BT1305-Genetic Engineering

Unit I: Basics of Recombinant DNA Technology

Role of genes within cell, genetic elements that control gene expression, restriction and
modifying enzymes, safety guidelines of recombinant DNA research.
Role of genes within cell genetic elements that control gene expression
Occasionally technical developments in science occur that enable leaps forward in our
knowledge and increase the potential for innovation. Molecular biology and biomedical research
experienced such a revolutionary change in the mid-70s with the development of gene
manipulation. Although the initial experiments generated much excitement, it is unlikely that any
of the early workers in the field could have predicted the breadth of applications to which the
technique has been put. Nor could they have envisaged that the methods they developed would
spawn an entire industry comprising several hundred companies, of varying sizes, in the USA
alone.
The term gene manipulation can be applied to a variety of sophisticated in vivo genetics
as well as to in vitro techniques. In fact, in most Western countries there is a precise legal
definition of gene manipulation as a result of government legislation to control it. In the UK,
gene manipulation is defined as the formation of new combinations of heritable material by the
insertion of nucleic acid molecules, produced by whatever means outside the cell, into any virus,
bacterial plasmid or other vector system so as to allow their incorporation into a host organism in
which they do not naturally occur but in which they are capable of continued propagation.
The definitions adopted by other countries are similar and all adequately describe the
subject-matter of this book. Simply put, gene manipulation permits stretches of DNA to be
isolated from their host organism and propagated in the same or a different host, a technique
known as cloning. The ability to clone DNA has far-reaching consequences, as will be shown
below.
Cloning permits the isolation of discrete pieces of a genome and their amplification. This
in turn enables the DNA to be sequenced. Analysis of the sequences of some genetically well-
characterized genes led to the identification of the sequences and structures which characterize
the principal control elements of gene expression, e.g. promoters, ribosome binding sites, etc. As
this information built up it became possible to scan new DNA sequences and identify potential
new genes, or open reading frames, because they were bounded by characteristic motifs. Initially
this sequence analysis was done manually but to the eye long runs of nucleotides have little
meaning and patterns evade recognition. Fortunately such analyses have been facilitated by rapid
increases in the power of computers and improvements in software which have taken place
contemporaneously with advances in gene cloning. Now sequences can be scanned quickly for a
whole series of structural features, e.g. restriction enzyme recognition sites, start and stop signals
for transcription, inverted palindromes, sequence repeats, Z-DNA, etc., using programs available
on the Internet.
From the nucleotide sequence of a gene it is easy to deduce the protein sequence which it
encodes. Unfortunately, we are unable to formulate a set of general rules that allows us to predict
a protein’s three-dimensional structure from the amino acid sequence of its polypeptide chain.
However, based on crystallographic data from over 300 proteins, certain structural motifs can be
predicted. Nor does an amino acid sequence on its own give any clue to function. The solution is
to compare the amino acid sequence with that of other better-characterized proteins: a high
degree of homology suggests similarity in function. Again, computers are of great value since
algorithms exist for comparing two sequences or for comparing one sequence with a group of
other sequences simultaneously. The Internet has made such comparisons easy because
researchers can access all the protein sequence data that are stored in central databases, which are
updated daily.
Any living cell, regardless of its origin, carries out a plethora of biochemical reactions.
To analyse these different reactions, biochemists break open cells, isolate the key components of
interest and measure their levels. They purify these components and try to determine their
performance characteristics. For example, in the case of an enzyme, they might determine its
substrate specificity and kinetic parameters, such as Kmand Vmax, and identify inhibitors and
their mode of action. From these data they try to build up a picture of what happens inside the
cell. However, the properties of a purified enzyme in a test-tube may bear little resemblance to
its behaviour when it shares the cell cytoplasm or a cell compartment with thousands of other
enzymes and chemical compounds. Understanding what happens inside cells has been facilitated
by the use of mutants. These permit the determination of the consequences of altered regulation
or loss of a particular component or activity. Mutants have also been useful in elucidating
macromolecule structure and function. However, the use of mutants is limited by the fact that
with classical technologies one usually has little control over the type of mutant isolated and/or
location of the mutation.
Gene cloning provides elegant solutions to the above problems. Once isolated, entire
genes or groups of genes can be introduced back into the cell type whence they came or into
different cell types or completely new organisms, e.g. bacterial genes in plants or animals. The
levels of gene expression can be measured directly or through the use of reporter molecules and
can be modulated up or down at the whim of the experimenter. Also, specific mutations, ranging
from a single base-pair to large deletions or additions, can be built into the gene at any position
to permit all kinds of structural and functional analyses. Function in different cell types can also
be analysed, e.g. do those structural features of a protein which result in its secretion from a yeast
cell enable it to be exported from bacteria or higher eukaryotes? Experiments like these permit
comparative studies of macromolecular processes and, in some cases, gene cloning and
sequencing provides the only way to begin to understand such events as mitosis, cell division,
telomere structure, intron splicing, etc. Again, the Internet has made such comparisons easy
because researchers can access all the protein sequence data that are stored in central databases,
which are updated daily.
The original goal of sequencing was to determine the precise order of nucleotides in a
gene. Then the goal became the sequence of a small genome. First it was that of a small virus
(φX174, 5386 nucleotides). Then the goal was larger plasmid and viral genomes, then
chromosomes and microbial genomes until ultimately the complete genomes of higher
eukaryotes (humans, Arabidopsis) were sequenced (Table 1.1).

Now the sequencing of large genomes has become routine, albeit in specialist
laboratories. Having the complete genome sequence of an organism provides us with fascinating
insights into certain aspects of its biology. For example, we can determine the metabolic
capabilities of a new microbe without knowing anything about its physiology. However, there
are many aspects of cellular biology that cannot be ascertained from sequence data alone. For
example, what RNA species are made when in the cell or organism life cycle and how fast do
they turn over? What proteins are made when and how do the different proteins in a cell interact?
How does environment affect gene expression? The answers to these questions are being
provided by the new disciplines of genomics, proteomics and environomics which rely heavily
on the techniques of gene manipulation, which are discussed in later chapters. A detailed
presentation of whole-genome sequencing, genomics and proteomics can be found in Primrose
and Twyman (2002).
The developments in gene manipulation that have taken place in the last 25 years have
revolutionized the study of biology. There is no subject area within biology where recombinant
DNA is not being used and as a result the old divisions between subject areas such as botany,
genetics, zoology, biochemistry, etc. are fast breaking down. Nowhere has the impact of
recombinant DNA technology been greater than on the practice of medicine.
 The first medical benefit to arise from recombinant DNA technology was the availability
of significant quantities of therapeutic proteins, such as human growth hormone (HGH). This
protein is used to treat adolescents suffering from pituitary dwarfism to enable them to achieve a
normal height. Originally HGH was purified from pituitary glands removed from cadavers.
However, a very large number of pituitary glands are required to produce sufficient HGH to treat
just one child. Furthermore, some children treated with pituitary-derived HGH have developed
Creutzfeld–Jakob syndrome. Following the cloning and expression of the HGH gene in
Escherichia coli, it is possible to produce enough HGH in a 10 litre fermenter to treat hundreds
of children. Since then, many different therapeutic proteins have become available for the first
time. Many of these proteins are also manufactured in E. coli but others are made in yeast or
animal cells and some in plants or the milk of animals. The only common factor is that the
relevant gene has been cloned and overexpressed using the techniques of gene manipulation.
Medicine has benefited from recombinant DNA technology in other ways (Fig. 1.1). New
routes to vaccines have been developed. The current hepatitis B vaccine is based on the
expression of a viral antigen on the surface of yeast cells and a recombinant vaccine has been
used to eliminate rabies from foxes in a large part of Europe. Gene manipulation can also be used
to increase the levels of small molecules within microbial cells. This can be done by cloning all
the genes for a particular biosynthetic pathway and overexpressing them. Alternatively, it is
possible to shut down particular metabolic pathways and thus redirect particular intermediates
towards the desired end-product. This approach has been used to facilitate production of chiral
intermediates and antibiotics. Novel antibiotics can also be created by mixing and matching
genes from organisms producing different but related molecules in a technique known as
combinatorial biosynthesis.

The impact of gene manipulation on the practice of medicine.

 Gene cloning enables nucleic acid probes to be produced readily and such probes have
many uses in medicine. For example, they can be used to determine or confirm the identity of a
microbial pathogen or to diagnose pre- or perinatally an inherited genetic disease. Increasingly,
probes are being used to determine the likelihood of adverse reactions to drugs or to select the
best class of drug to treat a particular illness (pharmacogenomics). A variant of this technique is
to use cloned cytochrome P450s to determine how a new drug will be metabolized and if any
potentially toxic by-products will result.
Nucleic acids are also being used as therapeutic entities in their own right. For example,
Antisense nucleic acids are being used to down-regulate gene expression in certain diseases. In
other cases, nucleic acids are being administered to correct or repair inherited gene defects (gene
therapy/gene repair) or as vaccines. In the reverse of gene repair, animals are being generated
that have mutations identical to those found in human disease. Note that the use of antisense
nucleic acids and gene therapy/repair depends on the availability of information on the exact
cause of a disease. For most medical conditions such information is lacking and currently
available drugs are used to treat symptoms. This situation will change significantly in the next
decade.
The early successes in overproducing mammalian proteins in E. coli suggested to a few
entrepreneurial individuals that a new company should be formed to exploit the potential of
recombinant DNA technology. Thus was Genentech born. Since then thousands of
biotechnology companies have been formed worldwide. As soon as major new developments in
the science of gene manipulation are reported, a rash of new companies are formed to
commercialize the new technology. For example, many recently formed companies are hoping
the data from the Human Genome Sequencing Project will result in the identification of a large
number of new proteins with potential for human therapy. Others are using gene manipulation to
understand the regulation of transcription of particular genes, arguing that it would make better
therapeutic sense to modulate the process with low-molecular-weight, orally active drugs.
Although there are thousands of biotechnology companies, fewer than 100 have sales of
their products and even fewer are profitable. Already many biotechnology companies have
failed, but the technology advances at such a rate that there is no shortage of new company start-
ups to take their place. One group of biotechnology companies that has prospered is those
supplying specialist reagents to laboratory workers engaged in gene manipulation. In the very
beginning, researchers had to make their own restriction enzymes and this restricted the
technology to those with protein chemistry skills. Soon a number of companies were formed
which catered to the needs of researchers by supplying high-quality enzymes for DNA
manipulation. Despite the availability of these enzymes, many people had great difficulty in
cloning DNA. The reason for this was the need for careful quality control of all the components
used in the preparation of reagents, something researchers are not good at! The supply
companies responded by making easy-to-use cloning kits in addition to enzymes. Today, these
supply companies can provide almost everything that is needed to clone, express and analyse
DNA and have thereby accelerated the use of recombinant DNA technology in all biological
disciplines. In the early days of recombinant DNA technology, the development of methodology
was an end in itself for many academic researchers. This is no longer true. The researchers have
gone back to using the tools to further our knowledge of biology, and the development of new
methodologies has largely fallen to the supply companies.
E. coli has always been a popular model system for molecular geneticists. Prior to the
development of recombinant DNA technology, there existed a large number of well-
characterized mutants, gene regulation was understood and there was a ready availability of a
wide selection of plasmids. Compared with other microbial systems it was matchless. It is not
surprising, therefore, that the first cloning experiments were undertaken in E. coli. Subsequently,
cloning techniques were extended to a range of other microorganisms, such as Bacillus subtilis,
Pseudomonas sp., yeasts and filamentous fungi, and then to higher eukaryotes. Curiously,
cloning in E. coli is technically easier than in any other organism. As a result, it is rare for
researchers to clone DNA directly in other organisms. Rather, DNA from the organism of choice
is first manipulated in E. coli and subsequently transferred back to the original host. Without the
ability to clone and manipulate DNA in E. coli, the application of recombinant DNA technology
to other organisms would be greatly hindered.
Restriction and modifying enzymes
Cutting DNA molecules
Before 1970 there was no method of cleaving DNA at discrete points. All the available
methods for fragmenting DNA were non-specific. The available endonucleases had little site
specificity and chemical methods produced very small fragments of DNA. The only method
where any degree of control could be exercised was the use of mechanical shearing. The long,
thin threads which constitute duplex DNA molecules are sufficiently rigid to be very easily
broken by shear forces in solution. Intense sonication with ultrasound can reduce the length to
about 300 nucleotide pairs. More controlled shearing can be achieved by high-speed stirring in a
blender. Typically, high-molecular-weight DNA is sheared to a population of molecules with a
mean size of about 8 kb by stirring at 1500 rev/min for 30 min. Breakage occurs essentially at
random with respect to DNA sequence. The termini consist of short, single-stranded regions
which may have to be taken into account in subsequent joining procedures.
During the 1960s, phage biologists elucidated the biochemical basis of the phenomenon
of host restriction and modification. The culmination of this work was the purification of the
restriction endonuclease of Escherichia coli K12 by Meselson and Yuan (1968). Since this
endonuclease cuts unmodified DNA into large discrete fragments, it was reasoned that it must
recognize a target sequence. This in turn raised the prospect of controlled manipulation of DNA.
Unfortunately, the K12 endonuclease turned out to be perverse in its properties. While the
enzyme does bind to a defined recognition sequence, cleavage occurs at a ‘random’ site several
kilobases away. The much sought-after breakthrough finally came in 1970 with the discovery in
Haemophilus influenzae of an enzyme that behaves more simply. That is, the enzyme recognizes
a particular target sequence in a duplex DNA molecule and breaks the polynucleotide chain
within that sequence to give rise to discrete fragments of defined length and sequence.
The presence of restriction and modification systems is a double-edged sword. On the
one hand, they provide a rich source of useful enzymes for DNA manipulation. On the other,
these systems can significantly affect the recovery of recombinant DNA in cloning hosts. For
this reason, some knowledge of restriction and modification is essential.
Host-controlled restriction and modification
Restriction systems allow bacteria to monitor the origin of incoming DNA and to destroy
it if it is recognized as foreign. Restriction endonucleases recognize specific sequences in the
incoming DNA and cleave the DNA into fragments, either at specific sites or more randomly.
When the incoming DNA is a bacteriophage genome, the effect is to reduce the efficiency of
plating, i.e. to reduce the number of plaques formed in plating tests. The phenomena of
restriction and modification were well illustrated and studied by the behaviour of phage λ on two
E. coli host strains.
If a stock preparation of phage λ, for example, is made by growth upon E. coli strain C
and this stock is then titred upon E. coli C and E. coli K, the titres observed on these two strains
will differ by several orders of magnitude, the titre on E. coli K being the lower. The phage are
said to be restricted by the second host strain (E. coli K). When those phage that do result from
the infection of E. coli K are now replated on E. coli K they are no longer restricted; but if they
are first cycled through E. coli C they are once again restricted when plated upon E. coli K. Thus
the efficiency with which phage λ plates upon a particular host strain depends upon the strain on
which it was last propagated. This non-heritable change conferred upon the phage by the second
host strain (E. coli K) that allows it to be replated on that strain without further restriction is
called modification.
Host-controlled restriction and modification of phage λ in E. coli strain K, analysed by efficiency
of plating (EOP). Phage propagated by growth on strains K or C (i.e. λ.K or λ.C) have EOPs on
the two strains, as indicated by arrows.
The restricted phages adsorb to restrictive hosts and inject their DNA normally. When the
phage are labelled with 32P, it is apparent that their DNA is degraded soon after injection and the
endonuclease that is primarily responsible for this degradation is called a restriction
endonuclease or restriction enzyme. The restrictive host must, of course, protect its own DNA
from the potentially lethal effects of the restriction endonuclease and so its DNA must be
appropriately modified. Modification involves methylation of certain bases at a very limited
number of sequences within DNA, which constitute the recognition sequences for the restriction
endonuclease. This explains why phage that survive one cycle of growth upon the restrictive host
can subsequently reinfect that host efficiently; their DNA has been replicated in the presence of
the modifying methylase and so it, like the host DNA, becomes methylated and protected from
the restriction system. Although phage infection has been chosen as our example to illustrate
restriction and modification, these processes can occur whenever DNA is transferred from one
bacterial strain to another.
Types of restriction and modification (R-M) system
At least four different kinds of R-M system are known: type I, type II, type III and type
IIs. The essential differences between them are summarized in Table.
 The type I systems were the first to be characterized and a typical example is that from E.
coli K12. The active enzyme consists of two restriction subunits, two modification (methylation)
subunits and one recognition subunit. These subunits are the products of the hsdR, hsdM and
hsdS genes. The methylation and cutting reactions both require ATP and S-adenosylmethionine
as cofactors. The recognition sequences are quite long with no recognizable features such as
symmetry. The enzyme also cuts unmodified DNA at some distance from the recognition
sequence. However, because the methylation reaction is performed by the same enzyme which
mediates cleavage, the target DNA may be modified before it is cut. These features mean that
type I systems are of little value for gene manipulation. However, their presence in E. coli strains
can affect recovery of recombinants. Type III enzymes have symmetrical recognition sequences
but otherwise resemble type I systems and are of little value.
Most of the useful R-M systems are of type II. They have a number of advantages over
type I and III systems. First, restriction and modification are mediated by separate enzymes so it
is possible to cleave DNA in the absence of modification. Secondly, the restriction activities do
not require cofactors such as ATP or S-adenosylmethionine, making them easier to use. Most
important of all, type II enzymes recognize a defined, usually symmetrical, sequence and cut
within it. Many of them also make a staggered break in the DNA and the usefulness of this will
become apparent. Although type IIs systems have similar cofactors and macromolecular
structure to those of type II systems, the fact that restriction occurs at a distance from the
recognition site limits their usefulness.
The classification of R-M systems into types I to III is convenient but may require
modification as new discoveries are made. For example, the Eco571 system comprises a single
polypeptide which has both restriction and modification activities. Other restriction systems are
known which fall outside the above classification. Examples include the mcr and mrr systems
and homing endonucleases. The latter are doublestranded DNases derived from introns or
inteins. They have large, asymmetric recognition sequences and, unlike standard restriction
endonucleases, tolerate some sequence degeneracy within their recognition sequence.
Nomenclature
The discovery of a large number of restriction and modification systems called for a
uniform system of nomenclature. A suitable system was proposed by Smith and Nathans (1973)
and a simplified version of this is in use today. The key features are:
 The species name of the host organism is identi-fied by the first letter of the genus name
and the first two letters of the specific epithet to generate a threeletter abbreviation. This
abbreviation is always written in italics.
 Where a particular strain has been the source then this is identified.
 When a particular host strain has several different. R-M systems, these are identified by
roman numerals.
Homing endonucleases are named in a similar fashion except that intron-encoded
endonucleases are given the prefix ‘I-’ (e.g. I-CeuI) and intein endonucleases have the prefix
‘PI-’ (e.g. Pl-PspI). Where it is necessary to distinguish between the restriction and methylating
activities, they are given the prefixes ‘R’ and ‘M’, respectively, e.g. R.SmaI and M.SmaI.

Recognition sequences
Most, but not all, type II restriction endonucleases recognize and cleave DNA within
particular sequences of four to eight nucleotides which have a twofold axis of rotational
symmetry. Such sequences are often referred to as palindromes because of their similarity to
words that read the same backwards as forwards. For example, the restriction and modification
enzymes R.EcoRI and M.EcoRI recognize the sequence:
5′-GAA T TC-3′
3′-CT T AAG-5′
Axis of symmetry
The position at which the restricting enzyme cuts is usually shown by the symbol ‘/’ and
the nucleotides methylated by the modification enzyme are usually marked with an asterisk. For
EcoRI these would be represented thus:
5′-G/AA* T T C-3′
3′-C TT A*A/G-5′
 For convenience it is usual practice to simplify the description of recognition sequences
by showing only one strand of DNA, that which runs in the 5′ to 3′ direction. Thus the EcoRI
recognition sequence would be shown as G/AATTC.
From the information shown above we can see that EcoRI makes single-stranded breaks
four bases apart in the opposite strands of its target sequence so generating fragments with
protruding 5′ termini:
5′-G 5′-AATTC-3′
3′-CTTAA-5′ G-5′
These DNA fragments can associate by hydrogen bonding between overlapping 5′
termini, or the fragments can circularize by intramolecular reaction (Fig. 3.2). For this reason the
fragments are said to have sticky or cohesive ends. In principle, DNA fragments from diverse
sources can be joined by means of the cohesive ends and, as we shall see later, the nicks in the
molecules can be sealed to form an intact artificially recombinant DNA molecule.
Not all type II enzymes cleave their target sites like EcoRI. Some, such as PstI
(CTGCA/G), produce fragments bearing 3′ overhangs, while others, such as SmaI (CCC/GGG),
produce blunt or flush ends.
To date, over 10 000 microbes from around the world have been screened for restriction
enzymes. From these, over 3000 enzymes have been found representing approximately 200
different sequence specificities. Some representative examples are shown in Table. For a
comprehensive database of information on restriction endonucleases and their associated
methylases, including cleavage sites.
 Occasionally enzymes with novel DNA sequence specificities are found but most prove
to have the same specificity as enzymes already known. Restriction enzymes with the same
sequence specificity and cut site are known as isoschizomers. Enzymes that recognize the same
sequence but cleave at different points, for example SmaI (CCC/GGG) and XmaI C/CCGGG),
are sometimes known as neoschizomers.
Under extreme conditions, such as elevated pH or low ionic strength, restriction
endonucleases are capable of cleaving sequences which are similar but not identical to their
defined recognition sequence. This altered specificity is known as star activity. The most
common types of altered activity are acceptance of base substitutions and truncation of the
number of bases in the recognition sequence. For example, EcoRI* (EcoRI star activity) cleaves
the sequence N/AATTN, where N is any base, whereas EcoRI cleaves the sequence GAATTC.
Number and size of restriction fragments
The number and size of the fragments generated by a restriction enzyme depend on the
frequency of occurrence of the target site in the DNA to be cut. Assuming a DNA molecule with
a 50% G+C content and a random distribution of the four bases, a fourbase recognition site
occurs every 44 (256) bp. Similarly, a six-base recognition site occurs every 4 6 (4096) bp and an
eight-base recognition sequence every 48 (65 536) bp. In practice, there is not a random
distribution of the four bases and many organisms can be AT- or GC-rich, e.g. the nuclear
genome of mammals is 40% G+C and the dinucleotide CG fivefold less common than
statistically expected. Similarly, CCG and CGG are the rarest trinucleotides in most A+T-rich
bacterial genomes and CTAG is the rarest tetranucleotide in G+C-rich bacterial genomes. Thus
different restriction endonucleases with six-base recognition sites can produce average fragment
sizes significantly different from the expected 4096 bp.

Certain restriction endonucleases show preferential cleavage of some sites in the same
DNA molecule. For example, phage λ DNA has five sites for EcoRI but the different sites are
cleaved nonrandomly (Thomas & Davis 1975). The site nearest the right terminus is cleaved 10
times faster than the sites in the middle of the molecule. There are four sites for SacII in λ DNA
but the three sites in the centre of the molecule are cleaved 50 times faster than the remaining
site. There is a group of three restriction enzymes which show an even more dramatic site
preference. These are NarI, NaeI and SacII and they require simultaneous interaction with two
copies of their recognition sequence before they will cleave DNA. Thus NarI will rapidly cleave
two of the four recognition sites on plasmid pBR322 DNA but will seldom cleave the remaining
two sites.
Safety guidelines of recombinant DNA research.
The Indian Recombinant DNA Safety Guidelines and Regulations
Contents of the Indian Recombinant DNA Safety Guidelines and Regulations:
1. INTRODUCTION
The new capabilities to manipulate the genetic material present tremendous potential and
find use in many novel experiments and applications. These developments have generated a
sense of concern among scientists working in biological areas and others to find ways as to how
safely the research in the field should be carried out and also possible means to regulate the work
involving pathogenic microorganisms and virulent genes. With the safety considerations in view,
the Department of Biotechnology is mandated to evolve the Recombinant DNA Safety
Guidelines. The Department has set up the Recombinant DNA Advisory Committee (RDAC) for
this purpose. On the basis of current scientific information a document on Recombinant DNA
Safety Guidelines has been brought out on the use of this technique in the area of research,
manufacture and applications. The booklet brings forth the salient features in the document as a
guide towards observance of the guidelines in research and also to meet the regulatory
requirements by those in production, testing and use of genetically modified organisms and
products.
2. RESEARCH
The guidelines cover areas of research involving
i) Genetically engineered organisms,
ii) Genetic transformation of green plants, animals,
iii) rDNA technology in vaccine development and
iv) Large scale production and deliberate/accidental release of organisms, plants, animals &
products derived by rDNA technology. The guidelines prescribe 4 levels of risk, while carrying
out experiments with microorganisms. Classification of organisms within these levels is based on
pathogenicity, local prevalence of disease and epidemic causing strains in India.
3. NOTIFICATION
Related to the incremental risk involved in the use of microorganisms, plants, animals in
experiments, the corresponding biosafety practices are required. Accordingly the notification
procedures are defined under 3 categories.
i) Exempt category-(self cloning experiments)
ii) Intimation to competent authority-(e.g. experiments involving nonpathogenic DNA vector
systems)
iii) Review and approval of competent authority-(e.g. Toxin gene cloning, antibiotic resistant
genes, etc.)
4. CONTAINMENT
Containment facilities are recommended for necessary safeguards while carrying out
experiments involving organisms belonging to 4 different biosafety levels.
a) PHYSICAL CONTAINMENT
Physical containment is to limit the spread of dangerous microorganisms by way of
1) Technique (Good Laboratory Practice)
ii) Safety equipments
iii) Laboratory design and facilities
b) BIOLOGICAL CONTAINMENT
Biological containment involves the use of the combination of vector and host in such a
way so that it
i) can limit the infectivity of vector to specific hosts and
ii) control host vector survival in the environment
The growth of whole plants will however require special environmental conditions which may be
achieved by using glasshouse containment.
Glasshouse containment A is appropriate to plant experiments involving no plant pathogen and
would be suitable for experiments involving non- pathogen DNA vector systems and
regeneration from single cells. (Notification to competent authority is needed).
Glashouse containment B is recommended for experiments involving:
i) genetically manipulated plant pathogens including plant viruses such as the propagation of
genetically manipulated organisms in plant, and
ii) the growth of plants regenerated from cells transformed by genetically manipulated pathogen
vector systems which still contain the pathogen. (Approval of competent authority is required
before commencement of activity).
5. RECOGNITION OF FACILITY
Application for recognition of research facility to carry out genetic manipulation should
be made to the Department of Environment before the commencement of work in the prescribed
proforma as per 7(2) of the rules on hazardous microorganisms/genetically engineered organisms
notified under the EPA 1986.
6. COMPETENT AUTHORITY
a) INSTITUTIONAL BIOSAFETY COMMITTEE (IBSC) (IN R&D CENTRES,
UNIVERSITY ETC.)
i) The IBSC shall be the nodal point for interaction within the institution for implementation of
the guidelines. As such, in the first instance it is necessary that the institutions intending to carry
out research activities involving genetic manipulation of microorganisms, plants or animals
should constitute the IBSC. Any research project which is likely to have biohazard potential (as
envisaged in the guidelines) during the execution stage or which involve the production of either
microorganisms or biologically active molecules that might cause biohazard should be notified to
IBSC. The on-site emergency plan should be prepared by the institution or occupier for each of
the above activities with the help of IBSC. IBSC will allow genetic engineering activity on
classified organisms only at places where such work should be performed as per guidelines. The
prescribed form-Notification of intention to carry on genetic manipulation-Part B should be
submitted to funding agency with comments of IBSC for financial support on experiments
falling under categories II, III & IV. Support on approved projects will be withdrawn in case of
deliberate violation or avoidable negligence of RDNA guidelines. In addition, it may attract
action under EPA.
ii) IBSC will provide half yearly reports on the on-going projects to RCGM.
iii) Authorisation for inter-state exchange of etiologic agents, diagnostic specimens and
biological products will be done by IBSC on standard proforma evolved for this purpose.
iv) Manipulation of plants under containment would be performed under the regulatory clearance
of IBSC. Development of organisms for agricultural and environmental applications should be
conducted in a step-wise fashion, moving where appropriate from the laboratory to the growth
chamber and green-house under containment conditions and good laboratory practice.
b) REVIEW COMMITTEE ON GENETIC MANIPULATION (RCGM) The RCGM will
have the function:
i) To review the reports in all approved ongoing research projects involving high risk category
and controlled field experiments.
ii) To visit site of experimental facilities periodically where projects with biohazard potential are
being pursued and also at a time prior to the commencement of the activity to ensure that
adequate safety measures are taken as per the guidelines.
iii) To issue authorisation for import and receipt of etiologic agents and vectors, germ plasms,
organelle, etc. needed for experimental work/training and research. (Applications should be
made in prescribed proforma).
7. LARGE SCALE EXPERIMENTS AND MANUFACTURE
 Regulations of large scale production and field testing of engineered organisms and
products including environmental release has been laid down under statutory provisions of
Environmental Protection Act 1986.
Experiments beyond 20 litres capacity for research as well as industrial purposes are
included in the category of large scale experimentation/operations. For such activities it is
recommended that one should seek approval of the competent authority (GEAC) on furnishing
the relevant details in a prescribed format (To be issued by GEAC with Department of
Environment). The following safety criteria are to be complied:
i) The host organism should not be a pathogen, should have extended history of safe use and
built in environmental limitations.
ii) The vector/insert should be well characterised and free from known harmful sequences, baited
in size as much as possible (with insert less than 10 base pair or below 30,000 M.W.) should be
poorly mobilisable, should not transfer any resistance markers. In cases, where the insert
sequence exceeds the above limit, toxicity screening should be made. The constructs should have
3 markers for bioprocess monitoring.
iii) The genetically manipulated organism should not be a pathogen. For large scale operations,
measures such as proper engineering for containment, quality control, personnel protection,
medical surveillance are recommended. Offsite contingency plans in event of unanticipated
effects of novel organisms/products on accidental release are to be worked out and appropriate
control measures are to be developed in consultation with the competent authority (State and
District level coordination committees of GEAC) to meet any exigency.
8. BIOLOGICALS PRODUCED BY rDNA TECHNOLOGY
The general regulations normally applicable for biologicals are relevant to the
recombinant DNA products. The specific relevant aspects to a particular product should be
discussed with the appropriate Govt. agency (Drugs controller) on a case by case basis.
i) A new licence for a product drug application would be required on products made of
recombinant DNA technology even if the product is considered to be chemically and physically
similar to the naturally occurring substance or previously approved product produced in
conventional system.
ii) A recombinant DNA product demonstrated to be identical to normally occurring substance
would not require toxicological and pharmacological data if the information is already available
at the dose levels intended for specific use. Otherwise the data on the above would be necessary
on such products.
9. RELEASE TO THE ENVIRONMENT & FIELD EXPERIMENTS
i) Depending on the type of organism handled & the assessment of potential risks involved
appropriate containment facilities must be provided to ensure safety and to prevent unwanted
release in the environment.
ii) It is important to evaluate rDNA modified organism for potential risk, prior to application in
agriculture and environment. Prior to introduction of microorganisms, properties of the
organisms, the possible interaction with other disease causing agents and the infected wild plant
species should be evaluated. An independent review of potential risks should be conducted on a
case by case basis to application.
iii) Pre-release tests of genetically engineered organisms in Agricultural applications should
include elucidation of genetic markers, host range, requirements for vegatative growth,
persistence and stability in small plots and experimental field trials for 2 year. Soil samples in
experiments under controlled containment conditions should be tested for the absence of viable
cells before disposal into the environment.
iv) Biowastes resulting from laboratory experiments, industrial operations should be properly
treated, so organisms are either destroyed or rendered harmless before disposal into the
environment.
10. GENETIC ENGINEERING APPROVAL COMMITTEE (GEAC)
i) Testing of genetically altered organisms, transgenic animals, plant material tested against
pathogens & products in the environment should follow regulatory guidelines seeking field use
permit from GEAC in the prescribed proforma (to be evolved by GEAC).
ii) The large scale planned release of organisms into the environment both for environmental and
agricultural applications should be done under licence. Applications to this effect should be made
to GEAC on the prescribed proforma which will be examined on case by case basis (to be
evolved by GEAC). The validity of approval is for a period of 4 years.
iii) Import for large scale use, export, manufacture, process, sell, use of any genetically
engineered substances or cells including food stuffs and additives that contains rDNA products
are subject to regulatory control by GEAC. Prior approval to this effect should be obtained on
applications in the prescribed proforma (to be evolved by GEAC).
11. A copy of the Recombinant DNA Safety Guidelines and the rules under EPA 1986 for the
manufacture, use, import and storage of hazardous microorganisms/genetically engineered
organisms or cells may be obtained on request.
1. Good Laboratory Practice
· Never do direct mouth pipetting of infectious or toxic fluids; use a pipettor.
· Plug pipettes with cotton.
· Do not blow infectious material out of pipettes.
· Do not prepare mixtures of infectious material by bubbling expiratory air through the liquid
with a pipette.
· Use an alcohol-moistened pledget around the stopper and needle when removing a syringe and
needle from a rubber stoppered vaccine bottle.
· Use only needle-locking hypodermic syringes. Avoid using syringes whenever possible.
· Expel excess fluid and bubbles from a syringe vertically into cotton pledget moistened with
disinfectant, or into a small bottle of cotton.
· Before and after infecting an animal, swab the site of injection with a disinfectant.
· Sterilize discarded pipettes and syringes in pan where they were first placed after use.
· Before centrifuging, inspect tubes for cracks. Inspect the inside of the trunnion cup for rough
walls caused by erosion or adhering matter. Carefully remove all bits of glass from the rubber
cushion. A germicidal solution added between the tube and the trunnion cup not only disinfects
the surfaces of both these, but also provides an excellent cushion against shocks that otherwise
might break the tube.
· Use centrifuge trunnion cups with screw caps or equivalent.
· Avoid decanting centrifuge tubes; if you must do so, afterwards wipe off the outer rim with a
disinfectant. Avoid filling the tube to the point that the rim ever becomes wet with culture.
· Wrap a lyophilized culture vial with disinfectant-wetted cotton before breaking. Wear gloves.
· Never leave a discarded tray of infected material unattended.
· Sterilize all contaminated discarded material.
· Periodically, clean out deep-freeze and dry-ice chests in which cultures are stored to remove
broken ampoules or tubes. Use rubber gloves and respiratory protection during the cleaning.
· Handle diagnostic Serum specimens carrying a risk of infectious hepatitis with rubber gloves.
· Develop the habit of keeping your hands away from your mouth, nose, eyes and face. This may
prevent self-inoculation.
· Avoid smoking, eating, and drinking in the laboratory.
· Make special precautionary arrangements for respiratory, oral, intranasal, and intratracheal
inoculation of infectious material.
· Give preference to operating room gowns that fasten at the back.
· Evaluate the extent to which the hands may become contaminated with some agents and
operations, forceps or rubber gloves are available.
· Wear only clean laboratory clothing in the dining room, library and other nonlaboratory areas.
· Shake broth cultures in a manner that avoids wetting the plug or cap.
2. AEROSOL MINIMIZATION
Because of their insidious nature, aerosols pose special problems in that the laboratory worker
may be unwillingly exposed. Procedures which can produce aerosols include:
· grinding
· blending
· sonicating
· resuspending packed cells or viruses
· inserting a hot loop into a culture
· centrifugation
· flaming an inoculation loop so that it splatters
· forceful ejection of fluid from a pipette or syringe
· opening a tube containing a lyophilized agent
· releasing the vaccum on a freeze dryer
· opening a tube within which the air pressure may differ from that of the room, such as may
occur when the tube is opened at a temperature different from that which it was sealed.
4. Glasshouse containment conditions for plant experiments
1. Glasshouse Containment A are: clearly marked with a biohazard sign indicating "glasshouse
containment "
i) Plants should be grown in a designated glasshouse or compartment, A
ii) Any other plants grown in the designated glasshouse or compartment must be handled under
conditions appropriate for the experimental plants.
iii) Plants should be managed by suitably trained personnel with the principles of good
glasshouse hygiene.
iv) The IBSC should consider whether any additional factors such as pest control, screening to
prevent ingress by vermin, birds and insects and destruction of surplus plants and seed are
relevant to the particular experiment.
2. Glasshouse containment B conditions will be specified by the committee (RCGM) and will
vary with the pathogen, being particulary dependant on its mode of dispersal, host range and
pathogenicity and they are to be worked out on case by case basis.
Special conditions may be needed in addition to those given under 'A' to prevent dissemination
of the genetically manipulated plant pathogen especially during transfer between glasshouse and
laboratory, during disposal of plants and equipment and through survival of pollen, seeds or
other biological vectors.
a) Need for negative pressure and air filtration double doors etc. in cases where airborne
dispersal is a potential hazard.
b) Need for effluent treatment plant where water borne dispersal is a hazard.
c) Need for suitable construction of glasshouse (floors, dwarfwalls, threshold at door etc.) in
cases where waterborne or soil borne dispersal are potential hazards.
d) Need to prevent pollination and seeding, or to contain pollen and seed in cases where pollen
and seed-borne dispersal is a potential hazard.
e) Need for measures either to prevent contamination of, or to decontaminate the clothing of
personnel or tools, pots, equipment etc., where mechanical transmissions are an above average
hazard.
f) Need to limit the growing of host plants in the vicinity of the containment facility and to
provide monitoring for escape. Inspection of a 'Glasshouse Containment B' facility by IBSC will
be required before approval.
Based on the universal code by which DNA encodes amino acids, we can make sense of
the constantly increasing amout of DNA sequence data as far as it encodes proteins. This code
was solved in 1966 and it has allowed researchers to find new genes and estimate the total
number of genes in the human genome. However, coding sequence covers only about 1.2% of
the human genome. New codes and grammatical rules need to be resolved in order to understand
the remaining 98.8% of the genome.
It is evident that genes are expressed in tightly controlled spatial and temporal patterns
but we do not know the code by which the expression is regulated. In this post-genomic era, the
next big goal is to decipher the genetic code of regulation of gene expression.
At the University of Helsinki the researchers have been interested in sequences which
regulate gene expression. The research group, led by professor Jussi Taipale, Ph.D, has defined
the binding specificities of several transcription factors. Transcription factors are DNA-binding
proteins which are required to activate gene expression. In collaboration biologists and computer
scientists designed a software called EEL (enhancer element locator) which searches genomic
sequence for regions where many transcription factors bind DNA side by side. Finding the same
region with high frequency of transcription factors in several species indicates that the DNA
element regulates gene expression. The researchers showed that the predicted regulatory
elements direct organ-specific expression of a marker gene in transgenic mice. Novel
experimental and computational methods enabled genome-wide analysis of regulatory elements
in several species.
Studying the control of gene expression is a growing area of research at the moment,
likely owing to its fundamental importance to many biological processes. The findings of the
Finnish scientists have implications to the study of cancer, evolution, development biology and
many other areas of biology. The work revealed a potential mechanism explaining why many
different genes are linked to cancer.
The researchers who contributed to the article were Outi Hallikas, Kimmo Palin, Natalia
Sinjushina, Reetta Rautiainen, Juha Partanen, Esko Ukkonen and Jussi Taipale. The work was
supported by the Academy of Finland, the BioSapiens network of excellence and the Regulatory
Genomics specific targeted research project of the European Union 6th Framework Program,
Magnus Ehrnrooth Foundation, Biocentrum Helsinki, University of Helsinki, Sigrid Juselius
Foundation and Finnish Cancer Research Organizations.
Regulation of gene expression (or gene regulation) refers to the cellular control of the amount
and timing of changes to the appearance of the functional product of a gene. Although a
functional gene product may be an RNA or a protein, the majority of known mechanisms
regulate protein coding genes. Any step of the gene's expression may be modulated, from DNA-
RNA transcription to the post-translational modification of a protein.
Gene regulation is essential for viruses, prokaryotes and eukaryotes as it increases the
versatility and adaptability of an organism by allowing the cell to express protein when needed.
The first example of gene regulation system was the lac operon, discovered by Jacques Monod,
in which protein involved in lactose metabolism are expressed by E.coli only in the presence of
lactose and absence of glucose.
Furthermore, gene regulation allows the presence in a multicellular organism of different
cells types arranged in a complex pattern, hence different transcriptomes despite them having all
the same genome and the generation of patterns by cellular differentiation and morphogenesis.
Regulated stages of gene expression
Any step of gene expression may be modulated, from the DNA-RNA transcription step to
post-translational modification of a protein. The following is a list of stages where gene
expression is regulated:
Modification of DNA
In eukaryotes, the accessibility of large regions of DNA can depend on its chromatin
structure which can be altered as a result of histone modifications which are directed by DNA
methylation, ncRNA or DNA binding protein.
Chemical
Methylation of DNA is a common method of gene silencing. DNA is typically
methylated by methyltransferase enzymes on cytosine nucleotides in a CpG dinucleotide
sequence (also called "CpG islands" when densely clustered). Analysis of the pattern of
methylation in a given region of DNA (which can be a promoter) can be achieved through a
method called bisulfite mapping. Methylated cytosine residues are unchanged by the treatment,
whereas unmethylated ones are changed to uracil. The differences are analyzed by DNA
sequencing or by methods developed to quantify SNPs, such as Pyrosequencing (Biotage) or
MassArray (Sequenom), measuring the relative amounts of C/T at the CG dinucleotide.
Abnormal methylation patterns are thought to be involved in carcinogenesis.
Structural
Transcription of DNA is dictated by its structure. In general, the density of its packing is
indicative of the frequency of transcription. Octameric protein complexes called histones are
responsible for the amount of supercoiling of DNA, and these complexes can be temporarily
modified by processes such as phosphorylation or more permanently modified by processes such
as methylation. Such modifications are considered to be responsible for more or less permanent
changes in gene expression levels.
Histone acetylation is also an important process in transcription. Histone
acetyltransferase enzymes (HATs) such as CREB-binding protein also dissociate the DNA from
the histone complex, allowing transcription to proceed. Often, DNA methylation and histone
acetylation work together in gene silencing. The combination of the two seems to be a signal for
DNA to be packed more densely, lowering gene expression.
Regulation of transcription
Regulation of transcription controls when transcription occurs and how much RNA is
created. Transcription of a gene by RNA polymerase can be regulated by at least five
mechanisms:
Specificity factors alter the specificity of RNA polymerase for a given prom Posttranslational
modification (PTM) is the chemical modification of a protein after its translation. It is one of the
later steps in protein biosynthesis for many proteins.

The bottom of this diagram shows the modification of primary structure of insulin, as described.
 A protein (also called a polypeptide) is a chain of amino acids. During protein synthesis,
20 different amino acids can be incorporated in proteins. After translation, the posttranslational
modification of amino acids extends the range of functions of the protein by attaching to it other
biochemical functional groups such as acetate, phosphate, various lipids and carbohydrates, by
changing the chemical nature of an amino acid (e.g. citrullination) or by making structural
changes, like the formation of disulfide bridges.
Also, enzymes may remove amino acids from the amino end of the protein, or cut the
peptide chain in the middle. For instance, the peptide hormone insulin is cut twice after disulfide
bonds are formed, and a propeptide is removed from the middle of the chain; the resulting
protein consists of two polypeptide chains connected by disulfide bonds.
Other modifications, like phosphorylation, are part of common mechanisms for controlling the
behavior of a protein, for instance activating or inactivating an enzyme.
PTMs involving addition of functional groups

The genetic code diagram showing the amino acid residues as target of modification.
PTMs involving addition include:
 acylation
o acetylation, the addition of an acetyl group, usually at the N-terminus of the protein
 alkylation, the addition of an alkyl group (e.g. methyl, ethyl)
o methylation the addition of a methyl group, usually at lysine or arginine residues. (This is a
type of alkylation.)
o demethylation
 amidation at C-terminus
 biotinylation, acylation of conserved lysine residues with a biotin appendage
 formylation
 gamma-carboxylation dependent on Vitamin K[2]
 glutamylation, covalent linkage of glutamic acid residues to tubulin and some other proteins.
(See tubulin polyglutamylase)
 glycosylation, the addition of a glycosyl group to either asparagine, hydroxylysine,
serine, or threonine, resulting in a glycoprotein. Distinct from glycation, which is regarded as a
nonenzymatic attachment of sugars.
 glycylation, covalent linkage of one to more than 40 glycine residues to the tubulin C-
terminal tail
 heme moiety may be covalently attached
 hydroxylation
 iodination (e.g. of thyroid hormones)
 isoprenylation, the addition of an isoprenoid group (e.g. farnesol and geranylgeraniol)
 lipoylation, attachment of a lipoate functionality
o prenylation
o GPI anchor formation
 myristoylation
 farnesylation
 geranylgeranylation
 nucleotides or derivatives thereof may be covalently attached
 ADP-ribosylation
 flavin attachment
 oxidation
 palmitoylation
 pegylation
 phosphatidylinositol may be covalently attached
 phosphopantetheinylation, the addition of a 4'-phosphopantetheinyl moiety from coenzyme
A, as in fatty acid, polyketide, non-ribosomal peptide and leucine biosynthesis
 phosphorylation, the addition of a phosphate group, usually to serine, tyrosine, threonine or
histidine
 polysialylation, addition of polysialic acid, PSA to NCAM
 pyroglutamate formation
 racemization of proline by prolyl isomerase
 tRNA-mediation addition of amino acids such as arginylation
 sulfation, the addition of a sulfate group to a tyrosine.
 selenoylation (co-translational incorporation of selenium in selenoproteins)
 sulfation
PTMs involving addition of other proteins or peptides
 ISGylation, the covalent linkage to the ISG15 protein (Interferon-Stimulated Gene 15)
 SUMOylation, the covalent linkage to the SUMO protein (Small Ubiquitin-related MOdifier)
 ubiquitination, the covalent linkage to the protein ubiquitin.
PTMs involving changing the chemical nature of amino acids
 citrullination, or deimination the conversion of arginine to citrulline
 deamidation, the conversion of glutamine to glutamic acid or asparagine to aspartic acid
PTMs involving structural changes
 disulfide bridges, the covalent linkage of two cysteine amino acids
 proteolytic cleavage, cleavage of a protein at a peptide bond
Case examples
 cleavage and formation of disulfide bridges during the production of insulin
 PTM of histones as regulation of transcription: RNA polymerase control by chromatin
structure
 PTM of RNA polymerase II as regulation of transcription: RNA polymerase II

External links
 deltaMasses: Differential PTM Detection after mass spectrometry
 Auto Motif Server: A Computational Protocol for Identification of Post-Translational
Modifications in Protein Sequences
Functional analyses for site-specific phosphorylation of a target protein in cells oter or set
of promoters, making it more or less likely to bind to them (i.e. sigma factors used in prokaryotic
transcription).
Repressors bind to non-coding sequences on the DNA strand that are close to or overlapping the
promoter region, impeding RNA polymerase's progress along the strand, thus impeding the
expression of the gene.
General transcription factors These transcription factors position RNA polymerase at the start
of a protein-coding sequence and then release the polymerase to transcribe the mRNA.
Activators enhance the interaction between RNA polymerase and a particular promoter,
encouraging the expression of the gene. Activators do this by increasing the attraction of RNA
polymerase for the promoter, through interactions with subunits of the RNA polymerase or
indirectly by changing the structure of the DNA.
Enhancers are sites on the DNA helix that are bound to by activators in order to loop the DNA
bringing a specific promoter to the initiation complex.

Posttranscriptional Regulation
 After the DNA is transcribed and mRNA is formed there must be some sort of regulation
on how much the mRNA is translated into Proteins. Cells do this by modulating the Capping,
Splicing, addition of a Poly(A) Tail, the sequence-specific nuclear export rates and in several
contexts sequestration of the RNA transcript. These processes occur in eukaryotes but not in
prokaryotes. This modulation is a result of a protein or transcript which in turn is regulated and
may have an affinity for certain sequences.
Capping changes the five prime end of the mRNA to a three prime end by 5'-5' linkage, which
protects the mRNA from 5' exonuclease, which degrades foreign RNA. The cap also helps in
ribosomal binding.
Splicing removes the introns, noncoding regions that are transcribed into RNA, in order to make
the mRNA able to create proteins. Cells do this by spliceosome's binding on either side of an
intron, looping the intron into a circle and then cleaving it off. The two ends of the exons are
then joined together.
Addition of poly(A) tail otherwise known as poly-adenylation. Junk RNA is added to the 3' end,
and acts as a buffer to the 3' exonuclease in order to increase the half life of mRNA.
In both prokaryotes and eukaryotes a large number of RNA binding protein exist, with often are
directed to their target sequence by the secondary structure of the transcript, which may change
depending on certain conditions, such as temperature or presence of a ligand (aptamer), some
transcripts act as ribozymes and self-regulate their expression.
Examples of gene regulation
Enzyme induction is a process in which a molecule (e.g. a drug) induces (i.e. initiates or
enhances) the expression of an enzyme.
The induction of heat shock proteins in the fruit fly Drosophila melanogaster.
The Lac operon is an interesting example of how gene expression can be regulated. Viruses
despite having only a few genes, possess mechanisms to regulate their gene expression, typically
into a early and late phase, using collinear systems regulated by anti-terminators (lambda phage)
or splicing modulators (HIV)
Circuitry Up-regulation and down-regulation
Up-regulation is a process which occurs within a cell triggered by a signal (originating internal
or external to the cell) which results in increased expression of one or more genes and as a result
the protein(s) encoded by those genes. Conversely down-regulation is a process resulting in
decreased gene and corresponding protein expression.
Up-regulation occurs for example when a cell is deficient in some kind of receptor. In
this case, more receptor protein is synthesized and transported to the membrane of the cell and
thus the sensitivity of the cell is brought back to normal reestablishing homeostasis.
Down-regulation occurs for example when a cell is overly stimulated by a
neurotransmitter, hormone, or drug for a prolonged period of time and the expression of the
receptor protein is decreased in order to protect the cell (see also tachyphylaxis).
Inducible vs. repressible systems
Gene Regulation can be summarized as how they respond:
Inducible systems - An inducible system is off unless there is the presence of some molecule
(called an inducer) that allows for gene expression. The molecule is said to "induce expression".
The manner in which this happens is dependent on the control mechanisms as well as differences
between prokaryotic and eukaryotic cells.
Repressible systems - A repressible system is on except in the presence of some molecule
(called a corepressor) that suppresses gene expression. The molecule is said to "repress
expression". The manner in which this happens is dependent on the control mechanisms as well
as differences between prokaryotic and eukaryotic cells.
Developmental biology
A large number of studied regulatory systems come from developmental biology.
Examples include:
The collinearity of the Hox gene cluster with their nested antero-posterior patterning. It
has been speculated that pattern generation of the hand (digits - interdigits) The gradient of Sonic
hedgehog (secreted inducing factor) from the zone of polarizing activity in the limb which
creates a gradient of active Gli3 which activates Gremlin which inhibits BMPs also secreted in
the limb resulting in the formation of an alternating pattern of activity as a result of this reaction-
diffusion system.
Somatogenesis is the creation of segmatation (somites) form a uniform tissue (PSM)
sequentially from anterior to posterior, this is a achieved in amniotes possibly by means of two
opposing gradients, Retinoic acid in the anterior (wavefront) and an oscillating gradient in the
posterior (clock) composed of FGF + Notch and Wnt in antiphase.
 Sex determination in the soma of a Drosophila requires the sensing of the ratio of
autosomal genes to sex chromosome encoded genes, which results in the production of sexless
splicing factor in females resulting in the female isoform of doublesex.
Theoretical circuits
Repressor/Inducer: a activation of a sensor results in the change of expression of a gene
negative feedback: the gene product downregulates its own production directly or indirectly,
which can result in keeping transcript levels constant/proportional to a factor inhibition of run-
away reactions when coupled with a positive feedback loop creating an oscillator by taking
advantage in the time delay of transcription and translation, given that the mRNA and protein
half-life is shorter positive feedback: the gene product up regulates its own production directly or
indirectly, which can result in signal amplification bistable switches when two genes inhibit each
other and have both positive feedback pattern generation
Methods
For DNA and RNA methods, see nucleic acid methods. For protein methods, see protein
methods.
Generally, most experiments investigating differential expression used whole cell extracts
of RNA, called steady-state levels, to determined which genes changed and by how much they
did, these are however not informative of where the regulation has occurred and may actually
mask conflicting regulatory processess (see post-transcriptional regulation), it is the most
commonly analysed (QPCR and DNA microarray).
When studying gene expression there are several methods to look at the various stages. In
eukaryotes these include:
The chromatin conformation of the region can be determined by ChIP-chip analysis by
pulling down RNA Polymerase II, Histone 3 modifications, Trithorax-group protein,Polycomb-
group protein or any other DNA binding element to which a good antibody is available.
Due to post-transcriptional regulation, transcription rates and total RNA levels differ
significantly, to measure the transcription rates nuclear run-on assays can be done and newer
high-throughput methods are being developed, using thiol labelling instead of radioactivity. [3]
[4]
Only 5% of the RNA polymerised in the nucleus actually exists and not only introns, abortive
products and non-sense transcripts are degradated therefore the differences in nuclear and
cytoplasmic levels can be see by separating the two fractions by gentle lysis .
 Alternative splicing can be analysed with a splicing array or with a tiling array (see DNA
microarray).
All in vivo RNA is complexed as RNPs. The quantity of transcripts bound to specific
protein can be also analysed by RIP-Chip, for example DCP2 will give an indication of
sequestered protein, ribosome bound gives and indication of transcripts active in transcription
(although it should be noted that a more dated method, called polysome fractionation, is still
popular in some labs)
Protein levels can be analysed by Mass spectrometry, which can only be compare to
QPCR data as microarray data is relative and not absolute. RNA and protein degradation rates
are measured by means of transcription inhibitors (actinomycin D or α-amanitin) or translation
inhibitors (Cycloheximide) respectively.