Search for Genetic Material | Biology Class 12 - NEET PDF Download

Search for Genetic Material

• Many scientists conducted several experiments providing evidence that DNA serves as the genetic material.

Transforming Principle

• Frederick Griffith introduced the concept in 1928 by studying Streptococcus pneumoniae bacteria, aiming to demonstrate bacterial transformation.

Two strains of pneumococcus bacteria were identified:

• S-strain (Smooth strain) - Virulent strain with a polysaccharide coat producing smooth colonies.
• R-strain (Rough strain) - Non-virulent strain lacking a polysaccharide coat producing rough colonies.

Griffith's Experiment:

• Injected live S-strain into mice resulting in fatalities due to infection.
• Injected live R-strain into mice leading to survival.
• Injected heat-killed S-strain into mice resulting in survival.
• Injected a combination of heat-killed S-strain and live R-strain into mice causing death due to infection.
• Griffith concluded that the R-strain transformed due to the "transforming substance" likely being the genetic material from the S-strain.

Biochemical Characterization of Transforming Principle

• Oswald Avery, Colin MacLeod, and Maclyn McCarty in 1944 determined the biochemical nature of the transforming substance observed in Griffith's experiment.
• They found that only DNase could destroy the transforming substance, indicating that DNA, rather than proteins or RNA, is the genetic material.

Oswald Avery, Colin MacLeod, and Maclyn McCarty (1944)

• Conducted an experiment to confirm DNA as the genetic material.

Alfred Hershey and Martha Chase (1952)

• Through their work, established that DNA serves as the genetic material by utilizing bacteriophages labeled with radioactive phosphorus and sulfur.
• They revealed that when viruses infected bacteria, only viral DNA entered the bacterium while viral proteins remained outside, supporting DNA as the genetic material.

Alfred Hershey and Martha Chase

The DNA

• The DNA is an acidic long polymer of deoxyribonucleotide.
• It forms two complementary strands that run antiparallel to each other.
• The strands are held together by hydrogen bonds between their opposite nitrogenous bases.
• It is made up of nucleotides which determine the length of the DNA.

Examples of DNA lengths:

• Human = 6.6 × 10⁹ BP
• E.Coli = 4.6 × 10⁶ BP
• Lambda phage = 48502 BP
• Ø X 174 phage = 5386 nucleotides (Single-stranded DNA)

Structure of the polynucleotide chain

• A polynucleotide is a polymer of nucleotides.
• Both DNA & RNA are polynucleotides.
• It consists of three components: a nitrogenous base, a pentose sugar, and a phosphate group.
• Nitrogenous bases are of two types: purines (Adenine and Guanine) and pyrimidines (Cytosine, Thymine & Uracil).
• Thymine is unique to DNA while Uracil is present in RNA instead of thymine.
• Pentose sugar is ribose in RNA and deoxyribose in DNA.
• The phosphate group makes the nucleotide acidic.
• The nitrogenous base links to the pentose sugar forming nucleosides through an N-glycosidic linkage.
• The phosphate bonds two nucleotides to create a dinucleotide via phosphodiester bonds.
• purines
• pyrimidines
• ribose
• deoxyribose
• N-glycosidic linkage
• phosphodiester bonds

Nitrogenous base sugar = Nucleoside Nucleoside phosphate group = Nucleotide

Nucleotide Structure

• Nitrogenous base + sugar = Nucleoside
• Nucleoside + phosphate group = Nucleotide

Structure of DNA

James Watson and Francis Crick, building on the work of Maurice Wilkins and Rosalind Franklin, proposed the iconic double helix model of DNA in 1953, supported by Erwin Chargaff's findings:

• The total amount of purines always equals the total amount of pyrimidines (A + G = T + C).
• The quantity of adenine is always equal to thymine, and guanine is always equal to cytosine (A=T and G=C).
• Adenine pairs with thymine through two hydrogen bonds, while guanine pairs with cytosine through three hydrogen bonds.

The double helical structure of DNA exhibits the following key characteristics:

• DNA comprises two polynucleotide chains with a sugar-phosphate backbone, where the bases face inwards.
• The two strands run in opposite directions (anti-parallel); if one strand goes 5' to 3', the other goes 3' to 5'.
• Bases in the two strands pair via hydrogen bonds, creating base pairs, with purines pairing with pyrimidines. This maintains a consistent distance between the strands.
• The double helix twists in a right-handed manner, with a pitch of 3.4 nm and around 10 base pairs in each turn.
• Each base pair stacks over the next, ensuring stability through hydrogen bonds and maintaining the helical structure.

Packaging of the DNA helix

• The DNA molecule is significantly longer than the size of a typical cell nucleus, measuring approximately 2.2 meters. To fit within the relatively small space of the nucleus, the long DNA strand undergoes compaction and packaging.
• In Prokaryotes, DNA is not enclosed within a nucleus but is organized into a structure known as the nucleoid. Here, the negatively charged DNA is structured into loops by positively charged non-histone proteins.
• In Eukaryotes, DNA is contained within a membrane-bound nucleus. Within the nucleus, DNA is wound around groups of eight positively charged histone proteins, forming nucleosomes. Each nucleosome comprises about 200 base pairs of the DNA helix. These nucleosomes further assemble into "beads-on-a-string" structures known as chromatin.
• Chromatin fibers are created by packaging chromatin, which eventually condenses into chromosomes during the metaphase stage of cell division.

The RNA

• RNA is the initial genetic material that appeared on Earth, playing a crucial role in the development of essential metabolic processes. It consists of a single polynucleotide chain and serves as the genetic material in retroviruses such as HIV. Additionally, RNA can act as a genetic material and a catalyst enzyme known as ribozyme. Due to its catalytic nature, RNA is reactive and hence unstable. Consequently, DNA has evolved from RNA, incorporating chemical modifications that enhance its stability.

DNA vs RNA

Properties of genetic material

• Genetic material should have the ability to replicate itself.
• It must maintain chemical and structural stability.
• There should be room for gradual changes (mutations) necessary for evolution.
• It should be capable of manifesting as 'Mendelian characters'.

Central Dogma

The Central Dogma describes the process by which genetic information is transformed into functional proteins, forming the foundation of living organisms. This concept was introduced by Francis Crick.

The sequence involves: DNA -> RNA -> Protein

However, in RNA viruses, this flow of information occurs in reverse: RNA -> DNA -> RNA -> Protein

DNA Replication

DNA replication is the process of duplicating a double-stranded DNA molecule from the parent DNA. In 1953, Watson & Crick proposed the Semi-conservative model of replication. This model suggests that the two parental DNA strands separate, with each serving as a template for the creation of new complementary strands. Upon completion of replication, each DNA molecule contains one original strand and one new strand.

• Messelson and Stahl's Experiment (1958): They conducted a study on E. coli using 15N, a heavy nitrogen isotope, as the exclusive nitrogen source. By transferring cells from 15N to regular 14N medium, they observed the DNA densities, concluding that DNA replication is semi-conservative.
• Taylor and Colleagues' Experiment (1958): They performed a similar experiment on Vicia faba (fava beans) with radioactive thymidine to analyze the distribution of newly synthesized DNA in chromosomes, affirming the semi-conservative replication of DNA in chromosomes.

Mechanism of DNA Replication

• In eukaryotes, DNA replication occurs during the S phase of the cell cycle.
• The process initiates at the Origin of Replication (ORI) point.
• Helicase enzyme aids in unwinding DNA by breaking hydrogen bonds, separating the strands for new strand synthesis.
• Single-stranded binding proteins stabilize the separated DNA strands.
• Topoisomerase enzyme helps release tension during unwinding.
• Due to the energy expense, the entire DNA strands cannot be separated simultaneously, resulting in the formation of a Y-shaped replication fork.
• An RNA primer is synthesized at the 5' end of the new DNA strand to initiate replication by DNA polymerase in the 5' -> 3' direction.
• DNA polymerase synthesizes one new strand (leading strand) continuously in the 5' -> 3' direction.
• The other new strand is formed discontinuously in small Okazaki fragments (lagging strand) in the 3' -> 5' direction.
• Okazaki fragments are joined by DNA ligase to form a complete new DNA strand.

S phase

• Specific phase in the cell cycle where DNA replication occurs in eukaryotic cells.

ORI

• Origin of Replication; the point where DNA replication initiates.

Helicase

• An enzyme responsible for unwinding the double-stranded DNA during replication.

Single-stranded binding proteins

• Proteins that stabilize the separated DNA strands during replication.

Topoisomerase

• An enzyme that helps relieve the tension generated during unwinding of DNA strands.

Replication fork

• The Y-shaped structure formed during DNA replication where the double-stranded DNA is unwound.

RNA primer

• A small RNA strand synthesized at the 5' end of the new DNA strand to initiate replication.

DNA polymerase

• An enzyme responsible for synthesizing new DNA strands during replication.

Okazaki fragment

• Short, newly synthesized DNA fragments formed on the lagging strand during DNA replication.

DNA ligase

• An enzyme that joins Okazaki fragments together to create a complete new DNA strand.

DNA Transcription

• Definition: DNA transcription is the process of copying genetic information from a single strand of DNA to RNA. During transcription, only a specific segment of DNA is transcribed into RNA, with the enzyme DNA-dependent RNA polymerase playing a key role.
• Template and Coding Strand: One strand of DNA with polarity 3’ → 5’ acts as a template for mRNA synthesis, while the other strand with polarity 5’ → 3’ is known as the coding strand, although it does not directly code for any protein.
• Components: The transcriptional unit consists of a promoter (where RNA polymerase binds), a structural gene (which produces mRNA), and a terminator (where transcription stops).

Mechanism of Transcription

• Initiation: Transcription begins at the promoter region where the sigma factor binds to RNA polymerase.
• Elongation: RNA polymerase adds nucleotides in a 5’ → 3’ direction to synthesize the RNA chain.
• Termination: Transcription concludes when the RNA polymerase reaches the terminator region and the rho factor binds to it, halting the process.

Additional Complexities in Eukaryotic Transcription

• Presence of Three RNA Polymerase Enzymes: Eukaryotic cells have RNA Pol I, RNA Pol II, and RNA Pol III involved in transcription.
• Post-Transcriptional Modifications: The primary transcripts (hnRNA) in eukaryotes contain both exons and introns. To form functional mRNA, capping (addition of a methyl guanosine triphosphate cap), splicing (removal of introns and joining of exons), and tailing (addition of adenylate residues) processes are essential.

Different Types of RNA

• mRNA (messenger RNA): Carries genetic information from DNA to the ribosome for protein synthesis.
• tRNA (transfer RNA): Transfers amino acids to the ribosome during protein synthesis.
• rRNA (ribosomal RNA): Component of the ribosome involved in protein synthesis.

RNA Types

• mRNA (messenger RNA): It serves as the blueprint for protein synthesis.
• rRNA (ribosomal RNA): This type of RNA has both structural and catalytic roles during translation, acting as a ribozyme.
• tRNA (transfer RNA): Also known as the adaptor molecule, tRNA carries specific amino acids for protein synthesis, interpreting the genetic code using its anticodon loop to match codons and its amino acid acceptor end for binding amino acids.

Translation Process Overview

DNA Translation involves the assembly of amino acids into a polypeptide chain or protein. The sequence of amino acids is dictated by the mRNA's codon sequence, with peptide bonds forming between adjacent amino acids.

Steps of Translation

• Charging of tRNA: Amino acids are attached to tRNA molecules by aminoacyl tRNA synthetase in a process called aminoacylation, ensuring tRNA is loaded with amino acids.
• Initiation: Translation commences at the 5' end of mRNA, where the small ribosomal subunit binds. The initiator tRNA with the anticodon UAC attaches to the start codon on mRNA. The large ribosomal subunit joins, creating A and P sites for tRNA binding.
• Elongation: Successive tRNAs bind to the A site, transfer their amino acids to the growing polypeptide chain, and shift to the P site. This cycle of amino acid addition and translocation continues.
• Termination: Translation halts when a stop codon (UAA, UAG, or UGA) is encountered. The synthesized protein is released, and the ribosome disassembles into its subunits.

Regulation of Gene Expression

Eukaryotes

• Regulation at the transcriptional level involves the formation of primary transcripts.
• Processing level regulation includes the control of splicing.
• Transport of mRNA from the nucleus to the cytoplasm is another regulatory mechanism.
• Translational level regulation is also significant in eukaryotes.

Prokaryotes

• Control of the rate of transcriptional initiation is crucial for gene expression in prokaryotes.
• Operons, such as the lac operon and trp operon, regulate metabolic reactions.
• Induction occurs when a substrate is added, activating the genes required for its metabolism.
• Repression happens when a metabolite is added, resulting in the shutdown of genes responsible for its production.

Lac Operon

The Lac Operon, proposed by Francois Jacob and Jacque Monod, controls lactose metabolism.

Three structural genes:

• The Lac z gene encodes Beta-galactosidase, which breaks down lactose into glucose and galactose.
• The Lac y gene codes for the Permease enzyme, enhancing cell permeability to lactose.
• The Lac a gene is responsible for Transacetylase production.

A regulator gene (i gene/ inhibitor gene):

• The regulator gene codes for a repressor protein that controls gene expression.

Inducer:

• Lactose acts as the inducer, enabling the transcription of structural genes to synthesize necessary enzymes.

Functioning of Lac Operon

When lactose (inducer) is absent:

• Without lactose, the regulator gene produces a repressor protein that binds to the operator region, halting RNA polymerase movement. This action prevents mRNA transcription from the structural gene, keeping it switched off.

When lactose (inducer) is present:

• When lactose is available, it binds to the repressor protein, rendering it inactive. Consequently, the repressor fails to bind to the operator region. This frees the operator gene, allowing the RNA polymerase to bind with the promoter gene, thereby switching on the lac operon. This activation leads to the transcription of structural genes.

Genetic Code

Genetic Code refers to the specific sequence of nucleotides present in the mRNA, carrying essential information for the process of Protein Synthesis, also known as Translation. This code is composed of sets of three nitrogen bases, termed codons, which collectively form what is known as the triplet code. Within the genetic code, there exist a total of 64 codons that correspond to the 20 naturally occurring amino acids. Notable scientists who have significantly contributed to this field include:

Salient Features of the Genetic Code

• The codon system operates based on a Triplet structure, with 61 codons dedicated to encoding amino acids and 3 codons serving as stop signals.
• There is a concept of degeneracy within the code, allowing some amino acids to be specified by more than one codon.
• Each codon uniquely codes for a single amino acid, ensuring clarity and specificity in the protein synthesis process.
• Codons are sequentially read on the mRNA without interruptions or punctuation marks, maintaining a contiguous flow of information.
• The genetic code is considered nearly universal, exemplified by instances like UUU consistently coding for phenylalanine across various organisms, from bacteria to humans.
• AUG serves a dual purpose, functioning both as the codon for methionine and as the initiator or start codon for protein synthesis.
• Specific codons, including UAA, UAG, and UGA, are designated as termination signals, marking the conclusion of protein synthesis.

Human Genome Project (HGP)

The Human Genome Project (HGP) commenced in 1990 with the objective of mapping the complete human genome, achieving its completion in 2003. This monumental endeavor was overseen by the U.S. Department of Energy, the National Institute of Health, and various global research entities.

Key Objectives of the HGP:

• Identification of all genes within human DNA.
• Determination of the sequence encompassing 3 billion base pairs in human DNA.
• Compilation of this genetic information in databases.
• Addressing ethical, legal, and social implications (ELSI) that might emerge from the project.

Methodologies Employed in the HGP:

• Expressed Sequence Tags (ESTs): Primarily focused on recognizing all genes expressed as RNA.
• Sequence Annotation: Involved a comprehensive blind sequencing approach of the entire genome, encompassing both coding and non-coding sequences. This process required the utilization of vectors such as Bacterial Artificial Chromosomes (BAC) and Yeast Artificial Chromosomes (YAC).

Process of the HGP:

• Extraction of whole DNA from a cell.
• Subdivision of DNA into random fragments of relatively smaller sizes facilitated by restriction enzymes.
• Cloning of these fragments in a suitable host (e.g., bacteria and yeast) utilizing specialized vectors like BAC and YAC for amplification (e.g., via PCR).
• Sequencing of these fragments using automated DNA sequencers (employing the Frederick-Sanger method).
• Arrangement of sequences based on overlapping regions present within them.
• Alignment of all sequences utilizing computer programs.
• Annotation of sequences and allocation to respective chromosomes.

Applications of Human Genome Project (HGP)

• Understanding biological systems is greatly enhanced through the knowledge of DNA sequences.
• The Human Genome Project's data has paved the way for innovative approaches in biological research.

DNA Fingerprinting

DNA fingerprinting is a method that identifies variations in specific regions of DNA known as repetitive DNA. These particular sequences contain short DNA fragments that are repeated multiple times. Initially developed by Alec Jeffrey, this technique utilizes satellite DNA as the foundation for DNA fingerprinting, showcasing a high level of polymorphism termed Variable Number Tandem Repeats (VNTR).

DNA Fingerprinting Process:

• Isolation of DNA.
• Digestion of DNA by restriction endonucleases.
• Separation of DNA fragments through gel electrophoresis.
• Transferring of separated DNA fragments to synthetic membranes like nitrocellulose or nylon.
• Converting double-stranded DNA into single-stranded by breaking the bonds.
• Hybridization using a labeled VNTR probe.
• Detection of hybridized DNA fragments: The autoradiogram displays various bands of different sizes post hybridization with the VNTR probe.
• Unique Banding Pattern: These bands create a distinctive pattern for an individual's DNA, varying from person to person.

The fundamental components of DNA fingerprinting include:

• Repetitive DNA: Involves non-coding repeated sequences within DNA.
• Satellite DNA: Represents highly-repeated short sequences found in repetitive DNA.

Applications of DNA Fingerprinting

• Utilized in determining paternity accurately.
• Identifying perpetrators in criminal cases through tissue samples such as in instances of rape or murder.
• Assessing population diversity and determining the phylogenetic status of animals.
• Diagnosing genetic diseases efficiently.