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Naqibullah Jogezai
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Date Posted: 03:04:47 12/23/05 Fri
Author Host/IP: ntc.net.pk/202.83.175.123
In reply to:
Naqibullah jogezai
's message, "Re: More Information About Biotechnology" on 03:43:20 12/22/05 Thu
>>
>>Welcome to BioTech! Our goal is to enrich the public's
>>knowledge of biology and chemistry. We aim to serve
>>everyone from high school students to professional
>>researchers. For more information
>>
>>
>>What is "BioTech"?
>>Located in Jinna Town Quetta at University of BUITMS,
>>BioTech is a hybrid biology/chemistry educational
>>resource and research tool on the World Wide Web.
>>BioTech is intended to be a learning tool that will
>>attract students and enrich the public's knowledge of
>>biology issues in the world today. At the same time,
>>BioTech is also a research tool for those already
>>involved in the broad subject of biology. By providing
>>information about resources, as well as avenues for
>>further exploration, we intend to open the doors of
>>biology resources to post-secondary students,
>>researchers, and faculty.
>>Our Mission:
>>Our mission is to make BioTech as useful a tool to a
>>high school student as it is to a postdoctoral fellow.
>>We aim to educate those who may not have as much
>>experience with biology and biotechnology while at the
>>same time providing quick access to biology-related
>>resources for those who are dealing with much more
>>specific and detailed information. We do not wish to
>>exclude anyone from this project -- we will assist
>>those who need assistance and merely open doors for
>>those who are interested in finding information on
>>their own.
>>Our Goal:
>>Our goal is to utilize the skyrocketing success of the
>>World Wide Web as a means of bringing information
>>about the broad scope of biology into view of as many
>>people from many educational levels as possible. We
>>intend to educate, facilitate, inform, and direct
>>attention to as many sources of biology-related
>>information as possible.Biotechnology:
>A new era for plant pathology
>and plant protection.
>
>Plant biotechnology ushers in a new era for plant
>scientists working to maintain healthy plants,
>optimize crop yields, and minimize pesticide usage.
>One of the ultimate aims of agricultural biotechnology
>is to feed an expanding world population. A recent
>survey by The Economist shows that the world
>population has increased by 90% in the past 40 years
>while food production has increased by only 25% per
>head. With an additional 1.5 billion mouths to feed by
>2020, farmers worldwide will have to produce 39% more
>grain (The Economist, March 25, 2000). These survey
>results aptly describe the food production challenges
>facing the global community of farmers and consumers
>in the new millennium and the dimension of the debate
>on the risks and benefits of developing genetically
>engineered crop plants to meet the increasing global
>food demand while preserving the environment.
>
>Genetic engineering has the potential to provide a
>cornucopia of beneficial plant traits, particularly an
>enhanced ability to withstand or resist attack by
>plant pathogens. New approaches to plant disease
>control are particularly important for pathogens that
>are difficult to control by existing methods. The
>percentage of crop losses caused by plant pathogens,
>insect pests, and weeds, has steadily increased to 42%
>worldwide, accounting for $500 billion dollars worth
>of damage (Oerke et al., 1994). In the United States
>alone, crop losses due to plant pathogens amount to
>$9.1 billion dollars, while worldwide, plant diseases
>reduce crop productivity by 12% (Food and Agriculture
>Organization, 1993). Worldwide, pesticide applications
>costing $26 billion dollars annually are applied to
>manage pest losses. Genetically engineered plants
>resistant to plant pathogens can prevent crop losses
>and reduce pesticide usage. This feature article
>provides a current perspective on four major areas of
>research and application of plant genetic engineering
>for resistance to plant pathogens.
>
> Enhancing resistance with plant genes: Scientists
>from all over the world are investigating the
>biochemical nature of, and the signals involved in, a
>plant’s reactions to pathogen invasion and disease
>development. Plant resistance genes and the genes
>involved in resistance reactions are being identified
>and engineered into crop plants to protect them
>against plant diseases. This rapidly advancing field
>of investigation is described in this feature under
>Enhancing a plant’s resistance with genes from the
>plant kingdom.
>
> Pathogen derived resistance: Plants can be protected
>from diseases with transgenes (genes that are
>engineered into plants) that are derived from the
>pathogens themselves, a concept referred to as
>pathogen-derived resistance. For example, plant viral
>transgenes can protect plants from infection by the
>virus from which the transgene was derived. Genetic
>engineering of plants for viral resistance is a
>thriving area of research and is described in this
>feature with special emphasis on research being done
>at Cornell University, Geneva, NY, under Genetic
>engineering: A novel and powerful tool to combat plant
>virus diseases.
>
> Antimicrobial proteins: Another area of
>investigation involves peptides and proteins with
>antimicrobial properties that when produced by plants
>have the potential to strengthen plant resistance to
>fungal and bacterial plant pathogens. Fungi, insects,
>animals, and humans all contain genes encoding
>antimicrobial compounds. This use of antimicrobials to
>improve plant resistance to pathogens is described in
>this feature with special emphasis on research being
>done at Cornell University, Geneva, NY, under Using
>antimicrobial proteins to enhance plant resistance.
>
> Plantibodies: Although plants have mechanisms to
>protect themselves against pathogen attack, in
>contrast to animals, there is no "immune system" per
>se in plants. With the advent of genetic engineering,
>plants can be engineered to express an antibody
>against a protein crucial for pathogenesis resulting
>in a level of immunity or resistance to the pathogen.
>This promising approach is described under
>Plantibodies: an animal strategy imported to the plant
>kingdom to fight back pathogens.
>
>Biotechnology is now a lightning rod for visceral
>debate, with opposing camps making strong claims of
>promise and peril. The debate involves not only
>scientific but also political, socio-economic,
>ethical, and philosophical issues (Wambugu 1999, Hails
>2000, Ferber 1999, Trewavas 1999, Sagar et al. 2000).
>
>This feature article provides a glimpse of the
>application of biotechnology to plant improvement. The
>dawn of a new era in plant pathology and plant
>protection is upon us. Biotechnology has rewritten the
>scope of scientific investigation, broadened the
>avenues to resistant plants, and challenged us to take
>safe and careful steps. Like any other new technology,
>much still needs to be done before the full potential
>of agricultural biotechnology is realized. As more and
>more plant biotechnology products become available,
>studies to evaluate the risks associated with
>biotechnology must be intensified. Findings from such
>studies must be easily accessible to the general
>public. The risks associated with this technology must
>be addressed and the benefits should be kept in mind.
>We are confronted with biotechnology’s vast
>perspective and this astounding view has expanded the
>very foundation of our understanding of life.
Recombinant DNA is DNA that has been created artificially. DNA from two or more sources is incorporated into a single recombinant molecule.
Treat DNA from both sources with the same restriction endonuclease (BamHI in this case).
BamHI cuts the same site on both molecules
5' GGATCC 3'
3' CCTAGG 5'
The ends of the cut have an overhanging piece of single-stranded DNA.
These are called "sticky ends" because they are able to base pair with any DNA molecule containing the complementary sticky end.
In this case, both DNA preparations have complementary sticky ends and thus can pair with each other when mixed.
DNA ligase covalently links the two into a molecule of recombinant DNA.
To be useful, the recombinant molecule must be replicated many times to provide material for analysis, sequencing, etc. Producing many identical copies of the same recombinant molecule is called cloning. Cloning can be done in vitro, by a process called the polymerase chain reaction (PCR). Here, however, we shall examine how cloning is done in vivo.
Cloning in vivo can be done in
unicellular prokaryotes like E. coli
unicellular eukaryotes like yeast and
in mammalian cells grown in tissue culture.
In every case, the recombinant DNA must be taken up by the cell in a form in which it can be replicated and expressed. This is achieved by incorporating the DNA in a vector. A number of viruses (both bacterial and of mammalian cells) can serve as vectors. But here let us examine an example of cloning using E. coli as the host and a plasmid as the vector.
Plasmids
Plasmids are molecules of DNA that are found in bacteria separate from the bacterial chromosome. They:
are small (a few thousand base pairs)
usually carry only one or a few genes
are circular
have a single origin of replication
Plasmids are replicated by the same machinery that replicates the bacterial chromosome. Some plasmids are copied at about the same rate as the chromosome, so a single cell is apt to have only a single copy of the plasmid. Other plasmids are copied at a high rate and a single cell may have 50 or more of them.
Genes on plasmids with high numbers of copies are usually expressed at high levels. In nature, these genes often encode proteins (e.g., enzymes) that protect the bacterium from one or more antibiotics.
Plasmids enter the bacterial cell with relative ease. This occurs in nature and may account for the rapid spread of antibiotic resistance in hospitals and elsewhere. Plasmids can be deliberately introduced into bacteria in the laboratory transforming the cell with the incoming genes.
An Example
(courtesy of David Miklos and Greg Freyer of the Cold Spring Harbor Laboratory, who used these plasmids as the basis of a laboratory introduction to recombinant DNA technology that every serious biology student — high school or college — should experience!)
pAMP
4539 base pairs
a single replication origin
a gene (ampr)conferring resistance to the antibiotic ampicillin (a relative of penicillin)
a single occurrence of the sequence
5' GGATCC 3'
3' CCTAGG 5'
that, as we saw above, is cut by the restriction enzyme BamHI
a single occurrence of the sequence
5' AAGCTT 3'
3' TTCGAA 5'
that is cut by the restriction enzyme HindIII
Treatment of pAMP with a mixture of BamHI and HindIII produces:
a fragment of 3755 base pairs carrying both the ampr gene and the replication origin
a fragment of 784 base pairs
both fragments have sticky ends
pKAN
4207 base pairs
a single replication origin
a gene (kanr) conferring resistance to the antibiotic kanamycin.
a single site cut by BamHI
a single site cut by HindIII
Treatment of pKAN with a mixture of BamHI and HindIII produces:
a fragment of 2332 base pairs
a fragment of 1875 base pairs with the kanr gene (but no origin of replication)
both fragments have sticky ends
These fragments can be visualized by subjecting the digestion mixtures to electrophoresis in an agarose gel. Because of its negatively-charged phosphate groups, DNA migrates toward the positive electrode (anode) when a direct current is applied. The smaller the fragment, the farther it migrates in the gel.
Ligation Possibilities
If you remove the two restriction enzymes and provide the conditions for DNA ligase to do its work, the pieces of these plasmids can rejoin (thanks to the complementarity of their sticky ends).
Mixing the pKAN and pAMP fragments provides several (at least 10) possibilities of rejoined molecules. Some of these will not produce functional plasmids (molecules with two or with no replication origin cannot function).
One interesting possibility is the joining of
the 3755-bp pAMP fragment (with ampr and a replication origin) with the
1875-bp pKAN fragment (with kanr)
Sealed with DNA ligase, these molecules are functioning plasmids that are capable of conferring resistance to both ampicillin and kanamycin. They are molecules of recombinant DNA.
Because the replication origin, which enables the molecule to function as a plasmid, was contributed by pAMP, pAMP is called the vector.
Transforming E. coli
Treatment of E. coli with the mixture of religated molecules will produce some colonies that are able to grow in the presence of both ampicillin and kanamycin.
A suspension of E. coli is treated with the mixture of religated DNA molecules.
The suspension is spread on the surface of agar containing both ampicillin and kanamycin.
The next day, a few cells — resistant to both antibiotics — will have grown into visible colonies containing billions of transformed cells.
Each colony represents a clone of transformed cells.
However, E. coli can be simultaneously transformed by more than one plasmid, so we must demonstrate that the transformed cells have acquired the recombinant plasmid.
Electrophoresis of the DNA from doubly-resistant colonies (clones) tells the story.
Plasmid DNA from cells that acquired their resistance from a recombinant plasmid only show only the 3755-bp and 1875-bp bands (Clone 1, lane 3).
Clone 2 (Lane 4) was simultaneous transformed by religated pAMP and pKAN. (We cannot tell if it took up the recombinant molecule as well.)
Clone 3 (Lane 5) was transformed by the recombinant molecule as well as by an intact pKAN.
Cloning other Genes
The recombinant vector described above could itself be a useful tool for cloning other genes. Let us assume that within its kanamycin resistance gene (kanr) there is a single occurrence of the sequence
5' GAATTC 3'
3' CTTAAG 5'
This is cut by the restriction enzyme EcoRI, producing sticky ends.
If we treat any other sample of DNA, e.g., from human cells, with EcoRI, fragments with the same sticky ends will be formed. Mixed with EcoRI-treated plasmid and DNA ligase, a small number of the human molecules will become incorporated into the plasmid which can then be used to transform E. coli.
But how to detect those clones of E. coli that have been transformed by a plasmid carrying a piece of human DNA?
The key is that the EcoRI site is within the kanr gene, so when a piece of human DNA is inserted there, the gene's function is destroyed.
All E. coli cells transformed by the vector, whether it carries human DNA or not, can grow in the presence of ampicillin. But E. coli cells transformed by a plasmid carrying human DNA will be unable to grow in the presence of kanamycin.
So,
Spread a suspension of treated E. coli on agar containing ampicillin only
grow overnight
with a sterile toothpick transfer a small amount of each colony to an identified spot on agar containing kanamycin
(do the same with another ampicillin plate)
Incubate overnight
All those clones that continue to grow on ampicillin but fail to grow on kanamycin (here, clones 2, 5, and 8) have been transformed with a piece of human DNA.
Some recombinant DNA products being used in human therapy
Using procedures like this, many human genes have been cloned in E. coli or in yeast. This has made it possible — for the first time — to produce unlimited amounts of human proteins in vitro. Cultured cells (E. coli, yeast, mammalian cells) transformed with the human gene are being used to manufacture:
insulin for diabetics
factor VIII for males suffering from hemophilia A
factor IX for hemophilia B
human growth hormone (GH)
erythropoietin (EPO) for treating anemia
three types of interferons
several interleukins
granulocyte-macrophage colony-stimulating factor (GM-CSF) for stimulating the bone marrow after a bone marrow transplant
granulocyte colony-stimulating factor (G-CSF) for stimulating neutrophil production, e.g., after chemotherapy and for mobilizing hematopoietic stem cells from the bone marrow into the blood.
tissue plasminogen activator (TPA) for dissolving blood clots
adenosine deaminase (ADA) for treating some forms of severe combined immunodeficiency (SCID)
angiostatin and endostatin for trials as anti-cancer drugs
parathyroid hormone
leptin
hepatitis B surface antigen (HBsAg) to vaccinate against the hepatitis B virus
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