BIOL 103 Final Mutations

• Study notes for BIOL103 Genetics and Development lectures by Vivian Irish

Lecture 1:

• Developmental biology studies how organisms change and how cells acquire different functions, relying on observation and manipulations.
• Genetics involves the study of genes and heredity.
• The genome comprises all DNA in a cell; DNA consists of nucleotides.
• Genes are DNA segments coding for proteins, and mutations alter the nucleotide sequence.
• Point mutations include missense (wrong amino acid), nonsense (stop codon), or silent mutations.
• Frameshift mutations alter the reading frame through insertions or deletions.
• Multicellular organisms have specialized, differentiated cells organized into tissues and organs.
• Genes govern development; alterations in genes lead to the evolution of new traits.
• Model systems facilitate laboratory study due to their size, ease of manipulation, or life cycle as similar processes are shared between organisms.
• Phylogenetic trees depict organism relationships using gene sequences.
• The scientific method relies on observation, hypothesis development, testing, and revision.

Lecture 2:

• Development occurs in space and time, requiring the correct sequence of events to form a complex organism and is controlled by genes.
• An allele refers to a specific genetic sequence variant.
• Genotype describes an individual's genetic makeup.
• Phenotype refers to observable traits resulting from genetics and environment.
• Loss of function (lof) means a gene loses its ability to produce a functional product; gain of function (gof) indicates a gene produces excess or abnormal product.
• "Catch It" shorthand for detecting where a gene product is expressed.
• "Break It" refers to eliminating gene function.
• "Move It" means expressing a gene in a new context.
• "Rescue It" indicates replacing a lost gene function.
• "Genetic Screen" refers to a process to find a mutation in a specific process or phenotype.
• Forward genetics involves unbiased mutation generation.
• Reverse genetics involves targeted gene mutation and phenotype analysis.
• Organisms can be haploid, diploid, tetraploid, or hexaploid referring to chromosome numbers in the nucleus.
• Gametes are haploid and possess a single copy of each chromosome.
• Diploid organisms have two alleles per gene, either homozygous or heterozygous.
• Mutant alleles are often denoted by lowercase letters or a "-" sign, while wild-type alleles are denoted by uppercase letters or a "+" sign.
• Dominant mutations cause effects in heterozygotes, while recessive mutations manifest in homozygotes.
• Loss-of-function mutations are usually recessive; dominant mutations can result from gain of function or haploinsufficiency.

Lecture 3:

• Differential gene expression is tissue-specific, guided by transcription factors.
• “Catch it” (or “Show it”) is shorthand for detecting where a gene product is expressed.
• “Break it” is shorthand for eliminating gene function.
• “Move it” is shorthand for expressing the gene in a new context.
• “Rescue it” is shorthand for replacing a lost gene function.
• A genetic screen is a procedure to generate and identify mutations of interest.
• Forward genetics involves unbiased mutation generation.
• Reverse genetics involves targeted gene mutation and phenotype analysis.
• A zygote is totipotent and can form all cell types.
• Cells become determined and are committed to specific fates and then differentiate.
• Differentiated cells can be induced to become pluripotent under certain conditions.
• Plant cells are totipotent.
• Mature cells can be induced to pluripotency, demonstrating nuclear equivalence.
• Stem cells are pluripotent.
• Embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC) can be used to make stem cells.
• iPSCs are formed by four transcription factors reprogramming.
• iPSCs can differentiate into specific cells or form organoids in vitro.
• Organoids have therapeutic potential in drug design disease modeling and organ replacement.

Lecture 4:

• Apoptosis is programmed cell death involved in tissue patterning.
• Apoptosis differs from necrosis; it involves a series of steps leading to engulfment by neighboring cells.
• It and cell proliferation are essential; disruption is detrimental.
• The genetic basis of apoptosis was discovered in C. elegans and is conserved in mammals.
• Double mutant analyses can order genes in a pathway.
• Cancer is caused by gene alterations and uncontrolled cell proliferation / loss of cell death.
• Mutations in proto-oncogenes (promote cell division) or tumor suppressors (inhibit cell division) are associated with cancer.
• Mutations can occur in somatic or germ line cells.
• Mutations in RAS, a human oncogene, lead to continuous activation.
• Humans / mice have three RAS genes susceptible to oncogenic mutations, all resulting in constitutive activation.
• Tumor suppressor genes, like Rb, can cause cancer through loss-of-function mutations.
• Knudson's two-hit hypothesis explains cancer predisposition- individuals inheriting one mutation need only acquire one additional mutation.
• Those without an inherited mutation need two mutations in the same gene.
• BRCA1 encodes for a DNA repair protein and its mutation leads to DNA damage and cancer.
• Cancers need both oncogene activation and tumor suppressor inactivation.

Lecture 5:

• Differential gene expression is the production of different gene products by different cells, triggered by various factors.
• Induction involves signals transmitted between cells
• Cytoplasmic segregation occurs during cell division.
• Asymmetric distribution of cytoplasmic determinants in egg cells, impacting gene expression exists.
• Organisms exhibit polarity along anterior-posterior, dorsal-ventral, and left-right axes; polarity also applies to cells.
• Cytoplasmic determinants establish body axes.
• The components can be mRNA or transcription factors. Localized determinants influence gene expression.
• Cytoplasmic determinants are asymmetrically divided, affecting daughter cell gene expression.
• Induction involves extracellular signals affecting neighboring cell fate.
• Ligand-receptor binding initiates signal transduction.
• Early animal development starts with fertilization and then the zygote undergoes cleavage to form a blastula.
• Gastrulation produces three germ layers: ectoderm, mesoderm, and endoderm.
• Neurulation (nervous system initiation) and organogenesis (organ system formation) follow gastrulation.
• Cell movements during these processes create new cell-to-cell interactions

Lecture 6:

• Early animal development starts with fertilization, followed by cleavage forming a blastula or blastocyst.
• Gastrulation produces ectoderm, mesoderm, and endoderm germ layers.
• Localized cytoplasmic determinants in eggs / embryos are crucial for body axis formation.
• Determinants, act differently in different organisms, they establish body axes.
• A gene contains a regulatory region, promoter, 5' UTR, coding region, and 3' UTR.
• Gene expression is regulated at multiple levels which include transcription, translation, mRNA stability, and protein stability.
• Enhancers are DNA regions binding transcription factors and control transcription.
• Repressors inhibit, and activators enhance transcription.
• Bilaterian species exhibit body segmentation arrayed from anterior to posterior during the last 600 million years.
• Cells use positional information in determining location through morphogens working directly on target cells.
• Morphogen concentrations confer different cell fates with the Bicoid gene product being well studied.
• Maternally deposited components such as Bicoid / nanos guide embryo development.
• Differential gene expression exists, and one regulated gene is hunchback, involved in anterior specification.

Lecture 7:

• Gastrulation forms the ectoderm, mesoderm, and endoderm germ layers.
• The ectoderm gives rise to the nervous system, epidermis, and glands.
• The mesoderm gives rise to connective tissue, cartilage, bone, muscle, blood, gonads, and kidneys.
• The endoderm gives rise to the gut, liver, and pancreas.
• After gastrulation the embryo undergoes neurulation forming the nervous system.
• Sonic hedgehog (Shh) encodes a developmental ligand and its defects disrupt ventral midline formation / cause cyclopia.
• Shh is involved also in limb development.
• Limbs have anterior-posterior, proximal-distal, and dorsal-ventral axes.
• Limb development initiates with mesenchyme and ectoderm cells forming a limb bud.
• Positional information comes from Hox genes initiating a cascade to activate FGF10 in limb buds.
• FGF10 is critical for limb bud formation and ectopic expression induces ectopic limbs.
• FGF10 induces FGF8 expression in the apical ectodermal ridge (AER).
• The AER is a major signaling center for limb outgrowth and the proximal-distal axis.
• The anterior-posterior axis of the limb depends on the zone of polarizing activity (ZPA).
• Shh is expressed in the ZPA and dictates posterior-anterior axis specification.

Lecture 8:

• Biology is underpinned by the central dogma: DNA transcribed into mRNA, translated into protein but the genome readout can be modulated.
• Chromatin is DNA with histones.
• Relaxed chromatin is accessible for transcription; condensed chromatin is not.
• Modifications, like histone acetylation (promotes relaxation) and methylation (condenses), mediate transitions.
• DNA methylation condenses chromatin.
• Changes in chromatin state can be heritable (epigenetic changes) and can be in response to environmental cues.
• Epigenetic marks are reset at meiosis.
• Regulatory processes is in response to noncoding RNAs.
• MicroRNAs (miRNAs) bind to mRNAs, repressing translation or causing degradation.
• miRNAs are derived from precursor RNAs. Small interfering RNAs (siRNAs) also bind to mRNAs, trigger the RISC complex, and repress action through destruction, or translational repression; siRNAs come from exogenous sources.
• RNA interference (RNAi) - introducing siRNAs to inactivate specific mRNAs.
• Transposable elements or transposons are DNA sequences that can move around the genome.
• Autonomous elements encode their own transposase, and non-autonomous elements rely on transposase from other source.
• Genome size differences relate to transposable element numbers; coding information amounts stay the same.
• Transposable elements disrupt genes or alter epigenetic landscapes.

Lecture 9:

• A pedigree describes inheritance.
• Pedigree analysis determines if mutations are recessive, estimates penetrance / expressivity.
• Penetrance reflects trait display; expressivity shows the trait's intensity.
• Gene mutations include frameshift, nonsense, missense, splice site, insertions, or deletions.
• Allele is each mutated version of a gene.
• Complementation tests assess if mutations are allelic: non-complementing mutations are allelic.
• Chromosomes consist of sex chromosomes / autosomes.
• Humans have 23 chromosome pairs: 22 autosome sets, one sex chromosome set.
• Meiosis forms haploid gametes in two steps.
• Meiosis I & II differ from mitosis where homologous chromosomes align, forming tetrads during synapsis, followed by crossing over / recombination at chiasmata.
• At anaphase I, homologous chromosomes separate. Nuclear envelope reforms with cytokinesis results in two daughter cells, undergoing meiosis II.
• In meiosis II, sister chromatids align / are pulled apart.
• Meiosis shuffles genetic material, halves chromosome complement from diploid to haploid, generates allele combinations.
• Meiosis allows for new combinations of alleles to come together during fertilization and reconstitution of the diploid genome.

Lecture 10:

• Linkage studies/analyses rely on pedigrees and inheritance patterns.
• Linkage refers to close genetic markers on a chromosome segregating together.
• Recombination shuffles alleles during meiosis proportional to marker distance.
• Genetic map distances measured in centimorgans (cM) named in honor of Thomas Hunt Morgan.
• Loci/gene positions in genetic maps correspond directly to the equivalent positions in the physical map of the chromosomes.
• Association mapping identifies molecular markers linked to traits and is carried out in genome-wide association studies (GWAS).
• Single nucleotide polymorphisms (SNPs) are molecular markers.
• Individuals vary in nucleic acid at SNP positions.
• SNP differences are detected by hybridization using SNP Chips.
• SNPs near each other are generally inherited together, co-segregating haplotype blocks.
• Linkage disequilibrium consists of non-random associations of alleles because of the fact that mutations occur over generations, and that recombination may not have had a chance to recombined the different markers
• GWAS are represented as Manhattan plots showing SNP trait association probabilities.
• By scoring thousands of SNPs in hundreds of patients / controls statistical power is achieved.
• Personalized medicine utilizes genetic variation for drug response prediction.
• Complex traits are due to multiple mutations / confounded by diet / environment.

Lecture 11:

• Sexual reproduction increases genetic diversity, unlike asexual reproduction.
• Sex determination varies: genetic determination involves sex chromosomes and environmental determination depends on external influences
• Sex-linked traits are controlled by sex chromosomes and they follow inheritance patterns different from those of autosomes, seen in pedigrees like hemophilia.
• In mammals, the Y chromosome confers maleness.
• Non-disjunction of chromosomes during meiosis yields aberrant sex chromosome arrangements.
• XXY individuals are male, XO individuals are female, demonstrating the necessity of the Y chromosome.
• Mapping experiments and DNA analysis defined the testis-determining factor on the Y chromosome: SRY.
• SRY encodes a transcription factor, and transgene insertion into female mice results in phenotypic sex reversal.
• SRY initiates a cascade producing testosterone controlling sexual differentiation.
• Androgens dictate male internal / external genitalia; absence leads to female development.
• Hormones bind to target receptors and affect action.
• Mutations in the testosterone receptor cause sex reversal / androgen insensitivity making genotypically male, but phenotypically female individuals.
• Dosage compensation normalizes X chromosome dosage in mammals, where females have 2 X chromosomes / males have 1.
• One X chromosome is inactivated and non-functional in females through Xist non-coding RNA.
• X chromosome inactivation results in a Barr body and occurs randomly around the 32-cell stage.
• Females are genetic mosaics as X inactivation is random.

Lecture 12:

• To test the role of candidate gene mutations, researchers perform "break it" and "move it" experiments, generating transgenic model organisms.
• Steps include engineering a DNA construct in bacteria and getting DNA into organism cells
• Targeted integration is followed by breeding to ensure the DNA construct is in every cell.
• Biotechnology manipulates DNA from various species to create pharmaceuticals, increase the level of nutrient contents and disease resistance.
• "Genetically modified organisms" (GMO's) refers to genetically engineered plants.
• Plant biotechnology is rapid and easy.
• Genetic engineering has produced GMOs such as Golden Rice which contains beta-carotene enzymes.
• Golden Rice contains beta-carotene, which is an A vitamin precursor.
• Rice is a food crop in third-world countries, and vitamin A deficiency leads to blindness in these regions; GMOs can improve nutrition.
• Human genetic engineering is feasible and includes somatic cell gene therapy.
• Somatic cell gene therapy engineers somatic cells to express a gene correctly.
• Germ line gene therapy, which involves transgenes into gametes, is ethically debated and is not being currently carried out. CRISPR-CAS9 revolutionizes transgenic organism engineering.
• CRISPR-CAS9 adapts a bacterial immune response; CAS9 nuclease cleaves double-stranded DNA at CRISPR-targeted sites.
• Guide RNA (gRNA) recognizes the PAM motif, the target sequence, and interacts with CAS9.
• Host cells repair double-strand breaks using non-homologous end-joining generating point mutations.
• Alternatively, introducing DNA and the cell will incorporate the altered DNA sequence into the genome.
• This process is easier and more efficient than other transgenic methods.