BIOLOGY: Circulation, respiration, skin and immune system Flashcards
Endothelium
- Single Cell Layer - Makes up the innermost surface of the cardiovascular system (heart and blood vessels) - Promote blood fluidity and the movement of WBC out of the bloodstream to the unaffected tissue - A selective barrier between blood and surrounding tissues
Diastolic Blood Pressure
-D=Diamtere - Heart muscle relaxed and ventricles fill with blood - Decreased blood volume - Arterial pressure decreases
Systolic Blood Pressure
-Top number - Heart contracts and pump blood into arteries -Increased blood volume - Arterial pressure increases
The function of the Lymphatic System
Collects protein-containing fluid leaked from blood capillaries and transports it back to the bloodstream
What is the series of a lipid droplet transported from the intestine to the bloodstream?
Interstitial fluid–> lymph capillaries–> lymph vessels –>lymph duct–> vein near the heart
An abnormal opening in the muscular wall separating the two ventricles of the heart causes the blood to flow from the left to the right ventricle. What does this cardiac defect cause?
- Increased blood flow into the pulmonary artery 2. Increased blood volume in the left atrium 3. Low blood oxygen levels in the systematic arteries
Asthma is the narrowing of respiratory airways. What happens in Asthma?
-INCREASES resistance to airflow, which would DECREASE the volumetric rate of forcibly exhaled air. -Traps air in the lungs, DECREASING the total volume of air that can be exhaled.
Regulation of the respiratory rate primarily relies on what?
It depends on the BLOOD PH of the blood (which is measured by chemoreceptors. -Receptors directly detect H+ in the blood, which is dependents on the Pp of Co2 in the blood via the BBS.
Temporary bronchoconstriction would have what effect on blood pH, and what would be the expected homeostatic response?
-Respiratory acidosis -Increased respiratory rate
An increase in blood CO2 results in what equilibrium shift in the BBS?
Equilibrium shifts to the right, increasing H+ (decreasing pH). –> Acidosis. Thus, increase respiratory rate.
What happens in hyperventilation?
-Increase the amount of CO2 to be removed from the blood -Shifting the equilibrium of the BBS to the LEFT -Decreasing H+ -Increased the blood pH back to NORMAL
Respiratory Alkalosis
Increase in blood pH
Intrapleural space
thin space between the lungs and the thoracic wall
What happens when oxygen-carrying capacity increases?
-The affinity of Hb for oxygen increases with O2 pressure -Hb exhibts positive binding cooperativity -Oxygen binding induces a chnage from the T-state (tense) to R-state (relaxed).
Positive Binding Cooperativity
is between binding sites in proteins and enzymes is indicated by a sigmoidal (S) shape of a kinetic graph
Negative Binding Cooperativity
Results if oxygen binding to one subunit stabilzed in the T state of other subunits
An upregulation of what enzyme would improve oxygen relsease into tissues? Explain.
Bisophosphoglycerate Mutase (BGP) -BGP converts 1,3BPG to 2,3BGP, which allosterically regulate Hgb -It decreases oxygen affinity by stabilizing the deoxyhemoglobin conformation. -It increases 2,3BGP –> Right shift in the O2 dissociation curve and favor oxygen delivery to tissues
Phosphoglycertae Kinase
interconverts 1,3BGP and 3-phosophoglycerate see glyucolysis FC on Uworld
Glyercerol-3-phosphate dehydrogenase
converts converts glyercerol-3-phosphate to 1,3-BGP
Phosphoglycerate Mutase
interconverts 3-phosphoglycerate and 2-phosphoglycerate -it acts on phosphoglycerates, not BGP
A left shift in the oxyhemoglobin curve signifies:
an increase of Hgb for oxygen, thus less oxygen delivery to tissues -Low carbonic acid, Co2, and H+ conc
Oxidative Stress
is caused by reactive oxygen species (ROS) which can cause hemolysis -compounds that remove reactive oxygen species can relieve the adverse effects of oxidative stress
Hematocrit
A lab measurement of red blood cell volume % of total blood volume -indicates the number of red blood cells within a blood sample
Hemolysis
induced by oxidative sress can reduce hematocrit
Cells of the Immune System
Desomosomes
Gap Junctions
Tight Junctions
-
Desmosome: mechanical strength, achnor cytoskeletons, specifically intermediate filaments
- found in muscle tissues & epithelial layers of skin
-
Gap junctions: Cytoplasmic Continuity
- connexons
- found in smooth & cardiac muscles or neural tissues
-
Tight junctions: watertight seals
- skin, GI, & testis
Functions of the Lymphatic Systems:
- lymph nodes: white blood cells remove & mount immune responses against pathogens from the lymph
- spleen: white blood cells remove pathogens 7 damaged or old blood cells from the blood
Types of T-Lymphocytes
Lymph Image
Innate Immunity
attacks any forgein substance
Adaptive Immunity
eliminates a specific pathogen
Expression of MHC proteins is solely dependent on a cell’s _______________________ , cells would still be able to display bacterial antigens on thier MHC proteins regardless of helper T cell ocunt
transcriptional & translational machinery
B Lymphocyte Activation
- Within the B lymphocyte, specialized antigen-binding peptides known as major histocompatibility class II (MHC II) proteins transport the antigen fragments to the cell surface, allowing the B lymphocyte to display the antigen fragments outside the cell.
Phagocytosis
Activation of macrophages by helper T Cells
Activation of Cytotoxic T cells by Helper T cells
Skin Sensory Receptors
Skin Functions
- The skin functions as a physical barrier to prevent the loss of fluid from the body while simultaneously blocking the entry of pathogens or harmful chemicals.
- The skin also contains receptors that gather and respond to sensory information from the surrounding environment.
- Ultraviolet radiation that strikes the skin induces the synthesis of a vitamin D precursor.
Thermoregulation is a major FUNCTION OF SKIN
- Body temp can be increased by: vasoconstriction of skin arterioles, shivering & piloerection (in hairier animals)
- Body temp can be decreased: by vasodilation of skin arteriols & sweating
- In cold enviroments of hairier animals, sympathetic signaling causes contraction of the arrector pili muscles, which causes piloerection (hairs standing upright). Piloerection, which impedes heat loss by trapping heat near the skin surface, is not an efficient means of heat retention in humans due to insufficient body hair.
Layers of skin
- Epidermal melanocytes prevent UV radiation from damaging the DNA of cells, hair is a keratinized derivative of skin that helps protect the body from external injury
-
Dermal sweat glands secrete sweat onto the skin surface to regulate body temperature
- connective tissues, blood vessels, hair follicles, sweat (sudiriferous) glands & oil (sebascous) glands
- The subcutaneous layer (hypodermis) is composed of adipose cells that insulate the body
- acts as a shock absorber
- protects internal organs
Epidermis in depth
- The outermost layer of the epidermis, the stratum corneum, is composed of 20–30 layers of these dead keratin-filled cells and functions as a physical barrier to protect the organism against pathogens, ultraviolet light, water loss, and injury due to abrasion or puncture
- Epidermal Langerhans cells are immune (dendritic) cells that recognize and ingest antigens before migrating to nearby lymph nodes to present these antigens to T cells, activating the adaptive immune response.
Reflex / Reflex arc
- relfex is an involuntary response to a stimulus that does not require input from the brain
- Refexes are mediated by reflex arcs, neuronal pathways that include a sensory
- Reflexes can be modulated (dampened or enhanced) by input from the brain. Descending signals from higher areas in the central nervous system (CNS) travel along spinal tracts to act on neurons in the reflex arc whose cell bodies lie within the spine.
Genetics & Evolution
Monohybrid Crosses to know COLD for test day:
Dihybrid heterozygous cross ratio
-
Homozygous parents (PP X pp) crosses results in:
- F1 genotype ratio: 100% heterozygous
- F1: phenotype ratio: 100% dominant (color)
- F2: mix of geno/phenotypes (25% dominant), (50% heterozygous, % 25% recessive)
-
Heterozygous parents (Pp X Pp) crosses result in:
- F1 genotype ratio: 1 PP: 2Pp: 1 pp
- F1 phenotypic ratio: 3 purple: 1 white
Dihybrid cross between two heterozygous: 9: 3: 3: 1 ratio
Test Cross:
- is used to determine an unknown genotype of the parent based on the phenotype of its offspring, aka sometimes called back crosses
- If all of the offspring (100%) are of the dominant phenotype, then the unknown genotype is likely to be a homozygous dominant
- If there is a 1:1 distribtuion of dominant to recessive phenotypes, the the unknown genotype is likely to be heterozyguos
*
Sex-linked Crosses
- Egg has Xs
- Sperm determines sex of the child
- Men with a sex-linked trait will have daughters who are all either carriers of the trait or who express the trait (if his partner also has an affected allele), and that a man can never pass down a sex-linked trait to his son
- for crosses between a female carrier of an X-linked recessive trait and an unaffected man (XXc & XY), only male children will express the trait (25%)
Recombination frequency
- tightly linked genes=0%
- weakly linked genes= ~ 50%, as expected from independent assortment
Hardy-Weinberg principle
allele fequency
-
allele frequency: how often an allele appears in a population
- remember: there will be twice as many alleles as individuals in a population-because each individual has two autosomal copies of each gene
- When gene frequencies of a population are not changing, the gene pool is stable, and evolution is ostensibly NOT occuring: 5 CRITERIA MUST BE MET FOR THIS TO BE POSSIBLE
- population is very large (no genetic drift)
- no mutations that affect the gene pool
- mating between individuals in the population is random (no sexual selection)
- no migration of individuals into or out of the population
- genes in the population are all equally successful at reproducing
given all of these assumptions, the population is said to be in hardy-weinberg equilibrium
patterns of selection
- stabilizing: loss of extremes, maintance of phenotype in a small window
-
directional: movement toward one extreme or the other (example an antibiotic, those colonies that exhibit resistance to this antibiotic will survive)
- another example: to adapt to a selevtive temperature (colder) will evolove thicker layer of fur to survive the ice age
- disruptive: movement toward both extremes with loss of the norm; speciation may occur
Natural selection
Modern synthesis model
Inclusive Fitness
Punctuated Equilibrium
- Natural Selection: certain traits that arise from chance are more favorable for reproductive success in a given environment and that those traits will be passed on to future generations
- Modern Synthesis Model: takes natural selection and explains that selection is for specific alleles, which are passed to future generations through formation of gametes, and that these favorable traits arise from mutations
-
Inclusive fitness: explains that the reproductive success of an organism is not only due to the number of offspring it creates, but also the ability to care for young (that can then care for others); it explains changes not only at the individual level, but based on the survival of the species (and that individual’s alleles within the species, including in other related individuals)
- The inclusive fitness of an individual is the sum of direct fitness (its own reproduction) and indirect fitness (its cooperative behavior to increase the number of offspring by close relatives). Therefore, inclusive fitness serves as a metric of an individual’s total evolutionary success.
- Punctuated Equilibrium: states for some species, little evolution occures for a long period, which in interrupted by rapid bursts of evolutionary change
Species
- defined as the largest group of organisms that can interbreed to rpoducce viable fertile offspring
- therefore, two populations are considered seperate species when they can no longer do so!
Genetic Recombination & Cross over
- occurs via crossover events (exchange of DNA segments between homologous chromosomes)
- Synapsis of the joining of the homologous chromosomes into tetrads occur during prophase I of meiosis and is required for crossing over to occur
- Crossovers increases genetic diversity by mixing maternal & paternal alleles into a single chromosome that is then inherited by the offspring
Mitosis & Meiosis
- Gamete formation requires cellular division by meiosis I & meiosis II, in which homologous chromosomes & sister chromatids, respectively, are seperated.
- In the absece of recombination, maternal & paternal alleles are seperated from each other during anaphase I, and identical alleles on each sister chromatid are seperated from each other during anaphase II
Sex linked traits & autosomal traits
-
Sex-linked traits
- arise due to the expression of alleles present on an organism’s sex chromosomes
- these traits tend to be present at greater rates in males than in females
-
Autosomal traits:
- arise due to the expression of alleles present on an organisms autosomes
- traits present in the same proportions in both male & female
Types of DNA mutations
- Mutations are changes in the DNA sequence that arise from errors in replication or from mutagen exposure (manmade or enviromental) that can alter the protein product of a gene.
- Truncated proteins result from nonsense mutations and frameshift mutations with a downstream stop codon (UAA, UAG, UGA) in the new reading frame.
Basic probability rules
Types of gated ion channels
- for a particular ion to go through a channel, the inside of the channel’s pore ust be lined by amino acid residues with charges opposite to the ion’s charge
- in addition, the ion must be briefly puled out of it’s hydration shell to pass from the aqueous enviroment outside the plasma membrane to the asquous enviroment inside the cell
- Example: if we have Ca+2 ion wanting to go through, its positive so the inside of the pore must be lined by amino acid residues that are negatively charged at physiological pH; such as Aspartate (D) and glutamate (E).
In advanced renal disease, all of the following components of physiological homeostasis would be disrupted except:
A. blood nitrogen levels
B. blood pH
C. Leukocyte production
D. Erythropoietin production
- Answer: C. leukocyte production
- For this question, choose which on that is not a function of the kidney
- Kidneys’ primary function is to maintain the salt and water balance of the blood. They also play a key role in regulating multiple aspects of physiological homeostasis (eg, blood pressure, waste removal, osmolarity, blood pH, erythrocyte production).
- Production of leukocytes (white blood cells) occurs in the bone marrow, not the kidneys. Leukocytes are immune system cells that protect the body against infectious agents and foreign antigens. Consequently, leukocyte numbers are regulated by the presence or absence of cytokines released during active infection.
Founder Effect (Bottlenecks)
- are sudden environmental changes that rapidly (not gradually) decrease the number of individuals in a population, often leading to reduced genetic diversity. If white mice in this population were lost due to a bottleneck event, their frequency would decrease in a short period (not over 100 generations).
- In addition, bottleneck events affect all members of the population randomly (regardless of genotype or phenotype). Therefore, a bottleneck event would also affect the frequency of black mice in the population.
Complete and incomplete Pentrance & Expressivity
Gel electrophoresis
- polyacrylamide gel electrophoresis (PAGE) is used to seperate DNA molecules by size using an electric field, not a pH gradient
- a native PAGE gel contains no denaturants or reducing agents, and allows double-stranded DNA molecules to traverse the gel in their native (unaltered) state
SDS-PAGE under reducing conditions
- Reducing substances are used to break disulfide bonds in protein
Isoelectric focusing:
- Uses pH gradient to separate proteins based on their isoelectric point
PCR
- polymerase chain reaction (PCR) is a thermal cycling technique used to amplify DNA fragments
- PCR reagents include a source DNA template (containig deoxyribonucleotides), GC-rich primer pairs, a thermostable DNA polymerase (not denatured at high temperatures to replicate the DNA template using a pool of supplied deoxyribonucleotide triphosphates- dNTPs), and a buffer solution with positively charged ions ((cations) to provide an optimal environment for DNA polymerase to function (ie, cations bind the negatively charged phosphates on the DNA backbone and those on dNTPs, neutralizing the negative charge of DNA and stabilizing primer-template binding)
In PCR, thermal separation (denaturation) of the DNA template is accomplished by exposing the sample to high temperatures. Subsequent cooling allows primers to anneal to the single-stranded flanking ends of the target region. DNA polymerase uses the primers to elongate the new daughter strands in the 5′ to 3′ direction. These steps are repeated to obtain millions of copies of the target DNA segment in a short time.
Inbreeding & outbreading
- inbreeding results in decreased heterozygosity (genetic diversity), reduced fecundity, and reduced fitness.
- species that mate with nonrelatives (outbred) increase their fitness because the introduction of new genetic material results in increased heterozygosity
Extinction
-
reasons of decline of small populations:
1. Inbreeding depression & genetic drift (ie, loss of genetic diversity, decreased fitness & reduced fecundity-number of offspring)
2. Pressure from random changes in demographic (birth & death) rates
3. Inability to adapt to changing environmental pressures (eg, decreased resources, increased predation, natural catastrophes)
Oogenesis
- In utero, oogonia (ovarian stem cells) of the female embryo rapidly multiply via mitosis to generate primary oocytes, which are surrounded by specialized cells that form a saclike structure known as a follicle.
- Female gametes must undergo meiosis to mature. Primary oocytes begin the first meiotic division but become arrested at prophase I until puberty.
- At puberty, hormonal changes during each menstrual cycle result in a single follicle being selected to continue meiosis I. Completion of meiosis I produces one haploid secondary oocyte and one small polar body that ultimately degenerates.
- The secondary oocyte begins the second meiotic division but is arrested at metaphase II.
- In the ovulation phase of the menstrual cycle, the follicle ruptures and the secondary oocyte is released into the abdominal cavity.
- The secondary oocyte enters the fallopian tubes, where it can be fertilized by a sperm cell. If fertilization occurs, the secondary oocyte will complete meiosis II to form one large ovum (fully mature) and a second polar body that degenerates.
Heterochromatin & Euchromatin
Heterochromatin (prevents gene expression)
Euchromatin (allows gene expression)
DNA & Gene Expression (D&GE)
Restriction Enzymes
What is a DNA palindrome?
- Restriction enzymes cleave DNA molecules at specific sites, known as restriction sites.
- Each enzyme has a unique restriction site that typically consists of a _DNA palindrome_ (in whcih the sequence of one stran is the reverse of its complement strand) and may cut the DNA such that an overhang, or sticky end, is left on the end of the strand.
DNA & Gene Expression (D&GE)
Gene Cloning
What are plasmids?
- How can a gene may be inserted into a plasmid?*
- how does bacteria exchnage plasmid?*
-
Plasmids—small, circular DNA molecules that can carry a small number of genes—are often used in laboratories to amplify and express genes of interest.
- commonly present in bacteria (can also be found in eukaryotes)
- bacteria exchange plasmids through:conjugation
-
A gene may be inserted into a plasmid by digesting the gene and the plasmid with the same restriction enzymes, followed by ligation (DNA LIGASE)
- same restriction enzyme to ensure that the plasmid & gene will aneall to each other
- PCR may be nexessary to introduce cut sites into the ends of the gene
DNA Annealing
PCR
DNA LIGASE
DNA & Gene Expression (D&GE)
Hybridization
- In genetic studies, hybridization is the annealing of two complementary nucleic acids.
- The presence of a particular mRNA within a specific cell type can be assessed by hybridization to a complementary probe.
DNA & Gene Expression (D&GE)
DNA Libraries
Coding/Noncoding DNA strands
RNA is procuded when?
- Sense strand=coding strand, antisense strand=anticoding/noncoding strand
- RNA is produced when RNA polymerase uses the antisesnse strand as a template to make a new nucleic acid
- During cDNA synthesis, reverse transcriptase uses mRNA as a template to synthesize a new single-stranded DNA molecule.
- Because mRNA has the same sequence as the sense strand of genomic DNA (using uracil instead of thymine), cDNA will have the same sequence as the antisense strand.
- Accordingly, cDNA hybridizes with single-stranded DNA that corresponds to the sense strand of genomic DNA.
Microarray analysis
- uses single stranded DNA to hybridize
- cannot use double stranded DNA becuase it’s already hybridized
transcription by RNA polymerase
Transcription & translation in the eukaryotic cell
DNA & Gene Expression (D&GE)
Expressing cloned genes
- RNA polymerase transcribes both exons and introns to form pre-mRNA.
- During RNA processing, introns are removed by splicing to yield mature mRNA.
- cDNA is generated from mature mRNA and does not contain introns, so it is not spliced during expression.
Modification of mRNA
- 5’ capping by 7-methylguanosine, 3’ polyadenylation, & splicing to remove introns
Splicing of pre-mRNA
Transcription factor function based on cell signaling
ELISA
DNA & GENE EXPRESSION (D&GE)
Transcription
Example mRNA: 5′ – UCAAGUGAUUCUCCU – 3′
What is the DNA coding strand product?
- RNA polymerase II reads the noncoding DNA (antisense or template) strand to produce a complementary mRNA transcript (reversed directionality) (with uracil replacing thymine).
- The sequence of the mRNA transcript is identical (same sequence & directionality) to the sequence of the coding DNA strand (again, with uracil rather than thymine).
- Answer to example above: 5’-TCAAGTGATTCTCCT-3’
DNA & GENE EXPRESSION (D&GE)
Transcription
Initiation
Elongation
Termination
- Transcription is the process of synthesizing RNA from template DNA and begins with RNA polymerase II binding to the gene promoter region (AT-rich sequence TATA BOX)
- RNA polymerase II reads the DNA strand in a 3′ to 5′ direction to generate a 5′ to 3′ pre-mRNA molecule.
- The pre-mRNA transcript undergoes 5′ capping (recognized by reibosome during translation &prevents degradation), the addition of a 3′ poly-A tail (prevents degradation & facilitates the export of the mature mRNA from nucleus to cytoplasm), and excision of noncoding regions (introns) to be converted into mature mRNA.
Modification of pre-mRNA
DNA & GENE EXPRESSION (D&GE)
DATA interpretation
Splicing of mRNA/Spliceosome
Splice donors & splice acceptors
- splicing is driven by spliceosomes, complexes composed of specific proteins & small nuclear ribonucleoproteins (snRNPs) containing small nuclear RNA (snRNA)
- The spliceosome removes introns from pre-mRNA by locating specific sequences within introns called 5′ splice donor sites and 3′ acceptor sites.
- Splice donor sites are located in the 5′ end of the intron next to the exon, and splice acceptor sites are found at the 3′ end of the intron adjacent to the exon.
DNA & GENE EXPRESSION (D&GE)
When does Splicing occur?
- splicing occurs during pre-mRNA processing, not during transcription
- processing (which includes splicing) of the pre-mRNA transcript into mature mRNA occurs in the nucleus
DNA & GENE EXPRESSION (D&GE)
Chromatin remodeling
Heterochromatin & Euchromatin
Histone deacetyalase inhibitor?
- Heterochromatin, or tightly packed chromatin, is composed of deacetylated histones (due to histone deacetylase activity) and is transcriptionally repressed.
- In contrast, euchromatin or relaxed chromatin is highly acetylated (due to histone acetylase activity) and transcriptionally active.
- Histone deacetyalase inhibitor: prevent the removal of acetyl groups, thus DNA more accessible. thus inc gene expression
Eukaryotic DNA organization
HISTONES & NUCLEOSOMES
DNA methylation and CpG sites
- Addition of methyl groups to DNA, which often co-occurs with histone deacetylation, leads to the silencing of gene expression.
- CpG sites, or CG dinucleotide sequences, are methylated by the enzyme DNA methyltransferase, not by histone deacetylase inhibitors.
DNA & GENE EXPRESSION (D&GE)
Northern blot detects what?
band intensity denotes what?
band position denotes what?
- Northern blots detect target RNA in a sample.
- The labeled RNA can be visualized as bands, where band intensity denotes the quantity of RNA expression and band position denotes the size (smaller molecules appear lower than larger ones).
Ribosomes translate mRNA
D&GE
Kinase & phosphatase
Post-translational modification
- Phosphorylation and dephosphorylation are post-translational modifications that alter the function or activity of proteins by changing their conformation.
- Kinases catalyze the transfer of phosphate groups from ATP or GTP to proteins
- Phosphatases catalyze the removal of phosphate groups via hydrolysis.
D&GE
Ribosomes
Eukaryotes & Prokaryotes, which are large/small subunits
- ribosome-mRNA complex translocates where inorder to_____________________to synthesize secretory, lysosomal, or integral membrane proteins.
- Ribosomes translate mRNA sequences into proteins in eukaryotes (80S ribosomes: 60S large subunit and 40S small subunit) and prokaryotes (70S ribosomes: 50S large subunit and 30S small subunit).
- The ribosome-mRNA complex translocates to the rough endoplasmic reticulum to synthesize secretory, lysosomal, or integral membrane proteins.
Nucleolus
Smooth ER
Golgi Body
-
Nucleolus: involved in the production & assembly of ribosmal subunits, when are then transported to the cytoplasm via nuclear pores
- neither ribosomes nor their individual subunits bind to nucleolus
-
Smooth ER: lacks ribosomes
- participates in carbs metabolism, drug detoxification & synthesis of lipids that will ether form part of the cell membrane (phospholipids) or be secreted from the cell (sterioids)
- Golgi Body: after translation, many proteins in the rough ER are transported to the Golgi body for further modification and subsequent packaging/distribution to the cell membrane, organelles (eg, lysosomes), and secretory vesicles.
D&GE
mRNA
PCR
Researchers can assess the half-lifes of mRNA isoforms via what mechanism?
Example attached
- Researchers can assess the half-lifes of mRNA isoforms by converting mRNA to cDNA via RT-PCR at varying time points, allowing for comparison of isoform concentrations.
- reverse transcription polymerase chain reaction (RT-PCR)—>yields thousands of copies
- cDNA is initially denatured into single strands using heat, and forward primers and reverse primers anneal to the denatured cDNA strands so that Taq polymerase can elongate the DNA sequence.
- The isoform with greater cDNA concentration at the final time point has the longer half-life.
Western blotting
DETECTS PROTEINS
EUKARYOTIC TRANSLATION INVOLVES 3 STAGES
- Initiation: The small 40S ribosomal subunit binds the 5′ cap to scan mRNA for the start codon (5’-AUG-3’) , an initiator “charged” transfer RNA (tRNA) (3’UAC5’) is recruited to the start codon, and the large 60S subunit binds the initiator tRNA at P site, marking the formation of translation complex
-
Elongation: The ribosome continues to elongate the polypeptide chain by reading each mRNA codon in a _5′ to 3′ direction._
- During this step, a new charged tRNA with a complementary anticodon enters the A site. The enzyme peptidyl transferase then transfers the growing polypeptide chain from the tRNA at the P site to the new tRNA at the A site by catalyzing the peptide bond between adjacent amino acids on these tRNAs
-
Termination: A stop codon is read in the mRNA at A site indicating the end of translation, and release factors induce peptidyl transferase to cleave the ester (not peptide) bond between the polypeptide and the final tRNA, causing translation complex dissociation.
-
DNA Replication
- During replication, DNA polymerase joins uncoupled deoxyribonucleoside triphosphates (dNTP) to the new DNA strand.
- each dNTP conposed of a base, a deoxyribose sugar, and three phosphate (PO4) groups.
- As a result, the exergonic release of a pyrophosphate molecule leads to the formation of a covalent phosphodiester bond between the last nucleotide of the growing strand and the incoming nucleotide.
- the 3’OH group from the last nucleaotide of the growing strand would attack the 5’ PO4 group of an incoming dNTP
cDNA cloning
Which enzymes are involved?
- In cDNA cloning, reverse transcriptase generates a single strand of cDNA from a target mRNA sequence.
- DNA polymerase synthesizes the second complementary DNA strand and amplifies the target cDNA sequence.
- The target cDNA can then be inserted into a plasmid vector via the actions of a restriction enzyme (cuts both plasmid and vector) and DNA ligase (joins the cDNA to the vector).
Nucleolus:
- in eukaryotic cells, the nucleolus is found within the nucleus and is the primary site of ribosomal RNA (rRNA) transcription by RNA polymerase I.
- Ribosomal proteins synthesized in the cytoplasm are transported into the nucleolus, where they combine with rRNA to form 40S and 60S ribosomal subunit precursors.
- These precursors are exported from the nucleus to fully mature in the cytoplasm.
Nucleaosomes & histones
binding/charges/amino acids bound
- The nucleosome is a structural subunit composed of DNA wrapped twice around a histone octamer (8-protein complex), which contains two molecules of H2A, H2B, H3, and H4.
- H1** is located outside the octamer core and serves as the “**linker” protein that secures the DNA wrapped around the nucleosome.
- Histone proteins are rich in positively charged arginine and lysine, which facilitate binding to negatively charged DNA.
SDS-PAGE
ployacrymal and agrose gels
- SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) is used to separate proteins by molecular weight.
- During the procedure, SDS coats proteins with a negative charge.
- An electric current is then applied, and smaller proteins travel through a polyacrylamide gel and toward the positive anode faster than larger ones, creating lanes of size-separated protein bands.
- (Note that this convention is opposite that used in electrochemical cells, in which the anode is negative.)
-
(larger ones=agrose gel)
- ethidium bromide is used as a stain to make DNA bands (not proteins) fluoresce; this effect occurs as a result of ethidium bromide’s ability to intercalate (“slip in”) between DNA base pairs.
Chromatids & Centromeres
Telomeres
- Centromeres join two sister chromatids and are essential for proper chromosome division during mitosis.
-
Telomeres are regions at the chromosome ends that are repeatedly truncated each time a cell divides.
- Telomeres contain repeats of only TTAGGG, a single DNA sequence of six nucleotides that is added by the enzyme telomerase. Multiple repeats of differing DNA sequences are not present in telomeres.
-
shorteded with each round of cell division
- However, only embryonic stem cells (not somatic cells) express telomerase and therefore have very long telomeres; this allows them to proliferate indefinitely in a controlled manner.
-
Both centromeres and telomeres are composed of heterochromatin, a transcriptionally inactive and tightly condensed complex of DNA wrapped around histones.
- hetechromatic regions are often gene-poor and contain repetitive DNA.
Sensory vs motor neurons
grey & white matter
- grey matter: unmyelinated neuronal cell bodies & dendrites
-
white matter composed of axons that allow long-distance communication between neurons, myelinated & unmylanted
- white matter of spinal cord: affarent (dorsal) axons carry sensory info to the brain & effarant (ventral) axons carry motor comands to the body
Blood Brain Barrier
- composed of endothelial cells held together by tight junctions, which limit paracellular transport.
- Substances can pass through the barrier by several mechanisms, depending on size & polarity:
- Carrier-mediated transport (faciliated diffusion & active transport) allows the passage of glucose, amino acids, and nucleosides into the CSF.
- Small, lipophilic molecules pass easily into the CSF through transcellular diffusion. (passive diffusion)
- non-polar hases such as CO2 & O2 as well as steriod hormones such as testosterone pass easily through epithelial cells & enter brain
- paracellular transport: small hydrophilic molecules is limited by the presence of tight junctions
- endocytosis is used to transport molecules in bulk
Action potential overview
what is the RMP restored by?
- resoted by the Na+/K+ Pump
- Resting membrane potential (RMP): The membrane potential of the resting neuron is −70 mV (ie, the inside of the neuron is 70 mV more negative than the extracellular space). The RMP is maintained by the Na+/K+ pump and K+ “leak” channels, which always allow passive diffusion of K+ ions across the membrane. Ion channels that open or close based on changes in membrane potential (voltage-gated channels) are closed at RMP.
- Threshold: An excitatory stimulus causes several nearby voltage-gated Na+ channels to open, allowing Na+ ions to rush into the cell. Consequently, the membrane potential becomes more positive than the RMP (ie, depolarized). If the neuron depolarizes to a certain threshold value, an AP is fired. If the threshold is not reached, no AP is fired and the RMP is restored.
- Rising phase: At threshold, the remaining voltage-gated Na+ channels open, resulting in the rapid depolarization of the local membrane. This rise in membrane potential triggers a positive feedback loop, opening more Na+ channels in adjacent segments to propagate the AP down the axon.
- Overshoot: The ongoing flood of Na+ into the cell causes the AP to reach its peak (overshoot), where the membrane potential is most positive.
- Falling phase: Voltage-gated Na+ channels close, and voltage-gated K+ channels open. K+ rushes out of the cell, causing membrane repolarization and restoring the RMP.
- Undershoot: Excessive K+ efflux causes the membrane potential to fall below the RMP (hyperpolarize), and the local membrane enters a refractory period (absolute or relative).
- RMP restoration: Both voltage-gated Na+ and K+ channels are inactive. The membrane potential returns to -70 mV and is maintained by the Na+/K+ pump until another action potential is initiated.
Electrical vs. Chemical Synapse
Electrical vs chemical synapse
-
Electrical synapses transfer information from one cell to another via passive ionic current flow through gap junctions.
- Due to this cytoplasmic continuity and the mere 2- to 4-nm gap between the pre- and postsynaptic membranes, the current passes almost instantaneously from one neuron to another.
- Cytoplasmic continuity also allows signals (ions) to flow bidirectionally across the synapse.
-
Chemical synapses use neurotransmitters to transfer information, which is a slower process (delay between pre- & postsynaptic
- unidirectional info transfer
- not continuous cytoplasm
Synaptic transmission via ligand-gated ion channels
- Although trends in neurotransmitter function exist, the postsynaptic response is ultimately determined by the type of receptor activated
- Receptors may be either ligand-gated ion channels or G-protein–coupled receptors.
Synaptic transmission via G protein-coupled receptors (GPCRs)
All Neurotransmitters & functions
Image of Oligodendrocytes & schawan cells
