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Topics 1. 2. 3. 4. 5. Molecular Organization of Genes Genes as Hereditary Carriers Basic Tools for Gene Investigation Recombinant DNA Technology Regulation of Gene expression: Prokaryotes and Eukaryotes Applications in Biotechnology
Molecular Organization of Genes
Cell is the fundamental unit of all living organisms. The deoxyribonucleic acid (DNA) serves as the life of the cell. This DNA is enveloped together to form highly condensed structures called as ‘chromosomes’. Every individual has its own specific pair of chromosomes. In humans, the count is 46. Almost all the cells in human body, expect the reproductive cells possess 23 pairs of chromosomes, which accounts to a total of 46. The reproductive cells, like sperm cells and egg cells have just 23 unpaired chromosomes. This enables them to form a diploid (2n condition) zygote, by receiving half (n) of the chromosomes from the mother's egg cell and the other half (n) from the father's sperm cell.
The chromosomes are of two types according to their nature. Body chromosomes direct the activities of the body whereas sex chromosomes determine the sex of the individual. Sex chromosomes are of two types X and Y.
A male baby receives an X chromosome from his mother and a Y chromosome from his father. A female gets an X chromosome from each parent.
‘Genes’ can be defined as the segments of DNA which are clumped on the chromosomes. In molecular terms, a gene can be defined well as the entire nucleic acid sequence that is necessary for the synthesis of a functional polypeptide or a protein molecule. A gene also includes all the DNA sequences which are necessary for the synthesis of a particular RNA transcript.
Even most of the genes are transcribed into messenger RNAs (mRNAs), which encode for the proteins; undoubtedly some DNA sequences are transcribed into RNAs that do
not encode proteins like tRNAs and rRNAs). Such types of DNA regions are commonly referred to as tRNA and rRNA genes, and the final products of these genes are RNA molecules and not proteins.
Genes are specifically referred as the ‘hereditary units’ and have the capability to establish definite characteristics of humans, such as skin colour or height.
Since the new born baby possesses one chromosome in each pair from each parent, two of every particular gene will be present. In some cases, the characteristics may result from a single gene, whereas in some others, it is the result of gene combinations. An innumerous number of gene combinations are possible as every individual is carrying 25,000 to 35,000 different genes.
Genes as Hereditary Carriers:
Heredity is the transient of genes from one generation to the next generation. The inheritance of parental characteristics helps to build a new individual, with the specific characteristics like a man with straight or curled hair, a girl with brown or blue eyes, as determined by the gene combinations. When the individual grows, along with the genes, the external environments he/she interacts with also mould his talents and interests.
The structure of the DNA molecule as suggested by Watson and Crick is indistinguishable among all living organisms, from unicellular one to the large developed multicellular ones like humans and plants. It consists of the three basic units: Deoxyribose sugar, phosphate, and four types of nitrogenous bases: adenine (A), thymine (T), guanine (G), and cytosine (C). A sugar, phosphate, and nitrogenous base together constitute a unit which is named as ‘nucleotide’. ‘Base pairing’ is a unique feature of DNA in which among the four bases, A always pair with T and G always with C. DNA forms double helical structure which is formed by two strands twisted together like a ladder. This gives the DNA molecule a live three dimensional structure.
Working of Genes:
The alternate sugar phosphate backbones are strung together in extremely coiled structures of DNA. Inside every tiny cell of the living organisms nearly 3 billion miles (if they are elongated straight without coils) long DNA thread is embedded. Thus the genes present on the DNA serve as the codes for manufacturing proteins, which are the essential chemicals that enable the body to work and grow.
The instructions for construction protein products like the enzymes which help in digestion of food, or contraction of muscles. DNA gets duplicated during cell division, and this information content is passed on in the form of genes to the newly formed daughter cells. Genes can serve as dominant or recessive. Even if there is only one copy of that gene is present in the pair and its effect is still present, we can conclude such a gene as a dominant one. If we need to declare an individual as possessing recessive characteristic, the gene must be present on both chromosomes of the pair. Such genes are termed as the ‘recessive’ genes. DNA molecule is the entirety of information needed to make a replica of any organism. If we consider the chromosomes as a big library, he DNA will be acting like a ‘book’, the ‘genes’ will be the sentences written in the book framed by the words of ‘codons’. The codons are also constructed with the alphabets of ‘nucleotide bases’. Guided by the base pairing rule, the four nitrogenous bases can be paired exactly in only one way. Hence the arrangement of base pairs in one strand will dictate the ordering of bases on the other strand of DNA. When a cell is getting duplicated, the two complementary strands unwind and each of them serves as the template (basis) for generating a new strand. As a result, two new strands which are identical to the original parental strands will be formed. One parental and its newly formed complementary daughter strand will wind together to form a newly synthesized DNA molecule. Thus two new DNA molecules, which are identical in base pairings to the original molecule, are formed at the end of duplication.
Nature of Codons:
The nitrogenous bases individually cannot be able to pass any information. Instead, they act as strings of three together, called as a ‘codon’. The four bases can be arranged in only sixty-four distinctive sets of three. In fact, DNA's store of information comprises more than just four bases arranged into sixty-four different codons, so that they can be arranged in innumerable number of ways. The prime function of the codons is to direct instructions for specifying and ordering amino acids. Proteins, the building blocks of our body contain amino acids as their fundamental units of structure. Amino acids also possess the pride of carrying out all the biological processes thus serving as the basic units of biochemistry. There are only twenty amino acids found in proteins, and the codes for ordering them are universal. This means that the sequence of the nucleotide letters to specify the amino acid is the same for almost all the living organisms. It is not specific for birds, mammals or humans. In real, the amino acids can be arranged in any order to construct thousands of proteins with exclusive functions. Certain proteins which are known as ‘enzymes’ act as catalysts for a number of reactions inside our body without which those reactions cannot occur. There are certain
other specific proteins called as ‘structural proteins’ which help to build the cells and tissues in our body. Some codons function as the units containing instructions to stop or start manufacturing a protein. Thus these codons present in the ‘genes’ not only utter the type of protein the organism needs to synthesize, but also about the arrangement of them inside the cells of the body. In simple terms, a single codon contains the instructions for the formation of one amino acid. The sequences of codons specify the production of a particular protein. If such codons are grouped together to form a set of specific proteins which can direct a particular characteristic of an individual, they are called ‘genes’.
Basic Tools for Gene Investigation:
The aggressive advance in the field of biotechnology is resulted from the wide spread applications of this tools of gene investigation. Important among them are (i)
DNA Sequencing: Using this method of DNA sequencing, the exact nucleotide sequence of a molecule of DNA can be determined. Sequencing process provides a wide range of information regarding the construction of a particular gene, the control of gene expression, and also the structure of the protein manufactured by it. Polymerase Chain Reaction (PCR): The polymerase chain
reaction leads to a billion fold augmentation of a segment of DNA. A single molecule of DNA can be amplified into quantities that permit the characterization and manipulation of genes contained in it.
The basic purpose of a PCR (Polymerase Chain Reaction) is to create a huge number of copies of a particular gene. This hugely amplified gene can be used as the starting template for the sequencing process. Basically, Polymerase Chain Reaction includes three important steps, which form the underlying principle of PCR. Three major steps occur in a PCR, which are repeated for 30 or 40 cycles. This is carried on an automated cycler, which can heat, also cool the tubes with the reaction mixture in a very short period of time.
Denaturation at 94°C:
During the denaturation process, the double strand of the duple DNA melts open to form the single stranded DNA. Also, all reactions carried out by the enzymes take a hault.
Annealing at 54°C:
The primers joggle around, caused by the Brownian movement. Abrupt cooling into 54°C allows the primers hybridize into a DNA strand. The primers are typically 20 to 30 nucleotides long.
Extension at 72°C:
This is the optimal temperature for the Taq DNA polymerase. This thermal stable polymerase comes from Thermus aquaticus, wchih is a thermophilic bacterium living in hot springs. The bases which are complementary to the template are coupled to the primer on the 3' side. The Taq polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side; bases are added complementary to the template. This powerful technique is being used to detect pathogens and hereditary diseases. It is very useful in forensics and also to resurrect genes from fossils. (iii)
Techniques of Blotting: Northern and Southern Blotting are used to separate and characterize DNA and RNA respectively.
Probes are often used to detect specific molecules from the mixture but the limitation is that probes cannot be applied directly to the gel. To overcome this problem, three types of blotting methods: Southern blotting, Northern blotting and Western blotting.
Southern blotting is a technique for detecting precise DNA fragments in a complex mixture of molecules. This technique was invented in 1970s by Edward Southern, hence got the name. This technique has been applied to detect Restriction Fragment Length Polymorphism (RFLP) and Variable Number of Tandem Repeat Polymorphism (VNTR).
Northern blotting is used for detecting RNA fragments, while souther blotting is used for DNA fragments. In the Southern blotting, DNA fragments are denatured with the help of alkaline solution while in the Northern blotting, RNA fragments are treated with formaldehyde to ensure linear conformation.
Western blotting is used to detect a particular protein present in a mixture. The probe used here is consequently not DNA or RNA molecules, but antibodies. Hence this technique is otherwise termed as “Immunoblotting". (iv) Restriction-enzyme analysis: Restriction enzymes are precise, molecular scissors that can cleave DNA at specific sites to generate discrete, gene-size fragments. These fragments can be rejoined in the laboratory also using a different set of enzymes. Restriction enzymes provided the research persons with a remarkable new tool for investigating gene organization, function and expression. (v) Solid phase synthesis of nucleic acids: In this method, precise sequence of nucleic acids can be synthesized de novo. Such synthesized nucleic acids are used to identify or amplify other nucleic acids.
Recombinant DNA Technology:
Recombinant DNA (rDNA) technology is a necessary one to learn about the significance of the field of biotechnology. DNA is the warehouse of all information required to construct an organism, in short DNA serves as the basis of life to every individual. DNA contains all the information needed to bring about life. This information is carefully stored in a series of structures from the nucleotides which together form triplet codons, and the codons join together to form the complete convention necessary for the synthesis of essential proteins and thus constructing the whole organism. The complexity of this passage of information lies in an intricate sequence of biochemical processes. As a result of these biochemical processes, the instructions contained in the genes are translated into the actual substances of which organisms need to be made. rDNA technology has its wide applications based on this important step of transforming the information contained in the genes. In the early 1970s, biochemists at Stanford University showed that genetic qualities could certainly be transferred from one organism to another. In this experiment, the DNA of one microorganism was recombined with the inserted DNA sequence of another. The resulting DNA strand will have a different modified character acquired as an edited version of the initial two genomes.
An example for rDNA technology:
The methods used in rDNA technology are practically simple. Example: • The specific sequence of codons called ‘gene’ which is responsible for the production of ‘penicillin’ antibiotic in a Penicillium fungus is isolated. • This isolated gene is pasted into the DNA of Escherichia coli, a bacterium that dwells in the human digestive tract.
The bacterial cells divide very rapidly making billions of copies of themselves, and each bacterium carries in its DNA an authentic copy of the gene for ‘penicillin’ production. Now, each new E. coli cell has inherited the fungus’ penicillin producing gene.
In order to transfer the gene embodying the instruction for penicillin production, a possible way is to cut the appropriate gene which encodes the antibiotic ‘penicillin’ from fungus DNA and paste or splice it into plasmid DNA.
Plasmid DNA is a special kind of extra circular DNA that can be used as a vehicle for this editing process to generate the new DNA.
The important molecular tools which acts like "scissors" are the class of enzymes called restriction enzymes. Nearly, over a hundred restriction enzymes are present in the body. Every restriction enzyme has its own way of cutting a specific base sequence of the DNA molecule and in a very particular way.
When these restriction enzymes or the molecular scissors are used singly or in various combinations, the segment of the fungus DNA molecule that specifies Penicillin production can be isolated. This DNA segment is "glued" to right position with the help of special enzymes called as DNA ligases. As a result of the action of this enzyme we will get an edited or recombinant DNA molecule.
When this recombinant plasmid DNA is inserted into E. coli, or any other bacterial cell capable of fast multiplication, the cell will be able to process the instructions stored in this recombinant DNA to assemble the amino acids for production of Penicillin.
The exclusive thing in this is the new instructions are passed on to the next generation of E. coli cells in the process known as gene cloning.
Using rDNA, it is possible to produce a number of substances of medical and economic value.
5. Regulation of Gene Expression: Prokaryotes and Eukaryotes
The salient features of genetic code are as follows: • The codon is triplet. 61 codons code for amino acids and 3 codons do not code for any amino acids, thus functioning as stop codons. • One codon codes for only one amino acid, hence it is specific and unambiguous. • Some amino acids are coded by more than one codon and hence we can consider the genetic code as ‘degenerate’. • The codon is read in mRNA in a contiguous fashion. There are no punctuations. • The code is nearly universal. For example, from bacteria to human, the triplet codon UUU would code for Phenylalanine (phe). Some exceptions to this rule have been found in mitochondrial codons, and also in some protozoans. • AUG has dual functions. It codes for Menthionine (met) and it also acts as an initiator codon.
Regulation of gene expression:
Regulation of gene expression can occur at various levels inside our body. If we consider the gene expression which results in the formation of a polypeptide, it can be regulated at several levels. In eukaryotes, the regulation could be exerted at 1. Transcriptional level (formation of primary transcript) 2. processing level (regulation of splicing) 3. Transport of mRNA from nucleus to the cytoplasm. 4. translational level The genes in the cell are expressed to perform a particular function or a group of functions. For example, if an enzyme called beta –galactosidase is synthesized by E. coli, it is used to catalyze the hydrolysis of a disaccharide - lactose into glucose and galactose.
The bacteria use them as the source of energy. Hence if the bacteria do not have lactose around them to be utilized from energy source, they would no longer require the synthesis of the enzyme of beta –galactosidase.
Hence to say in simple terms, the metabolic, physiological or environmental conditions have a control over regulating the expression of genes. The development and differentiation of embryo into adult organisms are also a result of the coordinated regulation of expression of several sets of genes.
In prokaryotes, control of the rate of transcriptional imitation is the predominant site for control of gene expression. In a transcription unit, the activity of RNA polymerase at a given promoter is in turn regulated by interaction with accessory proteins, which affect its ability to recognize start sites. These regulatory proteins can act both positively as ‘activators’ and negatively as ‘repressors’.
The accessibility of promoter regions of prokaryotic DNA is in many cases regulated by the interaction of proteins with sequences termed operators.
The operator region is adjacent to the promoter elements in most operons and in most cases the sequences of the operator binds a repressor protein. Each operon has its specific ‘operator’ and specific ‘repressor’. For example, lac operator is present only in the lac operon and it interacts specifically with lac repressor only.
Working mechanism of ‘lac’ operon:
The elucidation of lac operon was a result of association between a geneticist, Francois Jacob and a biochemist Jacque Monod. They were the first to elucidate a transcriptionally regulated system. In lac (lactose) operon, a polycistronic structural gene is regulated by a common promoter and regulatory genes. Such arrangement is very common in bacteria and is referred to as operon. To name few such examples, lac operon, trp operon, ara operon, his operon, val operon etc. The lac operon consists of one regulatory gene (the i gene) - here the term i does not denote the inducer, relatively it is derived from the word inhibitor and three structural genes (z, y and a). The i gene codes for the repressor of the lac operon. The z gene codes for the beta –galactosidase (beta –gal), which is primary responsible for the hydrolysis of disaccharide, lactose into its monomeric units, galactose and glucose. The y gene codes for permease, which increases the permeability of the cell to beta –galactosidase.
The ‘a' gene encodes for a transcetylase. Hence all the three gene products in lac operon are required for the metabolism of lactose. In most other operons as well, the genes present in the operon are needed together to function in the same or related metabolic pathway.
Lactose is the substrate for the enzyme beta–galactosidase and it regulates switching on and off the operon. Hence it is termed as inducer. In the absence of a preferred carbon source such as glucose, Iactose is provided in the growth medium of the bacteria, the lactose is transported into the cells through the action of permease. A very significant point is that a very low level of expression of lac operon has to be present inside the cell all the time, otherwise lactose cannot enter the cells. The lactose then induces the operon in the subsequent manner.
The repressor of the operon is synthesized (all the time consecutively) from the i gene. The repressor protein binds to the operator region of the operon and prevents RNA polymerase from transcribing the operon.
In the presence of an inducer such as lactose or allolactose, the repressor is inactivated by the interaction with the inducer. This allows RNA polymerase access to the promoter and transcription proceeds. Essentially the lac operon can also be visualized as regulation of enzyme synthesis by its substrate.
Regulation of lac operon by repressor is referred to as negative regulation. Lac operon is under the control of positive regulation as well.
Regulation in Eukaryotes:
A human cell, a eukaryotic cell, contains approximately 35,000 genes. Some of these are expressed in all cells all the time and they are called ‘housekeeping genes’ which are responsible for the regular metabolic functions like respiration occurring in all cells. When a cell enters a particular pathway of differentiation some housekeeping genes are expressed. Some are expressed all the time in only those cells that have differentiated in a particular means. For example, a plasma cell expresses continuously the gene for the antibody it synthesizes. Some are expressed only as conditions around and in the cell changes the way the arrival of a hormone may turn on (or off) certain genes in that cell. Gene expression is regulated in several methods in eukaryotes. a) Transcription Control: This is the most common type of gene regulation by turning on and off of mRNA formation. b) Post-Transcriptional Control: Regulation of the processing of a pre-mRNA into a mature mRNA c) Translational Control: This regulates the rate of Initiation d) Post-Translational Control: Regulation of the modification of inactive protein to form an active protein
Applications in Biotechnology:
Biotechnology is the recent branch of science which involves the techniques of using the living organisms or the enzymes produced from them in order to form products which are useful to mankind.
One of the most important applications of biotechnology is the genetically engineered crop- based agriculture. Bacteria, plants, fungi or animals whose genes are altered are called as genetically modified organisms (GMO). GM plants can be made useful in a number of ways. GM crops will have tolerant capacity to the unfavorable conditions like cold, drought, salt and heat. The nutritional value of food can also be enhanced. Moreover, GM plants increased the efficiency of mineral usage from the soil, thus preventing the early exhaustion of soil fertility. Gene modification also yield tailor made
plants which serve as alternative resources to the industries in the form of starches and pharmaceuticals.
Bt Cotton plant:
Bacillus thuringiensis is a bacterial strain secreting proteins that are capable of killing particular insects such as beetle, mosquitoes, flies and tobacco budworm. Protein crystals are formed by this during a specific phase of growth and they contain the toxic insecticidal protein. It is inactive in the bacillus, but once consumed by an insect, it becomes active, binds to the midgut epithelial cells, creates pores which causes cell swelling and lysis and eventually leads to death of tiny insects. Specific Bt toxin genes are isolated from the Bacillus thuringiensis and incorporated into crop plants such as cotton. The toxin is coded by a gene named cry. For example, the proteins encoded by the genes cryIAc and cryIAb control the cotton bollworms and the latter kills the corn borer.
Gene therapy can be defined as a collection of methods that allow the correction of a defect in gene diagnosed in an embryo. Treatment for the disease is done by the insertion of genes into the cells and tissues of the individual. It involves the delivery of a normal gene into the individual to take over the function of the non-functional gene
Transgenic animals are those whose DNA are manipulated to possess and express a foreign gene. Transgenic pigs, sheep, rats, rabbits, cows have been produced, but majority of the existing transgenic animals are mice.
Significance of Transgenic Animals:
Using transgenic animals, the regulation of genes in regulating a particular disease can be studied. Medicines used to treat human diseases contain biological products that are very expensive. Such biological products like proteins can be produced inside the body of the transgenic animals. They can also be used for checking the effectiveness of any vaccines before they are administered for humans. Toxicity of any drugs can also be confirmed in case of transgenic animals in a very less time.
Points to Remember:
• • This DNA is enveloped together to form highly condensed structures called as ‘chromosomes’. Total number of chromosomes in humans is 46. Almost all the cells in human body, except the reproductive cells possess 23 pairs of chromosomes, which accounts to a total of 46. • • A diploid (2n condition) zygote, by receiving half (n) of the chromosomes from the mother's egg cell and the other half (n) from the father's sperm cell. Chromosomes are of two types according to their nature, body chromosomes which directs the activities of body whereas sex chromosomes (X and Y) which determine the sex of the individual. • • • • • • • • • • A gene can be defined well as the entire nucleic acid sequence that is necessary for the synthesis of a functional polypeptide or a protein molecule. Heredity is the transient of genes from one generation to the next generation. DNA forms a three dimensional double helical structure which is formed by two strands twisted together like a ladder. ‘Base pairing’ is a unique feature of DNA in which among the four bases, A always pair with T and G always with C. Genes can serve as dominant or recessive. The four bases can be arranged in only sixty-four distinctive sets of three called as a ‘codon’. Certain proteins which are known as ‘enzymes’ act as catalysts and certain proteins are ‘structural proteins’ which help to build the cells. A single codon contains the instructions for the formation of one amino acid. DNA sequencing helps to determine the exact nucleotide sequence of a molecule of DNA. Polymerase Chain Reaction (PCR) leads to a billion fold augmentation of a segment of DNA. A single molecule of DNA can be amplified into quantities that permit the characterization and manipulation of genes contained in it.
• • • • • • •
Three major steps occur in a PCR, which are repeated for 30 or 40 cycles. Denaturation at 94°C, Annealing at 54°C, and Extension at 72°C. Northern and Southern Blotting are used to separate and characterize DNA and RNA respectively. Southern blotting is a technique that was invented in 1970s by Edward Southern, for detecting precise DNA fragments in a complex mixture of molecules. Northern blotting is used for detecting RNA fragments, while southern blotting is used for DNA fragments. Western blotting is used to detect a particular protein present in a mixture which is otherwise termed as “Immunoblotting". Plasmid DNA is a special kind of extra circular DNA that can be used as a vehicle for this editing process to generate the new DNA. Restriction enzymes are the class of enzymes which act like "scissors", and every restriction enzyme has its own way of cutting a specific base sequence of the DNA molecule and also in a very particular way.
In Recombinant DNA (rDNA) technology, the DNA of one microorganism was recombined with the inserted DNA sequence of another and the resulting DNA strand will have a different modified character got as an edited version of the initial two genomes.
• • • •
DNA segment is "glued" into right position with the help of special enzymes called as DNA ligases. Genetic code is nearly universal. AUG has dual functions. It codes for Menthionine (met) and it also acts as an initiator codon. Regulation of gene expression in eukaryotes can be done at Transcriptional level (formation of primary transcript), processing level (regulation of splicing), Transport of mRNA from nucleus to the cytoplasm and translational level.
• • • •
Francois Jacob and a biochemist Jacque Monod were the first to elucidate a transcriptionally regulated system called the ‘lac operon’. In lac (lactose) operon, a polycistronic structural gene is regulated by a common promoter and regulatory genes. Housekeeping genes are responsible for the regular metabolic functions like respiration that occurs in all cells. Bacteria, plants, fungi or animals whose genes are altered are called as genetically modified organisms (GMO).
• • •
Proteins encode by the genes cryIAc and cryIAb control the cotton bollworms and the latter kills the corn borer. Gene therapy can be defined as a collection of methods that allow the correction of a defect in gene diagnosed in an embryo. Transgenic animals are those whose DNA are manipulated to possess and express a foreign gene. E.g. Transgenic pigs, sheep, rats, rabbits, cows