Chapter 2 Flashcards
how did Briggs & King perform a somatic-cell nuclear transfer in amphibians?
they took a nucleus from somatic cell and transferred it to an unfertilized, enucleated egg
-heated a glass needle and used it as a micropipet to transfer the diploid nucleus
-only worked if it was a BLASTOMERE
what is the process of cloning mammals?
- extract egg form oocyte donor & remove spindle
- extract utter cells from nuclear donor and grow until G1 stage
- transfer donor cell into enucleated egg
- shock with electricity to cause bilayers to fuse
- grow until blastocyte forms and transfer to surrogate mother
embryo looked like nuclear donor
what is nuclear totipotency?
all cells have the info needed to direct the formation of a whole individual
-nucleus of any cell can drive development
what are epigenetic markers?
there are different ways DNA is wrapped to turn genes on & off
-does not alter DNA, just allows for silencing to occur
what is differential gene expression?
each type of cell has a complete genome, but each type activates different genes
-unused genes are still retained, just silenced, only small % of genes are used
what are two exceptions to differential gene expression?
gametes: only use half
red blood cells: ditch nucleus to carry O2
describe the structure of a nucleosome? what are the names of the histone proteins?
8 histone proteins w/ two wrapped DNAs
-H2A, H2B, H3, H4 (x2)
-H1 is a linker histone
what happens when histone tails are methylated? acetylated?
methylated: wound tight, no transcription
acetylated: unwound & spaced, transcription
what does the promoter do?
RNA poly2 binding site (reads DNA)
-contains TATA box (-30)
-binds basal TFs
what are exons? introns?
exons: kept in DNA, leave the nucleus to make proteins
introns: cut out of DNA
what is the sequence for the polyadenylation signal?
AATAAAA on 3’ end
what does the enhancer do?
bind gene specific TFs
-helps to stabilize RNA poly2 at promoter
what is the start codon?
AUG / ATG
what are the stop codons?
UGA
UAA
UAG
how does the enhanceosome work?
mediator complex brings far away aparts of DNA close together to allow for transcription to occur
-contains a series of TFs determined by cytoplasmic determinants
-histones must be acetylated by histone acetyl transferases
where in the DNA do TF’s bind to?
major groove of DNA at the bases
-not the phosphate sugar backbone
what two things do TF’s require?
DNA binding domain
Protein interaction domain (must be able to interact w/ enzymes)
what are the 6 families of TFs?
homeodomain (hox, POU, Lim, Pax)
basic helix-loop-helix
basic leucine zipper
zinc-finger
nuclear hormone receptor
sry-sox
what does hox do?
determine where you are in the body & what structure to build
what five things are needed for transcription?
- basal TFs
- gene specific TFs
- mediator complex
- transcription elongation complex
- RNA poly2
what does the carboxyl & amino termini do?
sensing domains (regulating) that respond to the environment
-sense what type and what they can do as a cell
describe what an enhancer trap is?
able to follow reporter gene when you add certain TFs to see what gets turn on
reporter gene (Ex: GFP) is used to make mRNA you are studying colorful
weak promoter + reporter gene + transposable element
what are the three ways DNA is regulated through methylation?
direct promoter silencing
imprinting
X-inactivation
what is direct promoter silencing?
adding a methyl group causes the base to grow and is unable to bind TFs
EX: fetus slowly inactivates epsilon-globin at 8 weeks by methylating it
what is imprinting?
able to see if the sperm-derived or egg-derived allele of the gene is expressed
-female vs male gonads have different affects to methylation
-both are needed, just imprinted differently (active or inactive in mom vs dad)
EX: Igf2 expressed in dad, Igf2r expressed in mom
-methylation turns gene off
does X-inactivation occur in somatic or germ line cells?
only in somatic cells
-germ line cells needs both X’s to be active
what is a barr body?
what the inactivated X gets converted into
what methylates the X-chromosome to make it inactive?
Xist RNA
-entire chromosome shutdown (turns off thousands of genes)
-even in females w/ aneuploidies, only one X is active (creates 2 barr bodies)
what is the chromatin of the barr body converted into?
heterochromatin
what is the difference between heterochromatin and euchromatin?
heterochromatin: highly condensed, gene-poor, not transcribed
euchromatin: less condensed, gene-rich, transcribed
who discovered X-inactivation? what was her hypothesis?
Mary Lyon (Lyon Hypothesis)
- early embryo: both X’s active
- later embryo (gastrulation): one inactive X
50/50 chance of mom vs dad’s X getting inactivated
once inactivated, the same X is inactivated in all of the cell’s progeny & gets passed down (irreversible)
why does one X get inactivated?
Dosage Compensation
-makes sure all members of the species have the same does of a gene
-males and female have ~equal amounts of X gene produces
what are some exceptions to dosage compensation?
lampbrush chromosomes (extra rRNA copies)
actin, homeobox, GFs, etc. (not equal in all species)
unequal crossing over (extra part of chromosome found in one part but not another)
trisomy (extra 21)
lethality of two active X’s
what are the two types of cells in a blastula? how does this relate to X-inactivation?
trophoblast cells (placenta) & inner cell mass (baby)
-paternal X contains lacZ & makes galactosidase (stains blue)
-maternal X shows up as pink
- day 4: both X’s active
- day 6: trophoblast cells inactivate paternal X (not random) –> placenta cells are pink
paternal X always gets turned off in placenta cells
how does Xist RNA inactivate an X? what type of RNA does it use?
Xist is transcribed at a higher level in one of the X’s
uses IncRNA (long, noncoding)
- acts like a rope to tie up the X chromosome
-does not code for a protein
- only works in cis conformation *
how was Xist found to be the inactivator of the X chromosome?
find it, lose it, move it
- right place at right time
- remove gene & see what happens (necessary & sufficient?)
- put on different chromosome to see if it serves same function
what percentage of genes escape X-inactivation?
15%
is the poly-A-tail and 7-MG cap added to DNA or RNA?
mRNA
what are the two ways RNA transcription is regulated?
RNA selection (censorship)
Differential Splicing
what is RNA selection (censorship)?
only certain genes are allowed out of the nucleus for transcription, other other genes are left in the nucleus (called hnRNA / nRNA)
how is censorship detected?
in situ hybridization
- take complementary RNA probe (antisense) and wash over fixed tissue
- if probe only glows in nucleus, mRNA was censored (not transcribed)
- if probe glows in nucleus & cytoplasm, mRNA is not censored
what is differential splicing?
different ways the introns can be cut out in different cells to make a variety of proteins
what is the untranslated region (UTR)?
in exons that are on the 5’ and 3’ ends that are skipped and not translated
what are consensus sequences?
regions that are the exact same in all species
EX: intron splice sites ( 5’ GU–A–AG 3’)
what cuts out introns?
splicesomes
- made up of snRNA’s and splicing factors
- forms when proteins accumulate at the 5’ and 3’ splice sites then they make contact with eachother
what are snRNA’s?
short RNAs, stay in the nucleus
snRNA + splicing factors = snRNPs
what are the four common types of splicing patterns?
cassette exon
mutually exclusive exon
alternative 5’ splice site
alternative 3’ splice site
how is a cassette exon spliced?
cassette exons function as either an intron or exon
- either it’s cut out with the introns, or left in
how is a mutually exclusive exon spliced?
either one cassette is cut out or the other, never both
how is an alternative 5’ splice site spliced?
it creates an early 5’ cut
- left with either the entire exon, or a very small piece
how is an alternative 3’ splice site spliced?
late 3’ cut
- left with either the entire exon, or a very big piece
describe Bcl-X and the impact of splice isoforms
example of alternative 5’ splice site
- small Bcl-X = cell death
- large Bcl-X = inhibits cell death and encourages tumor growth
describe muscle hypertrophy and the impact of a misplaced splice
example of alternative 5’ splice site
- proteins normally turn off muscle growth to allow for bone & muscles to work together
-misplaced RNA = deficiency in myostatin
(leads to too much muscle)
- read incorrectly and uses part of intron when it’s not suppose to, creates an early termination codon
-makes a short, nonfunctional myostatin protein
describe DSCAM in drosophilia and the impact of splice isoforms
example of mutually exclusive exons
- DSCAM is a cell adhesion molecule that helps in cell migration for dendrite communication
- different forms of DSCAM allows for more connections b/w dendrites (overlaps)
- having the same type of DSCAM repulse each other
what are two ways RNA translation is regulated? describe them
longevity = if RNA is going to be used by ribosomes
localization = certain ribosomes in certain types of regions can read DNA
how does structure of RNA impact longevity?
diamond ring structure cannot be translated
- maskin protein binds CPEB and brings the 5’ & 3’ ends together, blocking translation
- allows for RNA to be kept for later
how do you activate translation in the diamond ring structure?
- a kinase phosphorylates CPEB and it releases maskin
- CPSF binds and extend the poly-A-tail
- PABP binds and recruits a ribosome to start translation
what type of RNA helps with longevity?
microRNA (miRNA)
- short, noncoding
how does microRNA inhibit translation?
- they form hairpin loops in the RNA
- microRNAs are then cleaved to make them single stranded
- microRNA then binds to RISC (RNA-induced silencing complex)
- if the microRNA sequence matches the RNA, the RNA is cleaved and destroyed
- if the microRNA is different, the RISC complex inhibits translation
what are the three ways RNA uses localization?
diffusion & local anchoring
localized protection
active transport
how does diffusion & local anchoring work for RNA localization?
Nanos freely diffuse in the cytoplasm, when they diffuse to the posterior pole, they are trapped by proteins that activate mRNA = translation
how does localized protection work for RNA localization?
mRNA accumulates at posterior pole (similar to nanos) but proteins block enzymes from destroying the mRNA
how does active transport work for RNA localization?
kinesin & dynein walk along microtubules in opposite directions allowing the mRNA to be transported to the correct ends
what are emergent properties in proteins?
integration of different proteins in the same cell at the same time causes the cell to do new things that it couldn’t do before
- “properties that become apparent and result from various interacting components within a system but are properties that do not belong to the individual components themselves”
primary structure of a protein
secondary structure
tertiary structure
quaternary structure
primary: order of amino acids in a chain
secondary: alpha helix or beta sheet
-connected by hydrogen bonds
-due to partial charge on O- and R+
tertiary: interactions b/w beta & alpha structures
-held together by hydrogen bonds, disulfide bonds, ionic bonds, and hydrophobic interactions
quaternary: multiple tertiary structures of many polypeptide chains
-multiple subunits form the complex
what are some functional protein modifications?
cleavage
adding groups (acetylation, methylation, carbs, phosphotase, kinase)
alterations from enzymes (proteases, coenzymes, substrates, cofactors, etc.)
what does cleaving the inhibitory domain of a protein do?
causes the protein to activate
what does cleaving the anchor protein do?
disrupts nanos
explain the precursors for NGF (nerve growth factor) and how the receptor can impact it’s function
NGF = active form, cleaved (activates neuronal cells)
proNGF = precursor form, uncleaved
trkA = normal NGF receptor
p75NTR
-if proNGF bind = death
-if NGF binds = normal function
how does delta-notch work?
JUXTACRINE INTERACTION
- delta binds notch
- notch bends
- protease cleaves internal section of notch
- notch binds to repressor & recruits TFs (p300)
- mediator complex forms & transcription starts
