bcm208 metabolic biochemistry topic 7: gene metabolism and expression
TRANSCRIPT
Learning Objectives
• Describe the structure of DNA• Understand the processes involved in DNA,
RNA and protein synthesis• Understand the process of gene expression• Understand how genes are regulated• Understand the basic concept of recombinant
DNA technology• Understand how plants are transformed using
Agrobacterium
Regulation of mRNA transcription in Eukaryotes
• Most active eukaryotic cells transcribe a common (basal) set of structural genes that maintain routine (household) cellular functions
• Cells express other specialised genes which give the cells there unique properties
Regulation of mRNA transcription in Eukaryotes
(cont.)• A number of diverse, highly
specific processes that activate or repress transcription in eukaryotic cells
• Generally transcription is mediated by proteins that are collectively classified as transcription factors
Regulation of mRNA transcription in Eukaryotes
(cont.)• Transcription factors bind to DNA
sequences (often called boxes)• There are some general regulatory
sequences, however, most genes have their own set of response elements
Common eukaryotic regulatory sequences
• TATA box: (or Hogness box) 8 nucleotides that includes a TATA sequence (-25 bp)
• “cat” box: CCAAT (-75 bp)• GC box: a sequence of repeated
GC nucleotides (-90 bp)
DNA cloning
• Why clone DNA? : To allow large scale amplification of identical molecules
• What for?: – Further analysis eg DNA sequencing– Expression of a gene– Insertion into transgenic organism
Restriction enzymes
• Cleave DNA at specific sequences• Isolated from bacteria (eg Eco RI
isolated from E. coli)• Natural role: cleave invading DNA
viruses
DNA libraries
• Obtain DNA fragments– cDNA– Genomic DNA digested with RE
• Digest plasmid DNA with RE• Ligate DNA fragments to digested
plasmid
• Transform E.coli: each cell contains different DNA fragment
• Generate colonies for individual cells
Applications of DNA libraries
• Looking for genes expressed in particular tissues (cDNA)
• Identifying genes using southern hybridisation (requires probe with complementary sequence eg derived from a similar gene from a different species)
• Western analysis can also be used to screen libraries (requires gene to be expressed in bacteria)
Electrophoresis
• Migration of DNA, RNA or protein through a matrix
• Molecules move due to charge: migrate toward +ve electrode (due to negatively charged phosphate groups in nucleic acids)
• Smaller molecules are able to migrate more rapidly that larger molecules
• Migration monitored by a visible dye
Agarose gel Electrophoresis
• Agarose: polysaccharide from seaweed• Used to separate DNA and RNA molecules
above 300 bp• Prepared by dissolving agarose powder (by
heating in microwave) in running buffer (usually Tris-Borate EDTA buffer or TBE)
• Agarose concentration range: 0.8% - 2%• Higher concentration resolve lower molecular
weight molecules more effectively
Visualisation of DNA and RNA
• Ethidium bromide most commonly used for nucleic acid visualisation
• Binds to nucleic acids and fluoresces when exposed to UV light
• Mutagen
Acrylamide gel electrophoresis
• Used for Proteins and DNA molecules smaller that 300 bp (eg DNA sequencing)
• Higher resolution• Pore size determined by acrylamide
concentration• Acrylamide monomer is a neurotoxin
Protein electrophoresis
• Protein often treated with sodium dodecyl sulphate (SDS) to interrupt inter-molecular bonding so that molecules run based on molecular weight
Expression of eukaryotic genes in prokaryotic systems
• Eukaryotic genes contain introns• To express eukaryotic genes in
prokaryotic cells, introns need to be removed
• This can be done by generating DNA copies of mRNA (which have had introns spliced out)
• This DNA copy of mRNA is called complementary DNA (or cDNA)