New Developments or Research in Genetic Cloning: Summary
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
