN RESISTANCE GENE (neo) that is driven by an autonomous promoter, are shown trapping an endogenous gene 'X'. Successful integration of the trap vectors into the embryonic stem (ES)-cell genome will confer neomycin resistance permitting selection whether the insertion has occurred intergenically or intragenically. a | The p3LSN enhancer-trap vector contains a truncated heat-shock inducible minimum (hsp68) promoter immediately upstream of lacZ. Insertion of the enhancer-trap vector close to the enhancer of gene X will lead to the transcription and translation of the lacZ reporter when the enhancer of gene X is activated. This vector usually generates hypomorphic rather than null mutations. a) Enhancer trap vectors

MOLECULAR BIOLOGY – Gene expression analysis Various reporter gene vectors for random integration into cell genomes (e.g. ES cells) leading to production of transgenic animals Splice acceptor site/ sequence (no promoter) random (intronic) integration Gene fusion ... Simultaneously reports expression and usually inactivates function (potential cytolocalization analysis in marked cells) - hypomorphic b | The pGT4.5 gene-trap vector contains a splice acceptor (SA) site immediately upstream of a promoterless lacZ gene. Its integration in an intron leads to a fusion transcript being generated from the upstream exon of gene X and lacZ upon transcriptional activation of gene X. b) Gene trap vectors

MOLECULAR BIOLOGY – Gene expression analysis Various reporter gene vectors for random integration into cell genomes (e.g. ES cells) leading to production of transgenic animals No splice acceptor site or promoter random (exon) integration Active promoter also leads to gene fusion thus reports and usually inactivates gene function (also potential cytolocalization analysis in marked cells) c | The p -gal promoter-trap vector needs to be inserted into the coding sequence of gene X to activate transcription of lacZ. On activation of gene X, a fusion transcript and protein between the upstream gene X sequence and lacZ will be generated. ( -gal, -galactosidase; -geo, -galactosidase–NeoR fusion; HSV-tk, herpes simplex virus thymidine kinase; h -actin, human -actin; pA, polyadenylation; PGK, phosphoglycerate kinase 1.) c) Promoter trap vectors

Targeting specific genes - BACTERIAL RECOMBINEERING MOLECULAR BIOLOGY – Gene expression analysis Targeting specific genes - BACTERIAL RECOMBINEERING e.g. creation of C-terminal GFP fusion reporter in mammalian cells Bacterial GENOMIC LIBRARY CLONE containing the gene to be targeted STOP 3’UTR PCR generated TARGETING construct GFP hygr neor E-coli prom Mammalian prom 50bp of homlogous sequence flanking STOP codon + plasmids encoding RECOMBINASE enzymes from bacteriophage  TRANSFORM the targeting construct into the genomic library bacterial clone STOP GFP hygr neor E-coli prom Mammalian prom RECOMBINASE enzymes recognise homologous sequences and induce DNA double strand breaks and strand CROSSOVER 3’UTR c | The p -gal promoter-trap vector needs to be inserted into the coding sequence of gene X to activate transcription of lacZ. On activation of gene X, a fusion transcript and protein between the upstream gene X sequence and lacZ will be generated. ( -gal, -galactosidase; -geo, -galactosidase–NeoR fusion; HSV-tk, herpes simplex virus thymidine kinase; h -actin, human -actin; pA, polyadenylation; PGK, phosphoglycerate kinase 1.)

Targeting specific genes - BACTERIAL RECOMBINEERING MOLECULAR BIOLOGY – Gene expression analysis Targeting specific genes - BACTERIAL RECOMBINEERING e.g. creation of C-terminal GFP fusion reporter Bacterial GENOMIC LIBRARY CLONE containing the gene to be targeted STOP 3’UTR PCR generated TARGETING construct GFP hygr neor E-coli prom Mammalian prom 50bp of homlogous sequence flanking STOP codon + plasmids encoding RECOMBINASE enzymes from bacteriophage  TRANSFORM the targeting construct into the genomic library bacterial clone c | The p -gal promoter-trap vector needs to be inserted into the coding sequence of gene X to activate transcription of lacZ. On activation of gene X, a fusion transcript and protein between the upstream gene X sequence and lacZ will be generated. ( -gal, -galactosidase; -geo, -galactosidase–NeoR fusion; HSV-tk, herpes simplex virus thymidine kinase; h -actin, human -actin; pA, polyadenylation; PGK, phosphoglycerate kinase 1.) Recombinase mediates the successful INSERTION/ INTEGRATION of the targeting construct into the genomic DNA library DNA creating the GFP FUSION REPORTER GENE STOP GFP hygr neor E-coli prom Mammalian prom 3’UTR Successfully recombined bacterial clones can be selected for with antibiotics

MOLECULAR BIOLOGY – Gene expression analysis Targeting specific genes - BACTERIAL RECOMBINEERING GFP FUSION REPORTER GENE DNA construct isolated and purified from bacterial DNA GFP hygr neor E-coli prom Mammalian prom 3’UTR TRANSFORM (TRANSFECT) GFP fusion construct into mammalian cells e.g. ES cells GFP hygr neor E-coli prom Mammalian prom 3’UTR STOP Low frequency mammalian ENDOGENOUS RECOMBINATION enzymes induce strand cross over Successfully recombination can be selected for with second antibiotic resistance gene c | The p -gal promoter-trap vector needs to be inserted into the coding sequence of gene X to activate transcription of lacZ. On activation of gene X, a fusion transcript and protein between the upstream gene X sequence and lacZ will be generated. ( -gal, -galactosidase; -geo, -galactosidase–NeoR fusion; HSV-tk, herpes simplex virus thymidine kinase; h -actin, human -actin; pA, polyadenylation; PGK, phosphoglycerate kinase 1.) The GFP FUSION REPORTER GENE for a specific gene has now replaced the endogenous copies of the original gene in the mammalian cell genome. These cells can then be used to create TRANSGENEIC animals The same approaches can be used to disrupt genes by insertion or deletion of DNA sequence to generate GENETIC KNOCKOUTS to asses specific gene function

MOLECULAR BIOLOGY – Gene expression analysis To what extent is the gene expressed? NORTHERN BLOT RNA DNA ... Southern blot

MOLECULAR BIOLOGY – Gene expression analysis PROMOTER exon 1 exon 2 exon 3 DNA ATG TAA AATAAA TRANSCRIPTION intron intron Pre-mRNA AAUAAA AUG UAA RNA SPLICING mRNA AUG UAA AAUAAA REVERSE TRANSCRIPTION TTATTT TAC ATT cDNA HIV

MOLECULAR BIOLOGY – Gene expression analysis cDNA synthesis Some reverse transcriptases initiate 2nd stand synthesis via addition of hairpins Ordinary 1st strand synthesis GENERATION OF A cDNA THAT CAN BE AMPLIFIED USING SPECIFIC PRIMERS BY PCR

MOLECULAR BIOLOGY – Gene expression analysis Quantitative RT-PCR (reverse transcription) RP49 control (house-keeping)

Real-Time PCR light-cycler MOLECULAR BIOLOGY – Gene expression analysis Real-Time PCR light-cycler SYBR- green dsDNA intercalation and flourescence

MOLECULAR BIOLOGY – Gene expression analysis Molecular Beacon Probes Potential for multiplex i.e. more than one probe per sample Diagram of molecular beacon. This beacon is 33 nucleotides long with a reporter dye attached to the 5' end and a quencher attached to the 3' end. The nine 5' bases are able to form base pairs with the nine 3' bases which brings the reporter and quencher in very close proximity. Therefore, when the reporter is excited by the appropriate light, its emission is absorbed by the quencher and no fluorescence is detected. The pink lines represent nucleotides that can form base pairs with the PCR product under investigation. As the PCR continues, the newly synthesized PCR products are denatured by high temperatures. As each strand of the product are separated, the molecular beacon also is denatured so the hairpin structure is disrupted. As the temperatures cool for the next round of primer annealing, the molecular beacon is capable of forming base pairs with the appropriate strand of the PCR product (Figure 3). Any molecular beacons that do not bind to PCR product reform the hairpin structures and thus are unable to fluoresce. However, molecular beacons that bind to PCR product remove the ability for the quencher to block fluorescence from the reporter dye. Therefore, as PCR product accumulates, there is a linear increase in fluorescence.

MOLECULAR BIOLOGY – Gene expression analysis Norhern blot, RT-PCR to study one (couple) gene(s) at once ... ... in genomic era?

MOLECULAR BIOLOGY – Gene expression analysis Genomic expression analysis using MICROARRAY (CHIP)

MOLECULAR BIOLOGY – Gene expression analysis

Chromatin remodeling methylase (CG) + MOLECULAR BIOLOGY – Gene expression analysis Chromatin remodeling methylase (CG) + histone deacetylases (HDACs) histone acetyltransferases (HATs) Nucleosomes consist of DNA (black line) wrapped around histone octomers (purple). Post-translational modification of histone tails by methylation (Me), phosphorylation (P) or acetylation (Ac) can alter the higher-order nucleosome structure. Nucleosome structure can be regulated by ATP-dependent chromatin remodellers (yellow cylinders), and the opposing actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Methyl-binding proteins, such as the methyl-CpG-binding protein (MECP2), target methylated DNA (yellow) and recruit HDACs. a | DNA methylation and histone deacetylation induce a closed-chromatin configuration and transcriptional repression. b | Histone acetylation and demethylation of DNA relaxes chromatin, and allows transcriptional activation.

ChIP on chip ChIP-seq Chromatin immunoprecipitation MOLECULAR BIOLOGY – Gene expression analysis ChIP on chip Chromatin immunoprecipitation combined with DNA microarrays (chip) or ChIP-seq Chromatin immunoprecipitation combined with DNA sequencing The ChIP–chip method can be used to study many of the epigenomic phenomena discussed in this Review. The example presented here shows how ChIP–chip can be used to study histone modifications. Modified chromatin is first purified by immunoprecipitating crosslinked chromatin using an antibody that is specific to a particular histone modification (shown in green). DNA is then amplified to obtain sufficient DNA. The colour-labelled ChIP DNA, together with the control DNA prepared from input chromatin and labelled with a different colour, is hybridized to a DNA microarray. The microarray probes can then be mapped to the genome to yield genomic coordinates.

ChIP on chip e.g. of transcription factor binding site discovery MOLECULAR BIOLOGY – Gene expression analysis ChIP on chip e.g. of transcription factor binding site discovery REST RE1 The ChIP–chip method can be used to study many of the epigenomic phenomena discussed in this Review. The example presented here shows how ChIP–chip can be used to study histone modifications. Modified chromatin is first purified by immunoprecipitating crosslinked chromatin using an antibody that is specific to a particular histone modification (shown in green). DNA is then amplified to obtain sufficient DNA. The colour-labelled ChIP DNA, together with the control DNA prepared from input chromatin and labelled with a different colour, is hybridized to a DNA microarray. The microarray probes can then be mapped to the genome to yield genomic coordinates. NEW TARGET GENES COMBINE WITH HISTONE ChIP on Chip for extra structure function data

MOLECULAR BIOLOGY – Gene expression analysis cDNA more RNA in original sample ... ... more times the particular fragment is sequenced quantifiable without knowledge of sequence (microarray needs to know the sequences)

ChIP-seq Chromatin immunoprecipitation combined with DNA sequencing MOLECULAR BIOLOGY – Gene expression analysis ChIP-seq Chromatin immunoprecipitation combined with DNA sequencing

MOLECULAR BIOLOGY – Gene expression analysis ... at the protein level immunolocalization Y ANTIBODY protein detection

MOLECULAR BIOLOGY – Gene expression analysis ANTIBODY

MOLECULAR BIOLOGY – Gene expression analysis MONOCLONAL ANTIBODY Monoclonal antibodies are produced by immunizing mice with antigen X, to stimulate the production of antibodies targeted against X.  The B-cells or antibody secreting cells are isolated from the mouse's spleen.  Antibody secreting B-cells that secrete antigenX-specific antibodies are then selected using a B-cell selection assay.  These B-cells are then fused with Tumour cells grown in culture, in the presence of PEG (poly-ethylene glycol). This results in a hybridoma. Hybridomas are then screened for antibody production against antigenX. Each hybridoma cell produces relatively large quantities of identical antibody molecules. Hybridomas are then allowed to multiply in culture,thus generating a large population of cells, each of which produces identical antibody molecules. polyclonal serum

POLYACRYLAMIDE-GEL ELECTROPHORESIS MOLECULAR BIOLOGY – Gene expression analysis POLYACRYLAMIDE-GEL ELECTROPHORESIS (PAGE) Figure 8-18a Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – Gene expression analysis RNA PROTEINS WESTERN BLOTTING DNA

MOLECULAR BIOLOGY – Gene expression analysis

MOLECULAR BIOLOGY – Gene expression analysis 1st dimension: Isoelectric focusing Figure 8-22 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – Gene expression analysis Two-dimensional electrophoresis

MOLECULAR BIOLOGY – Gene expression analysis Figure 8-23 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – Gene expression analysis PROTEOMICS What is the difference on B? identification by mass spectroscopy

MOLECULAR BIOLOGY – Gene expression analysis Figure 8-21 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – Gene expression analysis GENOME TRANSCRIPTOME PROTEOME

MOLECULAR BIOLOGY – Gene expression analysis Good Luck with molecular biology! DNA PROTEINS RNA SAGE, CAGE, PAGE ... GENOMICS TRANSCRIPTOMICS PROTEOMICS RT-PCR Real-Time PCR Inverse PCR Nested PCR RFLP AFLP

Potential revision Class next time!! Alexander Bruce Ph.D. Aleksandar Mihajlovic M.Sc. Vasanth Thamodaran M.Sc. USUAL TIME & PLACE 10 am in lecture theatre