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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

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Introduction to molecular biology

Introduction to molecular biology…

(…in one hour!!)

Stephen Edwards


Overview
Overview

  • Overview of the cell

  • Different sizes/functions

  • Organised structure

  • Bacterial genetics are different



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
DNA function

  • Carries the blueprint for life

  • Duplication for new cells

  • Make proteins for biological functions:


Gene structure
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




Translation
Translation

  • Uses mRNA as template to make proteins

  • Occurs in ribosomes

  • One codon corresponds to one amino acid


Proteins
Proteins

  • Huge proportion of cell (after water)

  • Many functions:

    • Structure (e.g. collagen in bone)

    • Enzymes

    • Transmembrane receptors

    • Hormones

  • Four levels of structure



Protein interactions
Protein interactions

  • Proteins can form interations:

    • Proteins (complexes, oligomers)

    • mRNA

    • DNA

  • Proteins can bind to each other depending on their relative charges and structures



Common terminology
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
Common abbreviations

  • EST expressed sequence tag

  • SNP single nucleotide polymorphism

  • ORF open reading frame

  • UTR untranscribed region


Experimental techniques
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
Future of molecular biology 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.

  • Personalised medicine

  • Target-specific drugs (e.g. adipose tissue)

  • Gene therapy

  • Comparative genomics


References
References 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.

  • 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 15;21(2):248-56. Epub 2004 Aug 27. Gene name ambiguity of eukaryotic nomenclatures.Chen L, Liu H, Friedman C.


Two hybrid system
Two hybrid system 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.


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