fund cell bio mod 2

mitosis

diploid nucleus (2n) undergoes chromosome duplication (4n) then undergoes mitosis (division) to produce identical x2 diploid nuclei (2n)

meiosis

dilpoid germ-line nucleus (2n) undergoes chromosome duplication (4n) then meiosis I (2n) then meiosis II (n) to produce 4 non-identical haploid cells

bivalent

4 sister chromatids stuck together in meiosis

meiosis I vs II

first separate the maternal and paternal homologs then separates the sister chromatids

crossing over during prophase I

exchange of genetic material that occurs between duplicated maternal + paternal homologs and facilitated by synaptonemal complex. the region of occurrence is called the chiasma.

cohesin

the “glue” at the centromere that degrades during anaphase allowing for pull apart

two methods of achieving genetic diversity

independent assortment + homologous recombination

likelihood of independent assortment relies on..

the proximity of genes on the same chromosome. farther apart= higher chance

reverse genetics

altering a gene to study the phenotype (genotype—> phenotype)

forward genetics

examination of the genetic cause of an altered/abnormal phenotype (phenotype—> genotype)

forward genetics screen

ENU mutagenesis causes DNA damage. genotypes of interest are selected and their genome is sequenced to find underlying mutation

RNAi definition + pathway

study gene function through targeted mutagenesis

Pathway: ds RNA—DICER—> ss siRNAs —> hybridize against target gene’s mRNA —> degradation

temperature sensitive mutants

organisms or proteins that function normally at a low (permissive) temperature but exhibit altered, defective function at a high (non-permissive) temperature, often due to protein instability

complementation tests

a genetic breeding experiment used to determine if two recessive mutations with the same phenotype lie within the same gene or different genes. If crossing two mutants yields wild-type offspring, the mutations are in different genes (complementation).

polymorphisms

common, inherited DNA sequence variations that distinguish individuals within a species, contributing to genetic diversity and phenotypic differences. most common is SNP (single nt).

haplotype block

are segments of DNA, often comprising multiple neighboring polymorphism, that are inherited together as a unit due to low recombination rates.

mendelian disorders

mutations in a single gene

—> dominant and recessive disorders

multigenic conditions

cumulative effects of mutations in multiple genes

Genome Wide Association studies (GWAS)

research approaches that scan entire genomes of large populations to identify genetic variants (commonly SNPs) statistically associated with specific diseases or traits

RNA sequencing process

RNA—> cDNA—> quantitative analysis of cell’s transcriptome

in-situ hybridization

reveals when and where a gene is expressed by hybridizing a fluorescently labeled, single-stranded probe to its complementary mRNA sequence

reporter genes

determines pattern of gene’s expression by replacing coding sequence with reporter gene (like GFP)

describe process of homologous recombination using embryonic stem cells

Plasmid with mutated target gene + antibiotic-resistance gene introduced into ES cells → integrates via homologous recombination, replacing normal sequence → only successfully recombined cells survive antibiotic selection.

Homologous recombination using ES cells: mouse application

Recombined ES cells injected into blastocyst → implanted in surrogate → chimeric mouse born → bred to get heterozygotes → heterozygotes crossed → homozygous knockout mice.

CRISPR

Guide RNA designed to match target DNA sequence → binds Cas9 protein → complex locates and binds complementary sequence in genome → Cas9 cuts both strands of DNA → cell's repair machinery introduces mutations (knockout) or incorporates new sequence (knock-in).

Conditional Knockouts (CKOs)

Target gene flanked by lox P sites (via homologous recombination in ES cells) → mouse bred with another expressing Cre recombinase in a tissue-specific or time-specific manner → Cre recognizes lox P sites → excises gene only in target tissue/time → knockout is conditional rather than lethal/ubiquitous.

explain why the same signal can produce different responses in different cells

each cell expresses a different set of effector proteins that decode the signal in their own way

fast vs slow responses

fast modify proteins that already exist to change cell movement, secretion & metabolism. slow require changes in gene expression and new proteins to change cell differentiation, growth & division

2 molecular switches (how proteins turn on/off)

phosphorylation & GTP-binding

phosphorylation

Protein kinases add a phosphate group (from ATP) to a target protein, activating it (usually). Protein phosphatases remove that phosphate group, reversing the effect.

—> serine/threonine kinases and tyrosine kinases

GTP-binding

G proteins are active when they have GTP bound and inactive when they have GDP bound. GEFs (Guanine Nucleotide Exchange Factors) activate G proteins by promoting the swap of GDP for GTP. GAPs (GTPase-activating proteins) inactivate G proteins by accelerating the hydrolysis of GTP back to GDP.

3 classes of cell-surface receptors

ion channel coupled, G-protein coupled, enzyme-coupled

ion channel coupled receptors (aka transmitter-gated ion channels)

When a signal binds, the channel opens and allows ions to flow across the membrane, driven by electrochemical gradients. This changes the membrane potential and produces an electrical current. fastest signaling mechanism. It's common at synapses and in the heart, and it converts chemical signals (neurotransmitters) into electrical signals.

G-protein-coupled receptors (GPCRs)

When a signal binds, the receptor activates a trimeric G protein, which then activates intracellular enzymes or ion channels and triggers a signaling cascade.

enzyme-coupled receptors

When a ligand binds, the enzymatic activity is turned on and triggers intracellular signaling pathways.

GPCR activation

Signal molecule binds → receptor changes conformation → G protein changes conformation → α subunit releases GDP, binds GTP → α subunit and β-γ complex dissociate → each activates downstream targets. Receptor stays active while ligand is bound → one receptor activates many G proteins (amplification).

GPCR inactivation

α subunit hydrolyzes GTP → GDP (within seconds) → α subunit inactivates and dissociates from target → reassembles with β-γ complex → inactive G protein restored and ready for reuse.

Cholera toxin

locks Gs α in ON state (can't hydrolyze GTP) → adenylyl cyclase runs continuously → impaired Cl⁻ transport → massive water secretion → severe diarrhea.

pertussis toxin (whooping cough)

locks Gi α in OFF state (can't exchange GDP for GTP) → adenylyl cyclase uninhibited → excess cAMP.

GPCR/ion channel heart application

Acetylcholine binds GPCR → activates Gi → β-γ complex directly opens K⁺ channels → increased K⁺ permeability → harder to electrically activate membrane → heart slows. GTP hydrolysis → G protein inactivates → K⁺ channel closes → normal heart rate restored.

2nd messengers

G proteins activate membrane enzymes (adenylyl cyclase, phospholipase C) → many second messenger molecules generated (cAMP, DAG, Ca²⁺) → major amplification step

generating cAMP

synthesized from ATP by adenylyl cyclase through a cyclization reaction that removes two phosphate groups and forms a ring. broken down by cAMP phosphodiesterase, which is inhibited by caffeine (cAMP does not get degraded!!)

cAMP and PKA

PKA is held in an inactive state by regulatory complex. when cAMP binds, active PKA is released and can phosphorylate many target proteins

fast cAMP pathway example

epinephrine binds an adrenergic GPCR → Gs activates → adenylyl cyclase makes cAMP → PKA activates → PKA phosphorylates phosphorylase kinase → phosphorylase kinase activates glycogen phosphorylase → glycogen breaks down into glucose.

slow cAMP pathway

cAMP activates PKA → PKA enters the nucleus → it phosphorylates transcription regulators → those regulators turn on target genes → new proteins are synthesized.

the big picture: GPCR, cAMP, PKA

• Ligand binds receptor (outside)

• GPCR activates G protein

• Gα activates adenylyl cyclase

• ATP → cAMP

• cAMP activates PKA

• PKA:

  • FAST → modifies proteins

  • SLOW → changes gene expression