303 Study Guide FINAL PDF

Chapter 1: Introduction to Genetics Compare & Contrast: Theory of Epigenesis Theory of Preformation Organisms develop from a fertilized Organisms develop from already small egg (egg&sperm) by several versions...

Chapter 1: Introduction to Genetics Compare & Contrast: Theory of Epigenesis Theory of Preformation Organisms develop from a fertilized Organisms develop from already small egg (egg&sperm) by several versions of organism also known as developments done which eventually homunculus (mini-adult) transform it into a fully grown adult The fertilized egg CONTAINS the organism homunculus – usually exist in the Ex: fertilized egg grows into eventually sperm or egg a fetus They eventually just grow in size What is the Cell Theory? All organisms r made up of cells which are the basic unit of life, cells come from cells o How did Louis Pasteur experimentally come to this conclusion? He DISPROVED Spontaneous generation: living organisms r created from nonliving components, he used a heat/bacteria experiment, bacteria grew as air was let in What is: o Descent with modification – species evolve, with one common ancestor. o Natural selection – mechanism of evolution: the organisms with best fit traits r more likely to survive & reproduce to pass on those genes o The theory of evolution – based on naturals selection, species over time inherit a varation of traits eventually making a variety of species Basic understanding of Mendel’s work: will be covered in detail in later chapters o Father of genetics, heredity, the law of segregation (alleles segregate during gamete formation), and the law of independent assortment (genes for different traits assort independently) What is the Chromosome theory of Inheritance- genes located on chromosomes leading to genetic continuity o What does it mean to be diploid vs haploid – diploid is 2 sets of chromosomes, one from each parent in somatic cells. Haploid is n, sperm/egg cells, only one set of chromosomes o How many chromosomes do we have in humans? Total, somatic, and sex ▪ Total: 46, in 23 pairs ▪ 22 pairs are autosomes ▪ One pair of sex chromosomes Be able to interpret Avery, MacLeod, & McCarty and the Hershey & Chase experiments o DNA was carrier of genetic info. In bacteria then Hershey & chase confirmed this in bacteriophages Know Chargaff’s Rules for base pairing and general structure of DNA o A=T and G=C Know characteristics of model organisms o Easy to grow/maintain o Many offspring o Clear genetic analysis Chapter 2: Mitosis and Meiosis Be able to identify cell structures and their general function in the cell Know the human chromosome number and that different species have different numbers of chromosomes. o Humans: diploid, 2n, 46 o 23 sister chromatids o One sex chromosomes Know all the steps of both Mitosis and Meiosis – Prophase: Chromosomes condense, spindle fibers form. Prometaphase: Nuclear envelope breaks down; spindle fibers attach to chromosomes. Metaphase: Chromosomes (mitosis) or homologous pairs (meiosis I) line up in the middle. Anaphase: *These are where sister chromatids separate vs homologous chromosomes* o Mitosis: Sister chromatids separate. o Meiosis I: Homologous chromosomes separate. o Meiosis II: Sister chromatids separate. Telophase: Chromosomes arrive at poles, nuclei reform. Cytokinesis: Separates cytoplasm; occurs after Mitosis/Meiosis. It is a separate phase which involves separation of the cytoplasm where Mitosis/Meiosis deals with separation of the genetic material o Which steps is the cell diploid vs haploid Mitosis: Diploid throughout. Meiosis: - Diploid in Meiosis I. - Haploid after Meiosis I and in Meiosis II. o Know human diploid number of chromosomes: Humans: diploid, 2n, 46 Bodily functions that rely on Mitosis? Growth and repair (e.g., skin cells, blood cells, tissue healing). Cell cycle: Where is DNA duplicated, what are general functions within the different stages? G1 (Gap 1): Cell grows, prepares for DNA replication. S (Synthesis): DNA is duplicated. G2 (Gap 2): Cell prepares for division. M (Mitosis): Division of genetic material. Understand the importance of Cohesin (the protein complex) and sister chromatid cohesion (the resulting process) Cohesin: Protein that holds sister chromatids together. Importance: Ensures proper separation during anaphase of mitosis and meiosis. How do we regulate the cell cycle and why is this important? Prevents uncontrolled cell growth (e.g., cancer) o What are cell cycle checkpoints?  G1/S Checkpoint: Ensures the cell is ready for DNA replication. G2/M Checkpoint: Ensures DNA replication was successful. Spindle Assembly Checkpoint: Ensures chromosomes are properly attached to spindle fibers. Meiosis: In what ways is genetic variation introduced? Crossing over (Prophase I): Exchange of genetic material between homologous chromosomes. Independent assortment (Metaphase I): Random alignment of homologous pairs. Formation of Gametes: Sperm vs Oocytes Sperm: Continuous production; four functional sperm cells per meiosis. Oocytes: One functional egg (oocyte) and three polar bodies due to unequal cytoplasm division. Chapter 3: Mendelian Genetics What is Hybridization? The process of crossing two individuals with different traits to study inheritance patterns. Why were pea plants a good model system? Easy to grow and breed, Distinct, observable traits (e.g., seed color, pod shape), Short generation time, Ability to self-pollinate or cross-pollinate. You don’t need to memorize the 7 different characteristics Mendel used- but if I were to provide a problem in which they are used be able to go through the cross and make a conclusion. I will indicate which trait is dominant vs recessive Be able to perform monohybrid and dihybrid cross using either a Punnett square or the forked- line method What do P, F1 and F2 mean when relating to genetics? P (Parental): Original breeding pair. F1 (First Filial): Offspring of P generation. F2 (Second Filial): Offspring of F1 generation. Know Mendel’s Postulates and logically why they make sense Unit factors in pairs: Traits are controlled by two alleles (one from each parent). Dominance and recessiveness: One allele may mask the other. Segregation: Alleles separate during gamete formation. Independent assortment: Alleles for different traits separate independently (applies to unlinked genes). Know general vocab for example: genotype, phenotype, gene, allele Genotype: Genetic makeup (e.g., AA, Aa, aa). Phenotype: Observable traits (e.g., purple flowers). Gene: Unit of heredity. Allele: Variant of a gene (e.g., dominant vs recessive). What is a test cross? Cross an unknown genotype with a homozygous recessive individual to determine the unknown genotype.  What are homologous chromosomes? Pairs of chromosomes, one from each parent, that carry the same genes but may have different alleles. o How do we determine if chromosomes are homologous? Homologous chromosomes have the same size, shape, and banding pattern. o Homologous chromosomes vs sister chromatids vs duplicated DNA etc. Homologous chromosomes: Pair during meiosis. Sister chromatids: Identical copies of a chromosome, joined at the centromere. Duplicated DNA: Refers to replicated chromosomes before division. If you are given a chi squared value and a chart/graph for the p-value be able to tell me what this means- is there a statistical difference between what we observed vs what we expected to occur? o Know how to determine the degrees of freedom: Number of categories - 1 o Know the difference between a null and alternative hypothesis in terms of statistics Smaller than 0.05 reject and bigger than 0.05 fail to reject (for probability). That’s why the charts are different shades of blue Pedigrees o Be able to read one- what are the different symbols? What is a proband, etc. Proband is the individual being studied or first identified in the pedigree. Chapter 4: Extensions of Mendelian Genetics How do alleles alter phenotypes in different ways?  Loss of Function: Mutation reduces or eliminates the function of a gene (e.g., nonfunctional enzyme). Gain of Function: Mutation enhances or introduces a new gene function (e.g., overactive protein). Neutral Mutation: Mutation has no observable effect on phenotype. What is incomplete dominance? Heterozygous phenotype is intermediate between the two species What is Codominance? Both alleles are expressed equally in the heterozygote Understand that there can be many, many different alleles possible at each locus Know how to predict probability of offspring phenotypes/genotypes for blood typing o How are A and B antigens created? Produced by enzymes encoded by Iᴬ and Iᴮ alleles, which modify a carbohydrate chain on red blood cells. o What is the Bombay Phenotype? Individuals lack the precursor molecule (H substance) needed to form A or B antigens, appearing as blood type O even if they carry Iᴬ or Iᴮ alleles. Understand lethal alleles and how this relates to seeing the phenotypes in the offspring. How does this result in differing phenotypic probabilities? They are mutations that cause death, often before birth. Phenotypic Ratios: Changes observed ratios (e.g., 2:1 instead of 3:1) because homozygous lethal combinations are not viable. Understand why and how phenotypes can be affected by more than one gene Epistasis general definition. Understand the concept of epistasis, how different genes can influence each other to create various phenotypes One gene masks or modifies the effect of another gene (e.g., coat color in Labrador retrievers). What is complementation analysis? Determines whether mutations causing a similar phenotype are in the same or different genes. o Why is complementation analysis used? Identifying whether two mutants affect the same pathway. o Be able to interpret complementation analysis. If the offspring have a wild-type phenotype, mutations are in different genes (complementation). If the phenotype is still mutant, mutations are in the same gene (no complementation). What is X-linkage? Genes located on the X chromosome. o What does Hemizygous mean? Males have only one X chromosome, so they express all X-linked traits (dominant or recessive). The difference between a sex-influenced and a sex-limited inheritance Sex-Influenced: Traits expressed differently in males and females due to hormonal differences (e.g., baldness). Sex-Limited: Traits appear only in one sex due to anatomical or physiological differences (e.g., milk production). What does expressivity refer to? Degree to which a trait is expressed (e.g., mild vs severe symptoms in a genetic condition). What are some ways that the environment can affect gene expression? Temperature: Fur color in Siamese cats. Diet: Phenylketonuria (PKU) symptoms depend on diet. Chemicals: Exposure to toxins can alter gene expression. Chapter 5: Chromosome Mapping  What does it mean for genes to be linked? Genes located close together on the same chromosome that tend to be inherited together. They don’t assort independently unless separated by crossing over during meiosis. Completely linked vs partially linked Completely Linked: Genes are so close together on the chromosome that crossing over does not occur. Only parental combinations are observed. Partially Linked: Genes are close but not tightly linked, allowing occasional crossing over, which creates recombinant gametes. How do linked genes differ from the typical Mendelian Genetics? In Mendelian genetics, genes assort independently (on different chromosomes or far apart on the same chromosome). Linked genes violate this by being inherited together unless recombination occurs. No crossing over vs crossing over: How do these affect independent assortment? Effect on Independent Assortment: Crossing Over: NCO: Independent Exchange of genetic No Crossing Over: assortment material between doesn’t Only parental allele occur; genes are homologous combinations are transmitted together. chromosomes in inherited. CO: Prophase I of Meiosis. Recombinati Genes appear on introduces Results in recombinant completely linked. new allele gametes, breaking the combinations , partially appearance of restoring the appearance complete linkage. of independent assortment. Understand what changes the frequency of crossing over Gene distance: Crossing over frequency is proportional to the physical distance between genes. - Closer Genes: Less frequent crossing over. - Farther Genes: More frequent crossing over. Chromosome Region: - Less frequent near centromeres. - More frequent near the ends of chromosomes (distal regions).  With given frequencies of recombination be able to identify the distance between 2 or 3 genes on a singular chromosome Recombination Frequency (RF): Percentage of recombinants reflects the distance in map units (mu) or centimorgans (cM). - 1% recombination = 1 mu. Example: If RF between Gene A and Gene B is 12%, then the distance is 12 mu. Double Crossover: physically what is it and what does it look like? Two separate crossover events occur between three loci on the same chromosome. Appearance: Chromatids show recombinant alleles at two distinct points. Example: Gene A - no crossover; Gene B-C - crossover observed. o With the percentage of times that you get cross over between A&B and then B&C be able to predict the probability of having these two exchanges happen simultaneously Multiply the frequencies of single crossovers (SCOs). Example: SCO A-B = 0.10 (10%). SCO B-C = 0.15 (15%). DCO Probability: 0.10 × 0.15 = 0.015 (1.5%). o From probabilities of different SCO, NCO, and DCO determine the sequence of the genes on a chromosome - Identify DCO Phenotypes: o These are the least frequent offspring. - Find the Middle Gene: o Compare DCO offspring to parental types; the trait that differs is the middle gene. - Example: o DCO Phenotypes: g sm h+ and g+ sm+ h. o Middle gene = h (different from parental). o Be able to identify the distance between the genes Draw the Chromosome Layout: Visualize gene order and recombination events. Add Frequencies: Combine SCO and DCO frequencies to find distances. Example: SCO (A-B) = 8%, DCO = 2%. Distance A-B = 8% + 2% = 10 mu. Repeat for all gene pairs to construct the map. Chapter 7: Sex Chromosomes What does it mean to be heterogametic? Is the heterogametic sex always the male? Heterogametic means producing two different types of gametes in terms of sex chromosomes.  - For example, in humans, males are heterogametic (XY), producing sperm with either an X or Y chromosome. - Females are homogametic (XX), producing eggs with only X chromosomes. The heterogametic sex is not always the male. In birds, moths, and some fish, the females are heterogametic (ZW), while males are homogametic (ZZ). What determines “maleness” and “femaleness” Maleness and femaleness are determined primarily by the presence or absence of the Y chromosome, specifically the SRY gene (Sex-determining Region Y). - If the SRY gene is present (usually on the Y chromosome), the individual develops as a male. - Without the SRY gene, the individual typically develops as a female. How did scientists study/figure this out? Disorders characterized by aberrant sexual development – Klinefelter, Turner If presented with a karyotype designation (Example: 47, XXY) understand what the different aspects mean- what information can you gather from (47, XXY)? The number before the comma indicates the total number of chromosomes. In 47, XXY, the person has 47 chromosomes (instead of the usual 46). The letters after the comma represent the sex chromosomes. XXY indicates two X chromosomes and one Y chromosome, a condition known as Klinefelter syndrome. People with Klinefelter are genetically male but may have some physical traits associated with females due to the extra X. Sexual differentiation: development of the embryo based on sex. Know generally how sexual differentiation happens in the embryo. In the beginning, all human embryos are hermaphroditic. The gonadal primordia (future gonads) appear as gonadal ridges, which can develop into either male or female gonads. Primordial germ cells migrate to these ridges and influence the development into either ovaries or testes. The gonadal ridges, capable of forming either sex's gonads, are called bipotential gonads. The determining factor is the Y chromosome: - Presence of the Y chromosome: Testes develop. - Absence of the Y chromosome: Ovaries form. If testes form, they secrete hormones that drive male sexual differentiation and prevent the development of female reproductive organs. Sexual dimorphism: primary vs secondary Primary – reproductive purposes Secondary – boobs/accessories or organs that distinguish sex Structural components of the Y chromosome- what is important about PAR, MSY, SRY?  The Y chromosome contains approximately 75 genes, far fewer than the 900-1400 found on the X chromosome. Some Y chromosome genes are homologous to the X chromosome, allowing pairing during meiosis. - The Pseudoautosomal regions (PARs) on the Y chromosome share homology with the X chromosome and facilitate recombination during meiosis. - The Male-specific region of the Y (MSY) is the non-recombining region of the Y chromosome but still contains **euchromatic regions** with functional genes, although some parts are homologous to the X chromosome. A crucial part of the MSY is the Sex-determining region Y (SRY), which contains the testis- determining factor (TDF). This transcription factor initiates male development. The Y chromosome also contains heterochromatic regions, which are areas that lack active genes. How the Y chromosome can affect phenotype beyond sex determination The Y chromosome affects male traits beyond just "maleness." For example, certain regions of the Y chromosome influence traits such as height, hair growth, and even fertility. Paternal Age Effects (PAE)- cancers, schizophrenia, autism, & others Correlation between smoking and complete loss of Y chromosome in blood cells. Y chromosome loss correlated to elevated cancer risk among male smokers & suppressed immunity -Supports idea that Y chromosome affects more than sex determination and male fertility Hypothetical vs actual sex ratio in humans Hypothetical – 1:1, equal Actual: assessed in two ways 1. Primary sex ratio- proportion of males to females conceived in population 2. Secondary sex ratio- proportion of each sex that is born easier to measure, but doesn’t account for disproportionate embryonic or fetal mortality Barr Bodies and Dosage compensation. If all but one of the X chromosomes are always silenced into Barr Bodies, then why are females with varying numbers of X chromosomes abnormal? A Barr Body is an inactivated X chromosome in females, visible in the nucleus of somatic cells. Females typically inactivate one of their two X chromosomes to balance X-linked gene dosage with males. Dosage compensation ensures that females (XX) and males (XY) express similar amounts of X- linked genes. While X-inactivation compensates for extra X chromosomes, genes in the pseudoautosomal region (PAR), X inactivation not immediate Not ALL genes on barr body completely inactive (~15%) o Lyon Hypothesis, Mechanism of X-inactivation, Xic, XIST, epigenetics The Lyon Hypothesis suggests that X-inactivation happens randomly in somatic cells early in development, and once an X chromosome is inactivated, all descendants of that cell will have the same X inactivated. Mechanism of X-inactivation: The Xic (X-inactivation center) contains the gene XIST, which produces RNA that coats and inactivates the X chromosome. Epigenetics also plays a role, as modifications to DNA (e.g., methylation) help maintain X- inactivation throughout a female’s life. Temperature controlled sex determination As opposed to chromosomal sex determination (CSD)/Genotypic sex determination (GSD) sex predetermined at conception by sex chromosome composition temperature-dependent sex determination (TSD) sex determined by incubation temperature of eggs during stage of embryonic development Changes in steroids (estrogens) and their enzymatic synthesis based on temperature. Relationship between the X and autosomal number in flies and C. elegans (not the specific numbers, but the concept) In organisms like C. elegans, sex determination is based on the ratio of X chromosomes to autosomes rather than the presence of a Y chromosome. There is no Y chromosome in C. elegans: - Hermaphrodites (XX) contain both testes and ovaries and can self-fertilize. - Males (X) only have testes. In this system, the genetic signal contributing to maleness comes from the X chromosome and autosomes, and the X-to-autosome ratio determines the sex of the organism. Chapters 8 and 9: Chromosomal Mutations What are types of extranuclear inheritance? - Organelle heredity: Transmission of genetic information through organelles such as mitochondria or chloroplasts. - Infectious heredity: Inheritance of traits from symbiotic or parasitic microorganisms present in the cytoplasm. - Maternal effect: The mother’s genotype directly affects the offspring's phenotype through substances in the egg cytoplasm. All types involve the transmission of genetic information through the cytoplasm, not the nucleus. What is the Endosymbiotic Theory Suggests that mitochondria and chloroplasts arose independently about 2 billion years ago from free- living protobacteria (primitive bacteria). Evidence for this includes: - Their DNA resembles bacterial DNA. - Mitochondria and chloroplasts have a unique genetic system, capable of organelle-specific transcription and translation. o Know that Mitochondria has DNA Each mitochondrion contains 5-10 DNA molecules. Introns are absent in mtDNA, with very few exceptions. - Mitochondria use unique mtDNA ribosomes for translation, but they rely on both mtDNA and nuclear (gDNA) genes to function properly. o Consequence of cellular respiration: ROS- why is this a negative consequence? - A consequence of cellular respiration is the production of reactive oxygen species (ROS), which are toxic and mutagenic, potentially leading to DNA damage. Know that Chromosome mutations and major chromosomal changes can cause genetic diseases. What are: Chromosomal rearrangements, aneuploidy, polyploidy, euploidy, etc? Chromosome mutations and structural changes can lead to genetic diseases. Types include: - Chromosomal rearrangements: Changes in chromosome structure (e.g., inversions, translocations). - Aneuploidy: Organism gains or loses one or more chromosomes but don’t gain a complete set (e.g., monosomy, trisomy). - Polyploidy: More than two sets of chromosomes (e.g., triploid, tetraploid). - Euploidy: A complete set of chromosomes in an organism. How does nondisjunction occur? What are different consequences that can happen? Which is more detrimental? Nondisjunction during the 1st or 2nd meiotic division? Nondisjunction occurs when homologous chromosomes or sister chromatids fail to separate during meiosis. - Consequences: Can lead to aneuploidy, such as trisomy (extra chromosome) or monosomy (missing chromosome). - First meiotic division nondisjunction is more detrimental, as it results in all four gametes being abnormal, while in the second division, only two are abnormal. Logically, why would monosomy or aneuploidy be detrimental? Consequences? Dosages, protein levels, individuals that are heterozygous for mutations, etc. Monosomy Aneuploidy Consequences – Consequences – - Gene dosage imbalance, where - Gain or loss of chromosomes (e.g., insufficient protein levels affect cellular trisomy, monosomy). function. - Leads to abnormal gene dosage, - Haploinsufficiency: A single functional disrupting normal protein production and copy of a gene is not enough to maintain pathways. normal function. - Example consequences: Trisomy 21 - Exposes deleterious recessive mutations (Down Syndrome), Turner Syndrome (no backup copy). (45,X). Polyploidy vs Autopolyploidy vs allopolyploidy Polyploidy: Presence of more than two sets of chromosomes (e.g., 3n, 4n). Autopolypoidy: Allopolyploidy: - Extra chromosome sets derived from a - Chromosome sets single species from different species through hybridization - causes: meiotic nondisjunction or - Fertile if failed cytokinesis chromosome sets from both species - results: Larger cell size and plant vigor, pair properly during but often sterile meiosis (e.g., wheat). Different chromosome aberrations o Deletions: Loss of a chromosome segment. Types: Terminal or interstitial. Ex) Cri-du-chat syndrome (deletion on chromosome 5). o Additions: Gain of extra genetic material. o Rearrangements: ▪ Translocation: Movement of a chromosome segment to a non-homologous chromosome. Types: Reciprocal or Robertsonian translocations. Example: Familial Down Syndrome (Robertsonian translocation). ▪ Inversions: A chromosome segment reverses its orientation  Types: Pericentric (includes centromere) or paracentric (excludes centromere). Consequences during meiosis - Paracentric Inversion (excludes centromere): o Can result in acentric chromatids (no centromere, lost during cell division) or dicentric chromatids (two centromeres, break during segregation). o Leads to nonviable gametes. - Pericentric Inversion (includes centromere): o Can cause duplication or deletion of chromosome segments in gametes. o Results in reduced fertility due to nonviable offspring. Familial Down Syndrome: probability of normal, carrier, trisomy, & lethal o Normal: ~1/6 o Carrier: ~1/3 o Trisomy 21 (Down Syndrome): ~1/6 o Lethal: ~1/3 o Copy number variations: Changes in the number of copies of a specific gene. Associated with conditions like autism and cancer. o gene redundancy - Refers to the presence of extra copies of a gene within the genome. - Can provide backup copies, enhancing survival if one copy is mutated or lost. - Example: Ribosomal RNA (rRNA) genes are often redundant to meet high demand for ribosome production. o gene amplification - An increase in the number of copies of a specific gene, leading to higher expression. - Often observed in cancer cells, where genes like MYC or HER2 are amplified to promote uncontrolled cell growth. Fragile sites: Areas on chromosomes prone to breakage. o Example: Fragile X Syndrome (CGG repeat expansion). Chapters 10 and 11: DNA Structure and Replication Central Dogma of Molecular Biology: DNA → RNA → Protein The structure of nucleotides o Nucleic acid components ▪ Nitrogenous bases - Purines: Adenine (A), Guanine (G) - Pyrimidines: Cytosine (C), Thymine (T) (in DNA), Uracil (U) (in RNA) - Purines have a double-ring structure, while pyrimidines have a single-ring structure. ▪ Sugar - Ribose (RNA): Has a hydroxyl group (-OH) on the 2' carbon. - Deoxyribose (DNA): Lacks the hydroxyl group on the 2' carbon. ▪ Phosphate: Attaches to the 5' carbon of the sugar.  o Nucleoside vs nucleotide ▪ Nucleoside: A nitrogenous base + sugar (ribose or deoxyribose). ▪ Nucleotide: A nucleoside + phosphate group. How to build DNA molecule from the nucleotides Nucleotides are linked by phosphodiester bonds between the phosphate group of one nucleotide and the sugar of the next How many hydrogen bonds connect the different sets of pairs? Two strands of DNA are held together by hydrogen bonds between complementary nitrogenous bases: A-T has 2 hydrogen bonds. G-C has 3 hydrogen bonds. Conservative vs semiconservative vs dispersive replication: - Conservative Replication: The entire DNA molecule is copied, and the original strand stays intact. - Semiconservative Replication: Each new DNA molecule has one original strand and one newly synthesized strand. (Correct model of DNA replication) - Dispersive Replication: Pieces of the original DNA are mixed with newly synthesized parts in both strands. Origins of replication o What are they? Specific sequences in the DNA where replication begins. o Why are they important? They provide a starting point for DNA replication. o How does replication move from an origin? Replication moves bidirectionally from the origin, creating replication bubbles that expand as DNA is replicated. Function of different proteins (enzymes) in DNA replication - Polymerase: Adds nucleotides to the growing DNA strand (DNA polymerase). - Helicase: Unwinds the DNA double helix ahead of the replication fork. - Primase: Synthesizes RNA primers to initiate DNA replication. - Ligase: Joins Okazaki fragments on the lagging strand. - Topoisomerase: Relieves strain by unwinding the DNA ahead of the replication fork. Steps of DNA replication 1. Initiation: Helicase unwinds the DNA, and primase synthesizes RNA primers. 2. Elongation: DNA polymerase adds nucleotides to the primer, creating a complementary strand. 3. Termination: Replication ends when the entire DNA is copied, and the RNA primers are replaced with DNA. Telomeres - What are they? Repetitive DNA sequences at the ends of chromosomes that protect the chromosome from degradation. - How do they function? They prevent the loss of important DNA sequences during replication. - How do we protect them? Telomerase extends the telomeres in germ cells and stem cells. - Why are they there? Telomeres prevent the loss of genetic material during DNA replication. - Telomerase: An enzyme that adds repetitive DNA sequences to the ends of chromosomes, maintaining telomere length. - Hayflick Limit: The number of times a somatic cell can divide before telomere shortening causes cell death.  Genetic Recombination - General Process: The exchange of genetic material between homologous chromosomes during meiosis, resulting in new combinations of alleles. - Purposes: Increases genetic diversity, ensuring better adaptation and survival. Chapter 12: DNA Organization DNA condensation o Why is it important? DNA needs to be compacted to fit inside the nucleus and to be properly segregated during cell division. o It’s accomplished by: ▪ Supercoiling: DNA is twisted into a compact form to fit inside the cell. ▪ What type of proteins regulate supercoiling? Topoimerases o Nucleosomes/Histones ▪ Histone composition - Histones are small, positively charged proteins. There are four core histones: H2A, H2B, H3, and H4, which form the nucleosome core. ▪ What is the role of H1? H1 is a linker histone that helps stabilize the DNA as it wraps around the nucleosome and contributes to further DNA compaction. Heterochromatin vs euchromatin o Is compaction permanent? Heterochromatin is usually permanently compacted and transcriptionally inactive, while euchromatin can be dynamically altered based on cellular needs. o What are regions of the DNA which are most often bundled into heterochromatin? Centromeres, telomeres, and regions of repetitive DNA sequences tend to be found in heterochromatin. Chromosome banding- - What we use it for: Banding patterns are used for identifying chromosomes, detecting structural abnormalities, and studying genetic diseases. - What it tells us: It helps map the relative locations of genes and specific genetic disorders. Different types of sequences within DNA o Centromere: A region of the chromosome that links sister chromatids and is involved in the segregation of chromosomes during cell division. o Satellite DNA: Repetitive DNA sequences found in regions like centromeres and telomeres, typically not coding for proteins. o Non-coding sequences: DNA sequences that do not code for proteins, including introns, regulatory elements, and non-coding RNAs. o Multiple copy genes: Genes that have more than one copy in the genome, often found in regions involved in ribosome production (e.g., rRNA genes). o Short interspersed elements: A type of retrotransposon, short repetitive DNA sequences that can be inserted into the genome. o Long interspersed elements (LINEs): Another type of retrotransposon, these are longer sequences that can move and replicate within the genome. What’s a pseudogene? A gene that is a non-functional copy of a functional gene, often due to mutations that prevent it from being expressed or transcribed. Chapter 13: Transcription and RNA Processing  The central dogma of biology – the theory that describes the flow of genetic info in cells from DNA → RNA → Protein RNA vs DNA RNA DNA - Ribose sugar - Deoxyribose sugar - Single-stranded - Double-stranded - Uracil - Thymine - Different kinds: mRNA, tRNA, etc. - Shape: double-helix o Different shapes: primary - Stable (straight/simple) vs *Uracil & Thymine are still both secondary (folded) pyrimidines, purines: Adenine and - Easily degraded b/c of extra Thymine* hydroxyl group o Ex) Covid vaccine issue: it was RNA, so the storage conditions are crucial/picky What are different types of RNA? o RIbosomal RNA – rRNA (translator) ▪ In prok/eukar ▪ Located in cytoplasm ▪ Translates genetic info in mRNA to a protein/pat of ribosome o Messenger RNA – mRNA (gossiper) ▪ In prok/eukar ▪ Located in nucleus AND cytoplasm ▪ Transcribes (copies) genetic code for proteins from DNA to ribosome o Transfer RNA – tRNA (mailman) ▪ In prok/eukar ▪ Located in cytoplasm ▪ Transfers amino acids into polypeptide chain o Small nuclear RNA – snRNA (parent teaching their kid manners) ▪ In eukaryotes ▪ Located in nucleus ▪ Helps process pre-mRNA into mature mRNA by splicing out introns, allowing mRNA to be translated properly o Small nucleolar RNA – snoRNA (rRNA pit crew) ▪ In eukaryotes ▪ Located in nucleus ▪ Processing & assembly (modifies) rRNA so it can form functional ribosomes o Micro RNA – miRNA (security) & small interfering RNA – siRNA (hitman) ▪ In eukaryotes ▪ Located in nucleus AND cytoplasm ▪ miRNA: inhibits translation of mRNA which degrades the target mRNA ▪ siRNA: silences specific genes which also degrades target DNA o Long noncoding RNA – liRNA (manager, does everything) ▪ In eukaryotes ▪ Located in nucleus AND cytoplasm ▪ A lot of things including regulator, guide, etc.  *Look at the mini illustration you made on paper to understand relationship* Transcription requirements 1. DNA template 2. Nucleotide triphosphate (rNTPs) 3. Machinery: RNA polymerase, etc. Differences between RNA production and DNA replication RNA Synthesis DNA replication - Length: short segments - Length: entire genome - Strand: one strand is used - Strand: both are used - Selection: not all genes are - Selection: all genes are transcribed at transcribed (copied) at once once - Promoters: things that tell us where a - Promoters: doesn’t rely on them that gene is, which gene to express at much what time → very crucial! How is RNA synthesized? Direction, which strand? o Direction: 5’ to 3’ o Template: 1 strand of DNA I used to make the RNA copy (designed by the promoter) o Non-template strand (coding strand): identical to RNA, but uracil instead of Thymine o We can use top or bottom strands, however, some processes identify which one is the template o Each gene’s mRNA is copied from one strand Know the general layout of a gene on the chromosome o What is the importance of each part? ▪ RNA coding region: gene locus of DNA that will end up in RNA transcript ▪ Promoter: tells us where to start, where to go, which template strand RNA polymerase & other proteins are gonna find it, once it’s going in the right direction, the RNA polymerase can start copying (transcribing) the template strand all the way through ▪ Transc. Termination site – tells us when to stop/be done transcribing Mechanism of transcription initiation o Prokaryotic: ▪ Sigma Factor Binding: The sigma factor binds to the core RNA polymerase, forming the holoenzyme (a group of different pieces of proteins) ▪ Promoter Recognition: The holoenzyme scans for the -35 (TTGACA Box) and - 10 (TATAA Box) consensus sequences in the promoter, then binds to those sequences upstream  ▪ DNA Unwinding: The holoenzyme binds tightly to the promoter and unwinds the DNA. Sigma factor will fall off. ▪ Initiation of RNA Synthesis: An rNTP complementary to the start site base pairs with the template, becoming the first nucleotide of the RNA. ▪ Elongation Begins: Two phosphates are cleaved from each new NTP, adding nucleotides to the 3' end of the growing RNA. ▪ Release of Sigma Factor: Once transcription is underway, the sigma factor is released, allowing RNA polymerase to continue along the DNA. The mRNA is built using the DNA template strand. In summary, transcription initiation begins when RNA polymerase (with sigma) binds to the promoter and starts RNA synthesis. Mechanism of transcription termination o Two major ways to terminate transcription: ▪ Rho Dependent Terminator site: Causes RNA polymerase to pause Rho utilization (rut) site: A cytosine-rich RNA sequence upstream of the terminator binds to the Rho protein. Rho breaks/unwinds RNA-DNA base pairs ▪ Rho Independent Contains inverted repeats in which DNA has sequences that when transcribed, form a hairpin loop in the RNA After the hairpin, there’s a string of 7-9 adenines on the DNA RHO DEPENDENT RHO-INDEPENDENT - Mechanism: The hairpin causes RNA polymerase to pause, and the weak A-U bonds allow the RNA to detach, terminating transcription. Eukaryotic promoter elements o Are there more RNA pols in eukaryotes or prokaryotes? ▪ Eukaryotes: RNA Pol I, II, and III (three) ▪ Prokaryotes: RNA Pol o What’s needed for Transcription initiation? ▪ RNA Polymerase II: Enzyme for mRNA synthesis. Partial unwinding of DNA at the transcription site. DNA bends at a right angle to position the DNA-RNA hybrid at the active site. Newly synthesized RNA exits through its own channel. Separate entrance channel for rNTPs.  ▪ General Transcription Factors (GTFs): Essential for all RNA pol II transcription (e.g., TFIID, TFIIB, TFIIF, TFIIE, TFIIH). ▪ Specific Transcription Factors: Regulate specific genes, enhancing or repressing transcription. ▪ Promoter Region: DNA sequence recognized by the transcription machinery (e.g., TATA box). Why is processing of RNA important? o Stability of the molecule: protection o Transport- processing is essential for transport of mRNA o Function- tRNA and rRNA need modifications o Translation- mRNA can only be translated if processed mRNA Structure o Purpose of: Noncoding vs Coding regions ▪ Noncoding regions are important for stability and regulation ▪ Coding regions contain the sequence to be translated into amino acids, building a protein o The Shine-Dalgarno Sequence ▪ Eukaryotes/Prokaryotes? Purpose? In prokaryotes, it marks the ribosome binding site o Eukaryotes ▪ 5’ cap: What makes it up? It is formed by the addition of an extra guanine nucleotide that is methylated at the 5’ end of the mRNA  What kind of linkage? 5’ to 5’ which marks the true end of the RNA ▪ Poly(A) Tail What is it? Long stretch of 50-250 adenine nucleotides (A's) added to the 3' end of an mRNA molecule during RNA processing in eukaryotic cells How is it made? formed by cleavage of the pre-mRNA, followed by template-independent addition of adenine nucleotides by polyadenylate polymerase, stabilizing the mRNA for translation. o Cleavage downstream of the consensus sequence then addition of the adenines by which protein? PAP (polyadenylate polymerase) ▪ RNA contains intervening sequences: Introns and exons Splicing: purpose? Removal of introns from pre-mRNA; exons sealed back together to form mature mRNA. o What is the spliceosome? small nuclear RNA (snRNA) and small nuclear ribonucleoprotein particles (snRNP) o What are important sites within the exon and intron needed for splicing? 5’ splice site, 3’ splice site, and branch point o What is the main splicing mechanism? Spliceosome mediated Alternative Splicing: purpose? A single pre-mRNA can be processed in different ways to produce alternative mRNAs o Alternative splicing vs multiple 3’ cleavage site Both processes increase mRNA diversity, but alternative splicing alters the coding sequence, while multiple 3' cleavage sites affect the mRNA’s 3' end. tRNA o What are tRNAs? What is the purpose? The adaptor for the mRNA sequence to the amino acid sequence which makes up a protein, transfers amino acids to form polypeptide chain o Which RNA pol transcribes tRNA? RNA Pol III o What are 2 important sites on the tRNA which are required for its function? ▪ Anti-codon sequence ▪ 3’ ammino acid attachment site o How are they processed? 1. Cleavage/trimming at the 5’ and 3’ ends 2. Removal of intron 3. CCA added to 3’ end for amino acid attachment 4. Modification of bases  rRNA o What type of RNAs mediate the modifications of rRNAs? Small nucleolar RNA (snoRNAs) which are similar to snRNAs mediating splicing o How are rRNAs originally transcribed? rRNAs in both prokaryotes and eukaryotes are made as 1 large precursor RNA and cleaved & trimmed into eh final multiple mature rRNAs RNAi RNA processing o Differences between miRNA and siRNA – silencer vs hitman miRNA: inhibits translation of mRNA which degrades the target mRNA siRNA: silences specific genes which also degrades target DNA o General steps of processing ▪ Initial transcription of the RNA. ▪ Cleavage by Dicer enzyme into small RNAs ▪ Formulation of RISC complex (RNA-induced silencing complex). ▪ Inhibition or cleavage and degradation of target mRNA, depending on the type of RNA. In Prokaryotes, transcription and translation can be coupled due to the absence of nuclear processing Why can Eukaryotes not couple transcription and translation? - because transcription occurs in the nucleus, and translation happens in the cytoplasm, requiring mRNA processing and export. Chapter 14: Translation and Proteins  What is the genetic code? o Definition: The set of rules by which information encoded in mRNA is translated into proteins. o Orientation: Written 5' to 3'. o Codon Structure: Each codon is composed of three nucleotides. o Non-overlapping and universal across all domains of life. What is the structure of an amino acid (with R designating the side chain)? Central carbon with an amino group, carboxyl group, hydrogen, and variable R group. o What is the purpose of having such a variety of R groups? Determines the chemical properties and function of each amino acid. Be able to read an mRNA sequence and use the codon chart to translate it: o What is the start codon? what does it code for? Typically AUG, codes for methionine. What is the wobble base? The third nucleotide base in a codon o What does this mean for tRNA quantity and pairing to the mRNA? allows flexibility in tRNA pairing and reducing the number of required tRNAs. How many possible reading frames are there? Three possible frames per strand. o Importantly, know that the start codon’s position ensures that the correct reading frame is chosen  ▪ o How do different numbers of nucleotide insertions or deletions affect the reading frame? Insertions or deletions can disrupt the reading frame. tRNA o Function - ▪ Of the whole molecule - Transfers amino acids to the ribosome for protein synthesis. ▪ Anticodon: Matches codons on the mRNA. ▪ CCA Acceptor Stem: Binds the amino acid. o Are there tRNAs which bind to stop codons? NO o Remember, the interaction between tRNA and mRNA follows the same rules as any interaction between nucleotide sequences. They’re anti-parallel and the base pairing rules remain the same. o What is inosine? How does it affect base pairing and the wobble effect? ▪ Inosine (I) flexible base pairing o How are tRNAs charged? What protein is responsible and where does it attach the amino acid? ▪ aminoacyl-tRNA synthetase on the 3’ CCA Translation o Which direction does peptide growth occur? From the N-terminus (amino group) to the C-terminus (carboxyl group). o Ribosome: ▪ Structure: Composed of two subunits: large and small. Prokaryotes: 70S ribosome (50S + 30S). Eukaryotes: 80S ribosome (60S + 40S). ▪ A, P, and E sites: A (Aminoacyl): Entry site for charged tRNA. P (Peptidyl): Holds tRNA with the growing polypeptide chain. E (Exit): Site where tRNA exits the ribosome after amino acid transfer. o General steps of translation- what happens at each step? ▪ tRNA charging: Amino acids are attached to their corresponding tRNAs by aminoacyl-tRNA synthetases ▪ Initiation Prokaryotes vs Eukaryotes  Eukaryotes: Use Met-tRNA Prokaryotes: Initiation begins (methionine tRNA) without with fMet-tRNA (formyl- formylation. methionine tRNA) o What factors are needed? Initiation factors (IFs in prokaryotes; eIFs in eukaryotes) o Where does that 1st tRNA bind within the ribosome? The first tRNA binds to the P site in the ribosome. Elongation 1. A charged tRNA enters the A site. 2. Peptide bond forms between the growing polypeptide (in the P site) and the new amino acid (in the A site). 3. Ribosome shifts (translocates), moving the tRNA to the next site: A → P → E. 4. The growing polypeptide chain remains in the P site during elongation. Termination o Release Factor (RF): Recognizes stop codons and promotes polypeptide release from the ribosome. Peptide Release o Polysomes: Multiple ribosomes translating the same mRNA simultaneously, increasing efficiency. Remember the different levels of protein structure organization - Primary Structure: Sequence of amino acids. - Secondary Structure: Formation of alpha helices and beta sheets due to hydrogen bonds in the peptide backbone. - Tertiary Structure: Three-dimensional folding based on R-group interactions (e.g., ionic bonds, hydrophobic interactions). - Quaternary Structure: Interaction between multiple polypeptide chains to form a functional protein.Chapter 15: Gene Mutations and DNA Repair Chapter 17: Transcription Regulation in Eukaryotes Compare gene regulation in bacteria and eukaryotes o Bacteria: Often uses operons for coordinated gene regulation, typically in response to environmental changes. o Eukaryotes: Regulation occurs at multiple levels (chromatin structure, transcription, post-transcription), allowing for complex and tissue-specific expression patterns. What are chromosome territories and transcription factories? o Chromosome Territories: Distinct regions in the nucleus where individual chromosomes reside, preventing tangling and facilitating efficient gene regulation. o Transcription Factories: Sites within the nucleus with high concentrations of transcription machinery, supporting efficient transcription. Effects of heterochromatin and euchromatin o Heterochromatin: Tightly packed, generally transcriptionally inactive.  o Euchromatin: Loosely packed, accessible for transcription. Histone modifications and chromatin remodeling o Acetylation: ▪ Histone Acetyltransferase (HAT): Adds acetyl groups to histones, loosening chromatin and enhancing transcription. ▪ Histone Deacetylases (HDAC): Removes acetyl groups, leading to tighter chromatin and reduced transcription. o Methylation: Addition of methyl groups can either activate or repress transcription, depending on the context. ▪ CpG Islands: DNA regions with high frequency of CpG sites; often found near promoters and subject to methylation. Chromatin Remodeling (SWI/SNF) o SWI/SNF Complex: Uses ATP to reposition or remove nucleosomes, making DNA more accessible to transcription factors. cis-acting DNA elements vs trans-acting factors o cis-Acting Elements: DNA sequences (like promoters or enhancers) that affect nearby genes. o trans-Acting Factors: Proteins or RNAs (like transcription factors) that can influence gene expression, often by binding to cis-elements. What is a promoter? DNA sequences that initiate transcription. o Core Promoter Elements: Includes TATA box, initiator (Inr) sequences; essential for transcription start. o Focused vs. Dispersed Promoters: ▪ Focused: Single transcription start site, leading to regulated, cell-type-specific expression. ▪ Dispersed: Multiple start sites, resulting in more consistent expression. o Proximal Promoter Elements: Upstream sequences that help control gene expression levels. The human metallothionein 2A gene and its regulation - encodes a protein important for metal detoxification and ion balance. What are enhancers, insulators, and silencers? o Enhancers: DNA sequences that increase gene transcription; can act at a distance. o Insulators: Block interaction between enhancers and promoters, restricting enhancer action to specific regions. o Silencers: DNA elements that decrease transcription. o How do insulators control enhancers? Insulators prevent enhancers from activating the wrong promoters by acting as physical barriers or by looping the DNA to isolate enhancer activity. Activators vs repressors o Activators: Bind to enhancers or other cis-elements to increase transcription. o Repressors: Bind to silencer regions or compete with activators to decrease transcription. Functional Domains in Proteins Purpose: Each domain contributes to specific functions, like DNA binding, activation, or interaction with other proteins. Examples: DNA-binding domains, activation domains, and repressor domains.  Formation of the RNA pol II transcription initiation complex: o Steps and Factors: ▪ General transcription factors (TFIID, TFIIB, TFIIF, etc.) bind sequentially to the promoter. ▪ Formation of a pre-initiation complex that recruits RNA polymerase II for transcription initiation. Chapter 18 and 19: Posttranslational Regulation in Eukaryotes/Epigenetics Alternative splicing o Purpose and Results: Produces different mRNA isoforms from the same gene, resulting in diverse protein functions. o Sex Determination in Drosophila: Differential splicing of Sxl and dsx genes controls male vs. female development. RNA decay and ways it happens - Exonucleolytic decay: Deadenylation followed by 5′ to 3′ or 3′ to 5′ degradation. - Nonsense-mediated decay: Targets mRNAs with premature stop codons. sncRNAs (RNAi) o Purpose? Regulate gene expression post-transcriptionally o miRNA vs siRNA ▪ miRNA: Endogenous, imperfect base pairing, represses translation. ▪ siRNA: Exogenous, perfect base pairing, cleaves target mRNA. o Production of these sncRNAs ▪ Processed by Dicer into small fragments. ▪ Loaded onto RISC for targeting mRNA. o Base pairing differences: ▪ Imperfect pairing represses translation (miRNA). ▪ Perfect pairing degrades mRNA (siRNA). lncRNAs o processing? Similar to mRNA, including capping, splicing, and polyadenylation o what are the functions in regulation? Regulate chromatin state, transcription, and post- transcriptional events. Factors affecting translation rates o Availability of initiation factors. o mRNA stability and codon usage. o Ribosome abundance and activity. Post-translational modifications: Alter protein function, stability, and localization. o Ex) Phosphorylation, acetylation, ubiquitination, glycosylation How localization of mRNAs affects gene expression – It spatially restricts protein synthesis, crucial for processes like cell polarity and migration. The example of migrating cells and their movement/localization - Localization allows proteins to be synthesized where they are needed, supporting the cell's movement and function in migration. - Migrating Cells: Movement depends on localized gene expression. - Localization Mechanism: o Zip Code: A specific mRNA sequence directs its localization to specific parts of the cell.  o Zip Code Binding Protein 1 (ZBP1): Binds to the zip code sequence and ensures the mRNA is transported to the right cellular region. Protein stability and degradation o Ubiquitination: Marks proteins for degradation via the proteasome What is epigenetics? Study of heritable changes in gene expression without altering the DNA sequence. Know that changes in chromatin structure can alter gene expression o What are molecular mechanisms that alter chromatin structure? ▪ Patterns of DNA methylation ▪ Chemical modification of histone proteins ▪ RNA molecules that affect chromatin structure and gene expression o Methylation: ▪ Characteristics: Addition of methyl groups to cytosines in CpG islands. ▪ Maintenance: Preserved during DNA replication. ▪ Effects: Typically represses transcription. Types of histone modifications: Acetylation, Methylation, Ubiquitination, Phosphorylation Epigenetic effects of RNA molecules o 4 models for lncRNA mechanisms of action: a) Decoy: Protein binds to lncRNA instead of the DNA b) Adapter: lncRNA helps proteins form a complex c) Guide: Localizes a protein to the proper locus d) Enhancer: Works with activators to increase gene expression What are some epigenetic changes associated with development? o Cell differentiation - Epigenetic mechanisms regulate gene expression, ensuring that specific genes are turned on or off as cells specialize into various types. o Transgenerational effects through paramutation - Paramutation involves interactions between alleles, leading to heritable changes in gene expression without altering the DNA sequence. o X inactivation - In female mammals, one X chromosome is epigenetically silenced in each cell to balance gene expression with males. o Genomic imprinting - Certain genes are expressed based on their parental origin due to epigenetic marks inherited from the mother or father. How does behavioral epigenetics affect an individual? Life experiences, especially early ones, can induce epigenetic changes  Ex) Maternal behavior can modify DNA methylation patterns in offspring, affecting stress responses and cognition. Early-life stress or nurturing alters histone modifications and gene expression How does the environment induce epigenetic changes? Environmental factors like diet, stress, or exposure to toxins can modify chromatin structure. Ex) High maternal nurturing leads to increased serotonin, which triggers hypomethylation and histone acetylation, enhancing gene expression for stress regulation. Chapter 20: Recombinant DNA Technology What are different applications for recombinant DNA technology? Vaccines Diagnostic Testing Gene Therapy DNA fingerprinting Agriculture- GMO What are the 4 main steps of creating recombinant DNA? o Step 1: DNA is isolated, then this is where it is identified and extracted to use from the organism and vector What are restriction endonucleases? Enzymes that cleave DNA at/near restriction sites (usually palindrome). *Type II is the most common b/c it cuts at site in predictable patterns* What are the 2 different outcomes of cleavage? Pros and cons of both? 5’ to 3’ Overhang: STICKY ENDS Good: higher chance of ligation accuracy, specific and organized, overhangs are complementary and can only pair one way Bad: not flexible b/c picky about restriction enzymes and incompatible sticky ends Blunt end: straight, no complementary end for base pairing  Good: universal, flexible, any blunt DNA fragment can join Bad: rely on DNA ligase entirely b/c lack of base pairs, disorganized which promotes incorrect orientation like transcription from a promoter Think about the cutting and pasting mechanism- does it have to go into the target vector in a particular orientation? In class we talked about the promoter Yes, Proper orientation is critical when inserting DNA into a vector, particularly for promoter function in transcription. Know that we can isolate a target DNA segment from mRNA cDNA (complementary DNA) can be synthesized from mRNA, allowing researchers to isolate and work with a specific DNA segment that corresponds to a gene's coding region (exons only) What process do we use to make cDNA from mRNA? Reverse transcription What are the differences between cDNA and gDNA? Where do both originate from? cDNA is synthesized from mature mRNA in a lab to use reverse transcriptase which will transcribe the mRNA to cDNA. gDNA (genomic DNA) is extracted from the chromosome of the organism. It is extracted from the nucleus. What process do we use to amplify a target DNA sequence? Polymerase Chain Reaction (PCR) Template: the blueprint, or DNA sample and target sequence that will be amplified Primers: binds to specific locations of template, defines the exact region that’ll be copied Polymerase: helps build new DNA strands by adding nucleotides to existing primer sequence Nucleotides: the building blocks of DNA, in charge of matching up with complementary pairs  Thermal cycler: goes thru different temperatures that are needed for DNA denaturation (separating of strands) and primer annealing (binding to template) and extension (polymerase activity) o Step 2: Then DNA cutting takes place, this is where you take specific enzymes to cut both the source and vector DNA in specific site. This then creates sticky or blunt ends. What are 3 reasons we use modified plasmids as a cloning vector? They’re small, self-replicate, and easy to purify What are some common features of plasmids? Drug resistance (selection) Cloning host Origin Multiple cloning sites or unique RE sites o Step 3: Next, ligation is where you use DNA ligase to bond the source. and vector DNA together, this forms recombinant DNA. What enzyme binds together our DNA fragments? DNA ligase o Step 4: The last step is introduction to the host cell, this is when you introduce the recombinant DNA into the host cell which then allows for the replication and expression of the foreign DNA.  This process will have to be done twice: first for cloning host, then for final host. Different techniques can be used for each. What are 3 different methods we can use for getting rDNA into the host organism? Exogenous = foreign* Transformation: uptake of exogenous DNA by bacterial cell Transfection: forced introduction into eukaryotic cells with chemicals, lipid-based, or electroporation Transduction: introduction of an exogenous genetic material by a virus Once we get the plasmid into the organism, there are a few ways we can confirm the expression. If cells survive with antibiotics, they are resistant meaning the plasmid was expressed LacZ: Disrupted LacZ: White colonies (β-galactosidase is not functional). Intact LacZ: Blue colonies What do we need to consider when choosing a final host? Study genetic product activity (growth rate) Introduce beneficial gene to new system Synthesize enough proteins for isolation Gel Electrophoresis What are the steps of agarose gel electrophoresis? Load samples into wells Apply electrical current Migration thru the pores separates dna/rna by SIZE Stain the gel with dye, which will then bind to dna/rna Fragments move toward which charge? Positive What do we use to visualize DNA fragments in the gel? Ethidium Bromide (dye) and UV light What are different ways of visualizing *Specific* targets Locating DNA Fragments with probes  There’s different types of probes, what kind of labels can we use to visualize the probes? Radioactive label Fluorescent label Enzyme label which can generate a light or color signal Difference between southern and northern blots Southern Blot: DNA Northern Blot: RNA What is FISH? How can we use FISH? fluorescence in situ hybridization – ex) zebrafish Visualizes specific DNA/RNA sequences in chromosomes or cells using probes Applications: Diagnosing gene expression’s chromosomal abnormalities and mapping genes PCR vs Quantitative PCR PCR: Amplifies DNA; qualitative results. qPCR: determines the amount of DNA amplified as the reaction proceeds using fluorescent markers. What are some applications of DNA sequencing? DNA fingerprinting Paternity tests Crime scene identification Know how Sanger Sequencing works Uses chain-terminating dideoxynucleotides (ddNTPs). →Lacks OH group, terminating DNA synthesis Generates fragments of varying lengths to determine the sequence. What are different ways we can manipulate the Genome? Knock-out (KO): delete part of a gene leading to a loss-of-function mutation Conditional KO: may induce the KO, but only conditionally Transgenic or Knock-In (KI): alter gene sequence to change the gene New technologies to accomplish this include designed nucleases and CRISPR What are the main steps in generating Knockout Mice? Design the target vector Transform embryonic stem cells with target vector (rDNA) and select cells Selected cells introduced into a mouse blastocyst via microinjection  What are the benefits of making a conditional knockout? Controls time during development Specific and controlled Flexible What is the Cre-lox system? creates conditional KOs by inserting loxP sites around a target gene. When combined with a mouse expressing Cre recombinase, the gene is removed where and when the Cre promoter is active. This allows precise control over gene knockouts in specific tissues, stages, or in response to chemicals. What are 3 examples of engineered nucleases? CRISPR ZNC TALENS Chapter 22: Applications of Genetic Engineering and Biotechnology What are genetically modified organisms? The alteration of an organism’s genome by manipulating DNA in vitro and then introducing that DNA into living cells has allowed scientists to generate new plants, animals, and other organisms with specific traits What are applications of biotechnology? using microbes to make important products (wine, bread, beer) Biopharmaceutical products Agriculture Engineered Animals Genetic Testing Genome Analysis Synthetic Genomes What is biopharming? Pharmacy + farming = pharming Using GMOs to produce pharmaceuticals like hormones, vaccines, or antibodies. How was molecular cloning utilized to artificially produce insulin? Scientists inserted the human recombinant insulin gene into bacteria, enabling large-scale, cost-effective insulin production. What are some cool examples of transgenic animals? Farm animals produce anti-clotting protein in milk Hens have egg white protein that helps treat liver disease What are different types of vaccines? What are the differences between them? Attenuated: contain live viruses that can no longer reproduce (common) Inactivated: killed samples of virus/bacteria Subunit: 1+ surface protein of virus/bacteria What was the earliest form of manipulating the genetic makeup of plants/animals? Selective breeding  What are some reasons why we generate transgenic crops? Resistance to insects/weeds, herbicide, nutritional value What do we mainly use transgenic animal and what is the other common use for transgenic animals? Study gene function Pharmaceutical production What are uses for Genetic Testing? Detect genetic disorders, ancestry, and carrier status. o What are different types of pre-natal genetic tests? Amniocentesis Chorionic villus sampling Non invasive o What is Restriction fragment length polymorphism analysis? What are different approaches to genetic testing? o What is a SNP? Point mutations/Small variations in DNA sequences that can indicate disease risk o What are ASOs? Synthetic DNA probes which detect SNPs ▪ how do we test ASOs? DNA is extracted from the sample, and a specific region is amplified using PCR. The amplified DNA is spotted onto a DNA-binding membrane strip. This strip is exposed to ASO probes designed to match either the normal or mutant sequence, with fluorescent markers attached. The probes hybridize only with matching DNA on the strip, and the fluorescent signal is analyzed to determine the genotype ▪ Understand the application of ASO testing through Pre-Implantation Diagnosis genetic analysis of single cells from embryos created by in-vitro fertilization (IVF) o What is a DNA Microarray? Spot single stranded DNA molecules (probes) onto a glass slide ▪ What are applications? Gene Scanning Gene-Expression Profiling Example: normal cells vs cancer cells arising from same cell type→ Resulting ‘heat map’ with different fluorescent at each spot/probe indicates the relative RNA expression of many genes ▪ Generally- what is this process? Detect and measure gene expression or mutations across thousands of genes simultaneously. Used in disease research and personalized medicine ▪ Know how to read microarrays What are some applications of genetic analysis?  Diagnosing, Whole Genome sequencing (WGS), Alternative Whole Exome Sequencing (WES)- What are Genome Wide Association Studies? What are overarching goals of GWAS? Large-scale studies identifying genetic factors linked to diseases, with goals like improving treatments and understanding inheritance patterns. Goal: identify genetic variations that may confer risk of developing the disease Chapter 24: Cancer Genetics Why do we care so much about Cancer research? Connect this to the huge number of individuals affected by it every year and the heterogeneity of the disease Cancer affects millions annually and is highly heterogeneous, varying across cell types and genetic alterations. Its complexity makes understanding and treating it a significant challenge. Know the significance of cancer being a disease on the somatic cell level Predominantly caused by mutations that arise in somatic cells Know that cancer is VERY RARELY a single mutation in single gene- relate this to what we talk throughout the power point and what evidence we have to explain why this is so. Evidence shows cancer results from multiple mutations in genes regulating cell growth, division, and repair. Single mutations rarely provide enough dysfunction to drive cancer. What is chemotherapy vs gene-targeted therapy, Think about what we know about these and what may be pros and cons of both? Chemotherapy: Uses drugs to kill rapidly dividing cells but can harm healthy cells (side effects). Gene-Targeted Therapy: Focuses on specific genetic mutations in cancer cells (fewer side effects but limited to cancers with identified targets). Know that cancers have 2 fundamental properties: Abnormal Proliferation Metastasis These properties vary based on cell type and genetic changes. o Understand that Cancer is a large number of complex diseases that behave differently depending on the cell types from which they originate and the types of genetic alterations. o How does this relate to the complexity of cancer? What is a common misconception about cancer? Relate this to the fact that people commonly think that there will be a singular cure to cancer in the future). That cancer is only one cure, there are so many different ways that cancer is obtained. There’s no "one-size-fits-all" cure because of cancer's diverse nature across individuals and types. 4 hallmarks of many: resisting cell death, no apoptosis takes place genome instability and mutation, can not repair damages tumor-promoting inflammation, hide from white blood cells which makes chemo less effective evading growth suppressors, growth without any signals  What is the Philadelphia Chromosome? How does it promote cancer development?’ A translocation between chromosomes 9 and 22 creates a fusion gene (BCR-ABL), producing a protein that drives uncontrolled cell division, linked to leukemia. Reciprocal translocation, ABL, linking it with other gene so they share promoter. It’s not activated by something it’s activated all the time/constantly on, its growing and proliferating which is bad cause that’s how cancer grows What is the cell cycle? What are the 3 main checkpoints? What are we monitoring at each? G1 checkpoint: see if the cell is big enough, and has the nutrients that it needs, and also that the DNA is not damaged before committing to the cycle G2 checkpoint: makes sure that the DNA replication was completed accurately during the s phase, makes sure there is no big DNA damages before entering mitosis M checkpoint: makes sure all chromosomes are attached to the spindle fibers correctly, and preventing incorrect chromosomes separate during anaphase o What does it mean to be in a quiescent state? Cells exit the cycle (G0 phase) to stop dividing temporarily or permanently. Metabolically active, but does not grow or divide o How does the cell regulate the cell cycle? The cell cycle is regulated by cyclins and CDKs, which control progression through phases, and checkpoints (G1/S, G2/M, Spindle Assembly) that ensure DNA integrity and proper division. Tumor suppressors like p53 halt the cycle for repairs, while oncogenes can disrupt control if mutated.  AGAROSE Apoptosis: Programmed cell death o What proteins facilitate breakdown of cellular components? Caspases o Know the general process of apoptosis Apoptosis is programmed cell death that removes damaged or unnecessary cells. The process involves activation of proteins like caspases, which break down cellular components. The cell shrinks, its DNA fragments, and it forms apoptotic bodies that are engulfed by nearby cells without causing inflammation. o What are proto-oncogenes and tumor suppressor genes? How do they differ? Proto-oncogene: Transcription Factors that stimulate expression of other genes/molecules that stimulate cell division Tumor Suppressor Genes: normally regulate cell cycle checkpoint or inactivation of apoptosis o What is the *dramatic wave* Guardian of the Genome? How is it controlled? What types of situations can increase its activation? What are some downstream responses? p53, responds to stress (e.g., DNA damage) by halting division or triggering apoptosis, tightly regulated by signals from within the cell (intrinsic pathway) or external signals (extrinsic pathway). Situations That Increase Activation: DSBs Chemical damage to DNA DNA-repair intermediates Downstream Responses: Cell-cycle arrest DNA repair Apoptosis What are the steps of metastasis? What happens at each step? Invasion, invasion of the local environment of the tumor Intravasation enters into the blood vessels Circulation, tumor cells move along the blood vessels to other parts of the body Extravasation, exiting the vessels Colonization, creates a new home to grow Chapter: CRISPR-Cas Know ways we use CRISPR-Cas systems o Genome editing o Research ▪ Patents ▪ Study of protein function o Biotechnology o Medicine ▪ Clinical trials to treat disease Endogenous function/How it contributes to coevolution of bacteria and viruses o CRISPR-Cas systems are part of bacteria's immune system to defend against viral attacks. They store short sequences from viral DNA as "spacers" in their genome (CRISPR loci). o Coevolution: Bacteria and viruses engage in an arms race where bacteria evolve CRISPR-Cas defenses, and viruses evolve strategies to bypass them. Benefits of using CRISPR-Cas vs other gene editing methods o CRISPR is more efficient, precise, and cost-effective than earlier methods like ZFNs and TALENs. o It uses short RNA guides to direct the Cas9 protein to specific DNA sequences, making edits faster and more precise https://medium.com/genaerrative/a-comprehensive-timeline-of-human-gene-editing- 664a93bed8cf What exactly is CRISPR and what organism was it first identified in? stands for Clustered Regularly Interspaced Short Palindromic Repeats It was first identified in E. coli in 1987 Are CRISPR loci common in prokaryotes? Yes, CRISPR loci are common in prokaryotes like bacteria and archaea. They serve as a defense mechanism against viruses What are spacer sequences? They are short DNA fragments from viruses that are stored in the CRISPR loci. They act as a "molecular memory," allowing the bacteria to recognize and defend against viruses that have attacked before. Hypothesis that CRISPR loci are “molecular memory” of previous viral attacks? Relate this to innate vs adaptive immunity Adaptive Immunity: The CRISPR system allows bacteria to "remember" viral DNA and attack it more efficiently on subsequent infections, similar to how our immune system remembers pathogens Understand the experimental process of the discovery of evidence towards CRISPR loci serving as a “molecular memory” of previous viral attacks In experiments, researchers showed that bacteria with specific spacer sequences could target and destroy viral DNA from previous infections, providing evidence for the "memory" hypothesis. 3 main steps of the CRISPR-Cas mechanism: o Spacer Acquisition: Bacteria capture viral DNA and integrate it into the CRISPR array. o crRNA Biogenesis: The CRISPR array is transcribed into RNA (pre-crRNA), which is processed into smaller crRNAs. o Target Interference: crRNA guides Cas proteins (like Cas9) to the viral DNA, which is then cleaved. Know at least one example of each type of CRISPR-Cas system- I’m providing an example and how they are different- you can go out and look up other cool examples! This stuff is super neat  - Type I: Involves a complex nuclease called Cascade, common in E. coli. - Type III: Found in organisms like Staphylococcus aureus, uses a Cas10-containing complex. - Type II: Focus on this one! It's the system used for genome editing, with Cas9 cutting the DNA at specific locations. What 3 processes is Cas9 involved in o PAM (Protospacer Adjacent Motif): A short sequence needed for Cas9 to recognize and bind to the DNA. o tracrRNA: A RNA that helps process pre-crRNA and guides Cas9. o crRNA: The RNA that binds to the target DNA sequence, guiding Cas9 to its location. Know the different components of Cas9, target viral DNA, importance of the PAM sequence, tracrRNA and crRNA o PAM Sequence: ▪ Acts as a "landing pad" for Cas9 to bind and initiate DNA cleavage. ▪ Ensures Cas9 targets only foreign DNA, as PAM is absent in bacterial CRISPR loci. ▪ Sequence: 5’-NGG-3’. o tracrRNA: ▪ Connects the crRNA to Cas9, enabling the crRNA to guide the system. o crRNA: ▪ Contains the sequence complementary to the target DNA. ▪ Guides Cas9 to the specific DNA location for cleavage. How did scientists alter the CRISPR-Cas9 system for their research in vitro? - By combining tracrRNA and crRNA into a single guide RNA (gRNA) - Engineered Cas9 to be programmable by designing gRNAs to target any DNA sequence adjacent to a PAM. - Used it in mammalian cells by introducing Cas9 protein and synthetic gRNA via plasmids or delivery systems like liposomes Why is CRISPR-Cas9 a better system to use when we’ve had the ability to edit genomes since as far back as 1989? o Efficient o Cost-effective o Precise & specific edits What do we need to utilize the CRISPR-Cas9 system in mammalian cells? Why would we need these different aspects compared to the endogenous system in bacteria or the in vitro system? - Cas9 Protein: Bacterial protein adapted for eukaryotic conditions. - gRNA: Custom-designed for the target DNA. - Delivery System: o Plasmids, viral vectors, or lipid nanoparticles for gene insertion. - Repair Template (optional for HDR): o DNA sequence designed for insertion, used in homology-directed repair. - These aspects are essential as mammalian cells differ significantly from bacterial systems, requiring optimized delivery and functional expressions. Understand the basic mechanism of NHEJ and HDR NHEJ (Non-Homologous End Joining): o Error-prone. o Repairs DSBs by directly ligating DNA ends. o Often leads to insertions or deletions (indels), disrupting gene function.  HDR (Homology-Directed Repair): o High-fidelity repair mechanism. o Requires a repair template with homologous DNA to guide accurate repair. o Used for precise genome editing, like inserting or replacing genes How did scientists further improve the CRISPR-Cas9 system in order to not only make deletions, but also alter the genomic sequence? o Engineered Cas9 variants with nickase activity to reduce off-target cuts. o Developed base editors for single nucleotide changes without DSBs. o Created prime editing, combining Cas9 and reverse transcriptase for targeted insertions or edits. How do NHEJ and HDR repair differ in the context of CRISPR-Cas9? NHEJ HDR Quick and efficient. Slower and requires a repair template. Induces mutations that knock out Allows precise gene insertion or genes correction. What different materials do we need for the HDR? Cas9 Protein: To create DSBs. gRNA: Guides Cas9 to the target site. Repair Template: DNA with homologous regions flanking the desired edit. Delivery Vehicle: Efficient delivery to the nucleus. What are limitations of CRISPR-Cas9? Why does this happen? Off-Target Effects: Incorrect cuts can disrupt non-target genes. Delivery Challenges: Ensuring components reach the right cells. Immune Response: Host immune system may recognize Cas9 as foreign. How can we combat CRISPR-Cas9 infidelity? - Careful sgRNA design using algorithms. - Modified Cas9 variants (e.g., high-fidelity Cas9). - Systems that only activate Cas9 under specific conditions (e.g., inducible Cas9). What are the different applications for CRISPR-Cas9 and example of each? - Basic Research: Study gene function. o Ex) Knockout experiments in model organisms. - Biotechnology: Improve crops or livestock. o Ex) Disease-resistant plants. - Medicine: Correct genetic diseases. o Ex) Clinical trials for sickle cell anemia. - Gene Drives: Spread genetic traits in wild populations. o Ex) Malaria-resistant mosquitoes. What are the 4 ways we can use the dCas9 to fine tune the system to our needs? o Altering Cas9 aa sequence o Web-based algorithms for sgRNA design o Alternative CRISPR systems not using Cas9 o Inducible activate Cas9