1Lecture 14: Gene expression --continuedB. Analyses of transcriptomeI. cDNA microarraysII. Oligonucleotide arraysIII SAGE (serial analysis of gene expression)VI MPSS (massively parallel signiture sequence)C. Protein expression patternImmunohistochemistry (p661-662)Reporter (p798; 661-662)Western blotMass spectrometryRead 348-354Fig. 10.6; 10.24; 10.25; 10.26; 10.28; 10.2919.18; 19.25; 19.272High throughput analyses of the transcriptomeDocumenting gene expression on a genome wide scaleTranscriptome: complete set of transcripts and their relative expression levels in a particular cell or tissue under defined conditions3I. cDNA microarrays45II. Oligonucleotide microarrays (Affymetrix GeneChip)o o ooomaskLight deprotectionOH OHoooToooT T6Sporulation gene expression profile in budding yeastChu et al., (1998) Science 282, 699-705Several classes of sporulation gene expression after transfer to sporulation media7Survey of 1116 genes during sporulation in budding yeastChu et al., (1998) Science 282, 699-7058Fig. 10.28MPSS: Massively Parallel Signature Sequencing89SAGE: serial analysis of gene expression10Lecture 14: Gene expression --continuedB. Analyses of transcriptomeI. cDNA microarraysII. Oligonucleotide arraysIII SAGE (serial analysis of gene expression)VI MPSS (massively parallel signiture sequence)C. Protein expression patternImmunohistochemistry (p661-662)Reporter (p798; 661-662)Western blotMass spectrometryRead 348-354Fig. 10.6; 10.24; 10.25; 10.26; 10.28; 10.2919.18; 19.25; 19.2711Fig. 19.251112Fig. 19.18121314Fig. 19.271415Fig. 10.291516RNA interference RNA interference post-transcriptional gene suppression (PTGS)RNAi movie www.nature.com/focus/rnai/animations/index.htmlFig. 19.81718RNAi mechanismRNAi mechanism• Cellular RNase recognizes dsRNA• Cleaves to small (23 bp) fragments• Fragments hybridize to transcripts• RNA-dependent RNA polymerase forms dsRNA• RISC nuclease chews up dsRNAGood RNAi resource: http://www.ambion.com/techlib/resources/RNAi/19RNAi mechanismRNAi mechanism• Function of RNAi likely used to detect:– genome-invading transposable genetic elements and double-stranded (ds) RNA viruses– Other abnormal gene expression• Initially characterized in the following:– C. elegans• Antisense injection resulted in predicted phenotype– Plants• Resistance to spread of
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