Molecular Basis of Inheritance REVISION NOTES

The Discovery of DNA:

• In 1869, Friedrich Miescher isolated a unique chemical substance from white blood cells called nuclein.
• Nuclein was found to have high phosphorus content and showed acidic properties, leading to its name nucleic acid.
• By the early 1900s, it was discovered that nuclein was a mixture of proteins and nucleic acids.
• There are two types of nucleic acids: DNA (deoxyribonucleic acid) and RNA (ribonucleic acid).
• The development of the double-helix model helped further the understanding of DNA and its role in genetics.

The Genetic Material is DNA:

• By the early 1900s, geneticists knew that genes controlled the inheritance of traits and were located on chromosomes composed mainly of DNA and proteins.
• Initially, geneticists thought that proteins were the genetic material that caused variations observed within species.
• It was thought that DNA was a small and simple molecule that varied little among species.
• However, over time, it was shown that DNA molecules are large and vary tremendously within and among species.
• Variations in DNA molecules are different than variations in shape, electrical charge, and function shown by proteins.
• Over a period of roughly 25 years (1928-1952), geneticists became convinced that DNA, not protein, was the genetic material.

Griffith’s experiments :

• In 1928, Frederick Griffith conducted an experiment on Streptococcus pneumoniae to find a cure for pneumonia, which was a common cause of death at that time.
• Griffith used two strains of bacteria: the virulent, smooth, pathogenic, and encapsulated S type and the non-virulent, rough, non-pathogenic, and non-capsulated R type.
• Griffith conducted four experiments on these bacteria and discovered that heat-killed strain S bacteria caused harmless strain R bacterium to change into deadly S strain bacterium.
• Griffith concluded that the R-strain bacterium must have taken up a "transforming principle" from the heat-killed S bacterium, which allowed R strain to get transformed into smooth-coated bacterium and become virulent.
• Griffith showed that the change was genetic and suggested that the transforming principle was genetic material from the heat-killed S bacterium.

Avery, McCarty, and MacLeod’s experiment:

• In 1944, Avery, MacLeod, and McCarty showed that DNA is the transforming principle, which can change harmless R-strain bacteria into deadly S-strain bacteria.
• They conducted experiments using purified DNA, RNA, proteins, and other materials from S cells to transform heat-killed S and R cells.
• Only DNA was able to transform R cells into S cells, while protein- and RNA-digesting enzymes did not affect transformation.
• DNA-digesting enzymes inhibited the transformation, suggesting that DNA caused the transformation.
• Despite the evidence, not all biologists were convinced that DNA was the genetic material.

Hershey - Chase Experiment:

• Hershey and Chase worked with bacteriophages, which are viruses that infect bacteria.
• They used radioactive isotopes of phosphorus and sulfur to label DNA and proteins, respectively.
• Viruses grown in the presence of radioactive phosphorus contained radioactive DNA but not radioactive proteins, while viruses grown in the presence of radioactive sulfur contained radioactive proteins but not radioactive DNA.
• Radioactive phages were allowed to infect E.coli bacteria, and the viral coats were removed with the help of a centrifuge.
• Bacteria infected by viruses with radioactive DNA were radioactive, indicating that DNA was the material that passed from the viruses to the bacteria.
• Bacteria infected by viruses with radioactive proteins were not radioactive, indicating that proteins from the viruses did not enter the bacteria.
• The experiment showed that DNA is the genetic material that is passed from virus to bacteria.

DNA packaging :

• The length of DNA double helix molecule in a typical mammalian cell is approximately 2.2 meters.
• The size of a typical nucleus is 10^-6 m, which is very small in comparison to the length of DNA.
• The long DNA molecule must be condensed, coiled and supercoiled to fit inside the small nucleus.
• The process of DNA condensation involves the packaging of DNA into a more compact structure known as chromatin.
• Chromatin consists of DNA wrapped around histone proteins.
• This compact structure further coils and condenses to form chromosomes during cell division.
• The packaging of DNA allows it to fit into the nucleus and also regulates gene expression.

Packaging in Prokaryotes

• Prokaryotes like E. coli do not have a well-organized nucleus
• The nucleoid is a small, circular, highly folded, naked ring of DNA in prokaryotes
• The nucleoid in E. coli is 1100P long in perimeter, containing about 4.6 million base pairs
• The nucleoid has to be packaged into a cell that is hardly 2-3P long
• The negatively charged DNA becomes circular to reduce the size to 350Pm in diameter
• The circular DNA is further reduced to 30Pm in diameter by folding and looping
• 40-50 domains (loops) are formed in the DNA
• Formation of loops is assisted by RNA connectors
• Each domain is further coiled and supercoiled, reducing the size down to 2P in diameter
• Coiling and packaging are assisted by positively charged HU (Histone like DNA binding proteins) proteins
• Enzymes like DNA gyrase and DNA topoisomerase I maintain the supercoiled state.

Packaging in Eukaryotes

• Eukaryotes have a well-organized nucleus with a nuclear membrane, nucleolus, and chromosomes.
• The DNA in chromosomes is associated with histone and non-histone proteins.
• The organization of DNA is more complex in eukaryotes.
• The charge of a protein is determined by the abundance of amino acid residues with charged side chains.
• Histones are a set of positively charged, basic proteins that organize themselves to form a unit of 8 molecules known as a histone octamer.

• Eukaryotes have well-organized nucleus with nuclear membrane, nucleolus and thread-like material in the form of chromosomes.
• DNA in eukaryotic chromosomes is associated with histone and non-histone proteins.
• Histones are a set of positively charged, basic proteins that organize themselves to make a unit of 8 molecules known as histone octamer.
• Negatively charged helical DNA is wrapped around the positively charged histone octamer, forming a structure known as nucleosome.
• One nucleosome contains approximately 200 base pair long DNA helix of which about 146 bp long segment is wound around each octamer and the remaining bp contribute as linker DNA.
• Nucleosomes are the repeating units of chromatin and look like 'beads-on-string' under an electron microscope.
• Six nucleosomes get coiled repeatedly to form solenoid that looks like coiled telephone wire.
• The chromatin is a 10 nm thick fiber packed to form a solenoid structure of 30 nm diameter and further supercoiling of solenoid tends to form a looped structure that further coils and condense at metaphase stage to form the chromosomes.
• Packaging of chromatin at higher levels requires additional set of proteins called Non-Histone Chromosomal proteins (NHC).

• Heterochromatin: condensed regions of chromatin in eukaryotic cells, localized near centromere, telomeres, and intercalated, genetically mostly inactive, stains strongly and appears dark, 2-3 times more rich in DNA than in euchromatin.
• Euchromatin: non-condensed regions of chromatin in eukaryotic cells, stains light, genetically very active and fast replicating, transcriptionally active.

DNA Replication 

• DNA molecule controls all activities of the cell and duplicates itself during cell reproduction.
• DNA performs two important functions: heterocatalytic and autocatalytic.
• Replication is the process by which DNA duplicates itself.
• DNA replicates through semiconservative mode of replication.
• Semiconservative replication begins at a specific point 'O' and terminates at point 'T', which is flanked by 'T' sites.
• Replication occurs in a replicon, which is the unit of DNA.
• Enzyme endonuclease nicks one of the strands of DNA at the point 'O', and DNA helicase breaks weak hydrogen bonds to unwind the DNA molecule.
• The unwinding is bidirectional and forms a 'Y' shaped replication fork.
• SSBP prevents the two separated strands from recoiling and facilitates the synthesis of new polynucleotide strands.

• DNA replication is a process of copying genetic information from one DNA molecule to another.
• It occurs in the S-phase of the cell cycle and is a semiconservative process.
• The process begins with the unwinding of the double helix by an enzyme called helicase.
• This creates a Y-shaped structure called the replication fork.
• New strands are synthesized by adding complementary nucleotides to the template strands.
• The leading strand is synthesized continuously in the 5'-3' direction, while the lagging strand is synthesized discontinuously in the form of Okazaki fragments.
• Okazaki fragments are joined by DNA ligase, and RNA primers are removed and replaced by DNA sequences by DNA polymerase.
• Finally, DNA gyrase (topoisomerase) forms the double helix to create two identical daughter DNA molecules.
• Each daughter DNA molecule contains one parental strand and one newly synthesized strand.

Experimental confirmation 

• In 1958, Meselson and Stahl conducted an experiment to verify the semiconservative nature of DNA replication.
• E. coli cells were grown in 14N medium and then transferred to 15N medium and allowed to replicate for several generations.
• Heavy DNA (15N) can be distinguished from normal DNA (14N) by centrifugation in a 6M CsCl2 density gradient, and a band is formed at the equilibrium point.
• The E. coli cells were then transferred to 14N medium, and after the first generation, a band for 14N-15N (hybrid) was obtained and recorded. After the second generation, two bands were obtained - one at the 14N-15N position and the other at the 14N position.
• The position of bands after two generations clearly proved that DNA replication is semiconservative.

Protein synthesis

• Proteins are important biomolecules that serve as structural components, enzymes, and hormones.
• Protein synthesis includes transcription and translation.
• Transcription is the process of copying genetic information from one strand of DNA into a single stranded RNA transcript.
• During transcription, synthesis of complementary strand of RNA takes place, except Adenine nitrogen base pairs with Uracil base instead of Thymine.

Central Dogma 

• The flow of genetic information from DNA to RNA to protein is known as the central dogma of molecular biology.
• This concept was proposed by F.H.C. Crick in 1958.
• In retroviruses or riboviruses, the central dogma is modified to include an enzyme called RNA dependent DNA polymerase that synthesizes DNA from RNA.
• The process of protein synthesis involves transcription, which is the synthesis of a complementary RNA strand from a DNA template, and translation, which is the synthesis of a polypeptide chain from the mRNA transcript.

Transcription

• During transcription, only one strand of DNA is copied into RNA and it acts as a template.
• RNA polymerase enzyme catalyzes the formation of RNA transcript.
• DNA is located in the nucleoid of Prokaryotes and in the nucleus of Eukaryotes.
• Transcription occurs in the nucleus of Eukaryotes, and translation occurs in the cytoplasm.
• DNA transfers information to mRNA, which then moves to ribosomes.
• Transcription occurs during G1 and G2 phases of the cell cycle.
• DNA has promoter and terminator sites.
• Transcription involves three stages: initiation, elongation, and termination.

Transcription Unit:

• Each transcription unit consists of a promotor, a structural gene, and a terminator.
• The promotor is located upstream of the structural gene and provides a binding site for RNA polymerase.
• The structural gene consists of two strands of DNA with opposite polarity. The strand with 3' to 5' polarity is the template strand and the other strand is the coding strand.
• The coding strand is the sense strand and has the same sequence as the mRNA, except that it has thymine instead of uracil.
• The terminator is located downstream of the coding strand and defines the end of the transcription process.
• RNA polymerase causes local unwinding of DNA duplex and joins ribonucleoside triphosphates to the template strand of DNA to form mRNA.
• As transcription proceeds, the hybrid DNA-RNA molecule dissociates, and fully formed mRNA is released when RNA polymerase reaches the terminator signal on the DNA.
• Bacteria do not require any processing of mRNA as they do not have introns.
• Eukaryotes have three types of RNA polymerases, which transcribe different types of RNA molecules.
• RNA polymerase-I transcribes r-RNA, RNA polymerase-II transcribes m-RNA and hnRNA, and RNA polymerase-III is responsible for transcription of t-RNA and snRNA.
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Transcription unit and the gene

• The DNA sequence coding for different types of RNA is called a gene.
• A segment of DNA coding for a polypeptide is called a cistron.
• A transcription unit containing a single structural gene is monocistronic, while a unit containing multiple structural genes is polycistronic.
• Eukaryotic structural genes have non-coding introns and coding exons.
• Processing of primary transcripts in eukaryotes involves splicing out introns and joining exons in a definite sequence by DNA ligase.
• Heterogeneous nuclear RNA undergoes capping and tailing to become fully processed mRNA.
• Capping involves adding a methylated guanosine triphosphate to the 5’ end of hnRNA, while tailing involves polyadenylation at the 3’ end.
• Fully processed mRNA is transported out of the nucleus through nuclear pores for translation.

Genetic Code

• DNA stores the information for protein synthesis in the sequence of its nucleotides (nitrogen bases).
• The 20 different types of amino acids used in protein synthesis are identified by the 4 types of nitrogen bases in DNA.
• The genetic code is a coded language that contains code words (codons), each representing a specific amino acid.
• A single nitrogen base in a codon can only encode for four different types of amino acids, but a combination of three nitrogen bases (triplet codon) can specify 64 different types of amino acids.
• The genetic code is a triplet code, with every three consecutive nucleotides in DNA constituting a triplet codon.
• The triplet code was first evidenced by Crick using "frame-shift mutation."
• Homopolymer experiments were conducted to decipher the genetic code, where synthetic m-RNA containing only one type of nitrogenous base (Uracil) was used to synthesize a small polypeptide molecule. This helped to identify codons for specific amino acids.
• DNA is the master molecule that controls protein synthesis by carrying the requisite information in its nucleotide sequence.
• The genetic code is a coded language that contains codons, each representing a specific amino acid.
• A single nitrogen base in a codon can encode for only four different types of amino acids, while a combination of three nitrogen bases (triplet codon) can specify 64 different types of amino acids.
• The genetic code is a triplet code, and every three consecutive nucleotides in DNA constitute a triplet codon.
• Dr. Har Gobind Khorana devised a technique for synthesizing m-RNA with repeated sequences of known nucleotides, which helped in deciphering all 64 codons of the genetic code.
• Replication and transcription are based on complementarity principle, but during translation, genetic information is transferred from nucleotides to amino acids.
• A change in the nucleotide sequence of DNA results in a change in the amino acid sequence of proteins, indicating that the genetic code directs the sequence of amino acids during protein synthesis.

Characteristics of Genetic code:

• Genetic code is a triplet code, where each codon of three consecutive bases specifies one particular amino acid.
• The base sequence in a codon is always in the 5' to 3' direction, and the code is non-overlapping, meaning that each single base is part of only one codon.
• The code is also commaless, with no gap or punctuation mark between successive codons.
• The code has degeneracy, with some amino acids being encoded by more than one codon due to the wobble hypothesis.
• The code is universal, meaning that the specific codon specifies the same amino acid in all living organisms.
• The code is non-ambiguous, with a particular codon always encoding for a specific amino acid and never for two different amino acids.
• The initiation codon is always AUG, which codes for amino acid methionine, and three codons UAA, UAG, and UGA serve as termination codons, which stop the process of elongation of the polypeptide chain. Codon is a part of DNA, while anticodon is a part of tRNA, and codons and anticodons pair during translation.

Mutations and Genetic Code

• The mutation is a sudden change in the DNA sequence.
• It results in the change of genotype expressed in terms of phenotype.
• Mutation, along with recombination, is the raw material for evolution as it results in variations.
• During mutation, loss (deletion) or gain (insertion/duplication) of a segment of DNA results in alteration in the chromosome.
• Point mutation is a type of mutation that occurs due to the change in a single base pair of DNA. Eg. Sickle cell anemia.
• Deletion or insertion of base pairs of DNA causes frame-shift mutations or deletion mutations.
• Insertion or deletion of one or two bases changes the reading frame from the point of insertion or deletion.
• Insertion or deletion of three or multiples of three bases (insert or delete) results in the insertion or deletion of amino acids, and the reading frame remains unaltered from that point onwards.

t-RNA- the adapter molecule

• tRNA is an adapter molecule that reads the codon and binds with the amino acid.
• tRNA has a cloverleaf structure with an anticodon loop that is complementary to the codon.
• The amino acid acceptor end of tRNA has unpaired CCA bases for amino acid binding.
• Each amino acid has a specific tRNA.
• Initiator tRNA is specific for methionine.
• tRNA does not exist for stop codons.
• In actual structure, tRNA looks like an inverted L in 3D.

Translation - protein synthesis 

• The translation is the process of protein synthesis where codons of mRNA are translated into specific amino acids forming a polypeptide on ribosomes.
• Translation requires amino acids, mRNA, tRNA, ribosomes, ATP, Mg++ ions, enzymes, elongation, translocation, and release factors.
• About 20 different types of amino acids are available in the cytoplasm, which serve as raw material for protein synthesis. DNAA controls the sequence of amino acids in proteins through the transcription of mRNA, which follows a specific genetic code for each amino acid.
• RNA molecules serve as intermediates between DNA and protein.
• Ribosomes are the sites of protein synthesis, consisting of large and small subunits that associate together during protein synthesis due to Mg++ ions.
• A ribosome has one binding site for mRNA and three binding sites for tRNA: P site, A site, and E site. Only the first tRNA-amino acid complex enters directly into the P site.
• Eukaryotic ribosomes have a groove between the subunits, which protects the polypeptide chain from cellular enzymes and mRNA from nucleases

Mechanism of translation (i.e. synthesis of polypeptide chain) 

Initiation of Polypeptide chain:

• Activation of amino acids requires ATP.
• Small ribosomal subunit binds to the 5' end of mRNA.
• Initiator codon (AUG) on mRNA starts translation.
• Initiator tRNA with methionine or formyl methionine binds to the AUG codon.
• Large ribosomal subunit joins with the small subunit with Mg++ ions.
• The initiator tRNA with activated amino acid occupies the P-site of ribosome, and the A-site is vacant.

Elongation of polypeptide chain

• Activated amino acids are added one by one to the first amino acid (methionine).
• Amino acid is activated using ATP and binds with amino acid binding site of tRNA.
• Condon recognition: Aminoacyl tRNA enters the ribosome at A-site and binds with codon by hydrogen bonds.
• Peptide bond formation: Amino acid on the first initiator tRNA at P-site and amino acid on tRNA at A-site join by peptide bond.
• Translocation: tRNA at A-site carrying a dipeptide moves to the P-site. The ribosome moves along with tRNA and mRNA, making the A-site vacant. The tRNA carrying dipeptide now gets positioned at P-site, and the next charged tRNA molecule carrying an amino acid is received at A-site.
• The process is repeated as amino acids are added to polypeptide.
• Third charged tRNA with its amino acid arrives at A-site of ribosome, and the polypeptide bond is formed. The second tRNA is discharged from P-site to E-site and leaves the ribosome.
• Ribosome moves over mRNA, and all codons on mRNA are exposed for translation.
• The process of arrival of tRNA-amino acid complex, peptide bond formation, ribosomal translocation, and removal of previous tRNA is repeated.
• It takes less than 0.1 seconds for the formation of the peptide bond.

Termination and release of polypeptide

• Stop codon (UAA/ UAG/ UGA) is exposed at the A-site of ribosome.
• Release factor binds to the stop codon, terminating the translation process.
• Polypeptide is released in the cytoplasm.
• Two subunits of ribosome dissociate and last tRNA is set free in the cytoplasm.

Untranslated regions (UTRs) on mRNA:

• UTRs are present at both 5’-end (before start codon) and at 3’-end (after stop codon).
• UTRs are required for efficient translation process.

mRNA:

• mRNA is also released in the cytoplasm.
• mRNA is short-lived and gets denatured by nucleases immediately.

Regulation of gene expression

• Gene expression is a multistep process that results in the formation of a polypeptide.
• Gene expression can be regulated at different levels in eukaryotes, including transcriptional, processing, transport, and translational levels.
• Genes in a cell are expressed to perform different functions, and their expression can be regulated by metabolic, physiological, or environmental conditions.
• The regulation or expression of several sets of genes is responsible for the development and differentiation of an embryo into an adult organism.
• Organisms regulate gene expression in response to changes in the environment, and different genes may be regulated by different mechanisms.
• Inducible enzymes are adaptive enzymes that certain bacteria like E.coli synthesize depending upon the substrate present. A set of genes is switched on when there is a necessity to metabolize a new substrate, and this phenomenon is called induction, which is a positive control.

Operon concept 

• Metabolic pathways are regulated as a unit, and in E.coli, lactose sugar induces the production of three enzymes necessary for lactose digestion.
• The three enzymes are controlled by a long segment of DNA known as the operon, which consists of an operator site and three structural genes.
• The action of the structural genes is regulated by the operator site with the help of a repressor protein, which is produced by the regulator gene.
• The gene expression depends on whether the operator is switched on or off, and switching on or off is achieved by a protein called repressor.
• The lactose or lac operon of E.coli is an inducible operon, and it consists of a regulator gene, a promoter gene, an operator gene, structural genes, and an inducer.
• The regulator gene controls the operator gene in cooperation with an inducer present in the cytoplasm and produces a protein called repressor protein.
• The promoter gene marks the site at which the RNA Polymerase enzyme binds, and the operator gene controls the functioning of structural genes.
• The structural genes catalyze mRNA production, which in turn produces polypeptides on the ribosomes that act as enzymes to catalyze lactose in the cell.
• There are three structural genes in the sequence lac-z(1), lac-y(2), and lac-a(3), and the enzymes produced are E-galactosidase, E-galactoside permease, and transacetylase, respectively.
• Inducer is a chemical in the cytoplasm (allolactose) that inactivates the repressor protein.
• When the lac operon is switched on, the inducer joins with the repressor protein, preventing it from binding to the operator gene.
• A small amount of lactose enters the cell even when the operon is switched off, and a few molecules of lactose act as an inducer and bind to the repressor protein.
• The repressor-inducer complex fails to bind to the operator gene, which is then turned on, and the structural genes produce all enzymes.
• Lactose acts as an inducer of its own breakdown.
• When the inducer level falls, the operator is blocked again by the repressor protein, and the structural genes are repressed/inactivated again.
• This is negative feedback.

Genomics

• Genome refers to the total genetic constitution of an organism or a complete copy of genetic information.
• Genomics is the study of genomes through analysis, sequencing, and mapping of genes, along with the study of their functions.
• The sequencing of yeast, Drosophila, and mouse genomes was done to facilitate comparative studies between humans and other organisms commonly used for genetic studies.
• Structural genomics involves mapping, sequencing, and analyzing genomes, while functional genomics deals with the study of functions of all gene sequences and their expression in organisms.
• Genomics research can be applied in different sectors, including medicine, biotechnology, and social sciences, to improve crop plants, human health, and livestock.
• Applications of genomics include gene therapy, developing transgenic crops, genetic markers for forensic analysis, and introducing new genes in microbes for producing enzymes, therapeutic proteins, and biofuels.

Human Genome Project 

• The Human Genome Project (HGP) began in 1990 and was completed in 2003.
• The project was aimed at mapping the entire human genome, storing information in databases, developing analysis tools, transferring related technologies to private sectors, and addressing legal, ethical, and social issues.
• The HGP allowed researchers to understand the blueprint for building and constructing the human genome, which will have a major impact on fields such as medicine, biotechnology, and life sciences.
• The project aimed to sequence the genomes of several other organisms such as bacteria, nematodes, yeast, fruit fly, plants, and mice, which will be useful for comparative studies of gene functions.
• The HGP estimated that humans have about 33,000 genes, and understanding how these genes are used may be the key to our complexity and evolution.

DNA Fingerprinting

• Genes on chromosomes determine an organism's traits and inheritance. Recombination and mutation create unique genetic make-ups, like a fingerprint.
• DNA profiling or fingerprinting uses DNA restriction analysis to identify individuals. The technique was developed by Dr. Alec Jeffreys in 1984.
• The technique is based on identifying variable number tandem repeats (VNTRs) in DNA, which are unique to individuals.
• The steps involved in DNA fingerprinting are isolation of DNA, restriction digestion, gel electrophoresis, Southern blotting, selection of a DNA probe, hybridization, and photography.
• DNA fingerprinting is used in forensic science to solve crimes, in disputed parentage cases, and in pedigree analysis in various species.