Gene Expression Overview

Beadle and Tatum's Research

• Focus: Beadle and Tatum focused on understanding the relationship between genes and enzymes. They used the haploid bread mold Neurospora crassa to study how mutations affecting specific metabolic pathways led to changes in phenotype.

• Minimal Medium: Minimal medium contains only the basic nutrients necessary for growth, forcing organisms to synthesize all other essential molecules. Beadle and Tatum used minimal medium to identify mutants that could not grow without specific supplements.

• Generating Mutants: They exposed Neurospora crassa to X-rays, a mutagen that can induce random mutations in DNA. This led to the creation of mutants that lacked specific enzymes required for the synthesis of certain amino acids.

• Arginine Mutants: By testing the ability of different mutants to grow on media with various combinations of amino acids, they found that mutations affecting arginine biosynthesis were blocked at different steps of the pathway. This supported the idea that each gene controlled the production of a specific enzyme.

Central Dogma of Molecular Biology

• DNA to Protein: The primary process by which DNA directs the synthesis of proteins is through the central dogma of molecular biology. This process involves transcription, where DNA is transcribed into messenger RNA (mRNA), followed by translation, where mRNA is translated into a protein.

The One Gene-One Enzyme Hypothesis

• Proposal: Beadle and Tatum's research led to the development of the One Gene-One Enzyme Hypothesis, positing that a single gene controls the production of a single enzyme. This hypothesis has been refined to the One Gene-One Polypeptide Hypothesis, recognizing that some genes encode proteins with multiple subunits.

Archibald Garrod and Metabolic Defects

• Contribution: Archibald Garrod, a British physician, was a pioneer in understanding the relationship between genes and metabolic disorders. He studied alkaptonuria, a rare inherited disease, and proposed that it resulted from a deficiency in a specific enzyme responsible for breaking down a certain molecule.

Genes, Enzymes, and Metabolic Pathways

• Metabolic Pathways: Metabolic pathways are a series of connected chemical reactions that occur within a cell to build up or break down molecules. Genes code for the enzymes that catalyze these reactions.

• Metabolic Defects: Metabolic defects arise when mutations in genes disrupt the production or function of enzymes involved in a specific metabolic pathway. This can lead to the accumulation of harmful substances or a deficiency in essential products.

• Alkaptonuria: In alkaptonuria, a metabolic defect prevents the breakdown of homogentisic acid, a molecule in the tyrosine degradation pathway. This results in the accumulation of homogentisic acid, which darkens urine and can cause joint problems.

Gene Expression: From DNA to Trait

• Traits and Proteins: In gene expression, proteins ultimately control the appearance of traits, such as coat color and skin color.

Regulatory Mechanisms of Gene Expression

• CRP and the Lac Operon: CRP (cAMP receptor protein) is a regulatory protein that activates transcription of the lac operon in E. coli when glucose is limited. cAMP binds to CRP, and the complex then binds to a specific site on the lac operon, promoting the binding of RNA polymerase to the promoter.

• Eukaryotic Gene Expression: Eukaryotic gene expression is more complex than in prokaryotes, involving multiple levels of regulation, including chromatin remodeling, transcription, RNA processing, and translation.

• Histone Modification:*

• Acetylation: Histone acetylation loosens chromatin structure, allowing for increased gene expression.

• Methylation: Histone methylation can either enhance or repress gene expression, depending on the specific amino acid residue that is methylated.

• Epigenetic Inheritance: Modifications to chromatin, such as histone acetylation and DNA methylation, can be inherited from one generation to the next, even without changes in the underlying DNA sequence. This is known as epigenetic inheritance.

• Enhancers: Enhancers are DNA sequences that can significantly increase the rate of transcription of a gene, even if they are located many kilobases away from the gene they regulate. They work by binding to regulatory proteins that interact with transcription factors.

• Rapid Adjustment of Gene Expression: MicroRNAs (miRNAs) can quickly regulate gene expression after transcription by binding to mRNA and degrading it or inhibiting its translation.

• General vs. Specific Transcription Factors: General transcription factors are involved in the initiation of transcription for all protein-coding genes. Specific transcription factors, on the other hand, bind to specific DNA sequences near the genes they regulate, thereby controlling their expression.

• Heterochromatin: Tightly compacted chromatin (heterochromatin) is typically transcriptionally inactive because it is inaccessible to transcription factors and RNA polymerase.

• mRNA Binding and Translation: Regulatory proteins binding to mRNA during translation initiation can inhibit or enhance protein synthesis, depending on the specific protein involved.

• Activators and Repressors: Activators are regulatory proteins that bind to DNA and enhance transcription, while repressors bind to DNA and inhibit transcription. These proteins interact with specific transcription factors, often forming complex regulatory networks.

• DNA Methylation Impacts: DNA methylation is commonly associated with gene silencing. Methylation of cytosine bases in DNA can attract specific proteins that inhibit the binding of transcription factors, leading to reduced transcription.

• Co-Expressed Eukaryotic Genes: Eukaryotic genes that are co-expressed often share regulatory elements, such as enhancers, that are bound by the same transcription factors, allowing for coordinated control of their expression.

• Chromatin Structure Regulation: Histone modifications, such as acetylation and methylation, primarily regulate gene expression by altering chromatin structure, making it more or less accessible to transcription factors.

Development and Pattern Formation

• Maternal Effect Genes: Maternal effect genes are genes expressed in the mother organism that provide essential products (like RNA and proteins) for the development of the embryo.

• Drosophila Homeotic Genes: Homeotic genes control the identity of body segments in organisms. Mutations in homeotic genes result in the development of body parts in the wrong location, leading to alterations in body plan.

• Bicoid and Pattern Formation: Bicoid is a maternal effect gene in Drosophila that controls the formation of the anterior-posterior axis of the embryo. Bicoid protein, a transcription factor, establishes a concentration gradient across the embryo, with higher levels in the anterior end. This gradient instructs cells in different parts of the embryo to develop into specific structures.

• Bicoid Research Findings: Research on Bicoid led to the groundbreaking discovery that maternal effect genes can establish spatial patterns in the embryo, providing critical information for development.

• Embryonic Lethals Embryonic lethals are mutations that cause an organism to die during the embryonic stage. These mutations can be used to study the function of genes that are essential for development.

• Segmentation Genes in Drosophila: Researchers have identified 120 genes in Drosophila that influence segmentation patterns. These genes act in a hierarchical manner, with gap genes, pair-rule genes, and segment-polarity genes controlling different aspects of segmentation.

• Evo-Devo: Evo-Devo (evolutionary developmental biology) is a field of study that examines the evolutionary relationships between development and the changes in body plans that occur during evolution.

• Bicoid mRNA Injection: When Bicoid mRNA is injected into various regions of a Drosophila embryo, it can re-establish a proper anterior-posterior axis, demonstrating the crucial role of Bicoid in pattern formation.

Transcription and RNA Processing

• RNA Polymerase: RNA polymerase is the enzyme responsible for synthesizing RNA from a DNA template during transcription. It moves along the DNA template strand, reading the sequence and adding complementary RNA nucleotides to the growing RNA transcript.

• 5' Cap and Poly-A Tail: The 5' cap and poly-A tail added to mRNA are essential for protecting the molecule from degradation and promoting its translation. The 5' cap is a modified guanine nucleotide added to the 5' end of the mRNA. The poly-A tail is a string of adenine nucleotides added to the 3' end.

• Codons and the Genetic Code: Codons are three-nucleotide sequences in mRNA that specify a particular amino acid. The genetic code is a dictionary that translates each codon into its corresponding amino acid.

• Primary Transcripts: In eukaryotic cells, primary transcripts (pre-mRNA) undergo several processing steps before they can be translated into protein. These steps include capping, splicing, and polyadenylation.

• DNA Template Strand: The template strand of DNA is the strand that is used as a template for RNA synthesis during transcription. It has the opposite sequence to the RNA transcript, but with thymine (T) replaced by uracil (U).

• Spliceosomes: Spliceosomes are complex molecular machines that remove introns (non-coding regions) from pre-mRNA and splice together exons (coding regions) to create mature mRNA.

Bacterial Gene Regulation: The Operon Model

• Operon: An operon is a group of genes that are transcribed together as a single unit in bacteria. The genes in an operon are typically involved in a related metabolic pathway.

• Corepressor: A corepressor is a molecule that binds to a repressor protein, changing its shape and allowing it to bind to the operator in the operon, inhibiting transcription.

Translation: Protein Synthesis

• tRNA Function: Transfer RNA (tRNA) molecules act as adapters, bringing the correct amino acid to the ribosome based on the codon sequence in mRNA.

• TATA Box: The TATA box is a conserved DNA sequence found in the promoter region of many eukaryotic genes. It helps to position RNA polymerase for transcription initiation.

• Aminoacyl tRNA Synthetase: This enzyme links the correct amino acid to its corresponding tRNA molecule.

• Reading Frame: The reading frame during translation is established by the start codon (AUG), which specifies the first amino acid in the protein.

• Multiple tRNA Recognition: More than one tRNA can recognize a single amino acid because the genetic code is degenerate, meaning that some amino acids are encoded by more than one codon.

Cancer Genomics and Microarray Technology

• Cancer Genome Atlas Project: This project aims to characterize the genetic and epigenetic changes associated with different types of cancer. By sequencing the genomes of cancer cells, researchers can identify mutations, deletions, and other genetic alterations that contribute to cancer development.

• Microarray Technology: Microarrays are tools that allow researchers to simultaneously measure the expression levels of thousands of genes. In cancer research, microarrays can be used to identify genes that are upregulated or downregulated in cancer cells, providing insights into the molecular mechanisms underlying cancer development.

Genomes: Size, Structure, and Evolution

• Bacterial and Archaeal Genomes: Most bacteria and archaea have genomes that range in size from about 1 to 10 million base pairs (Mb).

• Multicellular Eukaryotic Genomes: Multicellular eukaryotes generally have much larger genomes, with gene density being lower (fewer genes per unit length of DNA).

• Noncoding DNA: A majority of the human genome (around 98%) is composed of noncoding DNA. This includes introns, repetitive sequences, and transposable elements.

• Pseudogenes: Pseudogenes are non-functional copies of genes that have been deactivated by mutations.

• Transposable Elements: Transposable elements (TEs) are DNA sequences that can move from one location in the genome to another. These elements can disrupt gene function or contribute to genome evolution.

• Reverse Transcriptase: Retrotransposons are a type of transposable element that uses reverse transcriptase to copy themselves from RNA into DNA before inserting into the genome.

• LINE-1 Elements: LINE-1 (Long Interspersed Nuclear Elements) are a type of retrotransposon that makes up about 17% of the human genome.

• Short Tandem Repeats (STRs): STRs are short, repetitive DNA sequences that are often used in DNA fingerprinting and genetic analysis.

• Multigene Families: Multigene families are groups of genes that have similar sequences and are thought to have arisen by gene duplication.

• Bacterial Genes: Free-living bacteria typically have around 1,000-10,000 genes.

• Genome Size vs. Phenotype: There is no direct relationship between genome size and phenotypic complexity in organisms. Some organisms have very small genomes but are complex, while others have large genomes but are relatively simple in structure.