Since genetic cloning is a very wide topic, the focus of my paper lies mainly on the new
discoveries which might be beneficial to human beings. The focus of the first section of
the paper is on the various cloning techniques geneticists use nowadays. They techniques
included range from the simplest and suitable for all situations, to complicated and
suitable for certain areas.
The second section of the paper, the longest section, discusses five of the many
researches performed over the last five years. The researches are arranged in descending
chronological order, dating from February 1997, to April 1992. These researches are
discussed because they all have one thing in common: they may be beneficial to human
beings later on. For example, the newest entry in my paper, and perhaps the one that
shocked the whole world, was the report about the first successful clone mammal from
non-embryonic cells. This will be helpful in the future for patients waiting for organ
transplants. Scientists will be able to clone a fully functional organ, and replace it
with the damaged one. The report on the cloning of the human's morphine receptor is
advantageous to us because this helps scientists to develop new analgesics.
The third section of the paper contains a brief discussion about the advantages and the
disadvantages of genetic cloning. It speculates how our future will improve due to the
technologies we are developing, and also the biggest drawbacks which might come from it.
The last part of the paper, is the explanation of complicated terms used in this paper.
The terms which will be explained are printed in bold terms throughout the paper. This
section, the glossary, is like the ones which appears in textbooks.
New Developments or Research in Genetic Cloning
Genetic cloning is one of the many aspects which has been recently introduced to improve
our quality of live. Researchers are trying to improve our lives everyday applying
genetic engineering onto our everyday lives. Cows can be genetically altered to produce
more milk, receptors in our body could be cloned to improve our health. The techniques
and new research reported in this paper is just one tree out of the whole forest of
genetic engineering.
Part I: Techniques of Genetic Cloning
Geneticists use different cloning methods for different purposes. The method used to
identify human diseases are different than the method used to clone a sheep. The
following are used techniques in genetic cloning.
Recombinant DNA
In recombinant DNA, the desired segment is clipped from the surrounding DNA and copied
millions of times. Each restriction enzyme recognizes a unique nucleotide sequence
wherever it occurs along the DNA spiral. This nucleotide sequence, known as the
recognition site is a short, symmetric sequence of bases repeated on both strands of the
double helix. After the segment is removed, the ragged, or "sticky" ends that remain
after cleavage by the restriction enzyme allow a DNA restriction fragment from one
organism to join to the complementary ends. This method allows a foreign DNA to be
cloned in a bacteria. The result will be identical clones of the original recombinant
molecule in hundreds of copies per cell.
Polymerase Chain Reaction (PCR)
The PCR is a method of gene amplification. It is a better method than bacterial cloning
because of its greater sensitivity, selectivity, and speed. Moreover, it does not
require bacterial vectors and rapidly amplifies the chosen segment of DNA in the test
tube without the need for living cells.
In this process, the DNA sequence to be amplified is selected by primers, which are
short pieces of nucleic acid that correspond to sequences flanking the DNA to be
amplified. After an excess of primers is added to the DNA, together with a heat-stable
DNA polymerase, the strands of both the genomic DNA and the primers are separated by
heating and allowed to cool. A heat-stable polymerase lengthens the primers on either
strand, therefore generating two new, identical double-stranded DNA molecules and
doubling the number of DNA fragments.
Positional Cloning
This method is used when scientists need to identify human disease genes. The overall
strategy is to map the location of a human disease gene by linkage analysis and to then
use the mapped location on the chromosome to copy the gene. There are two essential
requirements for mapping disease genes. Firstly, there must be sufficient numbers of
families to establish linkage and, second, adequate informative DNA markers. Once
suitable families are identified, the investigators determine if diseased people in the
family have particular DNA sequences at specific locations that healthy family members do
not. A particular DNA marker is said to be "linked" to the disease if, in general,
family members with certain nucleotides at the marker always have disease and family
members with other nucleotides at the marker do not have the disease. The marker and the
disease gene are so close to each other on the chromosome that the likelihood of
crossing-over is very small.
Once a suspected linkage result is confirmed, researchers can then test other markers
known to map close to the one found, in an attempt to move closer and closer to the
disease gene of interest. The gene can then be cloned if the DNA sequence has the
characteristics of a gene and it can be shown that particular mutations in the gene
confer disease.
Cloning by Nuclear Transfer
This method has been used in mammals to provide a valuable tool for embryonic study and
as a method to multiply "elite" embryos. In this method, two different cells are
involved: an unfertilised egg and a donor cell. The donor cells are obtained by culture
of cells from a mammalian embryos over a period of several months. This enabled the
culture to consist of many genetically identical cells. To illustrate this method, sheep
are used as an example. An all white breed sheep gave the donor embryo, while the
Scottish Blackface ewes provided the recipients eggs. By micromanipulation the
chromosomes were removed from the eggs before the nucleus of the donor cell was
introduced by cell fusion. An electric current is used to trigger the egg to begin
development. These new embryos were then transferred to recipient sheep to discover if
they were able to develop to lambs. When the lambs were born, they were genetically
identical female white lambs.
Complementation Cloning by Retroviral Technique
An efficient mammalian cDNA (complementary DNA) cloning process has been developed that
utilizes retroviral cDNA expression libraries. Complentation cloning of bacterial and
yeast genetic systems has produced a lot of information for researchers. This system, in
addition to cloning genes, is also helpful in analysing the structure-function
relationship of known proteins. One advantage this system has over others is that with
the retrovirus expression system, because of its wide range of target cells, allows it to
clone surface molecules genes.
Part II: New Research In Genetic Cloning
Late February, 1997: First Cloned Mammal
In late February, Dr. Ian Wilmut and his research team from the Roslin Institute in
Edinborough made a major scientific breakthrough: they cloned a sheep from non-embryonic
cells. To create this cloned sheep the research team focused on stopping the cell cycle.
They then take the cells from the udder of a Finn Dorset ewe. In order to stop the
cells from dividing, the scientists put these cells in a culture with very low nutrition
concentration.
While this was happening, Dr. Wilmut and his team used the nuclear transfer technique
(mentioned in part I) to continue. An unfertilized egg cell is taken from a Scottish
Blackface ewe. The first step is to remove the egg's nucleus, while leaving the
cytoplasm intact. They then place the nucleus along side the cell from the Finn Dorset
ewe. An electric pulse was used to fuse them together, and a second one to imitate the
burst of energy at fertilization, triggering cell division. About five to seven days
later, the embryo was implanted into the uterus of another Blackface ewe.
September 1996: Purification and Molecular Cloning of Plx1
Cdc2, a protein which controls mitosis in a cell, is negatively regulated1 by
phosphorylation on its threonine-14 and tyrosine-15 residues. Cdc25, a protein which
dephosphorylates these residues, undergoes activation and phosphorylation by multiple
kinases at mitosis. Plx 1, a kinase that associates with and phosphorylates the NH3
(amino) end of Cdc25, was purified extensively from Xenopus egg extracts. Dr. Kumagai
and his colleagues in C.I.T. (California Institute of Technology) found that cloning its
cDNA revealed that Plx 1 is related to the Polo family2 of protein kinases. Cdc25
phosphorylated by Plx1 reacted strongly with MPM-2, a monoclonal antibody to mitotic
phosphoproteins. The team concluded that Plx1 may be a mechanism for coordinating the
regulation of Cdc2 with the progression of mitotic processes such as separating
chromosome.
November, 1995: Positional Cloning of Clock Gene, timeless
In November, 1995, Michael W. Young and his colleagues from the Laboratory of Genetics
in Rockefeller University used positional cloning to clone timeless (tim) in the fly
Drosophila. The Drosophila's gene timeless (tim) and period (per) interact, and both are
required for production of circadian rhythms. Tim is a clock gene which controls
circadian behavioural rhythms3, such as the sleep-wake cycle in humans and insect
locomotor activity cycles. The molecular cloning of the gene tim has allowed the
detection of circadian cycles in tim RNA expression. The research revealed a strong
relationship between per and tim and suggest a rudimentary intracellular biochemical
mechanism4 regulating circadian rhythms in the fly Drosophila.
January, 1995: Genetically Altered Bacteria Which Makes Ethanol From Xylose
Researchers have achieved a key step in efforts to develop genetically engineered
bacteria that can produce ethanol efficiently from plant biomass for use in alternative
transportation fuels. Scientists at the Department of Energy's National Renewable Energy
Laboratory in USA have genetically modified the bacterium Zymomonas mobilis so that it
also makes ethanol from the five-carbon sugar xylose. In its natural form, this
bacterium produces ethanol from the six-carbon sugars glucose fructose and sucrose.
Right now, ethanol is produced by yeast fermentation of glucose. These Z. mobilis
bacterium makes ethyl alcohol in five to 10 % yield than yeast. The team which made this
discovery, molecular biologist Stephen Picataggio and his colleagues, spliced two operons
from an E-coli into the genome of the Z. mobilis bacteria. One of these operons encodes
xylose assimilation, while the other encodes the pentose metabolism enzymes. The
modified bacteria grows on xylose and ferments it efficiently to ethyl alcohol. The
teams work will also improve the ethanol-producing abilities of E. coli because it is now
able to produce ethanol from pentose sugars and hexose sugars.
July 1993: Morphine Receptor Cloned
In July, 1993, a team led by Dr. Lei Yu, associate professor of medical and molecular
genetics at Indiana University School of Medicine, decoded the amino acid sequence for
the morphine receptor that is located on the surface of nerve cells.
The group isolated the sequence from a rat brain cDNA library, and since homology
between the rat and human sequence is expected to be high, Dr. Yu performed
straightforward biological technique5 to isolate the human sequence from the genome.
The most promising application of this work will be the ability to design new analgesics
that are more potent than morphine but lack the side effects caused by it. From this
research, another possibility is to find a powerful analgesic that does not become
quickly tolerated by the body as morphine does. This could bring relief to people who
suffer from chronic pain. However, the most immediate advantage of this discovery is the
ability to screen new pharmacologic compounds for their similarity to the ? receptor far
more quickly and accurately than conventional methods. The research could also have
serious significance for the understanding of narcotic addcition and how to treat people
efficiently who have become addicted to these drugs.
August 1992: The Cloning of a Family of Genes that Encode Melanocortin Receptors
Proopiomelanocortin (POMC) is expressed primarily in the pituitary and in limited
regions in the brain and periphery. It is processed into a large and complex family of
peptides with different biological activities. The three major activities include the
regulation and production of a hormone called adrenal glucocorticoid and aldosterone,
control melanocyte growth and pigment production, and analgesia.
Roger Cone of Oregon Heath Sciences University cloned the murine and human melanocyte
stimulating hormone receptors (MSH-Rs) and a human ACTH receptor (ACTH-R). The cloning
of these receptors allowed the researchers to define the melanocortin receptors as a
subfamily of receptors coupled to guanine nucleotide-binding proteins that may include
the cannabinoid receptor. Also, from the information they found in their experiment,
they found that the melanocortin receptor is the smallest guanine coupled receptor
identified to date. (in 1992)
April 1992: Cloning of the Interleukin-1 Converting Enzyme
Interleukin-1 mediates a wide range of immune and inflammatory responses. The active
cytokine is generated by proteolytic cleavage of an inactive precursor. A complementary
DNA encoding a protease that carries out this cleavage has been cloned. Recombinant
expression in cells enabled the cells to process precursor IL-1 to the mature form.
Sequence analysis indicated that the enzyme itself may undergo proteolytic processing.
The gene encoding the protease was mapped to a site frequently involved in rearrangement
in human cancers. This discovery, by Douglas Cerretti and his team provides new insight
in this field of biology and offers a new target for the development of therapeutic
agents.
Part III: Brief Discussion about the Advantages and Disadvantages of Gene Cloning
Advantages
The most appealing aspect of genetic cloning is that it will improve our lifestyle.
Fatal diseases such as AIDS could be cured by genetics. Through X-ray crystallography,
new drugs could be manufactured to stop mutation of proteins. Our quality of food will
increase, because farmers will only sell produces of the highest quality.
Disadvantages
There are a few disadvantages that will be the result of genetic cloning. The problems
will mainly arise in the agricultural area. Since future livestocks might be cloned,
that means that they will have identical immune systems. If an epidemic spread through
the animals, most of the animals, if not all, will be killed by the disease or virus in
which they are not immune to.
Huge improvements of our lifestyles have been made possible because of technological
advancements. Biotechnology, such as genetic cloning, could have dramatic impacts on
human beings in the near future. Millions of people will be benefitted if these
knowledge is put into good use.
Part IV: Glossary of Terms
primers: An already existing DNA chain bound to the template DNA to which nucleotides
must be added during DNA synthesis.
restriction enzyme: A degradative enzyme that recognizes and cuts up DNA that is
foreign to a cell.
cDNA (complementary DNA): DNA that is identical to a native DNA containing a gene of
interest except that the cDNA lacks noncoding regions because it is synthesized in the
laboratory using mRNA templates.
operon: A unit of genetic function common in bacteria and phages and consisting of
regulated clusters of genes with related functions.
genome: The complete complement of an organism's genes; an organism's genetic
material.
homology: Similarity in characteristics resulting from a shared ancestry.
analgesia: The insensibility to pain without loss of consciousness.
proteolytic processing: the hydrolysis of proteins or peptides with formation of
simpler and soluble products (as in digestion)
Notes
1 Akiko Kumagai. "Purification and Molecular Cloning of Plx , a Cdc25-Regulatory Kinase
from Xenopus Egg Extracts." Science 273 (1996): 1377.
2 Akiko Kumagai. "Purification and Molecular Cloning of Plx , a Cdc25-Regulatory Kinase
from Xenopus Egg Extracts." Science 273 (1996): 1377.
3 Michael Young. "Positional Cloning and Sequence Analysis of the Drosophila Clock
Gene, timeless." Science 270 (1995): 805.
4 Michael Young. "Positional Cloning and Sequence Analysis of the Drosophila Clock
Gene, timeless." Science 270 (1995): 805
5 Lei Yu. "? Receptor Cloned From Rat Brain cDNA Library." Molecular Pharmacol: (44)
1993: 8-12
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