Docstoc

BIOTECHNOLOGY)

Document Sample
BIOTECHNOLOGY) Powered By Docstoc
					                                                 BIOTECHNOLOGY)

  Since Mendel’s work was rediscovered in 1900, geneticists have made startling advances which led to a new era
of DNA technology. Beginning in the mid-1970s, a revolution in the field of biology occurred as the development
of recombinant DNA technology led to radically new research approaches. Recombinant DNA technology has
many practical applications. One of the rapidly advancing areas of study today is genetic engineering, the
modification of DNA of an organism to produce new genes with new characteristics. In fact, genetic engineering
has launched a revolution in biotechnology, the use of living organisms to perform practical task. Biotechnology
includes genetic engineering and other techniques that make use of natural biological systems to produce a
product or to achieve an end, desired by human beings. Since the dawn of civilization, humans have bred plants,
animals to produce a particular phenotype. The biochemical capabilities of microorganisms have also been
exploited for a very long time. Today through genetic engineering, bacteria now produce drugs that promote
human health, proteins that are useful as vaccines and nucleic acids for laboratory research. Genetically
engineered bacteria have been used to clean up environmental pollutants, increase the fertility of the soil, and kill
inect pest. Biotechnology also extends beyond unicellular organisms; ways have been found to alter the genotype
and subsequently phenotype of plants and animals, including humans.

GENETIC ENGINEERING

        Genetic engineering allows the insertion f a foreign gene into cells, and this makes them capable of
producing a new and different protein.

Cloning of a Gene: Recombinant DNA (rDNA) or chimaeric contains genes from two or more different sources. To
maker rDNA, first a vetor is selected. By means of vector rDNA is introduced into the host cell. One common type
of vector is plsmid. Plsmids are small accessory rings or DNA, which is not part of bacterial chromosome and can
replicate independently. Making many identical copies of a molecule is called cloning. Phages are viruses which
can inject their DNA into bacteria for replication. The piece of DNA, (foeign gene) of phage combines with plamid.
The modified plasmid is called rDNA. When bacteria divide, the plasmids are replicated. Eventually there are many
copies of the plasmid and therefore many copes of the foreign. The gene is now said to have been cloned.

         Genetic engineering kin bacteria can be divided into fice stages. The order of stages are:

    (a) Obtain a copy of the required gene from the DNA of the donor organism.
    (b) Place the gene in a vector i.e. plasmid.
    (c) Use the plasmid to introduce the gene into the host cell (bacterium).
    (d) Select the bacteria which have taken up the DNA i.e. foreign DNA.
    (e) Clone the gene.
Use of Restriction

Endonucleases and Plasmids in rDNA Techoogy

          A specific piece of DNA containing the gene of interest must be cutout of a chromosome and “pasted”
into a bacterial plasmid. The cutting tools of recombinant technology are bacterial enzymes, these are restriction
endonucleases or restriction enzymes. In 1970 hamilton O. Smith, at John Hopkins University, isolated the
restriction enzymes. Restriction enzymes occur naturally in bacteria, where they stop viral reproduction by cutting
up viral DNA. They are called restriction enzymes because they restrict the growth of viruses. Bacteria produce a
variety of restriction enzymes. These cut the DNA at very specific sites characterized by specific sequence of four
or six nucleotide arranged symmetrically in the reverse order. Such sequence are known as plaindromic sequence
or recognition sequence. For example EcoRl, is a commonly used restriction enzyme, when double stranded DNA
has AATT sequence of basses at cleavage site.

          There are several hundred restriction enzymes and about a hundred different recognition sequences
known. In the figure (23.2) here, step (1) Shows a piece of DNA containing two copies of a recognition sequence.
In this case, the restriction enzyme will cut the DNA strands in the four places within each recognition sequence
where the bases A and G lie next to each other. (The places where DNA is cut are called restriction sites.) (2) The
result is a set of souble-standed DNA fragments with single-stranded ends, called “sticky ends”. Sticky ends are
the the key to joining DNA restriction fragments originating from different souces, even from different species.
These short extensions will form hydrogen-bonded base pairs with complementary single stranded stretches of
DNA.

          Step (3) shows a piece of DNA (pink) from another source. Notice that the pink DNA has single-stranded
ends identical in base sequence to the sticky ends on the blue DNA. The pink, “foreign” DNA has ends with this
particular base sequence because it was cut form a larger molecule with the same restriction enzyme used to cut
the blue DNA. (4) The complementary ends on the blue and pink fragments allow them to stick together by base
pairing. (The hydrogen bonds that hold the base pairs together are not shown.)

        The union between the blue and pink DNA framents shown in step 4 (fig. 23.2) is only temporary,
because only a few hydrogen bonds hold the fragments together. The union can be made permanent, however,
by the “pasting” enzyme DNA ligase. This enzyme, which the cell normally uses in DNA replication catalyzes the
formation of vovalent bonds beteen adjacent nucleotides, sealing the breaks in the DAN strands. (5) The final
outcome is recombinant DNA, a DNA molecule carriny a new combination of genes.

Genes can be Clones in

Recombinant Plasmids

         This figure 23.3 illustraes a way to make many copies of the gene using the techniques we have been
discussing.

        In step (1), the biologist isolates two kinds of DNA: the bacterial plasmid that will serve as the vector, and
eukaryotic DNA containing the gene.

        In step (2), the researcher treats both the plasmid and the human DNA with the restriction enzyme. The
enzyme cuts the plasmid DNA at one specific restriction site. It also cuts the human DNA, generationg many
thousands of fragments; one of these fragments carries the insulin gene. In making the cuts the restriction
enzyme creates sticky end on both the human DNA fragments and the plasmid.

         For simplicicity the figure here shows the step-by-step processing of one human DNA fragment and one
plasmid, but actually millions of plasmids and human DNA fragments are treated simultaneously.

         In step (3) the human DNA is mixed with the cut plasmid. The sticky ends of the plasmid base pair with
the complementary sticky ends of the plasmid base pair with the complementary sticky ends of the molecules by
covalent bonds, and the result is recombinant DNA plasmid containing insulin gene.

          In step (5), the recombinant plasmid is added to a bacterium. Under the right conditions, the bacterium
will take up the plasmid DNA from solution by the process of transformation. The last step shown here, step (6), is
the bacterium, with its recombinant plasmid, is allowed to reproduce. As the bacterium forms a cell clone, any
genes carried by the recombinant plasmid are also cloned.

Expression of Recombinant DNA

Plasmid library: Bacterial cells take up recombimids, especially if they are treated with calcium chloride to make
them more permeable. Thereafter, as the host cell reproduces, a bacterial clone forms. Each of the bacteria
contains the foreign gene, which is behaving just as if it were in its original cell. The investigator can recover either
the cloned gene, or a protein product from this bacterial clone (Fig. 23.4a).

          Plasmids can be used as vectors for carrying DNA, and so can viruses, such as the bacteriophage known
as lambda (Fig. 23.4-b). After lambda attaches to a cell, the DNA is released from the virus and enters the
bacterium. Here it may direct the reproduction of many more viruses. Each virus in th3e bacteriophage clone
contains a copy of the foreign gene. Notice that a clone is a large number of molecules (i.e. cloned genes) or
viruses (i.e. cloned bacteriophages) or cells (i.e. cloned bacteria) that are identical to an original specimen.

Using a Genomic Library

Phage library: A genome is the full set of genes of an individual. A genomic library is a collection of bacterial or
bacteriophage clones; each clone contains a particular segment of the DNA from a foreign cell. When you make a
genomic library, an organism’s DNA is simply sliced up into pieces, and the pieces are put into vetors (i.e. Plasmids
or viruses) that are taken up by the host bacteria. The entire collection of bacterial or bacteriophage clones that
result therefore contains all the genes of that organism.

         In order for mammalian gene expression to occur in a bacterium, the gene has to be accompanined by
the proper regulatory regions. Also, the gene sgould not contain introns (non coding region) because bacterial
cells do not have the necessay enzymes to process primary mRNA. It is possible to make a mammalian genome
that lacks introns. The enzyme called reverse transcriptase can be used to make a DNA copy of all the mature
mRNA molecules from a cell. This DNA molecul, called complementary DNA (cDNA), does contain introns.

Use a particular probe to search a genomic library for a certain gene: A probe is single-stranded nucleotide
sequence of RNA that will hybrize (pair) with a certain piece of DNA. Methods for detecting genes all depend on
base paring between the gene and a complementary sequence on another nucleic acid molecule, either DNA or
RNA. When at least part of the nucleotide sequence of a gene is already known or can be guessed, this
information can be used to advantage. Taking a simplified example, if we know that our hypothetical gene V
contains the sequence TAGGCT, a biochemist can use nucleotide labeled with a radio active isotope to synthesize
a short RNA molecule with a complementary sequence (AUCCGA) (fig.23.6) This short labeled nucleic acid
molecule is called probe because it is used to find a specific gene (or other nucleotide sequence) within a mass of
DNA. In practice prove molecule would be considerably longer than six nucleotide. Location of the probe is
possible because the probe is either radioactive or fluorescent. As illustrated in figure 23.7 bacterial cells, each
carrying a particular DNA fragment, can be plated onto ager in a Petri dish. After the probe hybridizes with the
gene of interest, the gene can be isolated from fragmen. Now this particular fragment can be cloned further or
even analyzed for its particular DNA sequence.

Replicating Small DNA Segments

         The polymerase chain reaction (PCR) was developed by Kary B. Mullis in 1983. It can create millions of
copies of single gene or any specific piece of DNA in laboratory glassware (e.g. a test tube). PCR is very specific. The
targeted DNA sequence can be less than one part in a million of the total DNA sample. This means that a single
gene among all the human genes can be amplified (copied) using PC.

         PCR takes its name from DNA polymerase, the enzyme that carries out DNA replication in a cell. It is
considered a chain reaction because DNA polymerase will carry out replication over and over again, until there are
millions of copies of the targeted DNA. PCR does not replace gene cloning; cloning is still used whenever a large
quantity of protein product is needed, or if the DNA needs to be in its accustomed configuration before being
sequenced.

          Before carrying out PCR, primaers-sequenes of about 20 bases that are complementary to the bases on
either side of the “target DNA” must be available. The primers are needed because DNA polymerase does not start
the replication process; it only continues or extends the process. After the polymerase copies the target DNA (Fig.
23.8).

        PCR has been in use for several years, and now almost every laboratory has automated PCR machines or
thermocycler to carry out the (thermostable0 DNA polymerase also know as Taq polymerase was extracted from
the bacterium Thermus aquatics, which lives in hot springs. This enzyme can withstand the high temperature used
to separate double-stranded DNA; therefore, replication need not be interrupted by the need to add more
enzyme.

Analyzing DNA

          DNA can be subjected to DNA fingerprinting, a process described in Figure 23.6. When the DNA of an
organism is treated with restriction enzymes, the result is a unique collection of different-sized fragments called
restriction fragments, which reflect specific sequence of nucleotide. Therefore, restriction fragment length
polymorphisms or RELPs, (pronounced “rif-lips”) exist between individuals. During a process called gel
electrophoresis (Fig. 23.11), the fragments can be separated according to their lengths, and the result is a No. of
bands that are so close together that they appear as a smear. However, the use of probes for genetic markers
results in a distinctive pattern that can be recorded on X-ray film.

         PCR amplification and analysis has been used (1) to diagnose viral infections, genetic disorders, and
cancer; (2) in forensic laboratories to identify criminals; and (3) to determine the evolutionary relationships of
various organisms. When the amplified DNA matches that of a virus, mutated disorder, or cancer is present. To
determine evolutionary relationship, it is often necessary to sequence the DNA. Sequencing mitochondrial DNA
segments helped to determine the evolutionary history of human populations. It has even made possible to
sequence DNA taken from a 17 to 20 billion year old plant fossil following PCR amplification. Nucleotide sequences
of DNA sequencer make use of computer.

DNA Sequencing Technique

Gel-electrophoresis

         This laboratory producer uses an electric field to move molecules through a viscous gel and separate them
according to their size, shape and net surface charge. Often the gullies sandwiched between glass or plastic plates
to form a viscous slab. The two ends of the slab are suspended in two salt solutions that are connected by
electrodes to a power source. When voltage is applied to the apparatus, the molecules present in the gel migrate
through the electric field according to their individual charge, and they move away from one another in the gel.
Later on, the molecules can be pinpointed by staining the gel after a predetermined period of electrophoresis.
DNA Fingerprints

         Except for identical twins, no two people have exactly the same sequence of bases in their DNA. By
detecting the differences in DNA sequences, scientists can distinguish one person from another. As you know, each
human has a unique set of fingerprints, a marker of his or her identity. Like all other sexually reproducing species,
each human also has a DNA fingerprint, which is a unique array of DNA fragments that were inherited from each
parent in a Mendelian pattern. DNA fingerprints are so accurate that even full siblings are readily distinguished
from one another.

         More than 99 percent of the DNA is exactly the same in all humans. But DNA fingerprinting focuses only
on the part that next. Throughout the human genome are tandem repeats – short regions of repeated DNA – that
differ substantially among people. For example, the five bases TTTTC are repeated anywhere from four to fifteen
times in tandem in different people, and three bases (CGG) are repeated five to fifty times in different people, and
three bases (CGG) are repeated five to fifty times in tanderm. By examining many tandm-repeat sites, researchers
found out that each person carries a unique combination of repeat numbers.

          Researchers detect differences at tandem-repeat sites with gel electrophoresis. In this case, it separates
DNA fragments according to their length. Size alone dictates how far each fragment moves through the gel, so
tandem repeats of different sizes migrate at different rates. A gel is immersed in a buffer solution, then DNA
fragments from individuals are added to the gel. When an electric current is applied to the solution, one end of
the gel takes on a negative charge (because of the negatively charged phosphate groups), so they migrate through
the gel toward the positively charged pole. They do so at different rates, and so they separate into bands according
to length. The smaller the fragment, the father it will migrate. After a set time, researchers can identify fragments
of different lengths by staining the gel or by specifically highlighting fragments that contain tandem repeats.

         Figure 23.12 shows a series of tandem-repeat DNA fragments that were separated by gel electrophoresis.
They are DNA fingerprints from seven individuals and from blood collected at a crime scene. Notice how much
their DNA fingerprints differ. Can you identify one of the patterns exactly matches the pattern from the crime
scene? DNA fingerprints help forensic scientists identify criminals and victims, and exclude innocent suspects. A
few drops of blood or cells from a hair follicle at a crime scene etc., on a suspect’s clothing often yield enough to
do the trick.

         Law enforcement officials have found an increasing number of uses for profiling in criminal cases, or
paternity cases.

DNA Sequencing

         For generation of different sized DNA fragment, two methods are generally used. One is Sanger’s method
in which dideoxyribonucleoside triphosphates are used to to terminate DNA synthesis at different sites. The other
method is known as Maxam-Gilbert method in which DNA threads are chemically cut into pieces of different sizes.

         DNA seqyencing (Fig. 23.14) is usually done by copying a stand of the cloned DNA in four different
reaction mixtures, using a modified form of DNA polymerase. In each reaction mixture the copies are made so that
the newly synthesized DNA chains are terminated randomly at positions corresponding to one of the four basis.
The lengths of the fragments from sequence of base in cloned DNA fragment to read of from one end to the other.

       The volume DNA sequence is now large that powerful computers must be used to store and analyze it.
DNA sequence is now completely automated, robotic devices mix the reagents and then load, run and read the
order of the nucleotide basis from the gel. This is facilitated by using chain terminating nucleotide that are each
labeled with a different coloured fluorescent dye; in this case, all four synthesis reactions can be performed in the
same tube, and the products can be separated in a single lane of a gel. A detector position near the bottom of the
gel reads and record the colour of fluorescent label on each band as it passes through a LASER beam. A computer
than reads and stores this nucleotide sequence.

Human Genome Project

         In 1990, the human Henome Project (HGP) led by the intemational Human genome Consortium, was
lauched to map the human genome. The genome is defined as a set of genes in a cell or living thing. The project
envisage mapping of the human genome in a period of 15 years. The consortium announced on April 12, 2003 the
finished sequence. The finished sequence is highly accurate, in the sense that there is an error probability of
around I letter for every 10,000. This means that we hae now an accurate map of over 99.99% of the human
genome i.e. over 3 billion DNA letters, leaving just under 0.01% i.e. I million DNA letters to be deciphered. The
Human Genome project involves four major lines of work:

Genetic (Linkage) maping of the human genome: To map the human genome, scientist combine linkage, pedigree
analysis and restriction fragment analysis.

Physical mapping of the human genome: This is done by breaking each chromosome into a number of identifiable
fragments with the help of restriction enzyme, and then determining the order of the fagments in the
chromosomes.

Sequencing the human genome: This is the process of determining the exact order of nucleotide pairs of each
chromosome.

Analyzing the genome of other species: Comparative analysis of the genes of other species e.g E. Coil, yeast, a
plant named Arabidopsis, Drosophila and mice, will help us to interpret the human data.

          The DNA sequence of human chromosome no. 22, one of the smallest human chromosome has been
completed in 1999. the human genome is 25 times larger than any other genome sequenced so far. The potential
benefit of having a complete map of the human genome are great. For basic science, the information will give
insight into such fundamental systeris as embryonic development and evolution. For human health, the
identification of genes will aid in the diagnosis, treatment, and prevention of may of the common disease like high
blood pressure, heart disease, diabetes etc.

Biotechnology Products

        Becteria, plants and animals are genetically engineered to produce biotechnology products. Free-living
organisms in the environment that have had a foreign gene inserted into them are called transgenic organisms.

Transgenic bacteria

         Recombinant DNA technology is used to produce bacteria that procuce in large vats called bioreactors. If
the foreign genes is replicated and actively expressed, a large amount of protein products can be obtained. The
biotechnology products now available include hormones and sililar types of proteins and vaccines.

Protection and Enhancement of Plants
         Genetically engineered bacteria can be used to promote the health of plants. For example, bacteria that
normally live on plants and encourage the formation of ice crystals have been changed from frost-plus to frost-
minus bacteria. Field tests showd that these genetically engineered bacteria protect the vegetative parts of plants
from frost damage. Also, a bacterium that normally colonizes the roots of corn plants has now been endowed with
genes (from another bacterium) that code for an insect toxin.. the toxin is expected to protect the roots from
insects.

Bioremediation

         Bacteria ca be selected for their ability to degrade a particular substance, and then this ability can be
enhanced by genetic engineering. For instance, naturally occurring bacteria that eat oil can be genetically
engineered to do an even better job of cleaning up beaches after oil spills. Industry has found that bacteria can be
used as biofilters to prevent air borne chemical pollutants from being vented into the air. They can also remove
sulphur from coal before it is burned and help to clean up toxic waste dumps. One such strain was given genes that
allowed it to clean up levels of toxins that would have killed other strains. Further, these bacteria were given
“sucide” genes that caused them to self-destruct when the job had been accomplished.

Chemical Production

         Organic chemicals ae often synthesized by having catalysts act on precursor molecules or by using
bacteria to carry out the synthesis. Today, it is possible to go one step further and to manipulate the genes that
code for these enzymes. For instance, biochemists discovered a strain of bacteria that is especially good producing
phenylalanine, an amino acid needed to make aspartame, the dipeptide sweetener better known as NutraSweet.
They are isolated, altered, and formed a vector for the appropriate genes so that various bacteria could be
genetically engineered to produce phenylalanine.

Mineral Processing

         Many major mining companies already use bacteria to obtain various metals. Genetic engineering may
enhance the ability of bacteria to extract copper, uranium, and gold from low-grade sources. Teasting of
genetically engineered organisms having improved blaching capabilities is in progress.

Transgenic Plants

Protoplasts: The only possible plasmid for genetically engineering plant cells belongs to the bacterium
Agrobacterium, which will infect many but not all plants. Therefore, other techniques have been developed to
introduce foreign DNA into plant cells that have had the cell wall removed and are called protoplasts. It is possible
to treat protoplasts with an electric current while they are suspended in a liquid containing foreign DNA. The
electric current makes tiny, self-sealing holes in the plasma membrane through which genetic material can enter.
Then a protoplast will develop into a complete plant.

         Presently, about 50 types of genetically engineered plants that resist insects, viruses, or herbicides have
entered small-scale field trails. The major crops that have been improved in this way are soybean, cotton, alfalfa,
rice, potato; and corn. Plants have been engineered to produce human proteins, such as hormnes, in their seeds. A
weed called mouseeared cress has been engineered to produce a biodegradable plastic (polyhydroxybutyrate, or
PHB) in cell granules.

        One type of antibody made by corn can diliver radio isotopes to tumor cells, and another made by
soybean can be ued as treatment for genital herpes (caused by herpes simplex 2 virus tiny, painful clisters appear
on genitals.) Plant made antibodies are inexpensive and there is little chance that contamination with pathogens
infect people.

Transgenic Animals

         Animals, too, are being genetically engineered. Because animal cells will not take up bacterial plasmids,
the method used to insert genes into their eggs is vortex (a whirling motion of a liquid) mixing. The eggs are placed
in an agitator with DNA and silicon-carbide needles, and the needles make tiny holes through which the DNA can
enter. Using this technique, many types of animal eggs have taken up bovine (cattle) growth hormone (bGH). The
procedure has been used to produce large fishes, cows, pigs, rabbits, and sheep. Genetically engineered fishes are
now lbeing kept in ponds that offer no escape to the wild because there is much concern that they will upset or
destroy natural ecosystems.

Gene Pharming

         The use of transgenic farm animals to produce pharmaceuticals, is being pursued by a number of firms. It
is advantageous to use animals because the product is obtainable from the milk of females. Genes that code for
therapeutic and diagnostic proteins are incorporated into the animals DNA, and the proteins appear in the animals
milk. In one instance a bull was genetically engineered to carry a gene for human lactoferrin, a drug for
gastrointestinal tract infections, and he passed the gene to many offsprings, among them were several females.

Procedure of Producing Transgenic Mammals

         DNA containing the gene of interet is injected into donor eggs. The fertilization takes place in vitro. The
zygotes are placed in the uterus of the host female where they develop. On maturation of the female offsprings
the product is secteted in the milk. Scientists have been able to genetically engineer mice to produce human
growth hormone in their urine instead of milk, as urine is produced in large quantities by all the individuals and it is
easier to extract.

Cloning of Transgenic Animals

         Cloning means making identical copies. When an embryo first grows from a fertilized egg, all of its cells
hace the same DNA and are much alike. Then different embryonic cells start using different parts of their DNA.
Their unique selections commit them to become liver cells, heart cell, brain cell etc.

          In 1997 in Scotland, a research group led by Wilmut at the Roslin institute in Scotland cloned a sheep. The
scientists first removed the haploid ncleus from an unfertilized sheep egg. They then inserted into the egg another
nucleus taken from an udder (the organ containing the mammary glands of sheep, cow, mare etc, having more
than one teat) cell. The egg become diploid, having a diploid nucleus containing two copies of every chromosomes,
just as fertilized egg would. The egg was then implanted into another female sheep. The lamb was named Dolly,
which developed into a healty adult and gave birth to a lamb of her own.

        In 1998 Ryozo Yamagimachi and his co-workers at the university of Hawii cloned three generations of
mice. They quickly transferred nuclei from mature cumulus cells from an ovary into unferilizded, enucleated egg.
(cumulus cell provide nutritional support to neighboring egg). Shortly afterward, they chemically activated the
eggs, which developed into fully formed mice. Scientists in Japan slipped nuclei from a cow’s cumulus cells and
oviduct cells into enucleated eggs. These were transferred to surrogate ( a female who bears a baby after
implantation embryo from another female) cow mother. Four cloned claves have survived. Cloning of human is
banned.
Gene Therapy

          Gene therapy is the transfer of one or more normal or modified genes into an individuals body cells to
correct a genetic defect or boost resistance to disease. It also includes the use of genes to treat various other
human illness such as cancer and cardiovascular disease. There are two main methods of gene therapy (a) ex vivo
(b) in vivo

Ex vivo method: During ex vivo ptherapy i.e. outside the living organism, the cells are removed from a patient
treated and returned to the patient. A retrovirus, which has an RNA genome, is often used as vector to carry
normal genes into the cells of the patient. The recombinant RNA from the retovirus enters a human cell, such as a
none marrow stem cell, reverse transcription occurs. It is the resulting DNA that carries the normal gene into
transcription occurs. It is the resulting DNA that carries the nrmal gene into human genome (fig. 23.16). A four year
old girl suffered from adenosine dreaminess (AD) deficiency. It is a lethal genetic disorder that severely limits the
ability of the body to fight disease. This disorder is caused by a mutation to a single gene that is expressed in our
white blood cells. It is called severe combined immunodeficiency syndrome (SCID). SCID is often called the “bubble
baby disease” after David, a youn d person who lived under plastic dome to prvent infection. AD is involved in
maturation of T and B cell. White blood cells were removed from her blood and infected with a retrovirus that
carried a normal gene for the enzyme. Then the cells were returned to the girl. This experiment was performed on
Sept 14, 1990. A few months later similar experiment was performed on a 8 years old girl.

         Since whit blood cells do not reduce, for several years these two girls were given generically engineered
white blood cells every few months. Genetically engineered stem cells are preferred because they are long lived
and their use may result in a permanent cure.

         Gene therapy is being used for treatment of hypercholesterolemia (excess cholesterol in blood) a
condition that develops when liver cells lack a reception for removing cholesterol from the blood. The high levels
of blood cholesterol make the patient subject fatal heart attacks at a young age. In a newly developed procedure,
a small portion of the liver is surgically excised and infected with a retrovirus containing a normal gene for the
receptor.

         Chemotherapy is cancer patients often kills off healthy cells as well as cancer cells. In clinical trails,
researchers have given genes to cancer patients that either make healthy cells more tolerant of chemotherapy of
make tumors more vulnerable to it. In one trial, bone marrow stem cells from about 30 women with late-stage
ovarian cancer were infected with a virus-carrying gene for multiple-drug resistance.

In Vivo Method

         Other gene therapy producers use viruses, laboratory grown cells, or even synthetic carries to introduce
hens directly into patients. If in vivo (inside the living organism) therapy is used, no cells are removed from the
patient. For example, liposome’s are microscopic vesicles that spontaneously form when lipoproteins are put in a
solution. Liposome’s have been coated with healthy cystic fibrosis genes and sprayed into patient’s nostrils as a
possible treatment for cystic fibrosis (It is characterized by an excessive secretion of very thick mucus from the
lungs, pancreas, liver, digestive tract. This mucus can interfere with berating, digestion and liver function, and
make the person vulnerable to pneumonia and other infections). Cystic fibrosis patient lack a gene that codes for
cytokinin, soluble hormones of the immune system, directly into the tumors of patients. It has been observed that
the presence of cytokinin stimulates the immune system of the body to remove cancer cells. During coromary
artery angioplasty a balloon catheter is sometimes used to open up a closed artery. Unfortunately the artery has a
tendency to close up once again. But investigators have come up with a new procedure. The ballon in coated with
a plasmid that contains a gene for vasculat endothelial growth factor. The expression of he hene, which promotes
the proliferation of blood vessels to bypass the obstructed area, has been observed in at least one person.

         Perhaps it will be used one day for in vivo therapy to cure hemophilia, diabetes, Parkinson disease, or
AIDS. To treat hemophilia, patient could get regular dose of cells that contain normal clotting-factor genes. Such
cells could be placed in organoids, artificial organs that can be implanted in the abdominal cavity. To cure
Parkinson disease, dopamine-producing cells could be grafted directly into the brain. These, dopamine-producing
cells could be grafted directly into the brain. These procedures will use laboratory-grown cells that have been
stripped of antigens (to decrease the possibility of an immune system attack).

Prenatal Screening for Genetic Defects

         Genetic screening has become widespread for developing fetus (foetus) a process known as prenatal (pre-
before, natal birth) screening. It provides the parents information about diseases, genetic disorders before birth.
Scientists use a variety of methods in prenatal screening.

Amniocentesis

        The perform amniocentesis physician first determines the position of the fetus with the helo of
ultrasound imaging in which sound waves are used to produce an image of the fetus. Then he carefully inserts a
medle through the mother’s abdomen into her uterus, avoiding the fetus. The physician extract about 10 ml of the
amniotic fluid, (the fluid which surrounds the fetus). The cells are cultured in the laboratory for several weeks. By
then enough dividing cells can be harvested so that varityping can be done and chromosomal abnormalities can be
detected. Amniotic fluid can also be used for DNA testing.

Genetic Counseling

         Genetic counseling is the giving of information and advice about the risk of genetic diseases and their
outcome. Tests are now available for large number of genetic diseases. For example chromosomal tests are
available for cystic fibrosis, neurofibromatosis (nodules allover the body surface) and Huntington disease
(inherited and fatal disease marked by progressive mental deterioration causing shaking of body). Blood can
identify carries of thalassemia (hereditary disorder of blood causing anemia) and sickle cell enzyme defects can
also be identified for certain invorn metabolic errors, such as Tay-sacchs disease (Lipid accumulation in brain cells,
mental deficiency, blindness, death in childhood). From this information, the physician can cometimes predict the
chances of a child having the disorder. Blood tests of the couple is performed for Rh factor. Chorionic villi sampling
and amniocentesis is dine to determine genetic disorder. If so treatment may be available even before birth or
parents may decide whether or not to end the pregnancy.

Tissue Culture

          Tissue culture is the growth of a tissue in an artificial liquid culture medium. In 1902 German bottaist
Harberlandt speculated entire plant could be produced by tissue culture. He said that plant cells are totipotent i.e.
each cell has the full genetic potential of the organism and therefore a single cell could become a complete plant.
In 1958 F.C Steward, a plants physiologist successfully genetated an entire carrot plant from a single cell derived
from a tiny piece of phloem from root. He provided the cells with sugars, minerals, vitamins and coconut milk
(later it was known that coconut milk contains cytokinin). When the cultured cells began dividing, they produced a
callus, an undifferentiated group of cells. Then the callus differentiated into shoot, roots and developed into
complete plant.
        Micropropagation is propagation or cloning, of plants by tissue culture. The three methods for micro
propagation are:

(1) Meristem culture (2) anther culture      (3) suspension culture.



Meristem culture: A meristenm is region where cell division is still taking place. If correct proportion of hormones
auxin and cytokinin are added to a liquid medium many shoots develop from a single shoot tip. The shoots are
called clonal plants as they are genetically identical and a meristem is virus free portion of the plant so meristem
culture produces virus free plants.

Anther Cloning: Anthers are cultured in a medium containing vitamins and growth regulators. The haploid tube
cells within the pollen grins divide, producing proembryos consisting of as many as 20 to 40 cells. The pollen grain
rupture releasing the haploid embryos. Then chemical is added for chromosomal doubling and the plants produced
are diploid but homozygous for all their alleles. This technique is a direct way to produce plants that express
recessive alleles.

Suspension culture: Rapidly growing cultures are cut into small pieces and shaken in liquid nutrient so that single
cells are small clumps of cells break off and form a suspension. These cells will produce the same chemicals as the
entire plants. For example cell suspension culture of Cinchona ledgeriana produce quinine.

Genetic Engineering of Plants.

         Since 1980, a plasmid of the bacterium Agro bacterium tumefactions, (which cause a tumor like disease
called crown gall in plants) has been main vehicle used to introduce foreign genes into broad leaf plants such as
tomatoes, tobacco and soybean. A technique known as disarming is used first or remove disease causing gene
from the bacterium. After the bacterium has been disarmed, it can be used as vector to shuttle desired genes into
plants cells. A part of its plasmids integrates into the plant DNA carrying the foreign genes (see fig 23.20) new
plants can be grown from these transformed cells in tissue culture. The cells develop into plantlets, which can be
grown in conventional way.

Many plant species are not natural host of the Agrobacterium. The other commonly used method, shoots
microscopic metal pellets coated with DNA into plant cell (see fig. 23.20) Then the cells are cultured and
propagated.

         In 1986 the gene for the enzyme luciferase, which cause the production of light in fireflies, was inserted
into tobacco plants. The plants expressed the gene and glowed in the dark.

          Since then the luciferase gene has been transferred to a variety of other species including frog, fish, and
the bacterium that cause T.B if the bacteria are resistant to antibiotics they express firefly gene and glow in the
dark. If the bacteria are not resistant hey sicken or die. As a result doctors can prescribe appropriate antibiotic for
the T.B patient.

Uses and Applications of Biotechnology

    (a) In Agriculture
    (i)     Introduction to foreign genes to form plants having resistance against insecticides, virus, herbicides.
    (ii)    Introduction of foreign genes for nitrogen fixation in crop plants
    (iii)   Transgenic plants have been produced.
        (iv)  Use of crops to produce drugs instead of food.
        (v)   Salt tolerant plant Aerabidopsis has been produced
        (vi)  Improved agriculture include.
              a. Herbicide resistant e.g wheat, rise, sugar beet, canola etc.
              b. Salt tolerant e.g cereals, rice, sugar etc.
              c. Drought tolerant includes cereals, rise, sugarcane
              d. Cold tolerant cereals, rise, sugarcane etc.
              e. Improved yield e.g cereals, rice, corn, cotton etc.
              f. Modified wood pulp e.g. Trees.
    (vii)     Improved food quality traits include.
              a. Fatty acid contents e.g. corn, soyabean
              b. Protein and starch contents e.g. cereals, potatoes, soyabean rice, etc.
              c. Amino acid content include corn, soyabean
              d. Disease protected include wheat, corn, potatoes.
(b)       In Medicine

(i)      Single gene transfers have allowed to produce various products such as heman hormones, clotting factors
and antibiotics.

(ii)           A type of antibody produced by corn can deliver radioisotopes to tumor cells. A type of antibody made
               soyabean can be used as treatment for genital herpes.

(iii)    Tobacco mosaic virus hace been used as a vector introduce himan gene in adult tobacco plant. Tobacco
         plants have been used to produce.
         (a) 10 grams α glycosidase an enzyme that can be used to treat a human lissome storage disease.
         (b) To produce antigens to trat Non-Hodgkins lymphoma (abnormal proliferation of lymphocytes) after
              being spryed with a genetically engineered virus.
(iv)     the human growth hormone, which is used to treat people who are growing more slowly than normal,
         was previously extracted from the pituitary glands of cadvers (a human corpse, used for organ transplant
         or dissection) and it took 50 glands to obtain enough for one dose. Now human growth hormone is
         produced in large quantity by biotechnology. Insulin, to treat diabetics, was previously extracted from the
         pancreatic glands of slaughtered cattle and ids; it was expensive and sometimes caused allergic reactions
         in recipients. And few of us knew of tPA (tissue plasminogen activator) a protein present in minute
         quantities that activates an enzyme to dissolve blood clots. Now tPA produced by biotechnology is used to
         treat heart attack victims by dissolving blood clots that are blocking the flow of blood in the coronary
         arteries of the heart. Clotting factor VIII treats haemophillia; human lung surfactant (surface proteins
         secreted by the lung cells) treats respiratory distress syndrome in premature infants; atrial natriurentic
         factor helps control hypertension . Several hormones produced by biotechnology are for use in animals.
         Farm animals can now be given growth hormone instead of steroids. Such animals produce a leaner meat
         that is healthier for humans.
(v)      Transgenic animals are used to relatively large quantities of rare and expensive proteins for use in
         medicine. Bacteria and viruses have surface proteins, and a gene for just one of these can be used to
         result can be used as a vaccine. A vaccine for hepatitis B is now available, and potential vaccines for
         malaria, and AIDS are in experimental stags. (table-23.1)
                          Table 23.1
Biotechnology Products: Hormones and Similar Types of
                            Proteins
Treatment of Humans                   For
Insulin                               Diabetes
Growth hormone                        Pituitary dwarfism
tPA (tissue plasminogen activator) Heat attack
Interferons                           Cancer
Erythropientin                        Anemia
(due to decrease of oxygen
kidneys release this hormone
Ceredase                                Gaucher disease (lipid
                                        stroring diseases)
Interleukin-2                           Cancer
Tumor necrosis factor                   Cancer
Cloting factor VIII                     Hemophilia
Human lungs surfactant                  Respiratory distress
                                        syndrome
Artial natriuretic factor               High blood pressure


Safety and Ethical Problem Raised by Biotechnology

         As our knowledge of human genetics increases, we must face a number of difficult ethical questions. On
one hand, as many people argue, the potential benefit for fighting disease provide a strong ethical reason for
obtaining this knowledge as fast as possible. On the other hand the potential for abusing the knowledge makes
people wary (cautions) of proceeding. Most people will agree that there was no ethical conflict in altering the DNA
of a bacterium or a virus, does that mean that there is no dilemma associated with altering the DNA of a plant, a
dog, a chimpanzee or a human.

         The use of DNA technology also involves risks for example; many a crop plants made and produce
offspring with closely related species. If a crop plant is genetically engineered to e resistant to herbicides, the
potential exits that the resistance gene will be transferred by mating for the crop to wild species.

        The use of DNA technology raises ethical questions and posses risks. There is a fine line between
acceptable and unacceptable changes to the DNA of humans and other species. Benefits and risks of DNA
technology must be considered carefully in evolutions of the potential impact to human society of particular use of
DNA technology.

EXERCISE – SECTION – I – OBJECTIVE QUESTIONS

1. Fill in the blanks.

(i) Cloning of a gene produces many _____ copies.

(ii) DNA from two different sources, combines to form ___.

(iii) The full set of genes of an individual is called _______.

(iv) A particular _____ can be used to search a genetic library for a certain gene.

(v) PCR takes its name from DNA _________

(vi) DNA polymerase is commonly known as _______.

(vii) There are __ billion base pairs in the human genome.

(viii)Organisms having a foreign gene inserted into them are called ____________.

(ix) Genetic counseling is the giving of information and advice about the risk of _____.
(x) The cells having full genetic potential of the organism are called ________.

2.       Mark the statements as “True” or “False”

(i)      EcoRI is not a restriction enzyme.
(ii)     A genomic library is collection of bacterial clones.
(iii)    DNA polymerase does not copy the target DNA.
(iv)     The plant cells with cell wall are called protoplasts.
(v)      Plant made antibodies are expensive
(vi)     Ex-vivo and In-vivo are the method of gene therapy
3.       Select the correct answer and encircle it.

(i)      The gene therapy is used to repair: (a) effective gene (b) faulty gene (c) expressive gene (d) suppressive
         gene
(ii)     Genes can be isolated from the chromosomes by cutting the chromosomes by enzymes called. (a)
         restriction nucleases (b) restriction exonucleases (c) restriction end nucleases (d) reverse transcriptase.
(iii)    PCR is done by a machine: (a) thermocycler (b) cycler (c) chemocycler (d) replicator
(iv)     DNA finger prings can be prepared from: (a) plasma (b) serum (c) lymph (d) blood
(v)      Urine is a preferable veicle for a product of: (a) ecology (b) physiology (c) morphology (d) biotechnology
(vi)     To cure Parkinson’s disease dopamine producing cells could be grafted directly into the: (a) blood (b)
         kidneys (c) liver (d) brain
(vii)    An undifferentiated group of cells is called: (a) thallus (b) callus (c) calyx (b) sepals
(viii)   A technique for the culturing of plant tissue is called: (a) anther culture (b) meristem culture (c) cells
         culture (d) cells suspension culture
(ix)     The enzyme that can be used to treat a human lysosome shortage disease is: (a) galactosidase (b) β-
         glactosidase (c) α-galactosidase (d) α- and β-glactosidase
(x)      The growth of a tissue in an artificial liquid culture medium is called (a) tissue culture (b) gene therapy (c)
         cloning (d) angioplasty
4.       Match the following columns.

         Column ‘A’                   Column ‘B’

(a)      Lambda phage         (i)     Replacing

(b)      A probe              (ii)    Replication

(c)      Genomic library      (iii)   To search the genetic library

(d)      DNA polymerase      (iv)     Pairs with DNA

                              (v)     Attaches to the host

5.       Drawn and label the diagram of:

         (a)       Cloning of a gene.

         (b)       Preparation of genomic library

         (c)       Genetic map of human X-chromosome

         (d)       Ex-vivo gene terapy in humans

                                           SECTION – II – SHORT QUESTIONS
1.        What is meant by the tem “genetic engineering”?
2.        What are restriction enzymes? How are they used recombinant DNA research?
3.        Make diagram of the process by which recombinant DNA molecules are usually constructed.
4.        How a gene library is constructed?
5.        Why is the PCR technique valuable?
6.        Distinguish between recombinant DNA technology and genetic engineering.
7.        Define DNA finger print. Briefly describe which portions of the DNA are used in DNA fingerprinting.
8.        Define the following: Fenetic engineering, biotechnology, plasmid, cloning, phage, genome, transgenic
          organism, genetherapy, totipotent, genome library.
                                        SECTION – III – EXTENSIVE QUESTIONS

1.      What is biotechnology? Describe cloning of a gene.
2.      Describe recombinant DNA technology, and mention the use of restriction end nuclease and plasmid in this
        technology.
3.      How a recombinatnt DNA expresses? How a gene of interst is location in the genome library?
4.      Describe in detail polymerase chain reaction (PCR).
5.      Describe process of DNA finger printing. What are the application of DNA fingerprinting?
6.      Discuss DNA sequencing technique with reference to electrophoresis.
7.      Give an account of the Human Genome Project.
8.      Write a not on biotechnology products.
9.      How a transgenic organism is made and how it is clones?
10.     Give a comprehensive account of gene therapy?
11.     Write notes on (a) amniocentesis (b) genetic counseling (c) tissue culture (d) genetic engineering in plants.
        (e) safety and ethical problems raised by biotechnology.
12.     Discuss use and applications of biotechnology.
                                                       ANSWERS

1.

(i)       Identical            (ii)    recombinant DNA

(iii)     genome               (iv)    Probe

(v)       Polymerase           (vi)    Taq polymerase

(vii)     three billion        (vii)   transgenic organisms

(ix)      genetic disease (x)          Totipotents

2.

(i)       F           (ii)     T       (iii)   F

(vi)      F           (v)      T

3.

(i)       b           (ii)     c       (iii)   a

(iv)      d           (v)      d       (vi)    d

(vii)     b           (viii)   d       (ix)    c
(xi)   a
4.

(a)    v     (b)   iv

(c)    iii   (d)   ii

				
DOCUMENT INFO
Shared By:
Categories:
Stats:
views:160
posted:3/19/2012
language:English
pages:16
Description: BIOTECHNOLOGY)