Detailed Notes -3-Chapter 5 -Molecular Basis Of Inheritance

5.7 TRANSLATION

1. Translation Process:

   • Translation involves the polymerization of amino acids to form a polypeptide.
   • The sequence of amino acids is determined by the sequence of bases in the mRNA.
   • Peptide bond formation between amino acids requires energy, and it occurs between charged tRNAs.
   • The ribosome, consisting of structural RNAs and proteins, serves as the cellular factory for protein synthesis.

2. Ribosome Structure and Function:

   • Ribosomes exist as two subunits, a large subunit, and a small subunit, in their inactive state.
   • The small subunit binds to mRNA to initiate translation, and the large subunit contains sites for amino acid binding and peptide bond formation.
   • The ribosome also acts as a catalyst (ribozyme) for the formation of peptide bonds.

3. Translational Unit and Untranslated Regions:

   • A translational unit in mRNA is the sequence flanked by the start codon (AUG) and the stop codon, which codes for a polypeptide.
   • mRNA contains untranslated regions (UTRs) at both the 5′ and 3′ ends, which are not translated but are necessary for efficient translation.

4. Initiation, Elongation, and Termination:

   • Translation initiation occurs when the ribosome binds to the mRNA at the start codon (AUG), recognized by the initiator tRNA.
   • The ribosome then moves through the mRNA in the elongation phase, adding amino acids one by one based on codon-anticodon base pairing.
   • Release factors bind to the stop codon, terminating translation and releasing the complete polypeptide from the ribosome.

5.8 REGULATION OF GENE EXPRESSION

1. Levels of Gene Expression Regulation:

   • Gene expression regulation can occur at various levels:
     • Transcriptional level (formation of primary transcript).
     • Processing level (regulation of splicing).
     • Transport of mRNA from nucleus to cytoplasm.
     • Translational level.

2. Functionality of Gene Expression:

   • Genes are expressed to perform specific functions or sets of functions.
   • Regulation of gene expression is influenced by metabolic, physiological, or environmental conditions.
   • Development and differentiation of organisms involve coordinated regulation of gene expression.

3. Gene Expression Regulation in Prokaryotes:

   • In prokaryotes, control of transcriptional initiation rate is the predominant site for gene expression control.
   • RNA polymerase activity at a promoter is regulated by interaction with accessory proteins.
   • Regulatory proteins can act as activators (positively) or repressors (negatively).
   • Promoter accessibility is regulated by protein interaction with operator sequences.
   • Each operon has a specific operator that interacts with a specific repressor protein (e.g., lac operator interacts with lac repressor in the lac operon).

5.8.1 The Lac operon

1. Discovery of the Lac Operon:

   • François Jacob and Jacques Monod elucidated the lac operon, a transcriptionally regulated system.
   • The lac operon consists of:
     • One regulatory gene (i gene) and three structural genes (z, y, and a).
     • The i gene codes for the repressor of the lac operon.
     • The z gene codes for beta-galactosidase (β-gal), responsible for lactose hydrolysis.
     • The y gene codes for permease, increasing cell permeability to β-galactosides.
     • The a gene encodes a transacetylase.
   • The structural genes are regulated by a common promoter and regulatory genes.

2. Function of Lac Operon:

   • Lactose acts as an inducer, regulating the switching on and off of the operon.
   • In the absence of glucose, lactose is transported into cells through permease.
   • Lactose induces the operon by inactivating the repressor, allowing RNA polymerase access to the promoter for transcription.
   • Regulation of lac operon can be viewed as regulation of enzyme synthesis by its substrate.

3. Regulation of Lac Operon:

   • Repressor synthesis occurs constitutively from the i gene.
   • Repressor protein binds to the operator region, preventing RNA polymerase transcription.
   • Inducer interaction with the repressor inactivates it, allowing transcription to proceed.
   • Regulation of lac operon by the repressor is termed negative regulation.

4. Additional Information:

   • Glucose or galactose cannot act as inducers for the lac operon.
   • Positive regulation of the lac operon exists but is not discussed at this level.

5.9 HUMAN GENOME PROJECT

1. Background and Launch of the Human Genome Project (HGP):

   • DNA sequence determines an organism’s genetic information.
   • The Human Genome Project (HGP) aimed to sequence the entire human genome.
   • Launched in 1990, it was a mega project involving significant resources and technological advancements.

2. Scope and Goals of the HGP:

   • Human genome contains approximately 3 billion base pairs.
   • Estimated cost of sequencing was $3 per base pair, totaling around $9 billion.
   • The data generated would require vast storage and computational resources.
   • Goals included identifying all human genes, determining DNA sequences, storing data in databases, and addressing ethical and social issues.

3. Project Management and Partners:

   • Coordinated by the U.S. Department of Energy and the National Institute of Health.
   • Wellcome Trust (U.K.) and other countries contributed to the project.
   • Completed in 2003, with significant contributions to understanding human biology and applications in various fields.

4. Methodologies and Approaches:

   • Two major approaches: ESTs (Expressed Sequence Tags) for gene identification and blind sequencing of the whole genome.
   • DNA isolated and fragmented, then cloned using vectors like BACs and YACs.
   • Sequencing done using automated DNA sequencers based on Frederick Sanger’s method.
   • Computer-based programs used for sequence alignment and annotation.

5. Challenges and Achievements:

   • Completion of chromosome sequencing, including chromosome 1 in 2006.
   • Mapping genetic and physical features using polymorphism data and microsatellites.
   • Significance in understanding human biology and potential applications in various fields like healthcare, agriculture, and energy production.

5.9.1 Salient Features of Human Genome 

1. Genome Size and Composition:

   • The human genome contains 3164.7 million base pairs (bp).
   • Average gene size is 3000 bases, with variations. The largest known gene is dystrophin at 2.4 million bases.

2. Gene Count and Functionality:

   • Estimated total genes at 30,000, much lower than previous estimates.
   • Nearly all nucleotide bases (99.9%) are identical across individuals.
   • Over 50% of discovered genes have unknown functions.
   • Less than 2% of the genome codes for proteins.

3. Repetitive Sequences:

   • Repetitive sequences constitute a large portion of the human genome.
   • These sequences repeat many times and are vital for understanding chromosome structure, dynamics, and evolution.

4. Chromosome Distribution:

   • Chromosome 1 has the most genes (2968), while the Y chromosome has the fewest (231).

5. Single-Nucleotide Polymorphisms (SNPs):

   • Scientists have identified about 1.4 million locations where single-base DNA differences occur in humans.
   • SNPs offer insights into disease-associated sequences and human history.

5.9.2 Applications and Future Challenges 

1. Biological Research Advancements:

   • The HGP enables a new approach to biological research, allowing scientists to study entire genomes systematically and on a large scale.
   • Researchers can investigate all genes in a genome, study gene expression in specific tissues or organs, or analyze complex gene networks.

2. Interdisciplinary Collaboration:

   • Progress in understanding biological systems will require collaboration among tens of thousands of scientists from diverse disciplines, both in the public and private sectors globally.
   • Expertise and creativity from various fields such as genetics, molecular biology, bioinformatics, and computational biology will be essential.

3. High-Throughput Technologies:

   • The availability of whole-genome sequences and high-throughput technologies facilitates large-scale data analysis and experimentation.
   • New technologies enable rapid and cost-effective sequencing, transcriptomics, proteomics, and metabolomics, accelerating research progress.

4. Systems Biology Approach:

   • Researchers can explore how tens of thousands of genes and proteins interact in interconnected networks to regulate biological processes.
   • Systems biology approaches provide insights into the complex dynamics of living systems and how they respond to internal and external stimuli.

5. Understanding Disease Mechanisms:

   • Genome-wide association studies (GWAS) help identify genetic variants associated with diseases, leading to insights into disease mechanisms and potential therapeutic targets.
   • Comparative genomics across species aids in understanding the genetic basis of diseases and evolutionary adaptations.

6. Ethical, Legal, and Social Issues (ELSI):

   • Addressing ELSI concerns related to genetic privacy, discrimination, and informed consent remains a critical aspect of genomic research.
   • Policies and regulations need to be developed to ensure responsible use of genomic data and technologies.

5.10 DNA FINGERPRINTING

1. Repetitive DNA: Repetitive DNA sequences, such as microsatellites and minisatellites, are isolated from genomic DNA using density gradient centrifugation. These sequences, which do not code for proteins, are highly polymorphic and form the basis of DNA fingerprinting.

2. DNA Isolation and Processing:

   • DNA is isolated from various tissues (blood, hair, saliva, etc.) of an individual.
   • The isolated DNA is digested using restriction enzymes, resulting in fragments of varying sizes.

3. Southern Blot Hybridization:

   • The digested DNA fragments are separated by gel electrophoresis.
   • The separated DNA fragments are transferred (blotted) onto synthetic membranes.
   • These membranes are then probed with a radiolabeled VNTR probe, specific to the repetitive DNA sequences.

4. Autoradiography:

   • The hybridized DNA fragments are detected using autoradiography, revealing a pattern of bands on the membrane.
   • Each individual exhibits a unique banding pattern, except for identical twins, due to variations in the lengths of repetitive DNA sequences.

5. PCR Amplification:

   • Polymerase chain reaction (PCR) amplification has enhanced the sensitivity of DNA fingerprinting.
   • PCR allows the amplification of DNA from a single cell, enabling DNA fingerprinting analysis with minimal sample requirements.

6. Applications:

   • Forensic Science: DNA fingerprinting is widely used in forensic investigations to identify suspects or victims based on DNA evidence from crime scenes.
   • Paternity Testing: It is employed to determine biological relationships between individuals, particularly in cases of disputed parentage.
   • Genetic Diversity Studies: DNA fingerprinting aids in studying population genetics, genetic diversity, and evolutionary relationships among species.
   • Medical Diagnosis: It has applications in medical diagnostics, such as identifying genetic disorders and predicting disease susceptibility.