Structure of DNA
* Nucleic acids are made up of polynucleotide chains, which are formed by several nucleotides that make up the structure of the DNA when bonded together.
* The length of DNA is defined by the number of nucleotides or pairs of nucleotides present in the DNA.
* It was Friedrich Meischer who first identified the DNA as an acidic material present in the nucleus in 1869.
* In 1953, James Watson and Francis Crick proposed the revolutionary simple double helix model for the structure of DNA.
Packaging of DNA Helix
* In prokaryotes, the negatively charged DNA is arranged in large loops and is held together by a few positively-charged proteins in a region known as the nucleotide.
* Eukaryotes contain a single molecule of negatively charged DNA packaged around a spool of positively charged, basic proteins known as histones.
* Histones are arranged to form a unit of eight molecules known as the histone ocatmer.
* A round the positively charged ocatmer, a negatively charged DNA molecule is wrapped to form a structure known as the nucleosome, which is held in place by the H1 histone.
* The nucleosome present in chromatin can be seen as a ‘beads-on-string’ structure when observed under an electron microscope.
* During the metaphase stage of cell division, the chromatin fibres coil further and condense to form chromosomes.
Human Genome Project-Objectives
* The Human Genome Project (HGP) was a thirteen-year project that began in 1990 and was completed in 2003.
* It aimed to determine the complete sequence of base pairs of DNA inside the human genome.
* It is the sequence of bases in our DNA that determines our genetic information.
* 99.9 percent of all nucleotide bases are exactly the same in human beings. Yet we differ from each other because our DNA sequences differ in certain places.
* SNPs or single nucleotide polymorphism are locations in DNA with single-base DNA differences.
* Repetitive sequences are those stretches of DNA sequences that are repeated many times, sometimes a hundred to a thousand times, in the human genome.
* The HGP also contributed to the rapid development of a new branch of biology called bioinformatics, which involves the application of statistics and computer sciences to the field of molecular biology.
Search for Genetic Material
* In 1869, Friedrich Meischer isolated an acidic material from the nucleus of a cell and called it nuclein-now known as nucleic acids.
* Gregor Mendel, Thomas Hunt Morgan, Walter Sutton and numerous other scientists has narrowed their search for genetic material to chromosomes.
* By around 1926, the search for the mechanism of inheritance of genetic material has reached the molecular level.
* In 1928, Frederick Griffith, conducted an experiment on Streptococcus pneumonia that demonstrated the transformation of genetic material.
* In 1944, Oswald Avery, Colin MacLeod and McCarty began research on the experiment that had been conducted by Frederick Griffith.
* Oswald Avery, Colin MacLeod and Maclyn McCarty concluded that DNA is the hereditary material in most living organisms.
Experiments on DNA Replication
* Scientists Watson and Crick, who had proved that DNA had a double helical structure, had also suggested that the DNA replication scheme is ‘semi-conservative’.
* According to semi-conservative replication:
• The two DNA strands separate and act as a template for the synthesis of two new complementary strands.
• After the replication process is completed, each DNA molecule ends up with one parental strand and one newly synthesized strand.
Meselson and Stahl conducted experiments on E.coli that validated the ‘semi-conservative’ scheme of DNA replication.
DNA Replication: Machinery and Enzymes
* DNA replication is a complex process that requires several enzymes such as helicases, topoisomerases, DNA polymeras and DNA ligase.
* It begins at a specific point called ‘origin of replication’.
* Replication takes place at a small opening in the double helix of DNA that divides into a Y-shaped structure known as a replication fork.
* The DNA-dependent DNA polymerase enzyme can catalyse the replication reaction in only one direction, five prime to three prime.
* The replication on a template strand that has three prime to five prime polarity, which is also known as the leading strand, is continuous.
* The replication on the other template strand with five prime to three prime polarity, also referred to as the lagging strand, is discontinuous.
Transcription Unit and the Gene
* Protein is synthesized in a cell in two phases, namely, transcription and translation.
* Transcription is a process by which genetic information in DNA is transferred to a molecule of messenger RNA.
* The segment of DNA which takes part in the transcription process is called the transcription unit.
* The transcription unit consists of three main regions, namely, the promoter, the structural gene and the terminator.
* The structural gene can be defined in terms of cistron or segment of DNA that codes for a polypeptide.
* The structural gene can be either monocistronic or polycistronic.
* In eukaryotes, moncistronic structural genes have exons, which are coding sequences intervened by introns.
Transcription in Prokaryotes and Eukaryotes
* The genetic information from DNA is transferred to mRNA through a process known as transcription.
* The mRNA, tRNA and rRNA are involved in the process of protein synthesis.
* Transcription occurs in three stages, which are initiation, chain elongation and termination.
* Prokaryotic transcription occurs in cytoplasm while eukaryotic transcription occurs in the cell’s nucleus.
* In prokaryotic transcription, only RNA polymerase is used whereas in eukaryotic transcription at least three RNA polymerase are involved.
* In prokaryotes, the mRNA does not require any processing while in eukaryotes the hnRNA undergoes processes such as capping, splicing and tailing.
Genetic Code
* The genetic code is an array of 64 codons in a gene that instructs a cell on how to synthesis a particular amino acid from the nucleotides of an RNA strand.
* A codon is a sequence of three nucleotides on an RNA strand that encodes a specific amino acid during protein synthesis.
* George Gamow proved that 3 nucleotides make up a single codon.
* Marshall Nirenberg and Heinrich Matthaei suggested triplet UUU codes for the amino acid phenylalanine.
Har Gobind Khorana formulated a chemical method to produce well-defined nucleic acids, lone strands of RNA with every nucleotide in exact position.
* The stop codons include:
UAA
UAG
UGA
Mutations And Genetic Code
* Mutation can be defined as a change in the genetic code, resulting in the loss or gain of a codon.
* Mutations are triggered by physical and environmental factors such as exposure to radioactive radiations, the ultraviolet rays of the sun or harmful chemicals.
* In the case of frame-shift mutation, two or more bases are either inserted or deleted from the DNA template.
* If three of multiples of three bases are deleted on inserted, it will result in the deletion or insertion of a codon.
Properties Of Genetic Material
* For a molecule to be genetic material it must fulfill four simple conditions. It should be:
• Structurally and chemically stable
• Provide scope for slow changes or mutation that is necessary for evolution.
• Able to express itself in Mendelian characters.
• Able to generate its own replica to become a genetic material.
* While most biomolecules such as proteins fail to fulfill these criteria, both DNA and RNA meet all these requirements.
* DNA serves predominantly as genetic material, whereas RNA acts as a genetic material in some microbes such as Tobacco Mosaic viruses and QB bacteriophages.
* DNA has thymine instead of uracil that is present in RNA, which makes it more stable.
* Even though the DNA is the preferred genetic material due to its stability, it is the RNA that is involved in the transmission of genetic information.
Translation
* Translation is the process of converting mRNA codon sequences into an amino acid polypeptide chain.
* Translation requires the activation of tRNA with amino acids in the presence of ATP and its own activating enzyme called aminoacyl RNA synthetase to form its respective aminoacyl-tRNA.
* The process of translation occurs in three phases:initiation, elongation and termination.
* In the initiation phase, the small subunit of the ribosome binds to the start codon AUG.
* In the elongation phase, the ribosome runs along the mRNA in the 5’ to 3’ direction, moving from codon to codon and adding subsequent amino acids to the growing polypeptide chain.
* When the mRNA reaches the stop codon, UAG, a release factor, binds to it, terminating the translation and releasing the polypeptide chain.
* After the termination, the small and large subunits of the ribosome dissociate and the mRNA degenerates.
Regulation of Gene Expression and Lac Operon
* Gene expression, which consists of transcription and translation, is regulated by the cell.
* In eukaryotes, regulation occurs at the following levels:
• Transcription or formation of mRNA
• Post-transcriptional modification or splicing
• Transport of mRNA from nucleus to cytoplasm
• Translation
* Gene regulation controls which protein must be synthesized and which protein need not and depends on the metabolic, physiological or environmental conditions in a cell.
* An operon is a gene which codes for several proteins and is regulated by common promoter and regulatory genes.
* The lac operon was described in detail by two people, a geneticist Francois Jacob and a biochemist Jacque Monod.
* The lac operon consists of one regulatory gene I and three structural genes z, y and a.
* The structural genes code for enzymes required for the metabolism of lactose.
Human Genome Project-Methodologies
* The Human Genome Project(HGP) allowed biological researchers to conduct advanced studies such as the study of all transcripts in an organ, tissue or tumor.
* HGP utilized two major methodologies of biological research:
* Expressed sequence tags (ESTs) or DNA sequence tags and sequence annotation.
* EST is the term for all genes that express as RNA.
* In EST or DNA sequence tag method, the focus was on identifying and isolating all genes that expressed as RNA.
* In sequence annotation methodology, the entire set of genome that comprised of all coding and non-coding sequences was indiscriminately sequenced and later different regions of the sequence were assigned with a function.
Genetic Material-DNA
* The truth about genetic material came to light through the experiments using bacteria and viruses.
* British bacteriologist Frederick Griffith was the first to conduct experiments with bacteria Streptococcus pneumonia.
* His experiment proved that R-strain bacteria had been ‘transformed’ by heat-killed S – strain bacteria.
* Oswald Avery, Colin MacLeod and Maclyn McCarty worked together and repeated Griffith’s experiments to sumrise that the transforming substance was not protein or RNA, but DNA.
* In 1952, Alfred Hershey and Martha Chase conducted their experiments with bacteriophages to prove that the genetic material that bacteriophages to prove that the genetic material that bacteriophage viruses passed on to the bacteria was DNA.
DNA Fingerprinting
* Small differences in base pair sequences of DNA make the phenotypic appearance of each individual unique.
* In human beings, 99% of DNA base sequences are identical and are known as the bulk genomic DNA.
* The remaining 1% DNA base sequences differ and are present as a small stretch of repeated sequences known as repetitive DNA.
* Satellite DNA is highly repetitive and consists of non coding sequences.
* Satellite DNA is classified as minisatellite DNA and microsatellite DNA.
* DNA polymorphism is the guiding principle behind genetic mapping and therefore it helps in the DNA fingerprinting technique.
* The DNA fingerprinting technique was developed by Alec Jeffreys.
* DNA fingerprinting technique helps in crime investigation, paternity testing, determining genetic and population diversity and studying evolution and speciation.
Transfer RNA
* Transfer RNA or tRNA is the smallest of all RNA molecules. tRNA is:
• Made up of about 75 nucleotides
• Formed of DNA
• Soluble in 1M-NaCl
* Each tRNA has G at its 5’ end and CCA at its 3’ primary end. In three dimensions, tRNA looks like and inverted, L-shaped molecule whereas it takes the shape of a clover leaf when examined as a two-dimensional structure.
* tRNA consists of four arms or sites:
• Ribosome Recognition Site
• Anticodon Site
• Enzyme Recognition Site
• Amino Acid Binding Site