Download BCM301 Food Biotechnology

BCM208 Metabolic
Biochemistry
Topic 7:
Gene metabolism
and Expression
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
The flow of genetic information
Structure of DNA
Structure of DNA (cont.)
Structure of DNA (cont.)
DNA Replication
DNA Replication (cont.)
DNA Replication (cont.)
DNA replication
Transciption
Transcription
Transcription
RNA processing
Decoding Genetic Information:
RNA and Protein
Translation
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Translation
Translation (cont.)
Translation (cont.)
Translation (cont.)
Regulating gene expression
Regulation of mRNA transcription
in Bacteria
Negative regulation
Positive regulation
Positive and negative regulation
A repressible operon
An inducible operon
lac operon (cont.)
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)
Eukaryotic gene regulation is
complex!!!
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
DNA cloning (cont.)
Enzymes used in recombinant
DNA technology
Restriction enzymes
• Cleave DNA at specific sequences
• Isolated from bacteria (eg Eco RI
isolated from E. coli)
• Natural role: cleave invading DNA
viruses
Recognition sequences
Sticky vs blunt ends
Plasmid cloning vector (pBR322)
Polylinker
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 TrisBorate 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)
Plant transformation
• Microprojectile bombardment
• Agrobacterium
Microprojectile
Bombardment
Plant transformation with
Agrobacterium tumefaciens
Plant regeneration
• Fig 29-19
Insect resistant
transgenic plants
• Fig. 29-21
Herbicide resistant
transgenic plants
• Fig 29-22