BIO3001 – Molecular Biology
Table of Contents
Animal Models & Molecular Techniques.............................................................................2
Signal Transduction............................................................................................................7
Transcriptional Control of Gene Expression......................................................................15
Post-Transcriptional Gene Control....................................................................................23
Cancer..............................................................................................................................30
Stem Cells, Asymmetry & Cell Death.................................................................................36
,Animal Models & Molecular Techniques
Gene analysis: Connecting knowledge about sequence, structure and biochemical activity of
encoded protein to its function in the context of a living cell or organism is important. This is
done by examining phenotypic consequences of mutations that inactivate a particular gene.
Classical genetics: isolation of a mutant that appears to be defective in the process
of interest. Genetic methods are then used to identify and isolate the affected gene.
The isolated gene can be manipulated to produce large quantities of the protein it
encodes for biochemical experiments and to design probes for studies of where and
when the protein is expressed in an organism.
Reverse genetics: identification and isolation of a gene of an interesting protein,
which is then altered and reinserted into an organism to study it effects.
Mutations can have different effects:
Recessive mutant: often causes a loss of function.
Dominant mutant: often causes a gain of function. Can be identified by mating two
homozygous strains (75% of F2 has mutant phenotype)
Dominant negative mutation: causes production of a protein that inactivated the
products of the wild-type gene. Produces phenotype similar to loss of function
mutation.
Cell isolation
Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry
which provides a method for sorting a heterogeneous mixture of biological cells into
two or more containers, one cell at a time, based upon the specific light scattering
and fluorescent characteristics of each cell. The wanted cells are conjugated with
antibody-associated fluorescent dye (fluorophore) and when a laser is pointed at the
mixture, light gets excited at a certain wavelength. Cells with an excitation above a
certain wavelength (wanted cells) are separated.
Different molecular genetic techniques can be used to isolate, sequence and manipulate
genes and to analyze when and where in the cell this protein is expressed.
Identification: done by genetic screening
Conditional mutations: used to study phenotypic expression of a mutation in
essential genes (usually lethal mutations, tricky to study)
o Temperature sensitive mutations: useful in bacteria and lower eukaryotes
(haploid yeast) that can grow at a range of temperatures. The mutant
phenotype is observed at the nonpermissive temperature and not observed
at the permissive temperature (usually higher), even though the mutant
allele is present. This temperature-sensitive mutant produces an altered
protein that works at the permissive temperature but unfolds and is
nonfunctional at the nonpermissive temperature. Mutant strains can thus be
maintained at permissive temperature, after transferring them to a
nonpermissive temperature for analysis of the mutant phenotype.
, Inbreeding: used to study recessive lethal mutations in diploids. Mutations can be
maintained in heterozygotes, of which the progeny contains recessive homozygotes.
Consequences of the mutation can be analyzed in the homozygotes.
Complementation analysis: restoration of the wild-type phenotype by mating two
different mutants. If two recessive mutations, A and B, are in the same gene, then a
diploid organism carrying one A allele and one B allele will exhibit the mutant
phenotype because neither allele provides a functional copy of the gene. In contrast,
if mutations A and B are in separate genes (complementing), then heterozygotes
carrying a single copy of each mutant allele will not exhibit the mutant phenotype
because a wild-type allele of each gene is also present.
Double mutations: can be used to determine the order in which proteins function
(biosynthetic pathways, signaling pathways) and their functionally significant
interactions.
o Suppressor mutations: when a mutation in protein A disrupts the ability to
associate with protein B in the same cellular process. Similarly, mutations in
protein B lead to small structural changes that inhibit its ability to interact
with protein A. normal functioning of protein A and B thus depends on their
interaction.
o Synthetic lethality: the deleterious effect of one mutation is increased by a
second mutation in a related gene. Mutation A for example inactivates a
process, of which another process can take over. Mutation B inactivates the
other process, of which the original one can take over. If both happen
simultaneously, the mutations become lethal.
Genetic mapping studies: genetic analysis based on gene position, the less
frequently recombination (crossing over) is observed to occur between two genes
on the same chromosome, the closer together they must be (linked/same gene vs
unlinked/different gene). Genetic markers allow for determination of the position of
unmapped mutations, determined by assessing its segregation during meiosis. The
more markers available, the more precisely a location can be mapped.
Isolation: obtain discrete, small regions of an organism’s DNA that constitute specific genes.
Needed to perform detailed studies of the structure and function of a gene at molecular
level. Uses recombinant DNA technology to obtain large quantities of DNA in pure form.
DNA cloning: the genome is cut into restriction fragments by restriction
enzymes/DNA ligases (endonucleases), DNA fragments of interest are then linked to
a vector (plasmid) DNA molecule that can replicate within a host cell. The inserted
DNA is replicated along with the vector, generating a lot of identical DNA molecules.
The isolated DNA fragment can be used for subsequent analysis
(sequencing/manipulation).
o Cutting: performed by restriction enzymes which recognize 4-8bp restriction
site sequences. Restriction enzymes generate fragments that have a single-
stranded tail at both ends (sticky ends), which are complementary to those
on other fragments and can therefore base-pair. The frequency of the cutting
and the fragment length depends on the length of the restriction site.
, o Insertion: DNA fragments are inserted into
vector DNA with the aid of DNA ligase, which
covalently joins the ends of a restriction
fragment and vector DNA that have
complementary ends (easier with sticky ends
than with blunt ends). Plasmids are circular,
bacterial dsDNA molecules that replicate
separately from a cell’s chromosomal DNA,
which are often used as vector. They contain
three regions essential for DNA cloning: a
replication origin (ORI), a marker that
permits selection (drug resistant gene), and
an insertion region for exogenous DNA
fragments. The versatility if an E. coli plasmid
vector can be increased by the addition of a
polylinker (restriction site), which is a
synthetically generated sequence containing
one copy of each different restriction site that
is not present elsewhere in the plasmid
sequence. This restriction site is cleaved by
two restriction enzymes in order to accept an
exogenous DNA fragment with the right sticky
ends (eliminates unwanted by products).
Shuttle vectors are capable of propagation in
different hosts. Bacterial cells will take up the plasmid DNA by the process of
transformation, which happens when cells are mixed with recombinant DNA
and subjected to a stress/heat shock (1 in every 10.000 cells transforms).
Sanger sequencing of the insert is performed to make sure it is the right one.
o Selection: rare transformed cells can be easily selected by use of a selectable
marker. If the plasmid contains a gene that induces resistance to a certain
antibiotic, transformed cells can be selected by growing them in antibiotic-
containing medium. All cells in the colony then contain cloned DNA.
o Isolation: a collection of DNA molecules each cloned into a vector molecule is
known as a DNA library, and the set of clones that collectively represent all
the DNA sequences in the genome is known as the genomic library. libraries
can be screened for the ability to express function proteins, allowing isolation
of genes that correspond to mutations identified in an experimental organism
(functional complementation)
cDNA cloning: expressed mRNAs are reverse-transcribed into complementary DNAs
(cDNAs), which are the used for cloning. A cDNA library is a set of cDNA clones
prepared from the mRNAs isolated from a particular type of cell or tissue. Used to
clone eukaryotic genes that require downstream RNA processing.
Polymerase chain reaction (PCR): powerful and versatile technique used to generate
large quantities of a specific known sequence and to manipulate DNA in the
laboratory. At each cycle, the number of copies of the sequence is increased
exponentially (doubled each cycle). All other sequences remain unamplified.
o Denaturation: done by heat (92-95 degrees), produces ssDNA
