LS 7A Notes PDF

17.2 Transcription and RNA Processing in Eukaryotes *In eukaryotes, additional levels of gene regulation can be provided by the way dna is packaged into chromosomes, mRNA processing, and the separation in space of transcription and translation. * Different types of cells express different genes EX:...

17.2 Transcription and RNA Processing in Eukaryotes *In eukaryotes, additional levels of gene regulation can be provided by the way dna is packaged into chromosomes, mRNA processing, and the separation in space of transcription and translation. * Different types of cells express different genes EX: Every cell in the body contains the genes that encode insulin, but only in pancreatic cells are they expressed. TRANSCRIPTION IS A KEY CONTROL POINT IN GENE EXPRESSION Transcriptional regulation in eukaryotic cells require proteins that interact with one another and with DNA sequences near the gene REGULATORY TRANSCRIPTION FACTORS: Two binding sites: One binds with a gene sequence in or near a gene called an enhancer, the other recruits a set of proteins called general transcription factors. GTF assembles near a short sequence in the promoter called the TATA box. Once bound to the promoter, the GTF attracts the RNA polymerase complex, which is the enzyme complex that synthesized the RNA transcript complementary to the template strand of DNA. Once it is in place, TRANSCRIPTION can begin. 100s of different RTF control the transcription of thousands of genes. ○ Some bind with enhancers and simulate transcription ○ Others bind with silencers and repress transcription RTF are highly diverse proteins that regulate one or a group of genes GTF are the same irrespective of the protein-coding gene ○ All protein-coding genes use the same RNA polymerase complex. Transcription of a gene with multiple silencers and enhancer depends on the presence of a particular combo of RTF, so it is called combinatorial control. RNA PROCESSING IS ALSO IMPORTANT IN GENE REGULATION Primary transcript undergoes several modifications: ○ Addition of 5’ cap ○ String of about 250 adenosine nucleotides to the 3’ end to the poly(A) tail.  ○ ! Necessary for the RNA molecule to be transported to the cytoplasm and recognized by the translational machinery. ○ Length of poly(A) tail helps determine how long the RNA will persist in the cytoplasm before being degraded. Eukaryotes: RNA splicing: ○ Long primary transcript consists of regions that are retained in the mRNA and expressed (exons) as well as regions interspersed with the exons and removed (introns). ○ Spliceosome: Joins the exons together in their original linear order and removes the introns to form the processed mRNA. ○ Alternative splicing: takes place because the spliceosome recognizes something as an exon in some primary transcripts but as an intron in others. May be produced in the same or different cells. Happens a lot i guess. RNA Editing: some RNA molecules can become a substrate for enzymes that modify some bases in the RNA… making it change its sequence and what it codes for. RNA Editing and whatnot ;) Lots and lots and lots of transcripts in the human genome undergo RNA editing. ○ Transcripts from the same gene can produce multiple types of proteins, even in a single cell. 17.3 Messenger RNA to Phenotype in Eukaryotes *processed mRNA must exit the nucleus before the translation step of gene expression can occur. The mRNA goes through the cytoplasm and there, there are multiple opportunities for gene regulation at the levels of mRNA stability, translation, and protein activity. SMALL REGULATORY RNAs PROMOTE mRNA DEGRADATION OR INHIBIT TRANSLATION Small regulatory RNA: plays an important role in the regulation of gene expression either by binding to regulatory proteins or directly to target mRNAs ○ Two important types: siRNA (small interfering RNA): miRNA (micro RNA): ○ Both are transcribed from DNA and form hairpin structures in the nucleus which are stabilized by base pairing in the stem. Enzymes in the cytoplasm recognize these structures and cleave the stem from the hairpin and cut the stem into small fragments that are two strands One of the two strands from each fragment are incorporated into RISC. The small regulatory RNA may result in the degradation of the mRNA transcript-inhibition of translation, or chromatin remodeling ○ Regulation by small regulatory RNA is widespread in eukaryotes TRANSLATIONAL REGULATION CONTROLS THE RATE, TIMING, AND LOCATION OF PROTEIN SYNTHESIS. *Another level of control of gene expression is translation of mRNA  Some RNA-binding proteins interact with molecular motors that transport the mRNA to particular regions of the cell. By either transport or repression, these proteins cause the mRNA to be translated only in certain places in the cell Cap structure is one of the main signals for translation initiation While some mRNAs from a gene are being translated, other mRNAs transcribed from the same gene may not have a translation initiation complex assembled. 3’ UTR and poly(A) tail are also important in translation initiation. Contact between a protein that binds them creates a loop in the mRNA, bringing the 3’ end of the mRNA close to the start site of translation. ○ Not all mRNA molecules are equally accessible to translation. PROTEIN STRUCTURE AND CHEMICAL MODIFICATION MODULATE PROTEIN EFFECTS ON PHENOTYPE Translation is completed-the resulting protein can alter the phenotype of the cell or organism by affecting metabolism, signaling, gene expression, or cell structure. *Post translational modification: changes to proteins that occur after they are translated. Folding and stability are key control points for some proteins. ○ Correct folding is important bc improperly folded proteins can bind together to form aggregates that are no bueno for cell function ○ Like Alzheimers, huntingtons Post Translational modification helps regulate protein activity. ○ Many proteins are modified by adding one or more sugar molecules to the side of amino acids which can alter the protein’s folding and stability. ○ A change in protein conformation affects protein function 17.4 Chromatin Remodeling and Epigenetics In eukaryotes, DNA is packaged in the form of chromatin ○ Chromatin: a complex of DNA, RNA, and nucleosome proteins that give chromosomes their structure. Chromatin must loosen to allow space for transcriptional enzymes and proteins to work ○ Chromosome remodeling: nucleosomes are repositioned to expose different stretches of DNA to the nuclear environment. Gene Expression can be influenced by chemical modification of DNA or histones Chromatin can be remodeled by posttranslational modification of histones around which DNA is wound. ○ Histone tails: strings of amino acids that protrude from the histone proteins in the nucleosome. Amino acids in the tails can be altered by the addition or removal of different chemical groups. Some modifications tend to activate transcription or repress transcription. ○ Histone code: the pattern of modifications of the histone tails, can affect the chromatin structure and gene transcription. DNA methylation recruits proteins that lead to changes in chromatin structure, histone modification, and nucleosome positioning that restrict access of transcription factors to promoters. ○ Heavy cytosine methylation is associated with transcriptional repression of the gene near the CpG island. Epigenetic mechanisms of gene regulation involve changes to how the DNA is packaged and not just to the DNA sequence itself. ○ Epigenetic modifications can be transmitted from parent to offspring, meaning these chemical modifications can be inherited. Imprinting: a process that silences one copy of a gene in an individual, while the other copy is expressed. ○ Some genes, the allele of the gene inherited from the mother is imprinted and silenced, so only the allele inherited from the father is expressed. Some of the genes that are imprinted affect the growth rate of the embryo. Gene Expression can be regulated at the level of an entire chromosome/ For most genes, there is a direct relation between the number of copies of the gene (the gene dosage) and the level of expression of the gene.  ○ Increased gene dosage=Increased level of expression: each copy of the gene is regulated independently of the other copies Dosage compensation: the differential regulation of X-chromosomal genes in females and in males X-inactivation: the process in female mammals in which dosage compensation occurs through the inactivation of one X chromosome in each cell. X-inactivation is a process in female mammals where one of the two X chromosomes in each cell is "turned off" to ensure females and males have the same amount of active X-linked genes. How it works: 1. Random choice: Early in development, each cell randomly chooses one X chromosome to inactivate (either the one from mom or dad). 2. Marking: A special RNA called XIST coats the chosen X chromosome, marking it for inactivation. 3. Shutting down: The marked X chromosome gets tightly packed (heterochromatin) so its genes can’t be used. 4. Permanent state: Once an X is inactivated in a cell, it stays inactive in all the cell’s descendants.  The Xist gene is necessary for x-inactivation ○ Deleted x-inactivation does not occur ○ Inserted into another chromosome: inactivates the chromosome. Gene expression in eukaryotic cells ○ Chromatin, transcription, RNA processing, mRNA, stability, translation, and post translation. Your lifestyle choices can affect gene expression in your own genome Dosage compensation: the differential regulation of X-chromosomal genes in females and in males 5.3 Regulation of Protein synthesis and sorting Most proteins are needed only in certain types of cells and only at certain times. ○ Contains signals that allow them to be sorted into particular cellular compartments where their main function carries out. Protein synthesis is regulated at multiple levels Gene regulation: the various ways in which cells control gene expression. ○ Determines when a gene is expressed, in which cell types, in what quality. Genes can be regulated at the level of the chromosome itself through transcription or translation, or even after the protein is made. Protein Localization: ○ Proteins have specific cellular destinations. ○ Signal sequences on proteins act as "barcodes" to ensure proper sorting and localization. Regulation of Protein Synthesis Gene Expression Control: ○ Regulation occurs at multiple levels: DNA accessibility. Transcription initiation. RNA processing (splicing and modifications). Translation. Post-translational modifications. ○ Each step offers control points for fine-tuning gene expression. Mechanisms: ○ Proteins can only be expressed when transcription machinery can access the DNA. ○ Transcription factors are often necessary to initiate transcription. ○ mRNA must be spliced, modified, and transported to the cytoplasm for translation. ○ Post-translational modifications (e.g., phosphorylation) activate proteins or modify their functions. Protein Sorting  Two Production Sites: ○ Free Ribosomes in the Cytosol: Proteins synthesized here are sorted after translation. Signal sequences determine the protein’s destination: Cytosol (no signal sequence). Mitochondria or chloroplasts (amino-terminal signal). Nucleus (internal nuclear localization signals). ○ Membrane-bound Ribosomes on the Rough ER: Proteins synthesized here are sorted during translation. These proteins may: Become transmembrane proteins. Reside in the ER or Golgi lumen. Be secreted or transported to lysosomes. Signal Recognition Particle (SRP): ○ SRP binds to the signal sequence and ribosome, pauses translation, and directs the ribosome to the rough ER. ○ Translation resumes, and the polypeptide is threaded into the ER lumen through a channel. ○ The signal sequence is cleaved, and proteins are sorted for their final destinations. 12.1 DNA Manipulation Reaction Enzymes cleave DNA at particular short sequences Cutting DNA molecules allows pieces from the same for different organisms to be brought together in recombinant DNA technology ○ Way to determine whether or not specific sequences are present in a segment of DNA, because techniques for cutting DNA depend on specific DNA sequences. ○ Allows whole genomes to be broken into smaller pieces for further analysis, such as DNA sequencing. Restriction enzymes: Any one of a class of enzymes that recognizes specific, short nucleotide sequences in double-stranded DNA and cleaves DNA at or near these sites. ○ About 1000 different kinds ○ Any kind can cut DNA from any organism Restriction sites: a recognition sequence in DNA cutting, which is typically four or six base pairs long; most restriction enzymes cleave double-stranded DNA at or near these restriction sites. ○ Where the enzyme finds this site in a dna molecule, it cleaves each strand exactly at the position indicated by vertical arrows. The sequence of the top strand is exactly the same as the sequence of the bottom strand. Palindromic: Reading the same in both directions, typical of restriction sites. Not at the center of the recognition sequence! ○ Each terminate a single stranded overhang When a certain restriction enzyme is used to break up a whole genome into smaller fragments, the specificity of restriction enzymes ensures that the DNA from each cell has the same set of fragments, and that any certain DNA sequence present in the cells is contained in a fragment of the same size DNA strands can be separated and brought back together again Renaturation: The base pairing of complementary single stranded nucleic acids to form a duplex; also called hybridization- opposite of denaturation. ○ Complementary strands come together again. ○ When a solution containing DNA molecules is gradually heated to a high temp, the strands denature. When the solution cools, the complementary strands renature. The amount of base pairing between two sequences affect the temperature at which they renature ○ Very closely related sequences renature at a higher temp than less closely related sequences.  The more closely two species are related, the more similar their DNA sequences. Probe: a labeled DNA fragment that can be tracked in a procedure such as a southern blot. ○ Can be used to determine whether or not a sample of double-stranded DNA molecules contains sequences that are complementary to it. ○ Any DNA fragment can be used as a probe 12.2 DNA Manipulation Recombinant DNA: DNA molecules from two (or more) different sources combined into a single molecule. ○ Involves cutting DNA by restriction enzymes, isolating the resulting DNA fragments by gel electrophoresis, and Ligationg the fragments with enzymes used in DNA replication. Recombinant DNA combines DNA molecules from two or more sources Complementary DNA: A DNA molecules produced by reverse transcriptase from an RNA template. Vector: in recombinant DNA, a carrier of the donor fragment, usually a plasmid. Plasmid: in bacteria, a small circular molecule of DNA carrying a small number of genes that replicates independently of the DNA in the bacterium’s circular chromosome. Transformation: The conversio of cells from one state to another, as from nonvirulent, when DNA is released to the environment by cell breakdown is taken up by recipient cells. In recombinant DNA tech, the into of recombinant DNA into a recipient cell. ○ A form of horizontal gene transfer Recombinant DNA is the basis of genetically modified organisms Transgenic organisms:alternative term for genetically modified organism GMO: an organism that has been genetically engineered, such as modified viruses and bacteria, labratory organisms, ag crops, and domestic animals. DNA editing can be used to alter gene sequences almost at will DNA editing: techniques used to “rewrite” a nucleotide sequence so that specific mutations can be introduced into genes to better understand their function or to correct mutant versions of genes to restore their functions. CRISPR: DNA sequences in Bacteria and Archaea that contain viral sequences and are used for defense; the basis of a DNA editing technique that allows researchers to modify any sequence in the genome ○ Name Describes the organization of viral DNA segments in the bacterial genome In genome editing, the CRISPR mechanism is used to alter the nucleotide sequence of almost any gene in any kind of cell. Almost any DNA sequence in an organism can be altered by means of a form of DNA editing called CRISPR, which uses modified forms of molecules found in bacteria and archaea that can cleave double-stranded DNA at a specific site. 11.1 The Cell Cycle Cell division is the process by which a single cell produces two daughter cells ○ Must be large enough and able to divide in two and contribute enough to either Cell cycle: The series of steps that take place as a eukaryotic cell grows, replicates its DNA, and divides to produce daughter cells. Prokaryotic cells divide by binary fission Binary fission: The process by which cells of bacteria and archaea divide to form two daughter cells. ○ Each daughter cell receives one copy of the replicated parental DNA. ○ ○ FtsZ encodes a protein that forms a ring at the site of constriction where the new cell wall forms between the two daughter cells. Prokaryotic cells divide by binary fission Cell division in eukaryotes is more complicated than cell division in prokaryotes  Mitosis: In eukaryotic cells, the division of the nucleus, in which the chromosomes are separated into two nuclei Cytokinesis: In eukaryotic cells, the division of the cytoplasm into two separate cells. ○ Tg these make miotic cell division The Cell proceeds in phases Cell cycle M Phase: The stage of the cell cycle consisting of mitosis and cytokinesis in which the parent cell divides into two daughter cells Interphase: The part of the cell between two successive M Phases During interphase, the cell prepares for division, like by doing DNA replication and cell growth. ○ DNA in the nucleus first replicates so each daughter cell receives a copy of the genetic material. ○ The cell increases in size so that each daughter cell gets sufficient amongst of cytoplasmix and membrane components to allow it to survive by itself. ○ Cytoskeleton organizes organelles to make sure they’re equally portioned. Interphase is three phases ○ S-Phase: entire content of the nucleus is replicated  ○ G1 phase: the gap phase of the cell cycle between the M phase and the S phase in which the sixe and protein content of the cell increase and regulatory proteins are made and activated in prep for s-phase and DNA synthesis ○ G2 phase: ^^ the size and protein content of the cell increase in prep for the m-phase mitosis and cytokinesis G0 Phase: the gap phase of the cell cycle in chich cells pause in the cell cycle between M phase and S phase; it may last for periods ranging from days to more than a year 11.2 DNA Replication DNA Replicates semiconservatively Replication fork: the site where the parental DNA strands separate as the DNA duplex unwinds. As each daughter strand is synthesized, the order of the bases in the template strand determines the order of the complementary bases added to the daughter strand DNA Replication Notes: Easy to Understand Key Concepts: 1. DNA Structure: ○ DNA is a double-stranded helix with strands running in opposite directions (antiparallel). ○ Bases pair specifically: A with T, and G with C. 2. Semiconservative Replication: ○ Each new DNA molecule has one original (parental) strand and one newly synthesized (daughter) strand. ○ Alternative (wrong) model: Conservative replication (where the original DNA remains intact, and a completely new molecule is formed). 3. Replication Fork: ○ A site where the two parental DNA strands separate to serve as templates.  ○ Unwinding creates a Y-shaped structure where replication occurs. Replication Enzymes: 1. Helicase: ○ Unwinds and separates the DNA strands by breaking hydrogen bonds. 2. Single-Strand Binding Proteins (SSBs): ○ Stabilize unwound DNA to prevent re-pairing. 3. Topoisomerase II: ○ Relieves the stress caused by unwinding the DNA. 4. Primase: ○ Synthesizes short RNA primers to start DNA synthesis. 5. DNA Polymerase: ○ Adds nucleotides to the 3' end of a growing strand. ○ Proofreads and corrects errors during replication. 6. Ligase: ○ Joins fragments of DNA on the lagging strand. DNA Synthesis: 1. Directionality: ○ DNA is synthesized in the 5' to 3' direction. ○ Polymerase can only add nucleotides to the 3' end. 2. Leading vs. Lagging Strand: ○ Leading Strand: Synthesized continuously in the direction of the replication fork. ○ Lagging Strand: Synthesized in short fragments (Okazaki fragments) moving away from the fork. ○ RNA primers are removed and replaced with DNA; fragments are joined by ligase. 3. Energy Source: ○ Energy for nucleotide addition comes from breaking the high-energy phosphate bonds of the incoming nucleotide triphosphates. Proofreading and Mutations: Proofreading Function: ○ DNA polymerase detects and removes incorrect nucleotides during replication. ○ Significantly reduces errors but doesn't eliminate them entirely. Mutations: ○ Errors that escape proofreading may lead to mutations. ○ Mutations can be harmful, neutral, or beneficial (source of genetic variation). Eukaryotic DNA Replication: 1. Fluorescent Labeling: ○ Demonstrated semiconservative replication in eukaryotic cells. ○ Chromosomes were labeled with fluorescent nucleotides to track replication. 2. Trombone Model: ○ Leading and lagging strands are synthesized simultaneously. ○ Lagging strand loops around so both strands are extended in the same direction. Summary of Key Processes: 1. Initiation: ○ Helicase unwinds DNA. ○ Primase lays down RNA primers. 2. Elongation: ○ DNA polymerase synthesizes new DNA strands. ○ Leading strand grows continuously; lagging strand grows in fragments. 3. Termination: ○ RNA primers are replaced with DNA. ○ Ligase seals the gaps. 4. Accuracy Mechanism:  ○ DNA polymerase proofreads and corrects errors, ensuring high fidelity replication. 11.3 Replication of Chromosomes Replication of DNA in chromosomes starts at many places almost simultaneously. Replication Speed: In eukaryotes, replication occurs at 50 nucleotides per second. Without multiple starting points, replication of large chromosomes would take months. However, it only takes a few hours due to simultaneous replication at multiple sites. Origins of Replication: Origins of replication (ori): Points where DNA synthesis begins. The opening of the double helix forms a replication bubble, with two replication forks moving in opposite directions. Replication Bubble Mechanics: Each replication fork has: ○ Leading strand: Synthesized continuously. ○ Lagging strand: Synthesized in fragments (Okazaki fragments). Enzymes involved: ○ Helicase: Unwinds the DNA. ○ Topoisomerase II: Relieves stress caused by unwinding. ○ Single-strand binding proteins: Stabilize unwound DNA. When replication bubbles meet, they fuse, and DNA ligase joins the ends. Circular DNA Replication: Found in bacteria, mitochondria, and chloroplasts. Starts at a single origin and proceeds bidirectionally until the forks meet. No shortening occurs because the DNA is circular. Linear DNA Shortening: Problem: On the lagging strand, the final RNA primer at the end cannot be replaced, leading to loss of ~100 base pairs each round.  Without a solution, chromosomes would shorten over time, eventually losing critical genetic information. Telomeres and Telomerase: Telomeres: Repeating sequences (e.g., TTAGGG in humans) at the ends of chromosomes, providing a buffer against loss of important DNA. Telomerase: ○ Restores telomeres after replication. ○ Contains an RNA template complementary to the telomere sequence. ○ Ensures the ends of chromosomes are maintained, preventing gradual DNA loss. 12.1 PCR: The polymerase chain reaction selectively amplifies regions of DNA. In the lab its hard to manipulate or visualize a sample containing just one or two copies of a DNA molecule. Common method for making copies of a piece of DNA is the polymerase chain reaction ○ Allows researchers to amplify or replicate a targeted region of a DNA molecule into as many copies as desired. ○ PCR is selective and sensitive ○ Takes place in small plastic tube containing a solution that includes: Template DNA:At least one molecule of double-stranded DNA containing the region to be amplied serves as the template for amplification. DNA Polymerase: the enzyme DNA polymerase is used to replicate the DNA All Four Nucleotides: Nucleotides with the bases A,T,G, or C are needed as the building blocks for the synthesis of new DNA strands. Two Primers: Two short sequences of single-stranded DNA are required for the DNA polymerase to start synthesis. Enough primer is added so that the number of primer DNA molecules is much greater than the number of template DNA molecules. The primer sequences are oligonucleotides produced by chemical synthesis and are typically 20-30 nucleotides long. ○ The primers flank the specific region of DNA to be amplified.  ○ The 3’ end of each primer must be toward he region being amplified so that when the DNA polymerase extends the primer, it creates a new DNA strand complementary to the targeted region. Polymerase Chain Reaction (PCR) PCR is a method to amplify specific DNA sequences through a cyclic process: 1. Denaturation ○ Heat the solution near boiling (~95°C) to break hydrogen bonds and separate the double-stranded DNA into single strands. 2. Annealing ○ Cool the solution (~50-65°C) to allow primers to bind (anneal) to complementary sequences on the template DNA. 3. Extension ○ Heat to the optimal temperature (~72°C) for DNA polymerase. ○ DNA polymerase extends primers by adding deoxynucleoside triphosphates (dNTPs) to synthesize new DNA strands. Cycles and Amplification ○ Repeated for 20–40 cycles. ○ Each cycle doubles the DNA quantity, resulting in exponential amplification (e.g., 2 → 4 → 8 → 16 → etc.). ○ Initially, non-specific DNA fragments may form; after a few cycles, most DNA fragments match the target sequence. Heat-Stable DNA Polymerase ○ Taq polymerase from Thermus aquaticus withstands high temperatures, eliminating the need to add fresh enzyme after each cycle. ○ Automated thermal cyclers control the temperatures, times, and cycles. Challenges in PCR Primer Issues ○ Incorrect sequences may prevent proper annealing. ○ Primers binding to multiple sites can produce unexpected fragments. Verification of Results ○ Gel electrophoresis is used to confirm amplification of the expected DNA size. Gel Electrophoresis Used to separate DNA fragments based on size:  1. Setup ○ DNA samples are loaded into wells of a gel (usually agarose) within a buffer solution. ○ An electric field is applied, with the negative electrode at the top (near the wells) and positive electrode at the bottom. 2. Migration ○ DNA is negatively charged (phosphate backbone) and moves toward the positive pole. ○ Small DNA fragments move faster through the gel's pores than larger fragments. 3. Visualization ○ DNA bands are visualized with dyes that fluoresce under UV light. ○ A ladder (known DNA fragment sizes) helps estimate fragment sizes. 4. Example ○ A 700-bp fragment amplified by PCR will appear as a single band at the 700-bp position in the gel if the experiment is successful. Applications and Limitations Gel electrophoresis can separate DNA from various sources (e.g., genomic DNA, PCR products). Protein molecules can also be separated using similar techniques. Unexpected results (e.g., no bands, incorrect size, multiple bands) require troubleshooting of primers, reagents, or conditions. This combination of PCR and gel electrophoresis is a cornerstone of molecular biology research. 12.5 Genome size and Packaging Gene number isnt a good predictor of biological complexity. Genome Sizes and Composition Measuring Genomes: ○ Kilobase (kb) = 1,000 base pairs ○ Megabase (Mb) = 1,000,000 base pairs ○ Gigabase (Gb) = 1,000,000,000 base pairs Viral Genomes: ○ Range: ~2 kb to 2.5 Mb. ○ The largest viral genome can be larger than some bacterial genomes. Bacterial and Archaeal Genomes: ○ Range: 0.2 Mb (e.g., Mycoplasma genitalium) to 10 Mb. ○ ~90% of the genome codes for proteins (information-dense). ○ Larger genomes → More genes → Greater metabolic capabilities. Eukaryotic Genomes: ○ No correlation between genome size and organismal complexity (C-value paradox). ○ Genome size in eukaryotes is highly variable due to: Polyploidy (multiple chromosome sets, common in plants). Noncoding DNA (introns, repetitive sequences, transposable elements). C-Value Paradox Definition: No clear relationship between genome size and organismal complexity. Reasons for Large Eukaryotic Genomes: 1. Polyploidy: Extra sets of chromosomes (e.g., wheat has six sets). 2. Noncoding DNA: Makes up the majority of the genome. Humans: ~2% of the genome codes for proteins; ~98% is noncoding DNA, introns, and repetitive DNA. Includes introns, repetitive sequences, and transposable elements. Repetitive DNA in Eukaryotes Types of Repeated Sequences: ○ Alpha Satellite DNA: Found near centromeres, essential for spindle fiber attachment. ○ Transposable Elements (TEs): DNA sequences that replicate and move within the genome. DNA Transposons: Move via DNA replication/repair. Retrotransposons: Move via RNA intermediates (reverse transcription). TEs are "selfish DNA," making up ~50% of the human genome. DNA Packaging Prokaryotic DNA Packaging: ○ DNA is circular and underwound (negative supercoiling). ○ Topoisomerase II helps create supercoils. ○ DNA forms loops (nucleoid structure) bound by proteins. Eukaryotic DNA Packaging: ○ Linear DNA packaged into chromatin: 1. Histones: Proteins form the core of nucleosomes. DNA wraps around histone proteins (positively charged, bind to negatively charged DNA). 2. Nucleosomes: Each contains ~147 bp of DNA wrapped around a histone core. 3. Multiple levels of chromatin compaction to fit DNA into the nucleus. Key Terms VNTRs: Variable Number Tandem Repeats; highly variable regions of DNA.  Topoisomerase II: Enzyme that helps relieve stress in DNA by supercoiling. Histones: Conserved proteins essential for DNA packaging. 13.4 Genome size and Packaging Chromosomal Mutations Definition: Mutations affecting large DNA segments, visible under a microscope. Effects: Alter gene copy number or the linear order of genes. ○ Can disrupt chromosome pairing and segregation in meiosis. Types of Chromosomal Mutations 1. Duplications: ○ Region of chromosome repeated. ○ Harmful in large duplications but small duplications can persist and contribute to evolution. ○ Less harmful than deletions. 2. Deletions: ○ Region of chromosome is missing. ○ Caused by replication errors or chromosome break repairs. ○ Essential gene deletions can persist if present in homologous chromosome. ○ Deletions including centromeres are usually lethal. 3. Inversions: ○ A chromosomal region is reversed in orientation. ○ Typically occur in noncoding regions. ○ Small inversions are common and play roles in chromosome evolution. 4. Reciprocal Translocations: ○ Segments from nonhomologous chromosomes exchange places. ○ Do not change gene number but disrupt gene dosage during meiosis. ○ Often occur in noncoding regions and typically do not disrupt gene function. Copy Number Variations (CNVs)  Definition: Differences in the number of copies of a genome region due to duplications or deletions. Prevalence: ○ 10-15% of the genome is subject to CNVs. ○ Average CNV length: 200-300 kb. Examples: ○ Amylase (AMY1) gene: Higher copy number linked to high-starch diets. Gene Duplication and Divergence Process: ○ Duplicate genes mutate over time, forming new functions. ○ Duplicated gene copy can evolve without harming the organism. Outcome: Gene families with related functions. ○ Example: Globin gene family on chromosome 11 (varies with developmental stages). ○ Example: Odorant receptor gene family (largest, ~400 genes). Tandem Repeats and DNA Typing Tandem Repeats: ○ Short sequences (2–50 base pairs) repeated in tandem. ○ Each individual has a unique tandem repeat genotype. Applications: ○ Basis of DNA typing or "DNA fingerprinting." ○ Small DNA samples (e.g., saliva, blood) are sufficient. ○ Used for forensic identification by analyzing multiple polymorphic loci. Key Concepts 1. Duplication vs. Deletion: ○ Duplications: Gain of DNA, less harmful. ○ Deletions: Loss of DNA, more harmful, especially if involving essential genes. 2. Gene Dosage: ○ Critical in maintaining genetic balance.  ○ Disrupted in reciprocal translocations and abnormal chromosomes (e.g., missing centromeres). 3. Meiotic Challenges: ○ Chromosomal mutations can lead to pairing/separation issues during meiosis. 4. Evolutionary Role: ○ Mutations like duplications and inversions contribute to species evolution. 13.2 The nature of Mutations Mutations: Overview and Causes Definition: Changes in DNA due to errors in replication or unrepaired damage. Causes: ○ Spontaneous errors in DNA replication. ○ Damage from reactive molecules, environmental chemicals, or radiation (e.g., UV light, X-rays). ○ Insertion of mobile DNA sequences ("jumping genes"). Nature: ○ Occur randomly, not in response to an organism’s needs. ○ Mutation rates vary across species, genomes, and DNA regions (e.g., hotspots). Types of Mutations 1. Germ-line Mutations: ○Occur in reproductive cells. ○Transmitted to future generations. ○Important for evolution. ○Average: ~70 new mutations per human zygote (mostly from fathers due to higher germ-cell divisions). 2. Somatic Mutations: ○ Occur in non-reproductive cells.  ○ Not inherited but passed to daughter cells during mitosis. ○ Responsible for phenomena like sectoring in plants and most cancers. Mutation Rates Per nucleotide per replication: ○ Low (~1 error per 10 billion nucleotides). ○ Higher in somatic cells compared to germ cells. Per genome per generation: ○ Influenced by genome size and the number of cell divisions. ○ Humans: High due to large genomes and many cell divisions. Hotspots: Specific DNA regions prone to mutation. Often linked to chemically modified cytosine bases used in gene regulation. Mutations and Cancer Cancers arise from sequential mutations in somatic cells. ○ Multiple mutations in key genes (e.g., APC, Ras, p53) can lead to malignancies. ○ Environmental exposures (e.g., chemicals, UV radiation) and genetic predispositions increase risk. ○ Germ-line mutations in cancer-associated genes (e.g., BRCA1, BRCA2) heighten inherited risk. Mutation and Evolutionary Impact Germ-line mutations contribute to evolution. Somatic mutations can impact individual fitness or cause diseases like cancer. Lederberg Experiment: Mutations and Natural Selection  Tested whether mutations arise spontaneously or in response to environmental stress. Method: ○ Grew bacterial colonies on antibiotic-free agar. ○ Transferred colonies (via replica plating) to antibiotic-containing agar. ○ Resistant colonies were traced back to original plates. Conclusion: ○ Mutations are random and pre-existing, not directed by environmental factors. Mutation and Disease Risk Genetic and environmental factors combine to influence disease risk. Example diseases: ○ Cancer: Risk from genetic mutations (e.g., BRCA genes) and environmental factors (e.g., smoking, UV exposure). ○ Heart Disease, Diabetes: Influenced by lifestyle choices (e.g., diet, physical activity). Genetic knowledge can inform better lifestyle decisions but does not eliminate environmental risks. 13.3 Small Scale Mutations: Definition of Mutation A mutation is a change in the nucleotide sequence of the genome. Types of mutations: ○ Small-scale changes: Affect one or a few bases. ○ Large-scale changes: Affect entire chromosomes. Point Mutations Definition: Changes in a single nucleotide. Causes: Errors in DNA replication or damage. ○ DNA repair mechanisms (e.g., proofreading by DNA polymerase) often correct errors before they become mutations. ○ If not corrected, mutations become permanent and are faithfully replicated in subsequent generations. Types of Point Mutations 1. Nucleotide Substitution (Point Mutation) ○ One base pair is replaced by another. ○ Effects: Synonymous (Silent) Mutations: Do not alter the amino acid sequence (e.g., GAG to GAA both code for Glu). Nonsynonymous (Missense) Mutations: Result in an amino acid change (e.g., GAG to GUG changes Glu to Val, causing diseases like sickle cell anemia). Nonsense Mutations: Create a stop codon, leading to premature termination of translation and usually nonfunctional proteins. 2. Insertions and Deletions ○ Small changes involving a few nucleotides. ○ Effects: If multiples of three nucleotides: Adds or removes whole codons, affecting only specific amino acids. If not multiples of three: Causes frameshift mutations, altering the entire reading frame downstream and resulting in nonfunctional proteins. Examples of Mutations and Diseases Sickle Cell Anemia: A missense mutation (Glu → Val) in the β-globin gene. Cystic Fibrosis: ○ Mutation: ΔF508 (deletion of three nucleotides, removing a phenylalanine). ○ Result: Improper protein folding and degradation of the CFTR protein. ○ Treatment: Drugs that stabilize the mutant protein. Frameshift Mutations Caused by insertions or deletions not in multiples of three. Consequences: ○ Alters codons downstream of the mutation. ○ Produces a completely different, usually nonfunctional protein. Transposable Elements (Transposons) Definition: Movable DNA sequences that can insert into or near genes. Effects: ○ Disrupt gene transcription, RNA processing, or the open reading frame. ○ Example: In maize, insertion of transposons can prevent pigment production, leading to yellow kernels. Mutations and Noncoding DNA Most mutations in noncoding regions have no detectable effects, as these regions do not encode proteins. Mutations in coding regions can have significant consequences, depending on the mutation type and location. Key Takeaways 1. Mutations are the raw material for evolution but can also lead to diseases. 2. DNA repair mechanisms prevent many potential mutations. 3. The impact of mutations depends on: ○ Their type (e.g., silent, missense, nonsense, frameshift). ○ Their location (coding vs. noncoding DNA). 4. Certain mutations, such as those in CFTR or β-globin genes, are well-studied examples of disease-causing genetic changes. 13.5 DNA damage and repair Overview Mutations result from unrepaired errors in DNA replication or DNA damage that is not repaired. DNA is damaged daily at tens of thousands of places in every cell. Cells have evolved DNA repair mechanisms to restore DNA and maintain a low mutation rate. Types of DNA Damage 1. Structural Damage: ○ Breaks in the sugar-phosphate backbone: Single-stranded breaks: Easier to repair. Double-stranded breaks: Harder to repair and can cause chromosomal rearrangements. ○ Thymine dimers: Caused by ultraviolet (UV) light. Cross-linking of adjacent thymine bases distorts the backbone. ○ Base loss: Loss of purines (A or G) occurs spontaneously (~13,000 per cell per day). 2. Chemical Alteration of Bases: ○ Alters base-pairing properties (e.g., by adding bulky side groups). ○ Mutagens (e.g., tobacco smoke) increase mutation probability by ~100-fold. 3. Sources of Damage: ○ Radiation: UV light, gamma rays. ○ Chemicals: Oxidizing agents (e.g., bleach, hydrogen peroxide), tobacco smoke. DNA Repair Mechanisms 1. Repair of Backbone Breaks: ○ DNA ligase: Joins ends of DNA. Uses ATP for energy. Seals both single- and double-stranded breaks. 2. Proofreading by DNA Polymerase: ○ During replication, removes mismatched nucleotides immediately. ○ Corrects ~99% of mismatched bases.  3. Mismatch Repair: ○ Fixes errors missed by DNA polymerase proofreading. ○ Steps: MutS protein: Recognizes mismatch. MutL and MutH proteins: Cleave and degrade the mismatched strand. DNA polymerase: Synthesizes corrected DNA. DNA ligase: Seals the nick in the backbone. ○ Defects in mismatch repair can lead to genetic predisposition to cancers, e.g., colon cancer. 4. Base Excision Repair: ○ Corrects damaged or abnormal bases. ○ Steps: Removes the damaged base. Cleaves the baseless sugar. Inserts correct nucleotide with a repair polymerase. 5. Nucleotide Excision Repair: ○ Removes damaged sections of DNA. ○ Handles bulky side groups and thymine dimers. ○ Defects in this pathway cause diseases like xeroderma pigmentosum (XP): Leads to extreme UV sensitivity and high skin cancer risk. Key Insights on Repair Mechanisms DNA repair ensures high fidelity of DNA replication and limits harmful mutations. Repair mechanisms reduce errors to a level that supports life. Not all mistakes are caught, resulting in genetic variation, which drives evolution. Important Points DNA repair is critical for: ○ Maintaining genome stability. ○ Preventing diseases like cancer. Imperfect repair systems allow mutations that contribute to diversity and evolution. 11.4 Miotic Cell Division General Overview Mitotic Cell Division: Process of mitosis (nuclear division) followed by cytokinesis (cytoplasmic division). ○ Basis for asexual reproduction in unicellular eukaryotes. ○ Essential for growth, development, and maintenance in multicellular eukaryotes. Goal: To ensure that daughter cells receive a complete and equal set of chromosomes. Chromosomes and DNA Organization Chromosome Pairs: Eukaryotic cells have homologous chromosomes (one from each parent). Condensation: DNA is condensed to fit into the nucleus, further condensed during mitosis to prevent tangling. Karyotype: Portrait of the number and structure of chromosomes in a species. ○ Humans: 46 chromosomes (22 homologous pairs + 1 pair of sex chromosomes). Ploidy: ○ Haploid (n): One complete set of chromosomes. ○ Diploid (2n): Two complete sets of chromosomes (e.g., human somatic cells). ○ Polyploid (4n or more): Multiple complete sets of chromosomes (common in plants). Sister Chromatids and Centromeres Sister Chromatids: Identical copies of a chromosome formed during S phase. Centromere: Region where sister chromatids are held together. Counting Chromosomes: Count the number of centromeres. Stages of Mitosis  1. Prophase ○ Chromosomes condense and become visible. ○ Centrosomes migrate to opposite poles. ○ Microtubules assemble into the mitotic spindle. 2. Prometaphase ○ Nuclear envelope breaks down. ○ Spindle microtubules attach to chromosomes at the kinetochores. ○ Dynamic instability helps position the chromosomes. 3. Metaphase ○ Chromosomes align at the metaphase plate (middle of the cell). ○ Symmetrical attachment of chromosomes ensures proper segregation. 4. Anaphase ○ Centromeres split, and sister chromatids separate. ○ Microtubules shorten, pulling chromatids to opposite poles. 5. Telophase ○ Chromosomes decondense. ○ Nuclear envelopes reform around separated chromosome sets. Cytokinesis Animal Cells: ○ Contractile Ring: Actin filaments form a ring at the cell's equator, contracting to divide the cytoplasm. Plant Cells: ○ Phragmoplast Formation: Overlapping microtubules guide vesicles to the cell's center to form a new cell plate. ○ The cell plate matures into a new cell wall, dividing the cell. Key Structures in Mitosis Microtubules: Form the mitotic spindle for chromosome movement. Centrosomes: Microtubule-organizing centers in animal cells. Kinetochores: Protein complexes at the centromeres where microtubules attach. Comparison of Animal and Plant Cell Mitosis Both: Use the mitotic spindle for chromosome segregation. Differences: ○ Animal cells use a contractile ring for cytokinesis. ○ Plant cells use a cell plate guided by microtubules. Summary of Outcomes End Result: Two genetically identical daughter cells with the same chromosome number as the parent cell. Significance: Maintains genetic stability during growth, repair, and reproduction. 11.5 Cell Cycle Regulation Notes on Cell Cycle Regulation and Cancer 1. Regulation of the Cell Cycle Purpose of Cell Division: Growth, repair, tissue renewal (e.g., skin, intestines); triggered by favorable conditions (e.g., nutrients, signals). Key Proteins in Regulation: ○ Cyclins: Regulatory proteins that control the cycle phases by fluctuating levels. ○ CDKs (Cyclin-Dependent Kinases): Enzymes always present but activated only when bound to cyclins. Cyclin-CDK Functions: ○ G1 Phase: Cyclins D & E + CDKs → DNA replication preparation. ○ S Phase: Cyclin A + CDKs → DNA synthesis initiation, prevents re-replication. ○ G2 & M Phase: Cyclin B + CDKs → Mitotic processes (e.g., spindle formation, nuclear envelope breakdown). 2. Checkpoints in the Cell Cycle Purpose: Ensure readiness at critical points; halt progression if conditions are suboptimal. Major Checkpoints:  ○ G1 Checkpoint: Prevents entry into S phase if DNA is damaged. ○ G2 Checkpoint: Prevents entry into mitosis if DNA is unreplicated or damaged. ○ M Checkpoint: Prevents mitosis completion if chromosomes are improperly attached to the spindle. Key Protein - p53: ○ Activated by DNA damage. ○ Phosphorylated p53 arrests the cycle for repair or triggers apoptosis. ○ Known as the "guardian of the genome." 3. Apoptosis (Programmed Cell Death) Role: Removes damaged, unnecessary, or harmful cells in a controlled manner. Mechanism: ○ p53 activates pro-apoptotic proteins (e.g., Bax). ○ Balance between Bax (pro-apoptotic) and Bcl-2 (anti-apoptotic) determines apoptosis. Importance: ○ Prevents unregulated cell growth (cancer). ○ Normal in development (e.g., shaping fingers) and tissue maintenance. 4. Cancer and Cell Division Causes: ○ Proto-Oncogenes: Normally regulate cell division; mutations convert them into oncogenes, leading to uncontrolled growth. ○ Tumor Suppressors: Proteins (e.g., p53) that inhibit division; mutations result in unchecked division. Features of Cancer Cells: ○ Divide without growth signals. ○ Resist inhibitory or apoptotic signals. ○ Invade local/distant tissues (metastasis). ○ Promote blood vessel formation for nutrients. Progression: ○ Requires multiple mutations in proto-oncogenes and tumor suppressors. ○ Benign Tumor: Slow growth, non-invasive. ○ Malignant Tumor: Rapid growth, invasive, metastatic. p53 and Cancer: ○ Mutations in p53 are common in cancers.  ○ Loss of p53 allows division despite DNA damage, leading to mutation accumulation. 5. Summary Proper cell cycle regulation involves cyclin-CDK complexes, checkpoints, and mechanisms like apoptosis. Disruptions in these controls (e.g., proto-oncogenes, tumor suppressors) can lead to cancer. Cancer development often involves a gradual accumulation of mutations.