A network of regulatory interactions governed by Hfq.

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Presentation transcript:

A network of regulatory interactions governed by Hfq. Hfq has been shown to bind each of the ncRNA (circled) and mRNAs (outer ring) facilitating the post-transcriptional gene regulation that responds to the indicated environmental stress. From the Feig Laboratory Wayne State University WEB Page

From the Feig Laboratory Wayne State University WEB Page Generic model through which regulatory ncRNAs can relieve translational repression of a complementary mRNA through strand displacement. Equivalent models can be used to explain translational repression as well. Secondary structures of DsrA (top) and rpoS mRNA 5'-UTR (bottom) The color coding of DsrA indicates the accepted domain structure. For rpoS, the open reading fram is shown in green and the region complementary to DsrA (bases 11 - 32 in DsrA) is shown in lavender. From the Feig Laboratory Wayne State University WEB Page

Principle of pyrosequencing http://www.s3.kth.se/mst/research/projects/beadbasedmicrofluidic/index.shtml

How to detect the gene expression? (1)　Northern Blot analysis (2)　Primer extension analysis　 (3)　S1 nuclease mapping (4)　Nuclease protection assay (5)　RT-PCR (6)　Large-scale cDNA sequencing (7) 　Microarray analysis (8) 　5’-, 3’- RACE (Rapid Amplification of cDNA Ends) (9) 　Reporter gene assay

Northern Blot Hybridization

Figure 4.12. Southern hybridization. (A) Transfer of DNA from the gel to the membrane. (B) The membrane is probed with a radioactively labeled DNA molecule. On the resulting autoradiograph, one hybridizing band is seen in lane 2, and two in lane 3.

Primer Extension Analysis

S1 Nuclease Mapping Analysis

Reporter Gene Assay Gene Gene product Assay lacZ β-galactosidase Histochemical test uidA β-glucuronidase lux Luciferase Bioluminescence GFP Green fluorescent protein Fluorescence Reporter Gene Assay

Luciferase gene Luciferase Activity (Promega)

PITX2-LACZ Staining of mouse embryo day 11 PITX2: bicoid-related transcription factor