Term
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Definition
contains DNA from 2 or more sources
once inside a host cell, replicated to produce clones |
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Term
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Definition
| insert gene from chromosomal DNA into vector DNA; insert recombinant vector into host cell w/o a vector. host cell divides |
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Term
| what are common vectors for gene cloning? |
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Definition
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Term
| what is responsible for cutting the genomic DNA that is then inserted into the vector DNA? where is it obtained froM? |
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Definition
restriction enzymes/endonucleases (make cuts at restriction sites)
made naturally by bacteria as protection against bacteriophages |
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Term
| what are the two possible outcomes of attempting to insert genomic DNA into vector DNA? |
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Definition
re-circularized vector w/o gene of interest inserted
recombinant vector w/ gene of interest inserted |
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Term
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Definition
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Term
| how do genomal and vector DNA combine? |
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Definition
| the sticky ends complement each other and hydrogen bond; DNA ligase catalyzes covalent bonds in the backbone to finalize |
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Term
| how can you tell if you've been successful at creating recombinant DNA? |
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Definition
vector carries a selectable marker- ampR genes code for B-lactamase, which degrades ampicilin, which normally kills bacterial cells. so grow host cells on ampicillin- those that survive must express B-lactamase and contain the genomic DNA.
also, the lacZ gene is disrupted when chromosomal DNA is inserted. lacZ codes for B-glactosidase which turns X-gal colorless. so blue colonies --> functional lacZ --> recircularized plasmid (not recombinant vector) |
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Term
| what are the 2 types of common DNA libraries (which are a collection of many recombinant vectors each w a fragment of chromosomal DNA)? |
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Definition
genomic- inserts derived from chromosomal DNA
cDNA- uses reverse transcriptase to make DNA from mRNA of interest (complementary DNA)- lack introns so easier to use |
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Term
| what are characteristics that electrophoresis sorts based upon? |
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Definition
| charge, size/length, and mass |
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Term
| what materials do you need to perform a PCR? |
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Definition
high concentration of 2 primers complementary to DNA sequences to be amplified
dNTPs
Taq DNA polymerase
buffer |
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Term
| what are the three cycles of PCR? what will the product after 30 of these cycles be? |
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Definition
denaturation, annealing, synthesis
2^30 copies of DNA sample |
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Term
| genomics: definition. 2 phases |
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Definition
molecular analysis of entire genome of a species
1. mapping of genome
2. functional genomics (what is turned on and off in particular cells) |
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Term
| what do BAC and YAC stand for? how many nucleotides can they take up? |
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Definition
bacterial artificial chromosomes - ~500,000 bp
yeast artificial chromosomes - ~2,000,000 nucleotides
(more than most plasmid or viral vectors) |
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Term
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Definition
| a collection of overlapping clones that allows researchers to ID order along the chromosome |
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Term
| describe the Sanger dideoxy chain termination sequencing overview |
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Definition
uses ddNTPs that are missing the 3'-OH group that thus terminate the chain
DNA polymerase makes complementary strand until ddNTP is inserted, at which point the chain terminates
after electrophoresis you can look at chain overlaps to determine gene sequence
can do all in one tube by using fluorescent dye |
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Term
| how does the microarray work? |
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Definition
have microarray chip with short sequences of known genes attached to different areas
isolate mRNA from cells of interest, add reverse transcriptase and fluorescent nucleotides to create flourescently labeled cDNA that then hybridizes to the known DNA on the microarray chip |
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Term
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Definition
| insulin is made of polypeptides A and B; both coding sequences are inserted into e.coli (into separate vector DNAs also containing ampR and B-galactosidase); use CNBr (cyanogen bromide) to cleave A or B chain from the B-hgal, then a disulfide bond forms linking A chain and B chain |
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Term
| what does the term bioremediation mean? |
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Definition
| use of microorganisms or plants to detox pollutants |
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Term
| what is a transgenic organism? |
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Definition
| has genes introduced using molecular techniques (is a genetically modified organism) |
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Term
| what is gene replacement? |
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Definition
| when a cloned gene recombines with a normal gene on one of two original chromosomes (creating a heterozygote) |
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Term
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Definition
| when a cloned gene contains a mutation that inactivates function or entire coding sequence of gene is deleted --> homozygote lacks gene function completely |
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Term
| what does molecular farming refer to? |
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Definition
production of medically important proteins in livestock mammary glands;
clone the human hormone gene into a plasmid vector next to the promoter of a gene specifically expressed in mammary cells; the vector is injected into oocyte, integrated into the the DNA, the egg is fertilized, implanted; milk containing the hormone is purified for the protein |
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Term
| how did cloning from a somatic cell (Dolly) work? |
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Definition
| fused diploid mammary cell + enucleated sheep oocyte= zygote that is then implanted |
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Term
| what were the minor differences between Dolly and her donor due to? |
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Definition
| differences in mitochondrial DNA and maternal effect genes |
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Term
| how does DNA fingerprinting work? |
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Definition
| use PCR to amplify short tandem repeat sequences (found at specific locations in the genomes of all species; the # of repeats @ each spot varies from one individual to the next) |
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Term
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Definition
| adenosine deaminase deficiency prevents proper metabolism of nucleosides, so that deoxyadenosine destroys B and T cells, leading to immunodeficiency |
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Term
| how was gene therapy used to treat ADA deficiency? |
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Definition
remove ADA-deficient lymphocytes from the person with the immunodeficiency
infect cell w/ retrovirus containing normal ADA gene. this retrovirus inserts its DNA into host cell chromosomes. then infuse ADA gene corrected lymphocytes back into patient. cured! |
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Term
| 4 ways in which cells can respond to positional info |
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Definition
| can divide, migrate, differentiate, or die |
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Term
| what is the Spemann graft? |
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Definition
| moved dorsal lip of one embyro to the ventral side of another embryo; demonstrated that an embryonic organizer can provide instructive cues |
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Term
| what are the two main mechanisms used to communicate personal info? |
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Definition
| morphogens and cell adhesion |
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Term
| what is the "french flag" analogy for morphogen gradients? |
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Definition
| morphogens act in a concentration dependent manner with a critical threshold concentration and are distributed asymmetrically (inducing changes) |
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Term
| what does the term "cell adhesion" refer to? |
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Definition
| the positioning of a cell within a multicellular organism is strongly influenced by the combo of contacts it makes w. other cells and the extracellular matrix |
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Term
| what are the four general phases for body formation (that correspond with differential gene expression of transcription factors)? |
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Definition
1. body organized along axis
2. body organized into smaller regions
3. cells organize to make body parts
4. cells become differentiated |
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Term
| what is the key process in the development of drosophila? |
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Definition
| creation of segmented body plan (into head, thorax, abdomen) |
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Term
| timeline of life cycle of drosophila |
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Definition
a. oocyte - 0 hours - axes established
b. embryo - 10 hours- segments established
c. then larva, pupa, adult 10 days after fertilization |
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Term
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Definition
an example of a morphogen
nurse cells are located near anterior end of oocyte
bicoid genes transcribed in nurse cells and mRNA transported into anterior end of oocyte
if it is mutated, results in larva with 2 posterior ends
in wild-type, bicoid is much more concentrated at anterior end; in mutant, bicoid is distributed uniformly. |
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Term
| bicoid mutation variability |
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Definition
if mutant is just all around deficient in bicoid, will have two tails, no head
if bicoid is added to anterior region, normal development
if bicoid is added to middle, there will be head in the middle, two tails
if bicoid is added to posterior end where it is already also in anterior end, will have two heads on either end |
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Term
| what is up with the torpedo gene? |
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Definition
its a maternal effect mutation affecting dorsal ventral polarity- even if the pole cells between wild type and torpedo deficient moms are swapped, it only matters whether the germ cells are implanted in a wild type or mutant mom
-Normal mom but torpedo deficient germ cells --> normal dorsal ventral acid
-torpedo deficient mom but wild type germ cells --> no dorsal ventral axis |
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Term
| what are three classes of segmentation genes? |
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Definition
| gap genes, pair-rules, segment-polarity genes |
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Term
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Definition
| several adjacent segments are missing |
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Term
| mutation in pair-rule genes |
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Definition
| alternating segments or parts of segments deleted |
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Term
| mutation in segment polarity genes |
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Definition
| portions of segments to be missing either anterior or posterior and adjacent regions to mirror each other |
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Term
| what type of genes promote phase 1 pattern development and activate gap genes? |
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Definition
maternal effect genes
then gap and maternal effect gene products act as transcription factors to activate pair genes, whose gene products act to regulate segment polarity genes |
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Term
| maternal effect, gap, pair rule, and segment polarity genes in drosophila |
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Definition
| bicoid, krupple/hunchback, fushi tarazu, engrailed |
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Term
| what does the colinearity rule state? |
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Definition
| order of homeotic genes along chromosome corresponds to their expression along the anteroposterior axis of body |
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Term
| what is the deal with MyoD |
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Definition
| causes fibroblasts to differentiate into muscle cells; found in all vertebrates; binds to an enhancer that is next to genes that are expressed only in muscle cells; allows RNA polymerase to initiate transcription |
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Term
| what is the term for highly influential proteins in cell differentiation? |
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Definition
| master transcription factors |
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Term
| what is responsible for red blood cells, platelts, dendrites (all myeloid cells), lymphoid cells, etc? |
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Definition
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Term
| is the inner cell mass, the source of embryonic stem cells, totipotent? |
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Definition
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Term
| what kind of stem cell is a hemopoetic cell? |
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Definition
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Term
| which types of cells are totipotent? |
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Definition
| only the fertilized egg (zygote) and the first few cells (of blastocyst) made by early cell division are totipotent |
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Term
| what types of cells are pluripotent? |
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Definition
| found in the inner cell mass of the blastocyst |
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Term
| what is the signature feature of stem cells? |
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Definition
| stem cells replicate indefinitely (self-renewal) but progenitor cells can only divide a limited number of times. |
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Term
| what type of stem cells are adult stem cells? |
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Definition
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Term
| what are homologous genes? |
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Definition
| a group of homeotic genes found in all animals so from the same ancestor. |
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Term
| what is the colinearity rule? |
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Definition
| order of homeotic genes along the chromosome corresponds to their expression along the anteroposterior axis of their body |
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Term
| what is the role of homeotic genes |
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Definition
| to encode homeotic proteins that function as transcription factors, activating transcription of specific genes that promote developmental changes |
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Term
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Definition
| encodes for homeodomain that is a chunk on the protein produced by homeogenes- homeodomain allows protein to function as a transcription factor |
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Term
| maternal effect genes, segmentation genes, examples |
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Definition
maternal effect: bicoid (anterior/posterior) and torpedo (dorsal/ventral)
gap: hunchback, krupple
pair-rule: fushi tarazu
segment polarity: engrailed |
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Term
| proteins bound to membranes |
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Definition
peripheral- noncovalently bounded to integral membrane proteins or to heads of phospholipids
integral membrane proteins
either transmembrane (region of protein in physically embedded in hydrophobic region) or lipid anchored (side chain of amino acid is attached covalently to lipid) |
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Term
| what does the term cotranslational protein sorting refer to? |
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Definition
| starts in cytosol, ends in rough ER; ER signal sequence is recognized by SRP which brings to SRP receptor in membrane, ribosome poops amino acid chain into ER lumen; signal peptodase chops off signal sequence |
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Term
| where does the synthesis of membrane components go down? |
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Definition
| lipids: in cytosolic leaflet of smooth ER, also in cytosol |
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Term
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Definition
N linked: adding carb to asparagine
O linked: only in Golgi; adding sugar to serine or threonine |
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Term
| what does the term crenation mean? |
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Definition
| shrinking in a hypertonic solution (in plants its called plasmolysis) |
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Term
| what is the name for the transmembrane protein facilitating the diffusion of water? |
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Definition
| aquaporin (is a type of channel) |
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Term
| 3 types of transporters (carriers) |
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Definition
| symporters, uniporters, antiporters |
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Term
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Definition
ATP driven ion pump that pumps Na+ and K+ against their gradients
3 Na+ exported for 2 K+ imported (antiporter) |
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Term
| what is the difference between pinocytosis and phagocytosis? |
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Definition
pino= small things, liquids, ions, proteins
phago- large complexes and cells |
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Term
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Definition
*BOTH break down mRNA
exonuclease is the enzyme, exosome contains the exonucleases
exonuclease traveles 5'-3' bc cap is removed at 5'
exosome traveles 3' to 5' (bc cap is not removed) |
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Term
| what is responsible for breaking down carbs, lipids, nucleic acids, proteins? |
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Definition
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Term
| what breaks down proteins |
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Definition
| proteosomes (with the help of ubiquitin) |
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Term
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Definition
substrate level phosphorylation
chemiosmosis |
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Term
| overview of glucose metabolism: pathways |
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Definition
| glycolysis, pyruvate breakdown, citric acid cycle, electron transport chain (oxidative phosphorylation) |
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Term
| what is produced by glycolysis of 1 glucose? |
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Definition
2 ATP (substrate level phosp)
2 pyruvate
2 NADH (go to ETC) |
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Term
| what does breakdown of 2 pyruvate make? |
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Definition
2 CO2
2 acetyl coA (go to citric acid)
2 NADH (go to ETC) |
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Term
| what does breakdown of two acetyl coA make? |
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Definition
2 ATP (substrate level phosph)
4 CO2
6 NADH, 2 FADH2 (go to ETC) |
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Term
| what happens during the ETC? |
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Definition
| oxidative phosphorylation- oxidation of NADH and FADH2 via ETC provides energy for ATP, O2 is consumed. |
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Term
| how is glycolysis controlled by feedback inhibition? |
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Definition
| overproduction of ATP binds to an allosteric site on fructokinase to inhibit it |
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Term
| slow arsenic poisining occurs at what stage? |
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Definition
| problems with pyruvate breaking down |
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Term
| acetyl coA is added to what to make citric acid? |
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Definition
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Term
| how is citric acid cycle controlled by feedback inhibition? |
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Definition
| oxaloacetate is a competitive inhibitor of succinate dehydrogenase |
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Term
| about how many molecules of ATP per glucose molecule broken down |
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Definition
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Term
| what can E. coli use under anaerobic conditions for ETC? |
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Definition
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Term
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Definition
| anywhere but glycolysis (so yes for pyruvate, acetyl coA, citric acid cycle) |
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Term
| where does glycerol enter? fatty acids? |
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Definition
| glyceraldehyde 3 phosphate, acetyl coA |
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Term
| what is the Warburg effect? |
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Definition
| cancer cells preferentially use glycolysis while decreasing oxidative phosphorylation |
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Term
what do the light reactions produce?
the calvin cycle? |
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Definition
ATP, NADPH, O2
use ATP and NADPH and CO2 to make sugars |
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Term
| what is the absorbtion vs the action spectrum? |
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Definition
absorbtion: wavelengths absorbed
action: rate of photosynthesis |
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Term
| where is the concentration of H+ highest in thylakoids? |
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Definition
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|
Term
| what is the product of the Calvin cycle? |
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Definition
| G3P (glyceraldehyde 3 phosphate) |
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Term
| 3 phases of the calvin cycle |
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Definition
| carbon fixation, carb production, regeneration of RuBP |
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Term
| what happens during carbon fixation stage? |
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Definition
| use rubisco to add co2 to RUBP |
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Term
| what happens during carb production stage? |
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Definition
| ATP and NADPH are used to make G3P |
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Term
| what happens during regeneration of RuBP? |
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Definition
| 10 of the 12 G3P you made are converted into RuBP using ATP |
|
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Term
| explain the carbon 14 graphs? |
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Definition
| if you know the level of carbon 14 present in the teeth, you can look at the chart to figure out what the person's teeth were formed. subtract four and you know what year they were born. |
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Term
| how is RAS changed in cancer? |
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Definition
| RAS promotes cell division when GTP is bound until RAS hydrolyses it into GDP; a mutation messing up RAS ability to hydrolyse GTP = signaling pathway on |
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Term
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Definition
| 2 fragments of genes fused together (chromosomal translocations) |
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Term
| what's up with retinoblastoma? |
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Definition
| you need 2 copies of mutant gene (if you start out with one, you get the disease earlier on in life) otherwise rarely and only late in life end up with two mutant alleles |
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Term
| what is abnormal methylation of CpG islands near promoter regions responsible for? |
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Definition
| loss of tumor supression gene function |
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Term
| what determines the precise starting point for transcription? |
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Definition
5'-TATA-3' box
along w transcriptional start site forms core promoter |
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Term
| proteins needed for transcription? |
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Definition
| RNA polymerase II, 5 GTFs (form transcription initiation site), then the mediator |
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Term
| what does glucocorticoid do? |
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Definition
| activates transcription of certain genes- binds to receptors that release chaparones and expose nuclear localization signals- glucocoricoid forms a dimer that enters nucleus and binds in front of promoter |
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Term
| what can you expect to find near promoters? |
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Definition
| CpG islands (which can become methylated, inhibiting transcription) |
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Term
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Definition
| binds with RISC (RNA induced silencing complex) to tag mRNA to degrade it or just not translate it |
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Term
| what is this stuff about iron toxicity an example of? |
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Definition
| iron regulatory protein can bind to mRNA that codes for ferritin when there is not enough iron present |
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Term
| what are the two sites open to bonding to of transcription factors? |
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Definition
| small effector molecules and DNA binding |
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Term
| what is fetal hemoglobin an example of? |
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Definition
| using methylation to only transcribe certain regions of DNA |
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Term
| 2 examples of epigenetic inheritance? |
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Definition
| X inactivation and genomic imprinting |
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Term
| how does cell know how many barr bodies to produce? |
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Definition
| counts # of X inactivation centers |
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Term
| how is a barr body inactivated? |
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Definition
the Xist noncoding RNA spreads along inactive chromosome
Tsix noncoding RNA represses Xist on active chromosome |
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Term
| what's up with maternal effect genes? |
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Definition
| maternal nurse cells surrounding the egg are what determines the PHENOTYPE (not genotype) of the developing organism |
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