New Developments or Research in Genetic Cloning: Summary

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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

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