Regulation of gene expression:
It
is the multistep process by which a gene is regulated and its product is
synthesized.
Thus, gene expression results in the formation of a Polypeptide. Gene expression process is regulated at different levels.
In eukaryotes, the regulation can be at different levels like-1)Transcriptional level (formation of primary transcript)2)Processing level (regulation of splicing)3)Transport of m-RNA from nucleus to the cytoplasm.4)Translational level.
• Genes of a cell are expressed to perform different function. for eg An enzyme beta galactosidase is syn It is used for hydrolysis of lactose to galactose and glucose.•
If
E. coli bacteria do not have lactose in the surrounding
medium as a source of energy, then enzyme β galactosidase
is not synthesised.
So,
it is the metabolic or physiological or environmental conditions that regulate
expression of genes.
The
development and differentiatien embryo into an adult organism, is also a result
of the coordinated regulation or expression, of several sets of genes.
Now
one has to understand a d know the mechanism by which the organisms regulate
gene expression in response to changes in th en ironment.
If
so, whether single mechanism exists for regulation of the expression of
different gene/ sets of genes or different genes are regulated by different
mechanisms
Certain
bacteria like E.coil adapt to their
chemical environment by synthesizing certain enzymes depending upon the 'tl
strate present
Such adaptive enzyme is called inducible
enzymes
A
set of genes will be switched on when there is necessity to metabolise a new
substrate.
This phenomenon is called induction and small molecule responsible for this, is known as inducer. It is positive control.
Operon concept:
•
It is a transcriptional control
mechanism of gene regulation. Francois Jacob and Jacques Monod (1961) explained
that metabolic pathways are regulated as a unit.
•
E. coli, when
lactose sugar is provided to the culture medium, cell induces production of
three enzymes necessary for digestion of lactose.
The
enzymes are: -
i) β -galactosidase: Digests
lactose into galactose and glucose.
iii)Transacetylase
(p-Galactoside acetyltransferase): Transfers an acetyl group from acetyl
CO-A to galactoside
Synthesis
of these three enzymes, is controlled by a long segment of DNA known as Operon.
It consists
of
an operator site 0 and three structural genes Z, Y and A.
The
action of structural genes is regulated by operator site with the help of a
repressor protein.
Repressor
protein is produced by the action of gene i (inhibitor) known as regulator
gene.
The
gene expression depends on whether operator is switched on or switched off.
Synthesis
of these three enzymes, is controlled by a long segment of DNA known as Operon.
It consists
of
an operator site 0 and three structural genes Z, Y and A.
The
action of structural genes is regulated by operator site with the help of a
repressor protein.
Repressor
protein is produced by the action of gene i (inhibitor) known as regulator
gene.
The
gene expression depends on whether operator is switched on or switched off.
Lac operon:• Lactose or lac operon of E. coli is inducible operon.
• The operon is switched on when a chemical inducer- lactose is present in the medium.
Lac operon consists of following components:
1)Regulator
gene (repressor gene)
2)Promoter
gene
3)Operator
gene
4)Structural
genes
5)Inducer
- It is not a component of operon
1) Regulator gene: This gene controls the operator gene in c ration with an inducer present in the cytoplasm.
Regulator gene comes before the promoter
gene. It may present immediately
adjacent to operator gene.
Regulator gene produces a protein called repressor p utefh. Repressor binds with operator gene and represses (stops) its action. It is called regulator protein.
2)Promoter gene: This gene precceds the operator gene. .It is present adjacent to operator gene. The promoter gene marks the site at which the RNA Polymerase enzyme binds. .
When the operator gene is turned on, the enzyme moves over the operator gene and transcription of structural genes starts. .Promoter gene base sequence determines which strand of DNA acts a template
3)
Operator gene: It comes before lie
structural genes. This controls the functioning of structural genes. It lies adjacent to the
Structural genes. lien operator gene is turned on by an inducer, the Structural
genes produce mRNA.
4)Structural gene: When lactose is added to the E. coli culture, the structural genes produce m-RNA which in tum produces polypeptides, on the ribosomes. The polypeptides formed, act as enzymes to produce lactose in the cell. There are 3 structural genes in the sequence lac-Z, lac- Y and lac-A. Enzymes produced are p-galactosidase, p-galactoside permease and transacetylase respectively.
5)Inducer: It is a chemical in the cytoplasm (allolactose) which inactivates the repressor. When lac operon is switched on, then inducer joins with repressor protein preventing the binding of repressor to the operator gene. So, the Operator gene becomes free and now enzyme RNA polymerase can move from promoter to structural genes via operator gene.
Genomics
The
term Genome (introduced by H. Winkler in 1920) is the total genetic
constitution of an organism. or
It
is a complete copy of genetic information (DNA) or one complete set of
chromosomes (monoploid or haploid) of an organism.
The
term Genomics (term coined by T.R. Roderick in 1986) is the study of genomes
through analysis, sequencing and mapping of genes along with the study of their
functions
The
sequencing of yeast, Drosophila and mouse genome was done in order to
facilitate comparative studies between humans and other organisms commonly used
for genetic studies, in laboratory.
Several
additional genomes are now either actively being sequenced or strongly
considered for sequencing. These include several microbes, bee, tomato and
other crops.
Genomics study may be classified into two types: -
Structural
genomics: It
involves mapping, sequencing and analysis of genome.
Functional
genomics: It
deals with the study of functions of all gene sequences and their expression in
orgamsms.
Application of genomics:
1)Structural
and functional genomics is used for different purposes in the improvement of
crop plant, human health and livestock.
2)The knowledge and understanding acquired from genomics research can be applied in a number of different
3)sectors, including medicine, biotechnology and social sciences.
4)It
helps in the treatment of genetic disorders through gene therapy.
5)Genomics
is used in agriculture to develop transgenic crops having more desirable
characters.
6)Genetic
markers developed in genomics, have applications in forensic analysis.
7)Genomics
can lead to introduce new gene in microbes to produce enzymes, therapeutic
proteins and even biofuels.
The
human genome project was initiated in 1990 under the International
administration of the Human
Genome Organization (HUGO).
1)This
project was coordinated by the US department of Energy and National institute
of health.
2)The
information is taken from international partners in the United Kingdg , france,
Germany, Japan, India and China.
3)The
Human Genome Project formally began in 1990 and was com ete in 2003.
4)The
human genome project is a multinational research project to determine the genomic structure of humans
The main aims of project are -
I)Mapping
the entire human genome at the level of nucleotide ,sequences.
II)To
store the information collected from the project in database.
III)To
develop tools and techniques for analysis of the data.
IV)Transfer
of the related technologies to the private se ors, such as industries.
V)Taking
care of the legal, ethical and social issues
which may arise from project.
HGP
(Human Genome Project) was closely
associated with rapid development of a new area in biology, called Bioinformatics.
1)The
work of human genome project as allowed
researchers to begin to understand the blueprint in building and constructing
the human genome.
2)As
researchers learn more about the
functions of genes and proteins, this knowledge will have a major impact in the
fields like medicine, Biotechnology and the Life sciences. Hence, HGP is very
important.
3)Human
Genome Project was to provide a complete and accurate sequence of the 3 billion
DNA base pairs that make up the human
genome and to find out the estimated number of human genes.
4)Now
about 33000 genes have been estimated to be present in humans.
5)The
project was also aimed to sequence the genomes of several other organisms such
as bacteria.
6)e.g.
E. coli, Caenorhabditis elegans (a free-living non-pathogenic nematode),
Saccharomyces cerevisiae (yeast), Drosophila (fruit fly), plants
(rice and Arabidopsis), Mus musculus (mouse), etc.
7)Complete
genome sequences of these model organisms will be useful for comparative
studies that will allow researchers to study gene functions in these organisms.
8)The
secret of our complexity may lie not in the number of our genes but how we use
them.
9)It
will lead to the understanding of gene structure and function in other species.
10)
Since we have many of the genes same as these of flies, round worms and mice,
such studies will lead to a greater understanding of human evolution.
DNA Fingerprinting:
1)Genes present on chromosomes are responsible for determining characters of the organism as well as for inheritance of characters.
2)Due to recombination of paternal and maternal genes, we differ from our parents.
3)Differences also arise due to infrequent mutations that occur during gamete formation (cell division).
4)Due to all these factors, every individual has its unique genetic make-up, which may be called its Fingerprint.
5) The technique developed to identify a person with the help of DNA restriction analysis, is known as DNA profiling or DNA fingerprinting.
6)The technique of finger printing was first given by British geneticist, Dr. Alec Jeffreys in 1984.
7)DNA fingerprinting technique is based on identification of nucleotide sequence present in this wonder molecule.
8)About 99.9% of nucleotide sequence in all persons, is same.
9)Only some short sequences of nucleotides differ from person to person in the population, every person shows unusual sequences of20-1 00 base pairs, which are repeated several times.
10) They are termed as Variable Number of Tandem Repeats (VNTRs).
11)The length of the regions having VNTRs is different in each individual and hence is the key factor in DNA profiling
Steps involved in DNA finger printing are as follows - 1. Isolation of DNA: The DNA must be isolated from the cell or tissues of the body (host). Only small amount of tissue like blood, hair roots, skin, etc. is required
2. Restriction digestion: The isolated DNA is treated with restriction enzymes. The restriction enzymes cut the DNA into small fragments having variable length .This phenomenon is called Restrlction agment Length Polymorphism (RFLP).
3. Gel electrophoresis: The DNA samples are, loaded for agarose gel electrophoresis under an electric influence. The DNA fragments having negatively charged move to the positive pole.The movement of these fragments depends on length of the fragments. This results in formation of bands.dsDNA splits into ssDNA by alkali treatment.
4. Southern blotting: The separated DNA fragments are transferred to a nylon membrane or a nitrocellulose filter paper by placing it oven ne gel and soaking them with filter paper overnight.
5. Selection of DNA probe: known sequence of single- stranded DNA is prepared. It is called DNA Probe.DNA Probe is obtained from organisms or prepared by cDNA preparation method.The DNA probe is labelled with radioactive isotopes.
6: Hybridization: Probe DNA is added to the nitrocellulose filter paper containing host DNA. The singlestranded DNA probe pairs with the complementary base sequence of the host DNA strand. As a result DNADNA hybrids are formed on the nitrocellulose filter paper. Remaining single stranded DNA probe fragments are washed.
7:Photography: The nitrocellulose filter paper is photographed on an X-ray film by autoradiography. The film is analysed to determine the presence of hybrid DNA.
Application of DNA fingerprinting- 1)In forensic science, DNA finger printing is used to solve problems of rape and some complicated murder cases.
2)DNA finger printing is used to find out the biological father or mother or both, of the child, in case of disputed parentage.
3) DNA finger printing is used in pedigree analysis in cats, dogs, horses and humans
