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