Download 8 How Cellular Information is Altered

8
How
Cellular Information
is
Altered
8.1. Introduction
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DNA Replication in Ch. 4.
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Exchange of genetic information from one
generation to another
Additional genetic information through
natural of artificial means
Some mechanisms causing alterations
in cells’ content of genetic information
8.2. Evolving Desirable Biochemical
Activities through Mutation and Selection
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Mutation: genotype change, irreversible
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Genotype: the sum of the genetic
consortium of an organism
Phenotype: the characteristics
expressed by a cell
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8.3. How Mutations Occur
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Point mutation: the change of a single base
(CAA:glutamine – UAA:stop)
Deletion mutation: alter the whole composition
of a protein
Insertion mutation: addition of about 700 to
1400 base pairs in length
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IS: insertion elements
Reversion: original wild-type phenotype
restored
8.2.2. Selecting for Desirable Mutants
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Natural (spontaneous) rates of mutation vary
greatly
Selectable mutation: mutants can survive
under a set of specific set of environmental
conditions
Direct selection: an example of direct
selection to find a mutant resistant to an
antibiotic or toxic compound
Indirect selection: isolate mutants that are
deficient in their capacity to produce a
necessary growth factor

Auxotrophic mutants would not grow on
such a simple medium unless it were
supplemented with the growth factor (ex:
lysine auxotroph, replicate plating method)
8.3. Natural Mechanisms
for Gene Transfer and Rearrangement
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Gene transfer
from one organism to another
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Large rearrangements
in chromosomal DNA
8.3.1. Genetic Recombination
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A process that brings genetic elements from two
different genomes into one unit.
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Three main mechanisms for gene transfer
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Transformation: free DNA taken by cells
Transduction: bacteriophage transfer DNA
Conjugation: DNA transfer between two intact
cells
8.3.2. Transformation (Figure 8.4)
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Competent cells are transformable bacteria
strain
E.coli are not normally competent, but
treating them with high concentrations of Ca++
coupled with temperature manipulation
induces competency of E.coli cells
Plasmid is autonomous, self-replicating,
double-strand piece of DNA that is normally
extrachromosal.
8.3.3. Transduction (Figure 8.5)
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Generalized transduction
Specialied transduction
Lysogenic cell: one carrying a
prophage or phage DNA incorporated
into chromosomal DNA
Phage lamda: temperature phage
Prophage → lytic cell
8.3.4. Episomes and conjugation
(Figure 8.6)
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Episome: DNA molecule that may exist
either integrated into the chromosome
or separate from it
(ex: F factor)
E.coli F(+) F(-)
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Sex pilus conduit for transfer copy of the F
plasmid to the F- cell
Hfr (high frequency recombination)
8.3.5. Transposons:
Internal gene transfer
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Gene or genes that have the ability to jump
from one piece of DNA to another piece of
DNA, or to another position on the original
piece of DNA
Many of the transposons encode antibiotic
resistance
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Can induce mutations
Bring once-separate genes together
Movement of genes between unrelated bacteria
Transposon mutagenesis
8.4. Genetically Engineering Cells
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The purposeful, predetermined manipulation
of cells at the genetic level – idea before
1970, now grasp of beginning college
students
Genetic engineering
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A set of tools
Not a scientific discipline
Manipulation of DNA outside the cell to create
artificial genes or novel combinations of genes
8.4.1. Basic elements
of genetic engineering
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Obtain the gene of insert (Figure 8.7)
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Shotgun cloning
Hybridization: Probe using chemical synthesis
Total chemical synthesis of a gene
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Insertion (Figure 8.8)
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cDNA synthesized from mRNA by reverse transcriptase
Vector: plasmid is typical vector
Restriction enzyme: cut DNA at prespecific site
Ligase
Construction of desired vector-donor DNA
Screen:
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selectable markers such as antibiotic resistance/ expression
Cloning and Amplificaton of genes
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Electrophoresis: agarose, SDS-PAGE
Immnunoblot (Western blot): Ag-Ab,
radioactive marker
Shotgun cloning (gene library/gene bank) –
radiolabeled DNA/RNA probes
complementary to the cloned gene
Phage displays & bacterial displays
PCR (polymerase chain reaction) – two short
primer sequences (<20mer nucleotides) and
Taq polymerase
8.4.2. Genetic Engineering
of Higher Organisms
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Transfection: introduction of foreign gene into
higher organisms
Agrobacterium tumefaciens: T-DNA
Biolistic process: plant, very successful
Electroporation
Protoplasts: without outer cell envelope
Protoplast fusion
A baculovirus – insect cell system
8.5. Genomics:
the set of experimental & computational tools which allow the genetic blueprints of life to be read.
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Human genome projects
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Functional genomics
Protemics (up-, down- regulation)
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DNA sequencing, mRNA & protein expression
Micorarrays: mRNA level detection
2D gel electrophoresis (pI & MW)
Bioinformatics
Metabolomics
Highly nonlinear, dynamic cellular system:
mathematical modeling