Introduction to molecular biology… (…in one hour!!) Stephen Edwards Overview Overview of the cell Different sizes/functions Organised structure Bacterial genetics are different Eukaryotic cell DNA Contained in the nucleus Arranged in 22 chromosomes, plus two sex chromosomes Two copies of each 99.9% identical to other humans, 98% to chimp! Around 2m DNA, enough to travel to sun and back 600 times! Therefore, very tightly packed 5' C-G-A-T-T-G-C-A-A-C-G-A-T-G-C 3' | | | | | | | | | | | | | | | 3' G-C-T-A-A-C-G-T-T-G-C-T-A-C-G 5' DNA function Carries the blueprint for life Duplication for new cells Make proteins for biological functions: Gene structure Genes must have: – Exons – Start site – Control region mRNA DNA is grouped into threes (codons) AGTTTTGGGCCCAAA Start and stop codons mRNA is then modified… …and travels out of the nucleus mRNA splicing Alternative splicing Translation Uses mRNA as template to make proteins Occurs in ribosomes One codon corresponds to one amino acid Proteins Huge proportion of cell (after water) Many functions: – Structure (e.g collagen in bone) – Enzymes – Transmembrane receptors – Hormones Four levels of structure Protein structure Protein interactions Proteins can form interations: – Proteins (complexes, oligomers) – mRNA – DNA Proteins can bind to each other depending on their relative charges and structures Gene expression regulation Common terminology Genome/proteome Genotype/phenotype Pseudogene Novel protein/gene Putative gene Locus/Allele/Chromosome region Dominant/Recessive Homologous Symbol cDNA Motif Dalton (Da or kDa) Common abbreviations EST SNP expressed sequence tag single nucleotide polymorphism ORF UTR open reading frame untranscribed region Experimental techniques Cut, change, knockout genes Transgenics Sequence genes and proteins Over-express proteins Microarrays We have used the yeast two-hybrid system to identify proteins that interact with the intracellular portion of the hepatocyte growth factor (HGF) receptor (Met) We isolated a human cDNA encoding a novel protein of 68 kDa, which we termed FAP68 This protein is homologous to a previously described FK506-binding protein-associated protein, FAP48, which derives from an alternative spliced form of the same cDNA, lacking an 85-nucleotide exon and leading to an early stop codon Here we show that epithelial cells, in which the HGF receptor is naturally expressed, contain FAP68 and not FAP48 proteins FAP68 binding to Met requires the last 30 amino acids of the C-terminal tail, which are unique to the HGF receptor Indeed, FAP68 does not interact with related tyrosine kinases of the Met and insulin receptor families FAP68 interacts specifically with the inactive form of HGF receptor, such as a kinase-defective receptor or a dephosphorylated wild type receptor Evidence In vivo, endogenous FAP68 can be coimmunoprecipitated with the HGF receptor in the absence of stimuli and not upon HGF stimulation Thus, FAP68 represents a novel type of effector that interacts with the inactive HGF receptor and is released upon receptor phosphorylation Free FAP68 exerts a specific stimulatory activity toward the downstream target p70 S6 protein kinase (p70S6K) Significantly, nonphosphorylated HGF receptor prevents FAP68 from stimulating p70S6K These data suggest a role for FAP68 in coupling HGF receptor signaling to the p70S6K pathway Future of molecular biology Personalised medicine Target-specific drugs (e.g adipose tissue) Gene therapy Comparative genomics References Molecular biology information – – – – – – ‘Biology’, Campbell and Reece (6 th Ed.), Very readable general biology textbook www.ebi.ac.uk/2can, good introduction to bioinformatics and molecular biology http://www.emc.maricopa.edu/faculty/farabee/BIOBK/BioBookTOC.html - online biology book http://biology-pages.info, good glossary/information site http://www.genomicglossaries.com/ http://www.gene.ucl.ac.uk/nomenclature/guidelines.html, defines the nomenclature for human genes Databases – – – – – – www.ebi.uniprot.org, excellent protein sequence database www.ncbi.nih.gov, numerous protein/genome databases www.ensembl.org, information on genes/proteins/exons of completed genomes http://www.ebi.ac.uk/embl/, European gene sequence databank Michael Y Galperin, The Molecular Biology Database Collection: 2005 update, NAR www.bioinfo.no/links, list of useful biological links Gene/Protein naming conventions – Bioinformatics 2005 Jan 15;21(2):248-56 Epub 2004 Aug 27 Gene name ambiguity of eukaryotic nomenclatures.Chen L, Liu H, Friedman C Two hybrid system ... represents a novel type of effector that interacts with the inactive HGF receptor and is released upon receptor phosphorylation Free FAP68 exerts a specific stimulatory activity toward the downstream target... We have used the yeast two-hybrid system to identify proteins that interact with the intracellular portion of the hepatocyte growth factor (HGF) receptor (Met) We isolated a human cDNA encoding... unique to the HGF receptor Indeed, FAP68 does not interact with related tyrosine kinases of the Met and insulin receptor families FAP68 interacts specifically with the inactive form of HGF receptor,