Mutations Flashcards
What do SMN1 mutations cause and how can this be treated?
cause spinal muscular atrophy; alternative splicing resulting in splicing out of the mutation-containing exon (exon 7)
what is caused by abnormal splicing of CD44?
tumor metastasis (fun fact: splice variants can be used as diagnostic and prognostic markers)
APC mutations cause (and, what is APC)
colon cancer; transcriptional regulatory protein
pRB mutations cause (what is pRB)
retinoblastoma and small cell lung cancer; transcriptional regulatory protein; inhibits cell cycle, and is inactivated by CDK phosphorylation (and cell can divide)
XP-B and XP-D mutations cause (and what are these proteins)
Cockayne’s syndrome, xeroderma pigmentosum, trichothiodystrophy; they are both subunits of TF2H and are transcriptional regulatory proteins. Specifically, XP-B opens the DNA at the promoter to permit initiation of transcription
p53 mutations cause
most cancers; p53 is a transcriptional regulatory protein
c-myc mutations cause
Burkitt’s lymphoma; c-myc is a transcriptional regulatory protein
RAR mutations cause
acute promyelocytic leukaemia; RAR is a transcriptional regulatory protein
SRY mutations cause
sex reversal; SRY is a transcriptional regulatory protein
PIT-1 mutations cause
dwarfism; PIT-1 is a transcriptional regulatory protein
XH-2 mutations cause
ATR-X (X-linked mental retardation); XH-2 is a transcriptional regulatory protein
What happens if there is decreased phosphorylation of E1F-4E?
does not bind the G-cap in the cytoplasm as tightly, resulting in overall decrease of cellular translation
Which snRNA recognizes the 5’ splice site (and what is the signal?)
U1 snRNA; signal is GU
Which snRNA recognizes the A-branch point?
U2 snRNA
What is the signal for the 3’ splice site, and what protein binds it?
AG; U2AF (U2 associated factor)
Explain the 2 biochemical steps of splicing
- the 2’ OH of the A at the branch point attacks the 5’ splice site phosphate
- The 3’ OH of exon 1 attacks the 5’ phosphate at the 3’ splice site.
What are the 2 enzymatic steps of 3’ polyA tail addition
- cleavage of 3’ OH
2. polyadenylation by hydrolysis of ATP –> AMP + PPi
What is one example of the result of alternative polyA site choice?
membrane bound (B cells) vs. secreted (plasma cells) forms of IgM
What is the polyA site consensus sequence?
AAUAAA
The polyA site consensus sequence is usually how many nucleotides upstream from the 3’ terminal polyA tract?
11-30
A single nucleotide change in the polyA site consensus sequence can cause which disease? (And what is the change?)
Alpha-thalassaemia (AATAAG instead of AATAAA)
What is one hypothesis for how alternative cleavage and polyadenylation activate oncogenes in cancer cells?
Shortening of the 3’ UTR may provide fewer target sequences for regulation of mRNA degradation by miRNA
What is the function of snRNA
splicing, located in the nucleus
What is the function of snoRNA
maturation of rRNA (forms ribosome), located in the nucleolus
what is the function of telomerase RNA?
template for telomere synthesis
What is the length of miRNAs, what is the precursor and what are some of their functions?
about 21 nucleotides long; derived from hairpin precursor; they bind to sites on the 3’ UTR of mRNA; an imperfect match results in degradation of mRNA OR repression of translation; a perfect match results in degradation of mRNA; they can also target the coding region or the 5’ UTR
What is the length of siRNAs, what is the precursor and what are some of their functions?
about 22 nucleotides long; derived from long double-stranded RNA; perfect match; target transposon or virus (mRNA degradation, transposon degradation, chromatin formation, transcriptional silencing)
What is the length of piRNAs, what is the precursor and what are some of their functions?
about 24-27 nucleotides long; long precursors in flies and mammals that are processed and can be amplified; target transposon (primary roles in spermatogenesis, germline, and control of mobile elements (transposons); transcriptional repression; may play role in epigenetic inheritance in worms)
What do drosha and dicer do
first, drosha processes newly transcribed primary miRNA in the nucleus –> becomes pre-miRNA. Dicer cleaves pre-miRNA into 21-25 nt strands that fold back in hairpin structures (with 2-base overhang on 3’ ends). Dicer also cleaves siRNA
What do argonaute proteins do
associated with RISC complex (RNA-induced silencing complex), cleave mRNA with miRNA, siRNA, piRNA
What are the functions of lncRNAs and what is their size
over 200 nucleotides long
regulate development of skeletal muscle, cardiac muscle, and immune response
control gene expression by:
- form complexes with protein, mRNA, miRNA
- affect DNA methylation
- histone modifications
- chromatin structure
How can antisense oligonucleotide therapeutics help treat diseases?
DNA oligonucleotides bind to mRNA (RNase is an enzyme in the cell that cleaves RNA-DNA hybrids–b/c they should not naturally occur in the cell)
bind at splice sites and force alternative splicing
siRNA binds to mRNA, RISC cleaves
anti-miRs bind to miRNA and inhibit function
mimic miRNA
A western blot measures _______ by using labeled _______.
protein
antibody to the protein
A southern blot measures _______ using labeled ________.
DNA
DNA or RNA hybrid probe
A northern blot measures ______ using labeled _______.
RNA
DNA or RNA hybrid probe
What are different types of vectors and what are they used for?
plasmid (25 kb, transformation)
bacteriophage (25 kb, infection, used for gDNA/cDNA libraries)
cosmid (combo of plasmid and phage, larger inserts of 45 kb are possible)
bacterial artificial chromosome (300 kb, genome sequencing, chromosome mapping)
yeast artificial chromosome (2 Mb, genome sequencing, chromosome mapping)
retrovirus (gene therapy)
How does gel electrophoresis separate DNA?
DNA is cut by restriction enzymes, then run through the gel, migrates toward the anode b/c DNA is negatively charged, the shorter fragments can travel faster through the gel b/c of smaller size
How does SDS-PAGE separate proteins?
Heat denatures proteins so they are unfolded, SDS adds negative charge, PAGE (polyacrylamide gel electrophoresis) separates by size (smaller proteins travel faster)
How does microarray analysis work?
Compares gene expression in different cell types
make 2 cDNA libraries, 1 from each cell type, using 2 different dyes
microarray chips contain hybridization probes, can contain 8600 genes
if gene expression equal in both cell types, color will be mix of the 2, more of 1 color indicates that this gene is expressed more in that cell type
What are the 3 stages and temperatures for PCR?
denature (95)
anneal (55)
elongate (72)
What is a Restriction Fragment Length Polymorphism, and what is a disease that can be diagnosed by this method?
When there is a mutation that destroys the normal site of cleavage of a restriction enzyme–apply enzyme and get different result, tells you there is a mutation (example: HbS mutation that causes sickle cell anemia destroys the MstII site)
cyclin proteins are needed for _________ of CDK
activation
The Kip/Cip family of CDKNs include the proteins __________ (encoded by the genes ________) and are __________ of all CDKs
p21, p27, p57
CDKN1A, B, C
inhibitors
The INK4 family of CDKNs include the proteins __________ (encoded by the genes ________) and are specific inhibitors of ________.
p16, p15, p18, p19
CDKN2A, B, C, D
CDK4/6-cyclinD1-3 complexes
p16 CDKN2A is a ___________ and mutations in this gene can cause what types of cancer?
tumor suppressor
melanoma, head and neck cancer, non-small cell lung cancer
What do mitogens/growth factors do?
signal at cell surface –> up-regulate transcription of cyclinD1-3 –> more cyclinD1-3-CDK 4/6 complexes –> inactivate Rb –> cell enters S phase
How many origin sites are in the human genome?
100,000
What are pre-replication complex formations, what is a major protein in the complex that opens the DNA helix for replication, and how do the pre-RC formations prevent re-replication?
multiprotein complexes that are loaded onto the origins in G1 phase
MCM DNA helicase
pre-RC can only be loaded in G1, and only activated in S (can’t be loaded and activated in the same phase)
Which to sets of protein kinases are required for pre-RC activation in S phase?
S-CDK and DDK
Where are the 2 major checkpoints located, what are their purposes, and what are the proteins that inhibit CDK cyclin complexes?
Before M –> checks for DNA damage after S phase, blocks entry into M (p53, Chk2, ATM, ATR)
During M –> checks for damage of spindle fibers and blocks exit from M if there is damage
Mutated p53 or Chk2 can cause what disease?
Li Fraumeni syndrome
What does Km describe?
The amount of substrate needed for the enzyme to function at 1/2 Vmax
What does Kcat describe?
The turnover number (the number of substrate molecules converted to product in a given unit of time on a single enzyme molecule when the enzyme is saturated with substrate
When Kcat/Km approaches ________ the enzyme is nearing “catalytic perfection)
10^8 M^-1s^-1
What is the TATA-box binding protein
TFIID
What TF is involved in both transcription initiation and repair?
TFIIH
What does RNA Pol 1 do
rRNA
What does RNA Pol2 do
mRNA, regulatory RNAs (snRNA, snoRNA, lncRNA), small non-coding RNAs (siRNA, miRNA)
What does RNA Pol3 do
tRNA
What proteins are involved in detection of double-strand DNA breaks?
ATM and ATR –> phosphorylate BRCA1 –> activated BRCA1 determines whether HR used to repair DNA double-strand breaks
What are the reasons DNA double strand breaks can occur naturally?
meiosis
class switching (B-cells and T-cell receptors)
DNA replication
retroviruses/retro-transposon insertion
What are some reasons DNA double strand breaks can occur (not naturally)?
ionizing radiation
medical imaging
What are the 2 pathways of repair of double-strand DNA breaks, and what are the proteins that determine which path to take?
Homologous recombination (determined by phosphorylated BRCA1)
NHEJ (non-homologous end joining, determined by 53BP1)
misregulated homologous recombination can lead to ___________
heterozygosity
about how many NHEJ “footprints” are there on an average 70-year-old somatic cell?
2,000
What types of DNA damage are repaired by direct reversal?
specific DNA strand break
alkylation (repaired by MGMT, silence by promoter methylation in 45% of human glioblastomas)
UV damage (though more frequently repaired by NER
Which type of DNA repair uses glycosylases?
base excision repair
What are 2 drugs that inhibit reverse transcriptase?
ddl
AZT
Which diseases are caused by accumulation of purines in the tissues?
gout
Lesch-Nyhan disease
