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Regulation of enzyme synthesis
The lac operon is an example of an inducible operon - it is normally off, but when a
molecule called an inducer is present, the operon turns on.
The trp operon is an example of a repressible operon - it is normally on but when a
molecule called a repressor is present the operon turns off.
Enzyme repression
Is the regulatory mechanism that inhibits
gene expression and decreases the
synthesis of enzymes.
Is a response to the overabundance of an
end-product of a metabolic pathway.
Is mediated by regulatory protein
‘repressors’, which block the ability of
RNA polymerase to initiate transcription.
Repressor: is an allosteric protein that
binds to the operator (after it binds to the
corepressor) region of the DNA and
blocks transcription
Operator: A specific region of the DNA
located at the beginning of the gene,
where the repressor protein binds
‘Corepressor’ is a small molecule that
combines with a repressor protein and
alters its conformation. Only the altered
repressor can bind to the operator
Enzyme Induction
• Turns on transcription of gene(s).
• An ‘inducer’ is a small molecule that initiates enzyme induction by combining with a
repressor protein and altering its conformation.
• The altered repressor is released
• ‘Inducible enzymes’ are those synthesized in the presence of inducer.
Genetic Engineering or Recombinant DNA technology
Applications of molecular biology have allowed scientists to modify the genetic
characteristics of organisms at the DNA level -- the most widely used of these techniques
is called recombinant DNA technology -- this technology is revolutionizing agriculture,
medicine and forensics.
Recombinant DNA technology -- procedures by which DNA from different
species can be isolated, cut and spliced together -- new "recombinant "
molecules are then multiplied in quantity in populations of rapidly
dividing cells (e.g. bacteria, yeast).
Hepatitis B vaccine is being made by yeast carrying a gene for part of the
hepatitis virus (viral coat protein), thus eliminating the need to use the whole
virus.
New recombinant DNA techniques can be also used to amplify DNA. Since each
person’s DNA is unique (except identical twins), the procedure is useful in the
field of criminology.
This technology is based on a number of important things:
1. bacteria contain extrachromosomal molecules of DNA called
plasmids. They are the cloning vectors (small, independently replicating
genetic elements used to replicate genes, and most are derived from plasmids or viruses)
2. bacteria also produce enzymes called restriction endonucleases
that cut DNA molecules at specific places -- DNA cut into many
smaller pieces called restriction fragments.
Restriction Enzymes There are many different kinds of
restriction endonucleases -- each cuts
DNA at a specific site defined by a
sequence of bases in the DNA called a
recognition site -- several hundred
endonucleases have been extracted from
bacteria and many are used in
recombinant DNA research.
Example: endonuclease called EcoRI
cuts DNA where the sequence GAATTC
occurs -- GAATTC is the recognition
site for this endonuclease -- cut occurs
between the G and the A so that the cut
is staggered and each fragment has 2
sticky ends.
Typical restriction enzymes recognize 4-, 6- or 8-base sequences.
The two strands have the same sequence if both are read 5’— 3’ or both read 3’—
5’.
Restriction enzymes make staggered cuts in the 2 strands of DNA (cuts are not
directly opposite to each other), leaving stretches of single-stranded DNA at the
end of DNA fragment called “sticky end” which is characteristic to that enzyme.
Sticky ends join to complementary stretches of single-stranded DNA by base
pairing (by hydrogen bonding).
Then DNA ligase enzyme is used to covalently link the backbone of the DNA
pieces and thus producing recombinant DNA molecule.
Cloning Vectors
Cloning vectors are
small, independently
replicating genetic
elements used to
replicate genes, and
most are derived from
plasmids or viruses
(bacteriophage).
Cloning: isolation &
incorporation of
certain gene or DNA
fragment in a vector to
be replicated
To clone a DNA
sample, the same
restriction enzyme
must be used to cut
both the vector and the
DNA sample.
Cloning vectors share four common
properties:
1.Ability to promote autonomous
replication.
2. Contain a genetic marker (usually
dominant) for selection.
3. Unique restriction sites to facilitate
cloning of insert DNA.
4. Minimum amount of nonessential DNA to
optimize cloning.
Gene Libraries:
A genomic library, also clone bank
or gene bank, is a collection of
DNA from a single organism,
ideally though not necessarily
containing its entire genomic DNA
sequence. The DNA from the
source organism of interest is
divided into multiple fragments
and packaged within cloning
vectors such that each carries a
portion of it. The vector DNA can
then be inserted into host
organisms - commonly a
population of bacteria - for
amplification and retrieval.
Plasmid vector
Each fragment of DNA containing
about one gene is carried by a
vector, either a plasmid within a
bacterial cell or a phage.
Creating a Genomic Library
1. DNA molecules of an organism of interest are isolated.
2. The DNA molecules are then partially digested by endonuclease restriction
enzymes, splitting the helix into small workable portions. Several different
restriction enzymes may be used at once, and the DNA molecules may be
digested for different lengths of time in order to ensure that the DNA has been
digested to manageable sizes.
3. DNA molecules are separated by size using agarose electrophoresis. Individual
DNA pieces are ligated into host vectors.
4. The hosts are kept in liquid media and can be frozen at -808C for a long period of
time for later experimental use.
Gene Library
Gel Electrophoresis
The process in which molecules (such as proteins, DNA, or RNA fragments) can be
separated according to size and electrical charge by applying an electric current to them
while they are in a gel. The current forces the molecules through pores in a thin layer of
gel, a firm jelly-like substance. The gel can be made so that its pores are just the right
dimensions for separating molecules within a specific range of sizes and shapes. Smaller
fragments usually travel further than large ones.
After certain time the gel is stained with ethidium bromide which will fluoresce under
UV light.
In Lane ‘A’ a restriction enzyme digestion of
a standard DNA sample where the size of
each band is known “Marker or Ladder”
Other lanes have purified sources of DNA
cut by one or more restriction enzymes.
The banding pattern of a given DNA is
reproducible since a given restriction enzyme
always cut at the same site.
By comparison with the standard, the
fragment size can thus be determined.
Uses of Gel electrophoresis:
• Comparative studies of 2 or more DNAs.
• Studies in the classification of microorganisms and their genetic relationship.
• To determine DNA fragment sizes after cutting the DNA with restriction enzymes
(a), or it might be necessary to check DNA that has been isolated and purified
MUTATION A mutation is a change in the base sequence of DNA. Such a change in the base sequence
of a gene will sometimes cause a change in the product encoded by that gene.
For example, when the gene for an enzyme mutates, the enzyme encoded by the gene
may become inactive or less active because its amino acid sequence has changed. Such a
change in genotype may be disadvantageous, or even lethal, if the cell loses a phenotypic
trait it needs. However, a mutation can be beneficial if, for instance, the altered enzyme
encoded by the mutant gene has a new or enhanced activity that benefits the cell.
Many simple mutations are silent (neutral); the change in DNA
base sequence causes no change in the activity of the product
encoded by the gene.
Types of mutations
The most common type of mutation involving single base pairs is
base substitution (or point mutation), in which a single base at
one point in the DNA sequence is replaced with a different base.
If the base substitution results in an amino acid substitution in the
synthesized protein, this change in the DNA is known as a
missense mutation.
By creating a nonsense (stop) codon in the middle of an mRNA
molecule, some base substitutions effectively prevent the
synthesis of a complete functional protein; only a fragment is
The bright “bands” on this gel
are DNA fragments produced by
restriction enzyme digestion.
The bands to the far sides are
DNA size markers.
synthesized. A base substitution resulting in a nonsense codon is
thus called a nonsense mutation.
Frameshift mutations are mutations in which one or a few nucleotide pairs are deleted
or inserted in the DNA. This mutation can shift the "translational reading frame-that is,
the three-by-three grouping of nucleotides recognized as codons by the tRNAs during
translation, which results in the production of an inactive protein.
Spontaneous mutations occur in the absence of any mutation-causing agents. Agents in
the environment, such as certain chemicals and radiation that directly or indirectly bring
about mutations are called mutagens. A wide variety of chemicals, many of which are
common in nature or in households, are known to be mutagens. Many forms of radiation,
including X rays and ultraviolet light, are also mutagenic.
In the microbial world, certain mutations result in resistance to antibiotics or altered
pathogenicity.
Mutagens
Chemical Mutagens
One of the many chemicals known to be a mutagen is nitrous
acid. Nitrous acid can convert the base adenine (A) to a form
that no longer pairs with thymine (T) but instead pairs with
cytosine (C).
Eventually, some AT base pairs of the parent will have been
changed to GC base pairs in a granddaughter cell. Like all
mutagens, it alters DNA at random locations.
Another type of chemical mutagen is the nucleoside analog. These molecules are
structurally similar to normal nitrogenous bases, but they have slightly altered base-
pairing properties. Examples, 2-aminopurine and 5-bromouracil. When nucleoside
analogs are given to growing cells, the analogs are randomly incorporated into cellular
DNA in place of the normal bases. Then, during DNA replication, the analogs cause
mistakes in base pairing, resulting in base-pair substitutions. Some antiviral and anitumor
drugs are nucleoside analogs, induding AZT (azidothymidine), one of the primary drugs
used to treat HIV infection.
Radiation
X rays and gamma rays are forms of radiation that are potent mutagens because of their
ability to ionize atoms and molecules.
Another form of mutagenic radiation is ultraviolet (UV) light, a nonionizing component
of ordinary sunlight. However, the most mutagenic component of UV light (wavelength
260 nm) is screened out by the ozone layer of the atmosphere.
The Frequency Of Mutation
The mutation rate is the probability that a gene will mutate when a cell divides.
Spontaneous mistakes in DNA replication occur at a very low rate, perhaps only once in
109 replicated base pairs (a mutation rate of 10
-9).
The occurrence of random mutations at low frequency is an essential aspect of the
adaptation of species to their environment, for evolution requires that genetic diversity be
generated randomly and at a low rate.
For example, a mutation that confers antibiotic resistance is beneficial to a population of
bacteria that is regularly exposed to antibiotics. Once such a trait has appeared through
mutation, cells carrying the mutated gene are more likely than other cells to survive and
reproduce as long as the environment stays the same. Soon most of the cells in the
population will have the gene.
Identifying Mutants
1. Positive (direct) selection e.g., penicillin resistant mutants can be identified by
exposure to penicillin
2. Negative (indirect) selection; detects mutant cells because they do not grow.
Auxotroph is a mutant organism or cell that requires growth supplements that could
normally be synthesized by wild-type strains.
Replica Plating
Replica plating is a very effective means of isolating mutants that require one or more
new growth factors.
Replica Plating
Identifying Chemical carcinogens
Ames Test:
The Ames test yields a number of growing bacterial colonies which is a measure
of the mutagenic activity (potency) of a treatment chemical.
This value is often expressed as the number of revertants per microgram of a pure
chemical (mutagen) or per gram of food containing that mutagen.
About 909 of the substances found by the Ames test to be mutagenic have also
been shown to be carcinogenic in animals.
Ames Test