Molecular Basis of Inheritance: NEET notes

1. The DNA: structure of polynucleotides

NCERT § "The DNA"

• DNA is a long polymer of deoxyribonucleotides, and its length is usually expressed as the number of nucleotides or base pairs. For example, bacteriophage φ×174 has 5386 nucleotides; bacteriophage lambda, 48502 bp; E. coli, 4.6 × 10⁶ bp; and the haploid human genome, 3.3 × 10⁹ bp.
• A nucleotide has three parts: a nitrogenous base, a pentose sugar (deoxyribose in DNA, ribose in RNA) and a phosphate group.
• The bases are purines (adenine and guanine) and pyrimidines (cytosine, thymine and uracil). Cytosine is common to DNA and RNA, thymine occurs in DNA, and uracil replaces thymine in RNA; thymine is 5-methyl uracil.
• A base joins the 1′ carbon of the sugar through an N-glycosidic bond to form a nucleoside. Adding a phosphate to the 5′-OH through a phosphoester bond makes a nucleotide.
• Nucleotides are joined by 3′–5′ phosphodiester bonds into a polynucleotide chain, which has a free phosphate at its 5′ end and a free 3′-OH at its 3′ end. Sugar and phosphate form the backbone; bases project from it.
• In RNA every nucleotide has an extra -OH at the 2′ position of ribose.
• Friedrich Meischer first identified DNA in 1869 as an acidic substance in the nucleus and called it 'nuclein'.

2. The double helix and central dogma

NCERT § "The DNA"

• X-ray diffraction data from Maurice Wilkins and Rosalind Franklin, together with base-pairing, led James Watson and Francis Crick to propose the double-helix model in 1953.
• Erwin Chargaff had shown that in double-stranded DNA the amounts of A and T are equal, and so are G and C, which fits with complementary pairing.
• The two chains run antiparallel: one is 5′→3′ and the other 3′→5′. The sugar-phosphate backbones lie outside and the bases stack inside.
• A pairs with T through two hydrogen bonds; G pairs with C through three. A purine always faces a pyrimidine, keeping the distance between the two strands uniform.
• The chains coil in a right-handed manner. Each turn of the helix is 3.4 nm long (the pitch) and holds roughly 10 bp, so neighbouring base pairs are about 0.34 nm apart.
• Stacking of one base-pair plane over the next adds stability to the helix on top of the hydrogen bonds.
• Francis Crick proposed the central dogma: information flows DNA → RNA → protein. In some viruses information flows in reverse, from RNA to DNA.

3. Packaging of the DNA helix

NCERT § "The DNA"

• Human diploid DNA is about 6.6 × 10⁹ bp; multiplied by 0.34 × 10⁻⁹ m per bp, the total length is about 2.2 m, far longer than a nucleus of about 10⁻⁶ m, so it must be packed tightly.
• In prokaryotes like E. coli there is no defined nucleus. The negatively charged DNA is held with some positively charged proteins in a region called the nucleoid, organised in large loops.
• In eukaryotes the DNA is packaged by histones, which are positively charged basic proteins rich in lysine and arginine, both basic amino acid residues.
• A histone octamer is made of eight histone molecules. Wrapping of the negatively charged DNA around it forms a nucleosome, which typically holds 200 bp of DNA.
• Nucleosomes are the repeating units of chromatin and appear as 'beads-on-string' under the electron microscope. Chromatin is coiled further into chromatin fibres, which condense at metaphase into chromosomes.
• Packaging beyond the nucleosome level needs another set of proteins, the non-histone chromosomal (NHC) proteins.
• Euchromatin is loosely packed, stains light and is transcriptionally active. Heterochromatin is densely packed, stains dark and is inactive.

4. The search for genetic material

NCERT § "The Search for Genetic Material"

• Griffith (1928) used Streptococcus pneumoniae. The S strain has a smooth, shiny mucous (polysaccharide) coat and is virulent; the R strain has rough colonies, lacks the coat and is harmless to mice.
• Mice died from live S but not from live R or heat-killed S. Yet a mixture of live R with heat-killed S killed the mice, and live S bacteria were recovered from them.
• Griffith concluded that R bacteria had been transformed by some 'transforming principle' from the dead S cells, which let them make the coat and become virulent. He did not identify the chemical.
• Between 1933 and 1944, Oswald Avery, Colin MacLeod and Maclyn McCarty purified the biochemicals from heat-killed S cells. Transformation continued in the presence of proteases and RNases but was stopped by DNase, showing DNA to be the transforming substance. Not every biologist accepted this.
• Alfred Hershey and Martha Chase (1952) grew bacteriophages either with radioactive phosphorus, which labels DNA (proteins lack P), or with radioactive sulphur, which labels protein (DNA lacks S).
• The phages infected E. coli; blending then removed the viral coats and centrifugation separated the bacteria from the free virus particles.
• Bacteria infected by ³²P phages were radioactive, while those infected by ³⁵S phages were not, the ³⁵S staying in the supernatant. So DNA, not protein, enters the bacterium and is the genetic material.

5. Properties of genetic material and the RNA world

NCERT § "The Search for Genetic Material" and § "RNA World"

• A genetic material must replicate itself, be chemically and structurally stable, allow slow changes (mutation) needed for evolution, and be able to express itself as Mendelian characters.
• In most organisms DNA is the genetic material; in some viruses such as tobacco mosaic virus and QB bacteriophage, RNA is.
• The 2′-OH in every RNA nucleotide makes RNA reactive, labile and easily degraded. RNA is also a catalyst, which adds to its reactivity.
• DNA is chemically less reactive and structurally more stable. Having thymine in place of uracil also gives DNA extra stability.
• Both nucleic acids can mutate, but unstable RNA does so faster; viruses with an RNA genome and a short life span therefore mutate and evolve faster.
• RNA can directly code for protein synthesis and express characters easily; DNA needs RNA for this. So DNA suits long-term storage of information, while RNA suits its transmission.
• RNA was likely the first genetic material. Early life processes evolved around RNA, which served both as genetic material and as catalyst; some reactions in living cells are still catalysed by RNA. DNA later took over storage because it is more stable, being double-stranded and able to resist change through repair.

6. Replication

NCERT § "Replication"

• Watson and Crick proposed semiconservative replication: the strands separate and each acts as a template, so every new molecule has one parental and one newly made strand.
• Meselson and Stahl (1958) grew E. coli for many generations in ¹⁵NH₄Cl so their DNA became heavy, then moved them to ¹⁴NH₄Cl. DNA was separated by density in a caesium chloride (CsCl) gradient.
• E. coli divides in 20 minutes. After one generation (20 min) all DNA had hybrid density. After two generations (40 min) there were equal amounts of hybrid and light DNA, as predicted by the semiconservative model.
• Taylor and colleagues (1958) used radioactive thymidine on Vicia faba (faba beans) and showed that DNA in chromosomes also replicates semiconservatively.
• The main enzyme is DNA-dependent DNA polymerase. E. coli, with 4.6 × 10⁶ bp, finishes replication in about 18 minutes, an average of about 2000 bp per second, with high accuracy.
• Deoxyribonucleoside triphosphates act both as substrates and as the energy source, from their two terminal high-energy phosphates.
• The helix cannot open fully at once, so replication happens in a small opened region called the replication fork. It begins at a defined site, the origin of replication.
• DNA polymerase adds nucleotides only in the 5′→3′ direction. So on the template with 3′→5′ polarity synthesis is continuous, while on the template with 5′→3′ polarity it is discontinuous, and the short pieces made there are later sealed together by DNA ligase.
• In eukaryotes DNA replicates in the S phase of the cell cycle. If replication is not followed by cell division, polyploidy results.

7. Transcription

NCERT § "Transcription"

• Transcription copies information from one DNA strand into RNA. Pairing follows complementarity, except that adenine pairs with uracil in RNA.
• Only one strand is copied. If both were used, the two RNAs would be complementary, pair into double-stranded RNA and not be translated, and the gene segment would give two different proteins, making the process futile and complicating the genetic machinery.
• A transcription unit has a promoter, a structural gene and a terminator. The template strand has 3′→5′ polarity. The other strand, the coding strand, has 5′→3′ polarity and the same sequence as the RNA, with T in place of U; it is displaced during transcription.
• By convention, the promoter lies upstream at the 5′ end of the structural gene, taken relative to the coding strand. It is where RNA polymerase binds. The terminator lies downstream at the 3′ end and marks where transcription stops.
• A cistron is a DNA segment coding for one polypeptide. Structural genes are mostly monocistronic in eukaryotes and polycistronic in bacteria.
• Eukaryotic genes are split: coding exons, which appear in mature RNA, alternate with non-coding introns, which do not.
• Bacteria have three main RNAs: mRNA gives the template, tRNA brings amino acids and reads the code, and rRNA has structural and catalytic roles in translation. One DNA-dependent RNA polymerase transcribes all of them.
• RNA polymerase binds the promoter and uses nucleoside triphosphates to elongate RNA. It associates briefly with the sigma (σ) factor to initiate and with the rho (ρ) factor to terminate. In bacteria mRNA needs no processing, and translation can start before transcription is finished since both occur in the cytoplasm.
• Eukaryotes have three nuclear RNA polymerases: RNA polymerase I makes rRNAs (28S, 18S, 5.8S), RNA polymerase III makes tRNA, 5S rRNA and snRNAs, and RNA polymerase II makes hnRNA, the precursor of mRNA.
• hnRNA is processed: introns are removed by splicing; capping adds methyl guanosine triphosphate at the 5′ end; tailing adds 200–300 adenylate residues at the 3′ end without a template. The processed mRNA then leaves the nucleus. Split genes and splicing are thought to reflect the ancient RNA world.

8. The genetic code and mutations

NCERT § "Genetic Code"

• George Gamow reasoned that 4 bases coding 20 amino acids need a code of three bases, since 4³ = 64 combinations covers 20.
• Har Gobind Khorana's chemical method for making RNA of defined base combinations (homopolymers and copolymers) and Marshall Nirenberg's cell-free protein-synthesis system helped crack the code. Severo Ochoa's enzyme (polynucleotide phosphorylase) polymerised RNA of defined sequence without a template.
• The code is a triplet: of 64 codons, 61 code for amino acids and 3 (UAA, UAG, UGA) are stop codons that code for none.
• Some amino acids have more than one codon, so the code is degenerate.
• Codons are read continuously in one direction without punctuation (comma-less).
• The code is nearly universal, for example UUU codes for phenylalanine from bacteria to humans. Exceptions occur in mitochondrial codons and in some protozoans.
• AUG has two jobs: it codes for methionine and it acts as the start (initiator) codon.
• A single base change can change one amino acid, as in sickle-cell anaemia. Inserting or deleting one or two bases shifts the reading frame from that point (frame-shift mutation); inserting or deleting three bases or a multiple of three adds or removes whole codons and keeps the downstream reading frame unchanged.

9. tRNA and translation

NCERT § "Genetic Code" and § "Translation"

• Francis Crick postulated an adapter molecule that could read the code and also bind amino acids; this is tRNA (earlier called sRNA, soluble RNA).
• tRNA has an anticodon loop with bases complementary to the codon and an amino-acid acceptor end. Each amino acid has its own specific tRNA, there is a special initiator tRNA, and there are no tRNAs for stop codons.
• Its secondary structure is drawn as a clover-leaf; in three dimensions it is a compact inverted L shape.
• Translation first needs amino acids to be activated using ATP and joined to their matching tRNA; this is charging of tRNA, or aminoacylation. Forming the peptide bond needs energy.
• The ribosome, the protein factory of the cell, consists of about 80 different proteins together with structural RNAs. An inactive ribosome exists as separate large and small subunits; translation starts when the small subunit meets an mRNA.
• The large subunit has two sites where amino acids carried by tRNAs are brought near enough to each other to form a peptide bond. The ribosome is also a catalyst: in bacteria, the enzyme is the 23S rRNA (a ribozyme).
• A translational unit in mRNA runs from a start codon (AUG) to a stop codon. Untranslated regions (UTRs) at the 5′ end before the start codon and at the 3′ end after the stop codon are not translated but are needed for efficient translation.
• Initiation: the ribosome binds the mRNA at AUG, which only the initiator tRNA recognises. Elongation: charged tRNAs bind their codons in turn by base pairing with their anticodons, and the ribosome moves codon to codon adding amino acids. Termination: a release factor binds the stop codon, ending translation and freeing the polypeptide.

10. Regulation of gene expression: the lac operon

NCERT § "Regulation of Gene Expression"

• In eukaryotes gene expression can be regulated at transcription, at processing (splicing), at transport of mRNA from nucleus to cytoplasm, and at translation.
• Genes are switched on and off as the cell's needs change. For example, E. coli makes beta-galactosidase, which breaks lactose into galactose and glucose, only when lactose must be used; without lactose it does not need the enzyme.
• In prokaryotes the main control point is the rate of transcription initiation. The promoter's accessibility to RNA polymerase depends on proteins binding operator sequences; these regulatory proteins can be activators or repressors.
• An operon is a set of genes transcribed together as a polycistronic unit under a common promoter and regulatory genes. Examples include the lac, trp, ara, his and val operons. François Jacob and Jacques Monod first described a transcriptionally regulated system, the lac operon.
• The lac operon has a regulatory i gene (the i comes from inhibitor, not inducer) and three structural genes: z codes beta-galactosidase, y codes permease, which increases the cell's permeability to β-galactosides, and a codes transacetylase.
• The repressor is made by the i gene all the time (constitutively). If no inducer is present, the repressor binds the operator, which stops RNA polymerase from transcribing the operon.
• Lactose (or allolactose) is the inducer. It binds to and inactivates the repressor, so the repressor cannot bind the operator, RNA polymerase reaches the promoter and the structural genes are transcribed.
• A very low basal level of lac operon expression is always present in the cell; without some permease, lactose could not enter the cell to induce the operon.
• Because a repressor switches the operon off, this control is called negative regulation.

11. Human Genome Project

NCERT § "Human Genome Project"

• The Human Genome Project (HGP) was launched in 1990 and completed in 2003. At an estimated cost of 3 US dollars per base pair for about 3 × 10⁹ bp, the projected cost was about 9 billion US dollars.
• Its goals included identifying all the approximately 20,000–25,000 human genes, finding the sequence of the 3 billion base pairs, storing the data in databases, improving analysis tools, transferring technology to industry and addressing ethical, legal and social issues (ELSI).
• The National Institutes of Health and the U.S. Department of Energy coordinated it. The Wellcome Trust (U.K.) was a major partner, and Japan, France, Germany, China and others contributed further.
• Two approaches were used: Expressed Sequence Tags (ESTs), which focus on genes expressed as RNA, and sequence annotation, which sequences the whole genome, coding and non-coding, and assigns functions later.
• DNA was broken into fragments, cloned in hosts using BAC (bacterial artificial chromosome) and YAC (yeast artificial chromosome) vectors, and sequenced by automated sequencers based on Frederick Sanger's method. The sequence of chromosome 1 was completed last, in May 2006.
• The human genome has about 3164.7 million bases. An average gene is 3000 bases long; the largest known gene, dystrophin, is 2.4 million bases.
• The estimated total is about 30,000 genes, much lower than the 80,000 to 1,40,000 estimated earlier. Nearly 99.9 per cent of nucleotide bases are the same in all people, and over half of the discovered genes have unknown functions.
• Proteins are coded by under 2 per cent of the genome, while repeated sequences make up a very large portion of it. The most genes (2968) are on chromosome 1 and the fewest (231) on Y. About 1.4 million single-base differences (SNPs, single nucleotide polymorphisms) were located.

12. DNA fingerprinting

NCERT § "DNA Fingerprinting"

• Since about 99.9 per cent of the sequence is the same between people, DNA fingerprinting compares the variable parts: specific regions of repetitive DNA.
• In repetitive DNA, a small stretch is repeated many times. In density gradient centrifugation it separates from bulk DNA: the bulk DNA gives a major peak, while the repetitive part gives small peaks, called satellite DNA.
• Satellite DNA is classed as micro-satellites, mini-satellites and so on by base composition, segment length and number of repeats. It normally does not code for any protein, but it is highly polymorphic and forms the basis of DNA fingerprinting.
• A DNA polymorphism is an inheritable variant (allele) at a locus that occurs in the population at a frequency above 0.01. Such variation is more likely in non-coding DNA, because mutations there have no immediate effect on reproductive ability and so build up over generations.
• The technique was developed by Alec Jeffreys, who used a satellite DNA probe showing very high polymorphism, the Variable Number of Tandem Repeats (VNTR).
• Steps: isolate DNA; digest it with restriction endonucleases; separate the fragments by electrophoresis; transfer (blot) them to a synthetic membrane such as nitrocellulose or nylon; hybridise with a labelled VNTR probe; and detect the hybridised bands by autoradiography.
• VNTRs belong to the mini-satellite class: a short sequence repeated in tandem, with a copy number that varies from chromosome to chromosome and is highly polymorphic, so VNTR size ranges from 0.1 to 20 kb. The resulting band pattern is characteristic of each person and matches only between identical (monozygotic) twins.
• Using the polymerase chain reaction (PCR) has raised the sensitivity so far that DNA from a single cell is enough for the analysis. Because the polymorphisms are inherited from parents, the method is used in paternity testing, forensic work and studies of population and genetic diversity.

Must-know facts

1. φ×174: 5386 nucleotides; lambda phage: 48502 bp; E. coli: 4.6 × 10⁶ bp; haploid human DNA: 3.3 × 10⁹ bp.
2. A=T with 2 hydrogen bonds; G≡C with 3 hydrogen bonds; strands antiparallel; right-handed helix.
3. Helix pitch 3.4 nm, about 10 bp per turn, 0.34 nm between base pairs.
4. Human DNA length is about 2.2 m; a nucleosome carries about 200 bp wound on a histone octamer.
5. Histones are rich in lysine and arginine; NHC proteins handle higher-level packaging.
6. Euchromatin is loosely packed and transcriptionally active; heterochromatin is dense and inactive.
7. Griffith 1928 (transforming principle); Avery, MacLeod, McCarty 1933–44 (DNase blocks transformation); Hershey–Chase 1952 (³²P DNA enters the cell).
8. Meselson–Stahl 1958 used ¹⁵N and CsCl gradient; after 20 min all hybrid, after 40 min half hybrid and half light.
9. E. coli replicates its genome in about 18 minutes at about 2000 bp per second.
10. DNA polymerase works only 5′→3′; synthesis is continuous on the 3′→5′ template and discontinuous on the 5′→3′ template, where DNA ligase joins the pieces.
11. Template strand has 3′→5′ polarity; coding strand has the same sequence as mRNA (T instead of U).
12. Bacterial RNA polymerase uses σ for initiation and ρ for termination.
13. Eukaryotic RNA pol I: 28S, 18S, 5.8S rRNA; pol II: hnRNA; pol III: tRNA, 5S rRNA, snRNA.
14. mRNA processing: splicing, 5′ capping with methyl guanosine triphosphate, 3′ tail of 200–300 adenylates.
15. 61 sense codons, 3 stop codons (UAA, UAG, UGA); AUG codes for methionine and is the start codon.
16. The code is triplet, degenerate, read contiguously without punctuation and nearly universal (exceptions in mitochondria and some protozoans).
17. In bacteria, 23S rRNA acts as a ribozyme for peptide bond formation.
18. lac operon: i (repressor), z (beta-galactosidase), y (permease), a (transacetylase); inducer is lactose or allolactose.
19. HGP 1990–2003; genome about 3164.7 million bp; about 30,000 genes; less than 2% codes for protein; chromosome 1 has 2968 genes, Y has 231; about 1.4 million SNPs.
20. DNA fingerprinting (Alec Jeffreys) uses VNTRs of 0.1–20 kb from mini-satellite DNA, detected by blotting, probe hybridisation and autoradiography.

Common traps

Key terms

Nucleoside

A nitrogenous base bonded to a pentose sugar, without phosphate.

Phosphodiester bond

The 3′–5′ link through phosphate that joins nucleotides into a chain.

Antiparallel

The two DNA strands run in opposite 5′→3′ directions.

Nucleosome

About 200 bp of DNA wrapped around a histone octamer; the repeating unit of chromatin.

Transformation

Change in a bacterium's genetic make-up after taking up genetic material from another cell.

Semiconservative replication

Each daughter DNA keeps one parental strand and gains one new strand.

Replication fork

The small opened region of the helix where replication is taking place.

DNA ligase

Enzyme that joins the short pieces made by discontinuous replication into one strand.

Template strand

The DNA strand with 3′→5′ polarity that RNA polymerase reads.

Coding strand

The strand with 5′→3′ polarity whose sequence matches the RNA, T in place of U.

Cistron

A segment of DNA that codes for one polypeptide.

hnRNA

Heterogeneous nuclear RNA, the unprocessed precursor of eukaryotic mRNA.

Splicing

Removal of introns and joining of exons in the primary transcript.

Codon

A triplet of mRNA bases that specifies one amino acid or a stop signal.

Anticodon

The tRNA triplet that pairs with a codon on mRNA.

Aminoacylation

ATP-driven attachment of an amino acid to its matching tRNA; charging of tRNA.

Ribozyme

An RNA molecule that acts as an enzyme, like 23S rRNA in bacteria.

Operon

Genes transcribed together under one promoter and shared regulatory elements.

SNP

Single nucleotide polymorphism: a single-base position that differs between individuals.

VNTR

Variable Number of Tandem Repeats: mini-satellite repeats used as the probe in DNA fingerprinting.