DNA Flashcards
sequence repeated at telomeres
TTAGGG
how long are telomeres?
10-15 kb
kinetochore
Protein scaffold where spindle fibers attach
primase
synthesizes rna primers during DNA replication, since DNA polymerase can’t start from nothing
telomerase extends the (leading/lagging?) strand
lagging (template)
because that’s the one that has issues with okazaki fragments.
telomerase - how does it extend the lagging strand?
by using its RNA component as a template to add TTAGGG to the end of the lagging strand, thus giving more space for synthesis of another okazaki fragment
replication on the lagging strand involves
primase (lays down primer)
polymerase
splicing
ligase
what direction does DNA proofreading go?
3’ to 5’ - done by “exonuclease proofreading” function of DNA polymerase
opposite direction of DNA replication. Polymerase senses error (a bubble that doesn’t pair properly) and its exonuclease component goes to excise the incorrect base to fix the problem; correct base is inserted via polymerase.
mismatch repair proteins - mechanisms of action? at what point do they go in?
MMR proteins go in if proofreading fails, and there’s a mismatch “bubble”
MMR binds bubble, also binds the nicked strand (NEW DNA is kNicked to “mark” it as being freshly-made). MMR scans along the nicked strand until it gets to the bubble, then fixes it by:
excising wrong base on both sides
polymerase inserts correct bases
ligase
xeroderma pigmentosum – defect in what?
nucleotide excision repair (big, bulky problems, like two bases dimerized by UV)
Damage within ONE strand of DNA fixed by one of 4 mechanisms ?
Proofreading
Mismatch repair
Nucleotide excision repair
Base excision repair
double strand break:
homologous can lead to –
translocation (wrong ends joined)
deletion/duplication (missing bp’s at joining)
double strand break:
NON-homologous ?
“eating away” a bigger portion at the double stranded breakpoint on the chromosome to reveal overhangs
overhangs help sister chromatid be used as template during repair
BRCA - mechanism broken?
double strand break repair – homologous
MUTYH - mechanism broken?
BASE excision repair
Lynch - mechanism broken?
mismatch repair
RISC -
stands for what?
function?
RNA-induced silencing complex
either brings RNA template to SILENCE mRNA
or
DOWNREGULATES TRANSLATION (incomplete RNA template match to mRNA)
carrier rate for Fragile X
1 in 150 females
affected male rate for Fragile X
1 in 4,000
what’s more common, Fragile X or Noonan?
Noonan is more than twice as common
FraX = 1 in 4,000
FraX CGG repeat cutoffs
45-54 = “gray zone”
next generation at risk for:
premutation
POI - premature ovarian insufficiency
tremor/ataxia (FXTAS)
56-200 = premutation; maternally only
at risk for POI, FXTAS
if female, at risk for kids with FraX
230+ = affected
mitigating factor in CGG repeats in FraX
AGG repeats every 10 or so repeats - helps prevent slippage (FMR1 promoter region)
FraX mechanism
expansion of CGG (mitigated by AGG every 10 or so)
increased methylation of CpG
turning off tx of FMRP = fraX protein
Huntington - mechanism?
CAG repeats (glutamine) within HTT (huntingtin gene) - within exon expand paternally
mechanism of toxicity not understood; potentially glutamine can aggregate in neurons
Huntington - repeat cutoffs
27-35 = at risk for child with HD 36-39 = reduced penetrance alleles 40+ = full penetrance
^ repeat length –> lower age at onset, but hard to predict
myotonic dystrophy - mechanism?
repeat expansion in 3’ UTR (polyglutamine)
loss of RNA processing factors
50+ repeats = pathogenic
polyglutamine repeat disorders (2)
Spino-Cerebellar Ataxia (SCA)
Huntington
Friedrich Ataxia - mechanism?
trinucleotide repeat disorder
RECESSIVE
gene: FXN / GAA repeat
sensitivity
specificity
PPV
NPV
analytic validity
clinical validity
clinical utility
Disease Disease
+ -
Test + – PPV CLINICAL VALIDITY
Test - – NPV + penetr + heterog
| |
sensitivity specificity
ANALYTIC VALIDITY
clinical validity is PPV/NPV, the pentrance of condition, and whether it’s genetically heterogeneous: ability to predict phenotype based on test
analytic validity is sensitivity/specificity for mutation(s) of interest
clinical utility: if know genotype, how useful is it for patient care?
analytic validity
clinical validity
clinical utility
analytic validity –> is mutation there?
clinical validity –> predict pheno?
clinical utility –> useful to patient if know mutation?
CLIA
Clinical Laboratory Improvement Amendments
- equipment
- turn around time
- personnel
- validation
- regular quality control
- interpretation !!
- comments on patient-specific results
outside US and research labs – not CLIA-certified
HexA - purpose
HexA enzyme is defective in Tay-Sachs.
Ashkenazi population has founder mutation(s?). Thus, DNA testing will pick it up 97% of time.
If person is non-AJ, may beed to do HexA enzyme testing, instead.
FraX – preferred test?
Southern Blot – because can visualize BIG del/dups
frequent/recurrent mutations in e.g., CF, sickle cell – assess by which testing technique, frequently?
oligo binding assay
- oligo to mutation binding
- oligo to normal sequence binding
- measure amount of bound DNA by ligating both oligos to addiitonal oligo. One of two will not ligate together properly. Then can assess differences in size between final oligos.
histones wraps ? bp of DNA as part of nucleosome
histone = 146 bp of DNA = beads on a string
nucleosome made up of —
146 bp of DNA,
2x ( H2A, H2B, H3, H4,) …. +H1
what modifications are added to histones as part of gene regulation? Do these modifications increase or decrease transcription?
acetyl groups, or methyl groups are added to Lysines of histones
acetyl groups increase transcription, methyl groups can decrease it
after beads -on-a-string, what is next level of DNA compaction? how wide is it?
fiber.
30 nm.
(each nucleosome is 11 nm)
after 30 nm fiber, the next sized DNA coil structures are are –
300 nm extended chromatin coil,
followed by
700 nm condensed (ready for mitosis)
3 checkpoints and what they monitor in Mitosis
G1/S - DNA integrity, cell size
G2/M - DNA synthesis/damage
M - spindle formation/attachment to kinetochore
cyclins/cyclin-dependent kinases - function? mechanism?
regulate cell cycle.
- cyclins - made and destroyed
- cdks - active only when cyclins around
cycles of phosphorylation/dephosphorylation of proteins necessary for various stages of cell cycle
tumor suppressor genes - mechanism?
regulate cell cycle
initiate apoptosis if errors
crossing over occurs during __ phase of Meiosis __
Prophase I of Meiosis I (not metaphase!!)
amino acid - parts?
amino group (NH3) carboxyl group (COOH) central carbon R group (H or bigger)
characteristics of genetic code
degenerate non-overlapping unambiguous e.g., AUG always met universal -- minor variation in prokaryotes, and within MITOCHONDRIA!!! e.g., AGA/AGG = Arg in nuclear, but STOP in mitochondria.
rRNA - significance, cytogenetic location
rRNA makes up the ribosomes (60S and 40S subunits)
located on short arms of acrocentric chromosomes
5S (subunit of ribo) rDNA is in tandem array on chr 1. (2,000 copies!)
Translation initiation
small ribosomal subunit (40S) loaded with:
- initiator t-RNA - met (distinct from other met tRNA)
- GTP
- initiation factors (eIFs) – can also act as helicases
complex binds 5’ cap of mRNA, scans till AUG codon
@AUG, eIFs leave/ large 60S subunit binds - ready for protein synthesis.
Translation - what happens at EPA sites?
A - “charged” tRNA enters with amino acid
P - existing aa – forms peptide bond with aa at A site, as everything shifts toward E
E - exit site - “uncharged” tRNA (with its aa detached) sits
is there a start tRNA? is there a stop tRNA?
yes start - met tRNA - it’s specific to start, and initiates translation
no stop tRNA - instead, a “release factor” goes into A site, and releases nacent polypeptide chain
non-sense mediated decay - function? mechanism?
degrades prematurely truncated proteins due to non-sense mutation (premature stop codon)
exon junction complexes (EJC) remain on mRNA after splicing out introns. typically, ribo comes across a few before reaching stop. yet if there is premature stop, a part of the ribo contacts the next EJC, and unleashes a clipping of the 5’ cap, thus leading to degradation of the mRNA.
purpose/structure of cap of mRNA
methyl group
GMP (via 5’ - 5’ bond; unusual)
phosphate group removal
function:
to mark mRNA as “eukaryotic” vs prokaryotic
stability
recruitment/transport to ribosome
function of 2’ hydroxyl (OH) group of RNA is –
function of 3’ OH, later –
(vs DNA is de-oxy…)
used during splicing of introns
splicing is via TWO consecutive transesterification reactions
- 2’ OH of conserved A on intron attacks 3’ end of left exon (exon junction) –> this forms lariat
- 3’ OH of this left exon then attacks 5’ end of right exon
- Exon Junction Complex (EJC) marks splice site as “complete”
CFTR - what is splicing-related mechanism, and how does it modify CF/symptom severity?
exon 9 splicing depends on length of U prior to it. Can have 7xU, 9xU, or 5xU. If have 5 U’s (aka 5T), this exon likely to be skipped and gives non-functional CFTR protein.
5T prevalence is 10%.
Alone, unlikely to cause disease, but if homozygous can cause male infertility. In combination with mild CF mutation, can act as severe mutation. 5T in combination with severe mutation may have CF symptoms.
alternative way of producing shorter protein, other than gene splicing
substitution editing (!!)
C–>U change ONLY in the RNA that produces a premature stop codon. Can be specific to organ system. e.g., ApoB is shorter in gut, vs liver.
A–> I (inosine, deaminated): I is read as G. – happens to 1,000 genes!
3’ processing
cleavage of some RNA off the end
capped by PAB protein –> signals “maturity” of mRNA
chance of a complex disease in a first degree relative = (rule of thumb)
risk to third degree relatives?
square root of population frequency
e.g., schizophrenia is 1% prevalence
thus, risk to FDR is sq rt (0.01) = 0.10 = 10%
which is true. Risk to FDRs for sz is 10-15%.
third degree relatives =~ population risk
3 factors that affect complex disease risk in a family
- age-at-onset (earlier than average?)
- less commonly-affected sex (females are less commonly affected than males)
- severity of condition
Cornelia de Lange - prevalence, features
CNS, facial (synophris) + arched, limb/bone defects, microcephaly, diaphragmatic hernia on ultrasound, GERD, short neck, cystic hygroma, low posterior hairline, hypertrichosis, long philtrum, thin upper lip, downturned corners, midface hypoplasia, 20% cleft palate, cryptorchidism, cesicoureteral reflux, micropenis, hearing loss, myopia, LONG eyelashes, LIMB ABNORMALITIES
check for: heart defects, gastrointestinal abnormalities
NIPTBL - chr5q - majority
SMC1A - X
SMC3 - chr10
almost 100% de novo; autosomal dominant
male to female ratio 1:1.3
McCune Albright - features, inheritance
- -one-sided cafe au lait spots, with ragged edges (vs smooth like in NF1)
- -fibrous dysplasia of bones, – many bone fractures
- –osteomas (1%)
- -endocrine features (thyroid)
due to somatic MOSAICISM, thus NOT INHERITED
Rett syndrome - features, inheritance
- almost exclusively affects girls, 1 in 12,000
- HAND-WRINGING/hands-to-mouth
- autism-like (social/emotional)
- random grimace/facial exressions
- inconsolable crying
- bouts of pain
- REGRESSION at ~18 months
- seizures (80%)
- microcephaly
- breath holding
- absent speech
often confused with Angleman, autism
MECP2 gene, X-linked, 95% de novo!!
Bardet-Biedl syndrome
rare 1 in 140,000
polydactyly obesity retinitis pigmentosa (one of first symptoms, in childhood) hypogonadism renal failre intellectual disability
14 genes - BBS10
autosomal recessive
derive heritability from disease concordance rates of MZ and DZ twins, if
MZ = 0.40
DZ = 0.25
Heritability = TWICE the difference of concordances
MZ=0.40
DZ=0.25
Heritability = (0.4-0.25)2 = 0.152 = 0.30 = 30%
heritability definition
portion of phenotypic variability that is attributed to genetics
e.g., phenotypic variability is 100
and genetic variance is 40
then heritability is 0.40 (40/100)
LD is NOT important in assessing linkage studies, but IS important for assessing association studies (T/F)
True. LD is NOT important in assessing linkage studies, but IS important for assessing association studies
Thus stuff like population substructure, admixture, etc, only influences association, but not linkage.
If find linkage signal, but NO association, maybe the linked marker is NOT in LD with the true locus. It can still be co-inherited, but it won’t be associated on a GWAS.
family studies good for –
estimating heritability
if marker is 3 cM from disease locus, and person does NOT inherit marker – chance of having inherited the disease locus (and thus having had a recombination event between locus and marker)?
0-3%
