Introduction to molecular biology

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Introduction to molecular biology

• (in one hour!!)
• Stephen Edwards

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Overview

• Overview of the cell
• Different sizes/functions
• Organised structure
• Bacterial genetics are different

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Eukaryotic cell
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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

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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'
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DNA function

• Carries the blueprint for life
• Duplication for new cells
• Make proteins for biological functions

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Gene structure

• Genes must have
• Exons
• Start site
• Control region

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mRNA

• DNA is grouped into threes (codons)
• AGTTTTGGGCCCAAA
• Start and stop codons
• mRNA is then modified
• and travels out of the nucleus

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mRNA splicing
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Alternative splicing
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Translation

• Uses mRNA as template to make proteins
• Occurs in ribosomes
• One codon corresponds to one amino acid

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Proteins

• Huge proportion of cell (after water)
• Many functions
• Structure (e.g. collagen in bone)
• Enzymes
• Transmembrane receptors
• Hormones
• Four levels of structure

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Protein structure
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Protein interactions

• Proteins can form interations
• Proteins (complexes, oligomers)
• mRNA
• DNA
• Proteins can bind to each other depending on
  their relative charges and structures

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Gene expression regulation
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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)

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Common abbreviations

• EST expressed sequence tag
• SNP single nucleotide polymorphism
• ORF open reading frame
• UTR untranscribed region

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Experimental techniques

• Cut, change, knockout genes
• Transgenics
• Sequence genes and proteins
• Over-express proteins
• Microarrays

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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.
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Future of molecular biology

• Personalised medicine
• Target-specific drugs (e.g. adipose tissue)
• Gene therapy
• Comparative genomics

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References

• Molecular biology information
• Biology, Campbell and Reece (6th 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 1521(2)248-56. Epub
  2004 Aug 27. Gene name ambiguity of eukaryotic
  nomenclatures.Chen L, Liu H, Friedman C.

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Two hybrid system