Chapter 3

 Scientific Aspects

 

 

3.1. The Cell :

Regardless of the complexity of the organisms, almost all life processes at molecular level are the same. Life starts from a cell in which all the chemical processes and reactions take place. The brain of the cell is the nucleus which contains the genetic material, “the DNA” molecules as illustrated in (Fig. 5). The molecule, which is the icon of the 20th Century, dictates all the complex chemical reactions in the cell :

 

3.1.1. DNA Molecules

The DNA structure was discovered in 1953 by two Cambridge scientists James Watson and Francis Crick who won the race to discover the structure of the DNA molecule, the blue print that builds and maintains all living things. They were awarded the Nobel Prize in Physiology and Medicine in 1962 for this discovery.

In simple terms DNA molecules structure as illustrated in (Fig 6), is composed of :

1. Sugar dexoyribose

2. Phosphate

3. Nitrogen (nucleoside) bases of which there are 4 kinds - Adenine complements Thymine and Guanine complements Cytosine

The structure resembles a twisted spiral staircase usually referred to as double helix, with sugar-phosphate backbone to the outside and are held together by hydrogen bonds between complementary bases resembling the steps.

  

The main functions of DNA molecule are :

1. Storage of genetic information, where all the information required to produce and maintain a unique organism are contained within DNA

2. Inheritance, The information stored in DNA is transmitted to all descendent.

3. Expression of genetic message, the information stored in DNA molecule is transcribed and translated into specific proteins that are required by the cell.

The way in which genetic information is stored, inherited and expressed is basically the same in all-living organism. This explains the uniformity of creation and uniqueness of creator.

DNA molecules performs two kind of reactions :

1- Replication of the DNA molecule: Is the reaction when the DNA molecule replicate itself during cell division, the mechanism of replication in simple terms is as follows :

* Replication start at a genetically specified point on the DNA molecule where specific enzyme (DNA Polymerase) catalyses this reaction by attaching to DNA molecule at that point. 

* The bond between A-T and G-C start to break

* A bubble formed at the site of breaking exposing the bases to pair with the nucleoside triphosphate, which are activated nucleotide building blocks.

* Each newly extended DNA strand is complementary to the original strand, which serve as a template.

* Each double strand now containing one template strand and newly synthesised strand.

* The newly synthesized strands on both templates travel simultaneously in opposite direction along the original DNA strands and once they meet at a point called terminus, the two completed strands separate.

* Both strands are identical but anti parallel, one strand runs from 5’-3’ and the other runs from 3’-5’.

These steps are illustrated in Fig (7).

 

2- Gene expression : Involve two steps :

* Transcription

* Translation

These two steps are crucial in protein synthesis.

3.2. Protein Synthesis

The nucleus responds to the demand of the cell’s messenger which receive signal from the outside, for example a hormone which ring a bell when it gets to the surface of the cell. The messenger carries the message to the nucleus to activate specific gene to synthesise specific protein. The synthesis of the protein is a major activity. Proteins form enzymes that catalyse cellular reactions, and contribute to the formation of antibodies, and all cells. Protein is required for all living things.

Protein synthesis starts with DNA. In general, protein synthesis is divided into 2-steps :

3.2.1. Transcription : the mechanism of transfer “transcription” of genetic code and polymerisation of ribonucleotide building blockes into molecule of RNA-either messanger RNA (mRNA), transfer RNA (tRNA) or ribosomal (rRNA). The information in DNA is  transcribed (rewritten) : C pairs G and A pairs with U (Uracil).

3.2.2. Translation : the mRNA molecule then carry the genetic code out of the nucleous into the cytoplasm to small factories “ribosomes”, where they translate these codes into amino acids. The amino acids connected to each other through peptide bonds to form specific protein.

The sequence of events is as follows :

* DNA Molecules consist of two strands held together by weak hydrogen bond. Transcription begin with detachment “unzipping” and the formation of a new single stranded mRNA (messenger RNA) which makes its way to the cytoplasm.

* mRNA, then translated inside small factory units “ribosomes” into amino acids. In some viruses an enzyme called reverse transcriptase, which reverse this process where a double stranded DNA formed from a messenger RNA. The discovery of this enzyme contributes immensely to genetic engineering and sequencing techniques.

* These amino acids are connected to each other through peptide bond to form a protein.

These events are illustrated simply in Fig (8)

  

3.3. Mutation

Mutation means a change in the sequence of a base pair of specific gene. This change has an inevitable effect on the protein encoded by that gene.

Mutations can occur spontaneously (spontaneous mutation) or may be induced experimentally by a mutagen. A mutagen is any chemical, physical factor or condition that brings about a change in the DNA material.

Mutation can occur on two levels.

1- chromosomal level  ®   chromosomal mutation

2- Gene level     ®     gene mutation

 

3.3.1.  Chromosomal Abnormalities:

Abnormalities of the chromosomes are usually classified into :

1- Numerical abnormalities, which refer to the abnormal number of the normal chromosomes in the somatic cells. Such as the case of Down’s syndrome which is caused by possession of an extra chromosome 21 as shown in Fig (3). The affected person has tripled chromosome 21 instead of diploid in the normal condition. Those individuals with this condition suffer from mental and physical abnormalities. Other examples are in conditions such as Klinefelter’s syndrome where affected individuals have 2 X-chromosomes in addition to Y chromosome. Those individuals are sterile and have tall stature. Also there might be less number of chromosomes as in the case of Turner’s syndrome where the affected individuals posses one X-chromosome and no Y-chromosome resulting in 45 chromosomes rather than normal 46 chromosomes.

The affected female would be sterile and have short stature.

2- Structural abnormalities, where the somatic cells contain one or more abnormal chromosomes. These abnormalities may result from chromosomal breakage, which result in loss or duplication of part of chromosome. Chromosome breakage is usually random. Structural chromosomal abnormalities, show some association with increased paternal and maternal ages, (Weatherall, 1985).

Genetic diseases and congenital malformations occur in approximately 2-5% of all live births, account for up to 30% of paediatric admission to hospital and are an important cause of death under the age of 15 years, (Weatherall, 1985). Genetic diseases produce a major burden on the health services. It is impossible to measure the Human misery caused by these disorders.        

  

3.3.2. Genetic Mutation

To understand the gene mutation it would be beneficial to refer to base pairings :

In DNA molecules there are four bases, Adenin (A) in one strand is specifically paired with thymine (T) on the other strand by two hydrogen bonds. Guanine (G) pairs with Cytocine (C) by three hydrogen bonds. This base pairing holds two linear DNA strands together as illustrated in Fig (6).

 

3.3.2.1. Concept of Gene Mutation

The cell functions are the consequence of its synthesised protein. Different kinds of protein are synthesised in response to different kinds of demands. Each of these proteins has a specific amino acid sequence. Hence any mutation in the gene affects the expression of protein - coding genes.

The diagram in Fig (9) illustrates how the alteration of a gene results in alteration of a phenotype as a result of changing the function of protein.

 

The consequence of a gene mutation falls into two categories :

1. Missense mutation : which causes the substitution of one amino acid for another as in Fig (10), where a transition mutation from AT to GC changes the codon from Lysin to glutamic acid. A change in one amino acid could have very serious consequences with regard to the function of the protein as in sickle cell anaemia, Where one amino acid glutamic acid is replaced by Valine Fig (11). A similar change in a sequence for another protein may not affect the function of the protein at all.

 

 

2. Nonsense mutation : which cause premature termination of synthesis of the polypeptide as in Fig (12). The changes in the base pairing in the DNA leads to changes in the mRNA codon. The codon instead of being translated into an amino acid, leading to growing polypeptide, will be changed to a chain-terminating (nonsense) codon (e.g. UAG). This will result in premature termination of translation. 

 

There are three kinds of gene mutation :

1. Substitution or rearrangement of the base pairs for another.

2. Addition of one or more base pairs.

3. Deletion of one or more base pairs.

These mutational changes are illustrated clearly in Fig (13)

 

Since we are dealing with lettering system we might draw this analogy used by (Moses, 1995), to make it simple for people not familiar with biochemistry.

A simple “message” in English encoded in a linear sequence might be :

TTHECATSATANDATETHERATANDRANOFF

It become more intelligible if a “reading frame” of three letters to each word is imposed upon the linear message as in Fig (14). You will get a complete sentence, which have a meaning and could be compared to a normal protein (Fig 14. A).

If a single error  (italic red colour) is introduced into the message as in (B), which we refer to in genetic as mutation. Then after segregation into three-letter frame, the sentence lost its meaning. This illustrates the delicate and the sophisticated manner that the DNA molecule works within. Given the fact that each Human cell contain 3 billions of genetic letter any error in any single letter will lead to mutation which sometime could be fatal and most times lead to different genetic diseases as we have seen in sickle cell anaemia Fig (11).

Currently there are more than 4000 genetic disease resulted from changes to a single gene. Most of these changes are rare, but many cause severe suffering and often lead to early death.

The number of people affected by genetic diseases worldwide is roughly 2% of all live births every year (Garvin, 1995). Most of the genetic mutations are maintained in the population by the passage of the genes from parents to offspring, or by steady input of new mutations. Not all the genetic anomalies run in families, some may result during the formation of gametes (sex cells), or in the early development of foetus or even exposure to radiation or other chemical agents.

THECATSATANDATETHERATANDRANOFF

 

 

Steve Jones in his brilliant book “The language of the Gene”, the winner of the Rhone-poulence prize of 1994 described genetics as follow : “genetics as a language, a set of inherited instructions passed from generation to generation. It has a vocabulary -the genes themselves- a grammar, the way in which the inherited information is arranged, and a literature, the thousands of instructions needed to make a Human being. The language is based on the DNA molecule”.

Bill Clinton President of the United States joined Tony Blair the Prime Minister of Britain in praising the feat at a satellite-linked press conference on 6/6/00. He announced “Today we are learning the language in which God created life. We are gaining ever more awe for the complexity, the beauty, the wonder of God’s most divine and sacred gift”. (The Daily Telegraph, 7/6/00).

 

3.4. Outline of Genetic Engineering :

The tool that makes the Human Genome project possible is genetic engineering. The tool that revolutionised the science of life and contribute immensely to the welfare of the Human being and promises to solve many of the greatest problems facing the Human being and environment, such as food shortage, diseases, pollution and energy crises. It relies mostly on microorganisms that can be genetically modified to serve certain purposes. Given the fact that microorganisms have a rapid growth rate and are highly versatile, this provides huge potential for new industries.

Genetic engineering Biotechnology alliance as used by (Wan Ho, 1999) will enable development of the 21st Century in all aspects of life, to compare with those of physics and chemistry’s contribution in the 20th Century.

As any other technology, Genetic engineering Biotechnology raises very profound questions relating to environment, economy, politics and religion. The questions that are always raised when talking about this technology are : is Genetic engineering biotechnology really bringing us cheap and effective drugs, disease free crops and clean environment as many biotech companies promise ? (Hamilton, 998), or is it posing threats to the environment, Human health and animal welfare ? (Reiss and straughan, 1996).

Is Genetic engineering a miracle or menace (Walgate, 1990), a curse or blessing, a Silicon Valley which is capable of getting the entire nations into a new era of prosperity or doom and gloom.

The answer to all these questions depends on how it is used and controlled.

In general there are two campaigns, one who champion novel inventions as a sign of a better future and this view held by many Biotechnology companies. On the other side, those who warn about the dangers of this technology and its misuse as explained by (Wan Ho, 1999) “Genetic engineering biotechnology is unprecedented intimate alliance between bad science and big business, which will spell the end of humanity as we know it, and of the world at large. Genetic engineering biotechnology is inherently hazardous; but the genetic determinist mentality that misinforms both practitioners and the public takes hold of peoples consciousness, making them act unquestioningly to shape the world to the detriment of Human beings and all its other inhabitants”. The impact of genetic engineering on shaping our life is also explained clearly by (Nossal, 1985). 

Recent surveys have found that people are almost equally divided in their expectations about genetic engineering/biotechnology. Two thirds believe it offers benefits, yet two thirds also believe it holds potential dangers (a large number of people believe both are true).

This may explain and reflect the lack of knowledge of this technology. This was confirmed by a study carried out by Food and Drink Federation in Sep. 1996. When they asked people how much they knew about biotechnology, only one third of adults in Britain claimed to have any knowledge at all; two thirds of adults knew nothing at all about biotechnology or never heard of it as illustrated in the graph (Fig 15).

   

Genetic engineering is an umbrella term, which cover a wide range of techniques involved in changing the genetic material-DNA Code- in a living organism.

The DNA-Code contain all the information stored in a long chemical molecule which determine the nature of the organism, whether it is an amoeba, a pine tree, an octopus a cow or human being.

The primary aims of Genetic Engineering are the alteration of Genetic Material of an organism by Scientists who aim to make a good product out of their design. This goal contributes largely to the welfare of Human beings and the environment in the last four decades, in particular forcing the microbes to make products they would not naturally make. The following example drawn from my experience with Human growth hormone production, which clearly demonstrate the importance of this technology to the well being of Human. Until recently (1984), the traditional production source of hGH was Human pituitary gland, so it was not widely available. To treat a child for 8 months, 400 brains are needed to extract the required hGH quantity. It is also possible to contaminate the product with prion (infectious protein), associated with serious brain disease Creusfeldt Jakob disease CJD (Ahmed, 1997 c).

The principle techniques are as follows :

1. Isolate the gene of interest (eg : Human growth hormone gene).

2. Isolate a suitable microbe to work with (eg :  E coli)

3. Isolate a circular genetic material (plasmid) from the microbe, which acts as a vehicle for transporting the desired gene.

4. Cut the plasmid and the gene with the same restriction enzyme, which cut at the same place (Hae III restriction enzyme is used in this example).

5. Ligate the gene with the plasmid to form recombinant plasmid.

6. Insert the recombinant plasmid back in the microbe.

by a process called transformation.

7. Fermentation and then extract the desired product as illustrated in the following example of Human growth hormone production (Fig 16)

 

3.5. Polymerase Chain Reaction (PCR) :

It is the technique that revolutionaries the Molecular Biology since the 1986 and was considered as strike of genius. This technique contributes immensely to the Human Genome project. It mainly involves the isolation of a fragment of DNA of interest and copy it millions of times in a very simple but extraordinary powerful and efficient technique. Now each laboratory has this small, but crucial instrument as prerequisite for any genetic engineering experiment. The technique involves the following steps :

1- The DNA segment of interest is chosen along the DNA molecule.

2- Two short oligonucleotides are flanged to both ends of the DNA fragment and act as primers for the DNA synthesis reaction.

3- Amplification of the flanged fragment carried out by DNA polymerase I enzyme. This enzyme is thermostable and is resistant to denaturation by high temperature, which is extracted from a bacterium that lives in hot spring Thermus aquaticus. This enzyme is an essential requirement for PCR technique.

4- Amplification process which involves 3 cycles of temperature profile:

* Denaturing temperature (94∞C) that breaks the double strands of DNA into 2 single strand, which both act as template.

* Annealing temperature (42 ∞C) at which the primers attach to the template.

* Polymerisation or extension temperature (74 °C), which is the actual DNA synthesis. Fig (17) illustrates these profiles. After 30 cycle millions of one single strand DNA are formed: 228 = 268 435 456 fragments could be generated in one run. For more details refer to the Web site (WWW.nhgri.nih.gov); and (Mullis and Faloona, 1987.

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