Lecture 27: Using Cloned Genes to Understand Molecular Defects

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since 4 October 1999

(version 25 September, 1999)

1. Sequence analysis:

Similarity to known proteins:

e.g., mouse and zebra fish T-locus :

SPNFGAHWMKAPVSFSKVKLTNKLNGGQ…IMLNSLHKYEPRIHIVRVGGPQRM

SPNFGAHWMKAPVSFSKVKLSNKLNGGQ…IMLNSLHKYEPRIHIVKVGGIQRM

Similarity to known motifs:

e.g., Asn X Ser

Zn finger

2. Analysis of expression patterns

Where and when is a gene expressed?

Use "northern blots" - like Southerns (see lecture 22), but run RNA instead of DNA on a gel.

3. Biochemical experiments

a. Generate large amounts of purified protein, using expression vectors

b. Use purified protein to raise antibodies for use in immunolocalization or purification of native protein from tissues

c. Test biochemical properties of purified or native protein.

4. Genetic studies, using model organisms

e.g., mice, flies, worms, yeast, bacteria.