posted Dec 23, 2010 9:12 PM by jaleelkabdul jaleel [ updated Dec 24, 2010 4:20 AM ]
Nucleic Acids: The polynucleotide chains of very high molecular weight are called nucleic acids. They are the genetic material of lining.
1. Deoxyribonucleic Acid (DNA):
# DNA is a long polymer of deoxyribonucleotides.
# the length of DNA is defined as the number of nucleotides present in it.
2. Structure of DNA:
* DNA is formed of number of nucleotides units.
* Each nucleotide has a nitrogenous base, a pentose sugar and inorganic phosphate.
* Nitrogen basis occur in DNA belonging to tw0o groups, purine and pyrimidine.
* DNA has two double ring of purines (Adenine-A and Guanine-G) and two single ring pyrimidine (Cytosine-C and Thymine-T)
* A nitrogenous base is linked to the pentose sugar through a N-glycosidic linkage to form a nucleoside.
* When a phosphate group is linked to 5 – OH of a nucleoside through phosphodiester linkage, a corresponding nucleotide is formed.
* The two chains are held together by two hydrogen bonds between adenine-A with Thymine-T and by three hydrogen bonds between Guanine-G with Cytosine-C.
* The two DNA chains are antiparallel that is, they run parallel but in opposite directions. In one chain the direction 5’ -> 3’ while in the opposite one it is 3’ -> 5’.
3. Double Helix Model of DNA.
* DNA is double helix and formed of two polynucleotide chains which are coiled with one another in a spiral.
* The nucleotides in a polynucleotide chain are linked together by phosphodiester bond.
* The two chains of DNA have anti parallel polarity 5’ -> 3’ in one and 3’ -> 5’ in other.
* Nitrogen bases of two polynucleotide chains form complementary pairs, A opposite to T & G opposite to C.
* The helix has a constant diameter of 20Ao (2nm) throughout its entire length.
* The pitch of helix in 3.4nm (34Ao) with roughly 10base pairs in each turn. The average distance between base pairs comes to about 0.34nm.
4. DNA as Genetic Model: (Griffith’s Experiment).
* It is the change in the genetic constitution of an organism by picking up genes present in the remains of its dead relatives.
* Transformation was first studied by S.F Griffith in1928 while studying on bacterium Streptococcus pneumoniaecalled pneumococcus.
* The bacteria has two strains – the smooth form(s) secretes a polysaccharide capsule which gives the colonies a smooth appearance and virulent. Another form is non-capsulated which gives the colonies a rough appearance and is not virulent (R)
*Griffith tested the virulence of the two strains by injecting the live R-type and live S-type separately into mice, he found that R-type bacteria were non-pathogenic while the S-type caused the death in the mice.
*Heat killed S-type bacteria into mice and they survived equally well
* In the last, he injected a mixture of heat killed “S” & live “R” simultaneously, the mice died with the symptoms of pneumonia. Living type S bacteria were recovered from their bodies.
S-type
This happened because something from the dead bacteria had entered the live ones & made them virulent.
* He concluded that the R-strain bacteria had been transformed by the heat killed S-type, which must be due to the transfer of the genetic material (Transforming Principle).
5. Hershey and Chase experiment.
Alfred Hershey and Marth Chase (1952) indicated that DNA is the genetic material and not the protein.
* Viruses that infect bacteria is called bacteriophage.
* The bacteriophage attaches to the bacteria & its genetic material enters the bacterial cell by dissolving the cell wall of bacteria.
* They grew some viruses on a medium that contained radioactive phosphorus
and other on medium that contained radioactive Sulphur.
* Virus grown in the presence of radioactive Phosphorus contained radioactive DNA but not radio active protein because DNA contains phosphorus but protein does not.
* Similarly viruses grown on radioactive Sulphur contained radioactive protein but not radioactive DNA because DNA dose not contain Sulphur.
* Radioactive phases were allowed to attach to E.coli bacteria then as the infection preceded the viral coats were removed from bacteria by agitating them in a blender. The virus particles were separated from the bacteria by spinning them in a centrifuge.
* Bacteria that were infected with virus had radioactive DNA were radioactive, indicating that DNA was the material that passed from the virus to bacteria. Bacteria that were infected with the viruses that had radioactive proteins were not radioactive. This indicates that proteins did not enter the bacteria from the viruses. DNA is therefore the genetic material that is passed from virus to bacteria.
6. Mode of DNA Replication
DNA replication occurs during S-phase of cell cycle.
Mechanism of DNA replication is as follows:
* The starting point where replication of DNA begins at a specific point were inter wind DNA segments start unwinding called origin of replication.
* Since the two strand cannot be separated in its entire length (due to very high energy requirement) replication occurs with small opening of the DNA-helix; the Y shaped structure formed is called replication fork.
* The DNA-dependent DNA-polymerases catalyze polymerization of the nucleotides only 5’ -> 3’.
* Consequently on one of the template strands (with 3’ -> 5’ polarity) the synthesis of DNA is continuous, while on the other template strand (with polarity 5’ -> 3’), the synthesis of DNA is discontinuous, i.e. short stretches of
DNA are synthesized known as Okazaki fragments.
* The discontinuously synthesized fragments are later joined by the enzyme DNA-ligase.
* As one strand grows continuously while the other strand is formed discontinuously DNA replication is semi discontinuous.
7. Transcription
* The process of copying genetic information from one strand of the DNA into RNA is called transcription.
* Principles of complementarities govern the process of transcription; the exception is that uracil is incorporated instead of thymine opposite adenine of template.
* Only a segment of DNA is transcribed and that only one of two strands is copied.
* Both the DNA strands cannot be copied in transcription because that will produce two types of proteins, one with correct sequence of amino acids and the other with reverse sequence of amino acids.
* Further if two complementary RNA’s are produced simultaneously, they would have a tendency to form double stranded RNA resulting in non translation of coded information into proteins. The whole exercise of transcription would then appear futile.
* Transcription Unit. The segment of DNA that takes part in transcription unit it has three components:
1) A promoter,
2) The Structural gene, and
3) A terminator.
Besides a promoter, eukaryotes also require an enhancer.
* Promoter is located upstream of structural gene. By convention it is called 5′ end (of coding strand which is 3’ end of template strand).
*Terminator region is present down stream of structural gene at the 3’ end (of coding strand which is actually 5’ end of the template strand).
* Structural gene is component of that DNA strand which has 3’ – > 5’ polarity (as transcription occur only in 5’ <– 3’ direction). This strand of DNA is called template strand.
* The other strand which has a polarity of 5’<– 3’ is displaced during transcription. This non-template strand which does not take part in transcription is also called coding strand or plus(+) because genetic code present in this strand is similar to genetic code (based on mRNA) except that uracil is replaced by thymine.
8. Genetic Code:
The relations hip between the sequence of amino acids in a polypeptide and nucleotide sequence of DNA or mRNA is called genetic code. There are 64 codons in the genetic code. Out of 64 codons UAA, UAG & UGA do not represent to any amino acid and are called non-sense or termination codons.
Salient features of genetic code:
• It is triplet (made of 3 letters).
• Non-Ambiguous & specific codons. One codon codes for only one amino acid and
• not any other.
• The code is degenerative. More than one codon can code for same amino acid. Example, Phenylalanine has two and arginine has six codons.
• The code is comma less. There is no punctuation between any of the codon triplets.
• The code is non-overlapping. It means that same latter cannot be used for two different codons.
• The code is nearly universal. Exceptions are these in mitochondrial codons, and in some protozoa’s
• AUG has dual functions. It codes for Methionine (met), and it also act as initiating codon.
9. Protein Synthesis:
Mechanism of Protein synthesis in E.coli is given below:
• The entire process of protein synthesis can be divided into two stages- Transcription and Translation.
• Amino acids are regarded as building blocks of proteins.
• Carboxyl group (-COOH) of one amino acid is bonded with amino group (-NH2) of another amino acid by a Peptide bond.
• Several amino acids are linked by Peptide Bonds to form Poly Peptide chain.
Transcription:
• The process of transmission of Genetic information from template strand of DNA to m-RNA is called Transcription .The site of transcription is nucleus.
• It requires DNA template, nucleotide triphosphates, RNA polymerase, & Mg or Mn ions.
• Transcription begins with uncoiling of the two strands of DNA. One strand of DNA acts as a template for m –RNA formation. By the action of RNA polymerase m-RNA is formed according to the triplet code of DNA by copying process.
• m–RNA comes out of the nucleus and attaches itself to the ribosomes.
• STEPS IN VOLVED IN TRANSLATION
• Attachment of m- RNA TO RIBOSOMES
• Activation of amino acids
• Initiation of amino acids
• Polypeptide chain elongation
• Polypeptide chain termination
TRANSLATION
• Arrangement of amino acids in specific sequence & formation of polypeptide chain according to the information present in m-RNA is called translation.
• Site of translation is ribosomes.
• Translation is a complex process which involves the participation of ribosomes, m-RNA, t-RNA& amino acids
Attachment of mRNA to ribosomes
• mRNA binds with 30 s ribosomal sub unit with its initiation codon AUG:
• At times GUG also acts as initiation codon.
• The group of ribosomes present on the mRNA molecule is called poly ribosome
ACTIVATION OF AMINOACIDS
• Amino acids are scattered in the cytoplasm , they are in inactive state.
• They are activated by specific aminoacyl t-RNA Synthetase enzymes,ATP is also required
• Amino acids + ATP AA – AMP complex +PP
Amino acyl t-RNAsynthetase
• These enzymes possess two binding sites one for amino acid and the other for its t-RNA.
• Initiation of polypeptide chain
• It requires amino acyl t- RNA complex ,m-RNA with initiation codon,30 s and 50 s subunits of ribosomes,GTP& initiation factors.
• With the help of anticodon UAC, t-RNA carries FORMYL METHIONINE and attaches to AUG codon of m- RNA and forms initiation complex .
• Both are linked together with hydrogen bonds.
• t-RNA is linked to m- RNA at decoding site.
POLYPEPTIDE CHAIN ELONGATIONFormyl methionine moves from decoding site. A second amino acid is brought to A site by another aminoacyl t-RNA.A peptide bond is formed between the amino acids
Water is formed in this reaction .The movement of dipeptide from A site to P site is called Translocation.
POLYPEPTIDE CHAIN TERMINATION
• Terminator or non sense codons (UAA, UAG, &UGA) present on the m-RNA strand signal the termination of polypeptide chain.
• The last m-RNA and t-RNA are also set free.
• Ribosomes dissociates into 30 S and 50 S subunits.
10. The Lac-Operon:
* The Lac-Operon consists of one regulatory gene (i), three structural genes(z,y,a), operator gene(o) & promoter gene(p).
* In Lac-Operon the regulator gene is called i-gene because it produces an inhibitor or repressor. The repressor binds to operator gene and turn off the Operon. It exerts a negative control over the working of structural genes.
* Structural genes are those genes which actually synthesize mRNAs. The Lac-Operon of E-Coli contains three structural genes (z, y, a).
* The ‘z’ gene codes for b-galctosides which is primarily responsible for the hydrolysis of the disaccharide, lactose into its monomeric units galactose and glucose. The ‘y’ gene codes for permease, which increases permeability of the cell to b-galctosides. The ‘a’ gene encodes a transacetylase.
* Operator gene is a gene which directory controls the synthesis of mRNAs over the structural gene. It is switched off by the presence of a repressor. An inducer can take away the repressor and switch on the gene. When switch on the structural gene synthesis of polypeptide chain, i.e., transcription and translation occur.
* Promoter gene acts as an initiation signal which functions as recognition centre for RNA polymerase provided the operator gene switch on. RNA polymerase is bound to the promoter gene. When the operator gene is functional, the polymerase moves over it and it reaches the structural genes to perform transcription.
* The repressor of the operon is synthesized from the i-gene. The repressor protein binds to the operator region of the operon and presents RNA polymerase from transcribing the operon. In the presence of an inducer such as lactose or allolactose, the repressor is inactivated by interaction with the inducer. This allows RNA polymerase access to the promoter and transcription proceeds.
* Inducer regulator switching on of the operon. The inducer of Lac-operon of Escherichia coli is lactose (actually allolactose).
Human Genome Project (HGP)
Human Genome Project was called a mega project. The project was a 13-years project coordinated by the U.S Department of Energy and the National Institute of Health. The Project was completed in 2003. HGP was closely associated with the rapid development of a new area in biology called as Bioinformatics.
Goals of HGP
Some of the important goals of HGP were as follows:
(i) Identify all the approximately 20,000-25.000 genes in human DNA;
(ii) Determine the sequences of the 3 billion chemical base pairs that make up human DNA;
(iii) Store this information in databases;
(iv) Improve tools for data analysis;
(v) Transfer related technologies to other sectors. such as industries;
(vi) Address the ethical, Legal, and social issues (ELSI) that may arise from the project.
Salient Features of Human Genome Project
1. Human genome has 3164.7 billion nucleotide bases.
2. The average gene size is 3000 base pairs with sizes vary much. The largest gene being dystrophin at 2.4 million base pairs.
3. The human genome consists of about 30,000 gene much lower than previous estimates to contain 80,000 to 1,40,000 genes.
4. The function of over 50% of discovered genes is unknown.
5. Less than 2% of the genome code for proteins.
1. 99.9% of the nucleotide bases are exactly similar in all human beings.
2. At about 1.4 million locations occur single nucleotide differences called SNPs (snips) or single nucleotide polymorphism. They have the potential to help find chromosomal locations for diseases associated sequences and tracing human history.
3. Repetitive sequences are nucleotide sequences that are repeated many times, sometimes 100 to 1000 times. They have the direct coding functions but provide information’s as to chromosome structure, dynamics and evolutions.
4. Chromosomes I has most genes (2968) and Y has the fewest (231).
Applications of Future Challenges of HGP
1. Detection of Cancers: Efforts are in progress to determine genes that will change cancerous cells to normal.
2. Healthy Living: It will indicate prospects for a healthier living, designer drugs, genetically modified diets and finally our genetic identity.
3. Knowledge of Interactions: it will be possible to study how various genes and proteins work together in an interconnected network.
4. Study of tissues: all the genes or transcripts in a particular tissue, organ or tumor can be analyzed to know the cost of effect produced in it.
DNA Finger Printing
1. Alee Jeffrey’s (1984) invited the DNA fingerprinting technique at Leicester University. U K. Dr. V. K. Kashyap and Dr. Lalji Singh started the finger printing technology in India.
2. DNA Finger printing is a technique to identify a person on the basis of his / her DNA specificity by the means of their digital / palmer print. Each person has a unique DNA finger print.
Principles of DNA Printing:
1. Based pairs of DNA by their differences, about 0.1% or 3 X 106 base pairs (out of 3 X 106 bp) provided individuality to each human being. DNA fingerprinting involves identifying differences in specific regions in DNA sequence called as Repetitive DNA because in these sequences a small stretch of DNA is repeated many times. These repetitive DNA are separated from the bulk genomic DNA as different peaks during density gradient centrifugation. The bulk DNA forms a major peak and the other small peaks are referred to as Satellite DNA
2. Depending upon length, base composition and numbers of repetitive units, satellite DNAs has subcategorizes like micro satellites and minisatellites.
3. Satellite DNAs show polymorphism.
4. If a variant at a locus is present with a frequency of more than 0.01 populations, it is called DNA polymorphism.
5. Variations occur due to mutations, therefore, DNA polymorphism is the occurrence of mutations in a population at high frequency.
6. Short nucleotide repeats in the DNA are very specific in each individual and vary in number from person to person but are inherited. These are Variable Number Tandem Repeats (VNTRs) also called minisatellites.
7. Each individual inherits these repeats from his / her parents which are used as genetic markers in a personal identity test.
Techniques of DNA Finger Printing:
1. The DNA is extracted from the nuclei of white blood cells or of spermatozoa or of the hair follicle, skin, bone, saliva, etc.
2. The DNA molecules are first digested with help of enzyme restriction endo-nuclease that cuts them into fragments. The fragments of DNA also contain the VNTRs.
3. The fragments are separated according to size by gel electrophoresis.
4. VNTRs are multiplied through PCR technique.
5. The separated fragments of DNA in the gel are copied on to a nylon paper by Southern Blotting Technique.
6. Special DNA-probes are made in the laboratories which contain repeated sequence of bases complementary to those on VNTRs. These probes are made radioactivity by labeling with radioactive isotopes. The radioactive DNA-probes bind to the repeat sequences on the nylon membrane.
7. An X-ray film is exposed to the nylon membrane to mark the places where the radioactive DNA has bound to the DNA fragments. These places are marked as dark bands when X-ray film is developed. This is known as autoradiography.
8. The dark bands on X-ray film represent the DNA fingerprints (DNA Profiles)
Application of DNA fingerprinting:
1. In forensic laboratories for identification of criminals.
2. Paternity disputes to find real parents can be solved by DNA fingerprinting.
3. It is useful in determining population and genetic diversities.
4. It is used to study the breeding patterns of animals facing the danger of extinction.
- Codon: Sequence of three nitrogenous bases on mRNA, that code for a particular amino acid.
- Anticodon: Sequence of three nitrogenous bases on tRNA that
- is complementary to the codon for the particular amino acid.
- Bacteriophage: Viruses which infect bacteria.
- Operon: A transcriptionally regulated system consisting of structural gene, operator gene, promoter gene and regulator gene.
- Nucleotide: A complex biomolecule formed by the condensation of pentose sugar, base and one to three phosphoric acids.
- Polymer: A large biomolecule formed by the linking small units the monomers.
- Inducer: A chemical substance which regulate switching on and off the operator gene besides also act as substrate.
- Splicing: Process of removal of introns and joining of exons in HnRNA.
- Template: A strand which acts as model for the synthesis of a new strand.
- Nucleosome: A repeating unit of chromatin structure formed by
- mistune octamer and wrapped around DNA.
- Central Dogma: Unidirectional flow of genetic information from
- DNA — RNA——-Proteins.
- Promotor: A site, at which RNA polymerase is held.
- Coding Strand: Strand of DNA which does not code for anything.
- Translation: Process of polymerization of amino acids to form a peptide under the direction of base sequences in m-RNA.
- Introns: Non-coding segments of eukaryotic structural gene.
- Exons: Coding segments of eukaryotic structural gene.
- Euchromatin: Light stained loosely packed transcriptionally active chromatin.
- Heterochromatin: Dark stained compactly packed, inactive.
- Semi conservative: Type of replication in which DNA molecule has one parental and one newly synthesized strand.
- Genetic Code: A triplet base sequence regulating the sequence of amino acids in a polypeptide