Chapter 10: Molecular Biology of the Gene

The Structure of the Genetic Material

The discovery of DNA as the genetic material was a pivotal moment in biology. Several key experiments established DNA's role and revealed its structure.

• Frederick Griffith: Demonstrated transformation in bacteria, showing that a "transforming principle" could transfer genetic information.

• Alfred Hershey and Martha Chase: Used bacteriophages to confirm that DNA, not protein, is the genetic material.

• Erwin Chargaff: Discovered that the amount of adenine equals thymine and cytosine equals guanine in DNA (Chargaff's rules).

• Watson and Crick: Proposed the double helix model of DNA structure.

• Wilkins and Franklin: Used X-ray crystallography to provide images that helped elucidate DNA's structure. Example: Franklin's Photo 51 was critical evidence for the helical structure of DNA.

• Structure of DNA: DNA is a double helix composed of two antiparallel strands of nucleotides. Each nucleotide contains a deoxyribose sugar, a phosphate group, and a nitrogenous base (adenine, thymine, cytosine, or guanine).

• Base Pairing Rules: Adenine pairs with thymine (A-T), and cytosine pairs with guanine (C-G) via hydrogen bonds.

• Nitrogenous Bases in DNA and RNA: DNA contains A, T, C, G; RNA contains A, U (uracil), C, G.

• Predicting Complementary Strands: Given one DNA strand, the complementary strand can be determined using base pairing rules.

DNA Replication

DNA replication ensures genetic information is accurately passed to new cells.

• Semi-conservative Replication: Each new DNA molecule consists of one old (parental) strand and one newly synthesized strand.

• Process of DNA Replication: Involves unwinding the double helix, complementary base pairing, and joining of nucleotides.

• Key Enzymes:

  • DNA polymerase: Synthesizes new DNA strands by adding nucleotides.

  • DNA ligase: Joins Okazaki fragments on the lagging strand.

The Flow of Genetic Information: DNA to RNA to Protein

The central dogma describes the flow of genetic information from DNA to RNA (transcription) and from RNA to protein (translation).

• Transcription vs. Translation:

  • Transcription: Synthesis of RNA from a DNA template.

  • Translation: Synthesis of a polypeptide (protein) from an mRNA template.

• Codon: A sequence of three nucleotides in mRNA that codes for a specific amino acid.

• Predicting RNA from DNA: Replace T with U in the RNA strand; use base pairing rules.

• RNA Polymerase: Enzyme that synthesizes RNA during transcription.

• Steps of Transcription:

  1. Initiation: RNA polymerase binds to the promoter.

  2. Elongation: RNA strand is synthesized.

  3. Termination: RNA polymerase reaches a terminator sequence and detaches.

• RNA Processing in Eukaryotes:

  • Addition of a 5' cap

  • Addition of a poly-A tail

  • RNA splicing (removal of introns, joining of exons)

• Types of RNA:

  • mRNA (messenger RNA): Carries genetic code from DNA to ribosome.

  • tRNA (transfer RNA): Brings amino acids to the ribosome; contains anticodon.

  • rRNA (ribosomal RNA): Structural and catalytic component of ribosomes.

• Ribosome Structure and Function: Ribosomes have A (aminoacyl) and P (peptidyl) sites for tRNA binding and peptide bond formation.

• Translation Steps:

  1. Initiation: Ribosome assembles on mRNA.

  2. Elongation: tRNAs bring amino acids; peptide bonds form.

  3. Termination: Stop codon is reached; polypeptide is released.

• Mutations:

  • Missense: Changes one amino acid.

  • Nonsense: Introduces a stop codon.

  • Frameshift: Insertion or deletion shifts the reading frame.

Genetics of Viruses and Bacteria

Viruses and bacteria have unique genetic mechanisms and play important roles in gene transfer and disease.

• Virus Structure: All viruses have a nucleic acid genome and a protein coat (capsid). Some have a membranous envelope.

• Lytic vs. Lysogenic Cycles:

  • Lytic: Virus replicates and lyses host cell.

  • Lysogenic: Viral DNA integrates into host genome and replicates with it.

• Animal Viruses: Most contain RNA genomes.

• Vaccines: Stimulate immune response to prevent viral infection.

• AIDS Virus (HIV): Enters host cells via specific receptors; reproduces using reverse transcriptase.

• Retrovirus: RNA virus that uses reverse transcriptase to make DNA from RNA.

• Prions: Infectious proteins causing neurodegenerative diseases.

• Gene Transfer in Bacteria:

  • Transformation

  • Transduction

  • Conjugation

• Plasmid: Small, circular DNA molecule in bacteria.

• R Plasmids: Carry antibiotic resistance genes; pose health risks.

Chapter 11: Control of Gene Expression

Gene Regulation in Eukaryotes

Gene expression is tightly regulated to ensure proper cell function and differentiation.

• Differentiation: Process by which cells become specialized; all cells contain the same DNA but express different genes.

• Histones and Nucleosomes: Histones are proteins around which DNA winds; nucleosomes are DNA-histone complexes.

• Chromatin Structure: Highly condensed DNA is less accessible for transcription; loosely condensed DNA is more active.

• Chemical Modifications:

  • DNA methylation: Usually represses gene expression.

  • Histone methylation: Can repress or activate gene expression.

  • Histone acetylation: Generally activates gene expression.

• X Chromosome Inactivation: One X chromosome in females is inactivated, forming a Barr body.

• Transcription Factors: Proteins that bind to DNA and regulate transcription.

• Major Control Point: Initiation of transcription is the most important regulatory step.

• Alternative RNA Splicing: Allows one gene to code for multiple proteins by varying exon combinations.

• miRNAs: Small RNAs that can degrade mRNA or block translation (RNA interference).

• Mechanisms of Gene Regulation: Include chromatin modification, transcriptional control, RNA processing, mRNA transport, translation, protein processing, and degradation.

Cloning of Plants and Animals

Cloning demonstrates that differentiated cells retain all genetic information.

• Clone: Genetically identical organism.

• Totipotent: Cell capable of developing into any cell type.

• Nuclear Transplantation: Nucleus from a differentiated cell is transferred to an enucleated egg.

• Genetic Identity in Cloning: Cloned individual has nuclear DNA from donor nucleus, not from donor egg or surrogate.

• Stem Cells:

  • Embryonic stem cells: Pluripotent, can become many cell types.

  • Adult stem cells: Limited differentiation potential.

The Genetic Basis of Cancer

Cancer arises from mutations in genes that control cell growth and division.

• Proto-oncogenes: Normal genes that promote cell division.

• Oncogenes: Mutated proto-oncogenes that cause uncontrolled cell division.

• Tumor-suppressor genes: Inhibit cell division; mutations can lead to cancer.

• Cancer Development: Usually requires multiple mutations over time; more common in elderly due to accumulation of mutations.

• Inheritance: Cancer-causing mutations must be present in gametes to be inherited.

• Key Genes: ras (oncogene), p53 (tumor-suppressor gene).

• Cancer Risk Factors: Include lifestyle, environment, and genetics; tobacco is the leading cause of cancer.

Chapter 12: DNA Technology and Genomics

Gene Cloning and Recombinant DNA

Gene cloning allows scientists to manipulate and study genes and proteins.

• Recombinant DNA: DNA molecules formed by combining DNA from different sources.

• Steps in Gene Cloning:

  1. Isolate gene of interest.

  2. Insert gene into plasmid (vector).

  3. Introduce plasmid into bacteria.

  4. Bacteria replicate, producing copies of the gene.

• Plasmids: Used as vectors to carry foreign DNA.

• DNA Ligase: Enzyme that joins DNA fragments.

• Restriction Enzymes: "Scissors" that cut DNA at specific sequences; DNA ligase is the "glue".

• Sticky Ends: Overhanging sequences that facilitate joining of DNA fragments.

• Nucleic Acid Probe: Labeled DNA or RNA used to detect specific sequences.

• cDNA: Complementary DNA made from mRNA using reverse transcriptase.

• CRISPR/Cas9: Genome editing tool that makes precise changes in DNA; popular for its accuracy and efficiency.

Genetically Modified Organisms (GMOs)

GMOs are organisms with artificially altered genes for research, medicine, or agriculture.

• Mammalian Cells: Can perform complex protein modifications not possible in bacteria or yeast.

• Applications: Production of insulin, growth hormone, and vaccines using recombinant DNA technology.

• Transgenic Organism: Contains genes from another species.

• GM Crops: Engineered for traits like pest resistance or improved nutrition (e.g., golden rice contains beta-carotene).

• Pros and Cons: Benefits include increased yield and nutrition; concerns include environmental impact and labeling.

• Labeling: GMO products are not required to be labeled in the U.S.

• Gene Therapy: Treats disease by correcting defective genes; has potential benefits and risks.

DNA Profiling and Biotechnology

DNA profiling identifies individuals based on unique DNA patterns.

• DNA Profiling: Analysis of DNA fragments to identify individuals.

• Steps:

  1. DNA extraction

  2. Amplification (PCR)

  3. Analysis (gel electrophoresis)

• PCR (Polymerase Chain Reaction): Amplifies specific DNA sequences.

• Gel Electrophoresis: Separates DNA fragments by size; shorter fragments move farther toward the positive electrode.

• STR Analysis: Uses short tandem repeats for identification.

• Applications: Forensics, paternity testing, and exoneration of the innocent.

• Other Biotechnologies: Gene therapy, proteomics (study of proteins).

Genomics

Genomics is the study of entire genomes, providing insights into evolution and gene function.

• Genomics: Study of whole genomes, including gene mapping and sequencing.

• Human Genome: Most DNA is non-coding (introns, regulatory sequences, transposable elements).

• Transposable Elements: DNA sequences that can move within the genome; common in humans.

• Gene and Protein Numbers: Humans have about 20,000–25,000 genes but can produce more proteins due to alternative splicing.

• Bioinformatics: Application of computational tools to analyze biological data.

• Proteomics: Study of the full set of proteins encoded by a genome.

• Significance: Genomics advances understanding of evolution, disease, and gene function.

Additional info: For equations, the central dogma can be represented as:

For PCR amplification:

where is the number of DNA molecules after cycles, starting from molecules.