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Processes of mitosis (2)
- mitosis - division of nucleus
- cytokinesis - division of cytoplasm
- division of nucleus
- asexual mode of cell replication producing genetically identical progeny.
- 5 stages
Stages of mitosis (5)
attachment of both kinetochores to same pole of sister chromatids (unstable)
attachment of one kinetochore to both poles of sister chromatids (unstable)
attachment of only one sister kinetochore to sister chromatids (unstable)
anaphase promoting complex (APC)
- activates separase by cleaving securin to degrade cohesin; allows sister chromatids to separate and segregate
- activated by cdk (cyclin dependent kinase)
- spindle checkpoint to make sure chromosome alignment is correct
proper attachment of kinetochores to sister chromatids, one to one, so that tension between centromeres makes stable attachment.
- stage I of mitosis
- chromosomes condense, centrioles migrate to poles
- Spindle forms outside of nucleus
- kinetochore assembles
- cohesin along arms degraded/removed
- stage II of mitosis
- nuclear envelope breaks down due to cdk (phosphorylates lamins)
- chromosomes attach to spindle
- stage III of mitosis
- chromosomes align at equatorial plane
- stage IV of mitosis
- sister chromatids separate (via separase)
- chromatids migrate to opposite poles
- cytokinesis starts
- stage V of mitosis
- chromosomes arrive at poles
- nuclear membranes reform (dephosphorylates)
- chromosomes de-condense.
- division of cytoplasm, 2nd process of mitosis
- contractile ring of actin and myosin in metaphase plate plane
- occurs from anaphase to telophase
- cell cycle after cytokinesis.
- G1 (gap 1, new round of cell division)
- S (genome replication)
- G2 (gap 2, cell prepares to divide)
- part of mitotic spindle
- microtubule organizing centers
- have a pair of centrioles
- start with one, replicate during interphase, move to new poles at prophase, end up in each daughter cell
- part of mitotic spindle
- link spindle pole to chromosomes (kinetochore - protein complex at centromere)
- shorten to drag chromatid to pole
- part of mitotic spindle
- give spindle symmetrical bipolar shape
- act in pole separation (motor proteins)
- part of mitotic spindle
- radiate from centrosome
- act in pole separation (outward force)
- spindle positioning
- protease that separates sister chromatids in anaphase
- activated by degredation of securin by Anaphase Promoting Complex (APC), which happens after chromosomes are attached to spindle
- process producing haploid gametes from diploid parental cell
- 2 rounds of chromosome segregation without DNA, named I and II. Each has 4 steps
- causes DNA coiling in DNA to make chromosomes in prophase
- with cohesin, makes chromosomes easier to drag.
- protein deposited during DNA replication
- holds sister chromatids together.
- Helps make chromosomes more compact and easier to drag. (with condensin)
Prophase I generally
- first phase of meiosis, 90% of cycle. Pairing of homologous chromosomes (maternal and paternal), recombination between them (linked by chiasmata).
- 5 steps.
sequence of meiotic recombination
- formation of double-stranded DNA break
- break resected to produce 3' overhangs
- a 3' end invades a homologous chromatid
- double Holliday junction
- resolvase cuts the joined part to allow separation
genetic exchange between two homologous sequences
Holliday Junction vs Chiasmata
- Holliday junctions involve single-stranded DNA
- chiasmata are with double stranded DNA
- both are connections between homologs
mitosis vs meiosis
- somatic cells vs germ cells
- chromosomes # maintained vs halved
- 2 daughter cells vs 4 gametes
- one S phase per division vs one S phase for 2 divisions
- sister chromatids divide at anaphase vs anaphase II (NOT I)
- no crossing over vs required cross-over
- no pairing of homologs vs synapsis of homologs
- conservative vs genetic variation
5 steps of prophase I
- duplicated, paired sister chromatids condense
- synaptonemal complex develops between sister chromatids
- recombination (2-3 per chromosome usu)
- chiasma observed, synaptonemal complex dissolves
- transition to metaphase I. Chromosomes re-condense.
- 2nd step of Meiosis I
- bivalents (complex of 4 chromatids) held together by chiasma, align at equatorial plate
- 3rd step of Meiosis I
- chiasma resolved, cohesions dissolve, sister chromatids attached only at centromere.
- Homologs (joined sister chromatids) migrate to opposite poles
- 4th step of meiosis I
- chromosomes dissociate from spindle
- nuclear membranes reform around daughter nuclei
- chromosomes may de-condense
- follows telophase I and brief interphase (no DNA replication)
- first step in Meiosis II
- nuclear envelope breaks down, chromosomes attach to new spindle
- 2nd step of meiosis II
- chromosomes align on equatorial plane
- 3rd step in meiosis II
- attachments between sister chromatids dissolved, separate to each pole
- 4th step in meiosis II
- chromosomes de-condense and nuclear membrane reforms
- followed by cytokinesis, creating four haploid cells
- failure to separate two chromosomes. Occurs at anaphase I or anaphase II
- leads to Downs Syndrome (ch 21)
2 mechanisms of meiosis to create diversity
- chromosomes randomly segregated
- non-sister chromatids (homologous chromosomes) exchange DNA in Prophase I
- double-stranded DNA break (endonuclease)
- resected for 3' overhangs invade homologous chromatids
- DNA synthesis makes a Holliday Junction
- resolvase cuts juncture
- recomb happens based on distance, closer the loci are the less likely they are to combine. Can be used to figure out genes on chromosome
produced during recombination of chromatids in Prophase I, when DNA synthesis on displaced 3' overhangs joins/replicates
post-recombination of chromosomes, some from each side (mom and dad) are on each chromosome
Mitosis vs Meiosis
- mitosis: somatic cells; one parent makes 2 daughters; diploid to diploid; DNA replication makes 1 division; sister chromatids separate at anaphase; no pairing of homologs; no crossing over; genetically identical progeny
- meiosis: germ cells; one parent to four gametes; diploid to haploid; DNA replication then 2 divisions; sister chromatids separate only at anaphase II; synapse of homologous chromosomes; cross-over; genetic variation in progeny
- protein that infinitely propagates an altered form of itself (transmissible)
- proteinaceous infectious particle lacking nucleic acid
- new biological principle of infection that violates principle that DNA or RNA required for reproduction/infection
amyloid in alzheimers
small protein fibers that form an insoluble mass, lots of these "plaques" in the brain
- phosphate group
- sugar (deoxyribose or ribose)
- nitrogenous base (purine or pyrimidine)
- DNA is a polymer of nucleotides linked by phosphodiester bonds
Purines vs Pyrimidines
- Purines: Adenine, Guanine
- Pyrimidines: Thymine, Cytosine
- polymer of nucleotides linked by phosphodiester bond. Sugar-phosphate backbone on outside, nitrogenous bases held together by H bonds inside
- Two complementary strands (H bonds), anti-parallel. Labeled due to sugar (5' vs 3')
- DNA in complex with proteins.
- 2 kinds: euchromatin and heterochromtin (2 kinds, facultative and constiutitive)
- highly condensed, devoid of genes, associated with low gene expression
- Can be facultative (heterochromatic in some cells, euchromatic in others) or constituitive (always heterochromatic)
less condensed, transcriptionally active genes
- 200 base pairs and 2 molecules each of histones H2A, H2B, H3 and H4. Octometric core.
- H1 packages them into 30nm fibers in looped domains.
- Most condensed in mitotic chromosomes.
- unique sequences
- regions of DNA that control discrete hereditary characteristics, usually corresponding to single protein or RNA
- Include exons, introns and regulatory sequences
- parts of DNA that repeat. Include
- retroviral-like elements (retrotransposon with long terminal repeats); non-retroviral retrotransposons (non LTR, many not able to transpose. Include LINES and SINES); DNA transposons (mobile DNA, no RNA); simple sequence repeats (satellites, mini-satellites, micro-satellites)
Grows in the same direction as unwinding. Produces one continuous new strand.
Grows in opposite direction from unwinding, needs many (Okazaki) fragments, ligated together to form a continuous strand.
replication factor catalyzing addition of new nucleotides at 3' end of growing chain. Can be multiples in higher eukaryotes
- replication factor
- unwinds DNA in front of replication fork opening to prevent tangling
single-stranded DNA binding proteins
binds unwound, single-stranded DNA and prevents it from re-annealing
DNA-dependent RNA primase generates a short piece of RNA that base-pairs with the template DNA and serves as a primer for DNA synthesis. RNA is ultimately replaced by DNA
seals gaps, like between Okazaki fragments to make a continuous DNA strand
assists in replication of chromosome ends (telomeres) where lagging strand synthesis is problematic
- gene expression regulation
- normally silences gene
length of haploid genome in base pairs
3 billion. ~ 1 meter, end to end
FOur phases of transcription cycle
initiation, pausing, elongation, termination
- sequence specific DNA binding proteins, can recruit others, to activate or repress transcription. Could be any factor that regulates.
- Can be cell-type specific or non-specific
- All cells have same DNA, so TFs determine what proteins get made
Transcription Factor Domains
- nuclear localization signal
- DNA binding domain
- protein interaction domains (allow TFs to form homo- and heterodimers)
- often trans-activation or trans-repression domains to directly promote or inhibit
- can have ligand/hormone binding domain
- Group of proteins that regulates transcription
- includes core promoter and proximal region.
response elements (2)
- DNA sequences TFs bind to.
- Can be palindromic or non-palendromic
Complex regulatory DNA sequences
- Adjacent response elements can form functional units together to control transcription (silencers or enhancers)
- interact and initiate a subset of expression patterns available to the gene. i.e. brain-specific enhancer vs gastrula-specific enhancer
- complex regulatory DNA sequence.
- Cell, tissue or developmental-specific because TFs are only expressed in the specific cell, tissue, or time.
- LOTS in cell, can be upstream, downstream or at a distance (due to looping, interact with promoter TFs and RNA polymerase pre-initiation complex
What controls the behavior of transcription factors?
the environment, external and internal to the cell. Signaling pathways, too (protein kinases that phosphorylate can stabilize or stimulate degredation)
How histones control chromatin
- Histones have nucleosomal histone N-terminal tails have very positive AAs (argenine and lysine), interact with negative DNA. All of this makes for tight winding, no transcription.
- Requires co-regulators, recruited by TFs, bind to methylated DNA, bind to histones, allow transcription.
- These genes are frequently mutated in cancer.
- N-terminal histone tail amino acids can be marked (methylated, phosphorylated, ubiquitination, acetyation) by a writer protein.
- activating marks vs repressive marks control activity. Reader protein reads the code, recruit proteins to regulate. An eraser removes histone modifications to reset the code.
- change of phenotype and gene expression. doesn't change DNA, reversible.
- histone modifications, Heterochromatin to euchromatin
what modifies histone tail interactions
- must neutralize or expose the charges
- recruit and activate
- histone acetyltransferases (HATs) or histone deacetylases (HDACs)
- Can also change postition of DNA to nucleosome.
- Both require ATP-dependent chromatin remodeling complexes
- Initiates transcription
- RNA Polymerase II (PolII) needs general TFs (TFII-D with TATA binding protein and TFII-H with helicase and protein kinase) and a mediator
RNA polymerase II
- part of pre-initiation complex
- made of protein subunits and a C-terminal tail (protein kinase phosphorylates the tail as the activation trigger). RNA Pol II unhooks and goes off on its own to transcribe
- part of pre-initiation complex
- bridge between gene-specific regulator proteins and pre-initiation complex
- general transcription factor, part of pre-initiation complex
- Contains TATA-binding protein to position complex just upstream of transcription site.
- general transcription factor, part of pre-initiation complex
- contains helicase to expose DNA template strand
- contains protein kinase to phosphorylate the C-terminal tail of RNA Pol II as the activation trigger
activation trigger of transcription
when TFII-H's protein kinase phosphorylates RNA Pol II's C-terminal tail, letting it disengage from the pre-initiation complex and go off on its own to transcribe
messenger RNA, codes for proteins
ribosomal RNA, form ribosomes and catalyze protein synthesis
transfer RNA, carry amino acids to ribosomes, crucial to protein synthesis
microRNA, regulate gene expression by blocking mRNA translation and marking it for breakdown
small interfering RNA, mark mRNA for degredation, compact chromatin structures
piwi-interacting RNA, protect germ cells from transposable elements
long non-coding RNA, regulate processes like X-chromosome inactivation
RNA processing (3-4 steps)
- primary coding RNA
- 1. 5' capping
- 2. RNA splicing
- 3. RNA cleavage and 3'polyadenylation
- Factors often found bound to RNA Pol II C-terminal tail, begins before transcription is complete
- 7-methylguanosine cap, attached to 5' end of RNA
- helps with splicing, transport out of the nucleus, stabilization and translation.
- Requirs Nuclear Cap Binding Complex and Cytoplasmic Eucaryotic Initiation Factor (eIF4E).
- Can be a target for cancer/virus
- introns are removed and exons are joined together. Rapid, precise, causes major problems if screwed up
- Spliceosome + ATP, can be on same mRNA (cis) or on different (trans).
- Requires sequences on 5', branch point, polypyrimidine tract, and 3' to match up.
- Bad splicing makes Narcolepsy
- normally attacks intron/exon junction, makes a lariat with a phosphodiester bond, attacks the next one, makes a phosphodiester between exons to release intron.
When only some exons are left in, others cut out. Makes many proteins from one gene
How is splicing regulated
- protein factors bind to introns and exons (splice regulatory elements, SREs) in RNA.
- splicing enhancers or splicing silencers.
- Currently masking splice sites as a form of therapy.
RNA Cleavage and polyA tail
signals encoded in DNA, so that during transcription proteins read them and cleave the RNA, then POST-TRANSCRIPTIONALLY add the Poly-A tail (NO STRING OF Ts ON DNA). Poly A polymerase (PAP) adds the As. PolyA binding proteins are required to leave the nucleus and be translated
Half-life of mRNA, and 2 mechanisms of decay
- regulatory proteins like transcription factors are short, maybe 30 minutes. Structural proteins like actin are long, maybe 15 hours.
- deadenylation-dependent: get rid of poly-A and shred from there
- deadenylation-independent: endonuclease cuts in half, degrade from inside.
- Degrades any defective mRNAs before translation (like nonsense-mediated decay to find early stops)
How mRNA moves in cytoplasm
eIF4E replaces 5' cap (nuclear CBC). eIF4G binds to both cap and tail, making a circle. CRITICAL TO TRANSLATION. Regulated by controlling amount of eIF4E (bound by 4E-BP, which is phosphorylated to let go).
protein kinase mTOR is the master regulator of growth and homeostasis, phosphorylates 4E-BP to free eIF4E, and activates/phosphorylates S6 protein kinase which phosphorylates for translation. Nutrient starvation and energy depletion block mTOR/translation. Proliferative signals activate it via lipid kinase, PI3K, protein kinase Akt.
Interfering RNA (RNAi)
- small interfering RNAs and microRNAs
- synthesized from double stranded RNA precursors (dsRNA), cleaved by a dicer then associate with RNA-induced silencing complex (RISC), with endonuclease argonaute.
- Inactivate mRNA by inhibiting translation and/or stimulating degredation.
- Used in the lab to knock down genes, study function and cure genetic issues. miRNA can have many multiple targets, regulates the most mRNA in vivo.
Protein structures: primary, secondary, tertiary, quaternary
- primary - amino acid sequence
- secondary - alpha helix, beta pleated sheet, loops
- tertiary - domain (fold/shape of whole protein)
- quaternary - multiple proteins together
basic groups of aa side chains
nonpolar, uncharged polar, acidic (negative charge), basic (positive charge)
can be parallel or antiparallel
- quaternary structure.
- Could be a homotetramer (four of the same), a heterotetramer (different), for example
prevent aggregation, help fold. If incorrectly folded, may be returned to chaparone to try again.
makes no difference. Codes for same aa, or is in a place that doesn't get transcribed.
results in single amino acid substitution
substitutes a stop codon for amino acid
insertions or deletions of nucleotides NOT in series of 3, changes how everythign is read.
protein folding diseases, like prion issues. Bovine spongiform encephalopathy, scrapie, alzheimers, huntingtons, etc.
fatty acids form surface film or micelle. Triacylglycerols form spherical fat droplets in cytoplasm. Phospholipids and glycolipids form lipid bilayers.
- stored energy
- hormone precursor
- membrane identity
- lipids from sphingosine (amino alcohol) linked to fatty acid acyl group.
- Enriched in neural tissue for signal transmission, cell recognition and as a metabolite
- contributes to mechanical and chemical stability fo plasma membrane
- carbohydrate-attached lipids
- extracellular surface of plasma membrane
- energy providers and cellular recognition
- GPI-anchors on proteins
Main substrate for ATP production
Functions and basic structure of carbohydrates
- ATP production
- structure (glycolipids and glycoproteins)
- receptors, cell signaling
- attach to proteins in ER and golgi
- posttranslational modification, attaches protein to carbohydrate. Does EVERYTHING
- structural, protective, lubrication, transport, immunology, hormones, enzymes, cell recognition/attachment, antifreeze, receptors, prtien folding, development, hemostasis
- posttranslational modification of protein
- transports cholesterol in blood
cellular functions of lipids
- organelle ID and shape
- second messenger and hormone
- energy storage
- classification of lipid
- fats/triglycerides, phospholipids, eicosanoids, glycolipids, sphingolipids.
- Saturated or unsaturated (cis double bond, reduces melting point).
- Hydrophilic carboxylic acid head, long hydrophobic hydrocarbon tail.
non-glycerides. Steriods (cholesterol - metabolite, keeps membranes fluid. Testosterone - hormone)
- glycerol with three acyl chains linked to glycerol via ester bond. Neutral fat, energy storage.
- With only two chains it would be a glycerophospholipid (biological membrane)
polar hydrophilic head group, 2 acyl tails. building blocks of biomembranes
- isoprene derived lipid
- part of all animal membranes
- transported by lipoproteins
- LDL/HDL to transport to/from liver
- precursor for steroid hormones
- causes atherosclerosis, inflammation, heart attack, stroke
- interacts with unsaturated phospholipids, modulating membrane properties.
types of microscopy (in order of resolution)
- Light microscopy (0.2um) (fluorescent proteins, green for living, immuno for dead)
- electron microscopy (0.1nm, 1000x better, dead things only)
- -Scanning Electron Microscopy (smaller, cheaper than TEM but 3D)
- -Transmission Electron Microscopy (like LM but HUGE, 2D)
Nucleus structure, function
- Structure: round/elliptical, nuclear envelope(double membrane, inner faces nucleus, outer contiguous with ER), nuclear pores, nucleolus
- function: control center, DNA and RNA synthesis (rRNA synthesis inside nucleolus)
Er structure, function (4-6)
- structure: membrane network throughout cell, RER with ribosomes
- function: RER (protein synthesis), SER (steroid and lipid synthesis), glycosylation, calcium storage, exocytic pathway
Golgi apparatus structure, function
- structure: cisternae (cis (ER) and trans faces)
- function: protein sorting, modification (glycosylation), dispatch. Exocytic pathway
Proteasome structure, function
- structure: cytoplasm and nucleus, non-membrane bound, large complex of proteins
- function: PROTEOLYSIS, break down tagged proteins (ubiquitin), create parts for antigen presentation
- 3 steps, tags proteins for breakdown by the proteosome, sometimes lysosome
- Includes abnormally folded proteins dislocated from ER
- includes foreign or viral proteins, parts used for antigen presentation
Lysosome structure, function
- structure: membrane-bound sphere full of hydrolases
- function:endocytosis (breakdown of nucleic acids, proteins and lipids), autophagy (organelles). Endycytic pathway
- Can use ubiquitin tag
breaking of peptide bonds, proteosome funciton
peroxisome structure, function
- structure: spherical, membrane-bound, full of oxidation enzymes
- function: oxidation to break down fatty acids, some organic. Free radicals to peroxide, peroxide to water.
mitochondria structure, function
- structure: outer membrane, inner membrane has cristae
- function: ATP generation via oxidative metabolism, electron transport chain
lysosomal storage disease
- class of disorders, lysosome can't break down proteins and lipids
- Most effect at neurons and glial ells
change in nucleolus size
what makes the cytoskeleton?
- microfilaments (actin filaments)
- intermediate filaments
- actin subunit
- polar, add to plus end
- flexible, easily bend, fragile, crosslink accessory proteins
- bind ATP
- just beneath plasma membrane
- SHAPE OF CELL SURFACE, WHOLE CELL LOCOMOTION (contractile ring in mitosis, amoeboid movement, tracks for myosin)
- Tubulin subunit
- bind GTP
- large rigid hollow cylinders, not easily bent, largest of the three filaments, stiff and hard to bend
- polarized, adds to the plus end
- tracks for kinesin and dyneins
- made at microtubule organizing center (MTOC), anchors minus end
- Centrosome is a MTOC
- function: position of organelles and direct intracellular transport, move chromosomes in mitosis, meiosis
- rope-like filaments, include keratins, neurofilaments, vimentin-like proteins and lamins
- no nucleotide
- great tensile strength
- NO MOTOR
- mechanical strength, intracellular scaffold, cell-cell junctions, shape to cells and resist pulling forces
- Only in "squishy" eukaryotes
3 types of molecular motors
- myosin: toward +, walk on actin (microfilament), muscle contraction
- kinesin: toward +. Largest, fastest. Walk on microtubules.
- dynein: toward -, walk on microtubules
molecular motor, walks on actin/microfilaments in + direction, Head binds/hydrolyzes ATP.
- molecular motor, walks on microtubules in + direction, takes cargo to periphery of cell
- made from many proteins, 2 gobular heads and long stalk/tail to bind cargo
- binds and hydrolyzes ATP
- help keep organelles positioned
- molecular motor, walks on microtubules in - direction, takes cargo to center of cell
- largest, fastest motors, big and short.
- binds and hydrolyzes ATP
- keeps organelles correctly positioned, mitotic spindle and movement of chromosomes. Produce movement by bending microtubules.
- toxin that binds and stabilizes microtubules.
- kills dividing cells, part of chemotherapy.
canine x-linked muscular dystrophy
failure to make actin accessory protein dystrophin, causes degeneration of muscle fibers, weakness and death
epidermolysis bullosa simplex
disruption of intermediate filaments, epidermis not well anchored to dermis, shears off and blisters
cyclin dependent kinase (Cdk)
- protein that promotes cell cycle progression by phosphorylating specific substrates
- different at different stages of cell cycle
- proteinaceous Infectious Particle that lacks nucleic acid
- PrP (prion protein) is protease resistant, coded for by PRNP, evolutionarily conserved.
- PrPc normal, alpha helix (no aa change)
- PrPsc infectious, beta sheets (no aa change)
- small protein fibers that form an insoluble mass
- prion diseases, alzheimers, etc.
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