Lectuer 28 Flashcards
Finding protein coding genes in genome
Look for ORF (AUG start) if greater than 300bp it is likely real thus some genes are missed by this method
How does introns affect coding genes localization
protein coding gene sin humans have an average of 9 introns
most exons are short and ORFs and indistinguishable
Best gene finding algorithms utilize what?
RNA sequencing info
Degree of fit models
Gene sequence similarity
Protein-Coding genes and Funcitons
Functional domains of proteins usually have conserved sequence motifs. There are databases of such motifs and their consensus sequence motifs (for instance InterPro) that facilitate such analyses.
*Using the BLAST algorithm, a new sequence can be tested for similarity to any known sequence. A match is often informative for function.
*Because of the degeneracy of the genetic code, similarity searches with predicted protein sequences work better than those with DNA sequences.
Metabolism genes
Metabolism genes make up the majority of the total number of genes while transcription and translation related genes are also present in significant number
RNA interference screening of unknown genes in Drosophila
Expressing shRNAfor any of 62 of these genes caused lethality, so around 25% of these unknown genes are essential for life.
Proteomics
Study of all proteins in a biological system
LC-MS/MS
A complex mixture of proteins is digested with a protease, and the many many resulting fragments are separated by LC into multiple less complex fractions.
*The peptides in each fractions get ionized, and each peptide gets analyzed for its mass and sequence.
*Using protein sequence databases, computational methods identify the proteins in the original sample.
*Thousands of proteins can be identified from 1-50 micrograms of total sample (10,000-150,000 cells).
BioID
Biotin Fusion proteins and then addition of Biotin promotes proximity-based biotinylation of proteins then isolation of biotinylated proteins then digest isolated proteins and MS identification
