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entire nucleic acid sequence that is necessary for the synthesis of a functional gene product
of the same origin, but in different species
very similar sequences that differed due to divergence/duplication
overall family of genes that are similar in sequence to each other
controls transcription of the gene, bound by transcription factors
upstream of sequence, binding site for transcription initiation factor and polymerase
Solitary vs. Duplicated Genes
- solitary -one copy per haploid genome
- duplicated - genes with similar seq nearby, gene families
Process of gene duplication (ex. tubulin)
- ancestral cell had one tubulin gene, which got duplicated into two (alpha and beta)
- Replication occurs, the duplicated gene accumulates mutations and diverges as time goes on
Example of tenderly repeated gene
ribosomal RNA encoding gene
Example of repetitious DNA
- simple seq DNA
- interspersed repeats (mobile elements)
Examples of interspersed repeats
- mobile elements -
- LTR retrotransposons
- non-LTR retrotransposons (SINEs/LINEs)
DNA transposons vs. Retrotransposons (basic mechanism)
- transposon - cut and paste
- retrotransposon - copy and paste
How does a retrotransposon jump from one gene to another?
- Donor DNA is transcribed into an RNA intermediate with the help of RNA polymerase
- Reverse transcriptase creates DNA intermediate from the RNA intermediate
- that DNA intermediate is then inserted into target DNA
2 ways transposons cause exon shuffling?
- (1) if two transposons jump onto either side of an exon, and cell cuts from beginning of first transposon till end of second transposon, the exon in the middle will be taken along to the new gene, and therefore shuffles exon and creates new gene
- (2) retrotransposon is transcribed but instead of stopping at the end, accidentally transcribe to the end of nearest exon, which is then copied into another gene = exon shuffling
Nuclear vs. non-nuclear genome
- nuclear- DNA and genes that make us up
- non-nuclear - genome in organelles, such as in mitochondria or chloroplasts
- (endosymbiont theory)
What does a karyotype show?
numbers, shape and size of chromosomes
Traditional vs. Modern methods for visualizing mitotic chromosomes
- traditional - Giemsa
- modern - FISH
Process of Flourescence in situ Hybridization (FISH)
- 1. use formaldehyde to freeze everything into place
- 2. prepare DNA probe with fluorescently labelled nucleotides
- 3. Denature DNA strand of interest so it is single-stranded
- 4. complementary strand will hybridize to fluorescent probe
- 5. Under microscope, we can localize the probe/sequence by looking at fluorescence
At which stage are chromosomes most condensed?
Structure of mitotic chromosome
two sister chromatids joined at the centromere, which telomeres at the end of chromosomes
Why is chromosome x-shaped in mitosis?
After replication, single chromosome becomes two chromosomes but the sister chromatids are held together at the middle until they are ready to let go
Compaction of chromosomes in interphase vs. metaphase
- interphase - highly extended structure (replication, transcription)
- metaphase- tightly packed chromosomes
Levels of DNA compaction
- DNA double helix
- beads on a string
- packed nucleosomes
- loop structures
- condensed chromosome
What is a nucleosome? Structure?
- basic repeating unit of chromatin, first step of compaction
- Structure - 8 histone proteins, with DNA wrapped around the core of histones, slightly less that 2 full turns around
Histone modifications - writing vs. erasing vs. reading
- Writing - enzyme puts modification on histone tail
- Erasing - proteins remove modification from tail
- Reading - proteins can bind to histone tails if that tail is modified with something
3 ways histones can modify function?
- Change chromatin compaction
- inhibit binding of certain proteins
- Facilitate binding of certain proteins
Function of H3k9me3 in heterochromatin formation
- H3K9 methyl transferase (writer) puts a methyl group on the K9, which allows binding of the protein HP1
- HP1 comes in and binds to Me3, HP1 attracts other HP1 proteins and oligomerizes, bringing the nucleosomes together and compacting the the heterochromatin
If there is a mutation that abolishes interaction between HP1 proteins during heterochromatin formation, what would be the effect on spreading?
heterochromatin will not be as close together or as compact because no oligomerization
What is a centromere?
region of chromosome that controls movement during cell division
What is the kinetochore?
protein structure at centromere that attaches the centromere to mitotic spindles
What is a telomere?
end of chromosome with repetitive sequence
Spreading of the Sir complex? What is it involved in?
- Involved in heterochromatinizing telomeres
- 1. Sir 2/3/4 complex brought to telomeres
- 2. Sir 4 protein binds to hypoacetylated tail
- 3. Sir 2 deacetylates nucleosomes, making more landing sites for sir 4 so that sir 4 can continue to spread
- 4. oligomerization occurs in the end -> condensed structure
Counterpart to Sir 2 and Sir 4?
- Sir 2- H3K9 HMT (instead of putting modification, it creates landing site by clearing out proteins)
- Sir 4- HP1 (reader of H3K9, in this case Sir 4 is the reader of the lack of acetylation in the tails)
How are chromatin loops made?
SMC protein complexes
How do histone modifications differ for eu/hetero chromatin?
- Heterochromatin = very condensed, so a lot of tri-methylation
- Euchromatin = much more open, more acetylation
3 ways histones can be modified?
- 1. Change chromatin compaction (if a modification neutralizes the positive charge on histones, that may reduce the affinity between histones and DNA, the more acetylation you have the less compacted the chromatin will be)
- 2. inhibit binding of certain proteins (a modification may get in the way of a protein trying to bind to histone/DNA)
- 3. facilitate binding of certain proteins (provides a flag for the protein to come and bind so that it will do its job correctly)
When is chromosome conformation capture technique used?
When you want to know what 2 regions of chromatin are close to each other so that you can localize chromosomes
Chromosome conformation capture technique?
- 1. cross-link chromosomes near each other, digest them, and then ligate to make a loop
- 2. uncross-link the DNA and purify it
- 3. create libraries with PCR
- 4. Illumine sequencing - cluster DNA clones by bridge PCR
- 5. cut/denature bridges so they are single stranded
- 6. use flourescently labelled nucleotides to read seq with a scanner
What does deamination of methyl-cytosine do?
- Changes C to T
- results in incorrect base pairing that needs to be repaired
At what cell cycle stage is homologous recombination most likely to happen?
- because this is when homologous chromosomes are held together very closely
Base excision repair
- DNA glycosylase removes incorrect T nucleotide
- site is left without a base
- APEI endonuclease recognizes this empty space and cleaves backbone
- DNA Pol beta adds correct nucleotide
- Ligase comes in and repairs it
Mismatch excision repair
- Endonuclease recognizes mistake
- calls for helicase and exonuclease
- DNA helicase separates the two strands
- DNA exonuclease removes 25 bp around mismatched region
- polymerase and ligase fixes it
Repair of T-T dimers by Nucleotide Excision Repair
- UV radiation creates thymine-thymine dimer, which bulges out of DNA strand
- Proteins recognize this building as a mistake
- Helicase opens up double helix
- Endonuclease nicks on both sides of damage and removes damaged DNA strand
- Leaves a gap, which is fixed by polymerase/ligase
Proofreading mechanism by polymerase
- Incorrect base is added, leading to structural change in polymerase b/c it recognizes structure isn’t right
- conformational change in polymerase removes the strand that is being polymerized out to exonuclease site
- Mispaired base is cleaved
- Strand returns to catalytic site, and polymerase continues adding nucleotides
Prevention of erroneous recombination via repeats
How can heterochromatin prevent homologous recombination?
What is fidelity?
correct sequence to what it was like before, restore the sequence perfectly
Why does non homologous end joining not have good fidelity?
because it makes a lot of mistakes, DSB does not leave perfect blunts all the time
Repair of DSB by non homologous end joining
- When there is no HR available, these proteins recognize the break and try to fix it
- * Binds to double stranded break
- * Makes this end blunt by removing the extra bases that are sticking out
- * Introduces random deletion inside repair area
Are nicks or double stranded breaks more likely to cause genome rearrangements?
DSB b/c one end of chromosome may be joined to another end of the chromosome, which leads to a lot of chromosomal tanslocation due to incorrect repair of double stranded breaks
Cancer cells have lots of mutations why don’t they die?
cancer cells overcome mechanisms that limit growth (such as telomere shortening)
Role of sigma factors
bind to promoter DNA and signal the RNA polymerase where to bind and start
What kind of factor is the sigma factor?
transcription initiation factor because it is released after initiation
What is processivity?
how long an enzyme can grab/hold onto something
Role of Rho factor?
has RNA helicase activity, disrupts the mRNA-DNA-RNA polymerase complex
Rho-dependent transcription termination
- Once RNA polymerase starts transcribing DNA, Rho protein attaches to recognition site on Rna in growing chain
- starts moving along RNA as polymerase moves forward
- polymerase pauses at the end
- Rho catches up, unwinds the DNA-RNA hybrid, which releases the RNA from the DNA
Imagine you isolated a Rho mutant that slows down its migration along RNA. What effect do you predict on transcription?
it would be copied farther along in genome because it will not be able to catch up with the RNA polymerase
Rho-dependent transcription termination
- Inverted repeat is followed by a string of A’s
- when the inverted repeat is transcribed into RNA, it folds into a hairpin loops, causing RNA polymerase to pause
- the hydrogen bonds between A/U break
- RNA transcript breaks off from template, terminating transcription
What is a promoter?
DNA that directs RNA polymerase to bind and begin transcription
What is an operator?
Specific DNA in a bacteria that controls the transcription of an adjacent gene
Is the trp operon positively or negatively regulated?
- Negative - lac repressor
- Positive - CAP protein (when bound to DNA, it activates transcription, when not bound there is no transcription)
Regulation of glutamine at high levels
- * Glutamine binds to sensor domain of Ntrb protein, it senses concentration of glutimaine in cells
- * In presence of glutamine, sensor domain prevents kinase domain to act, Ntrb protein not phosphorylated and its inactive
- * Second component - NtrC : two domains
- * Regulatory (contains aspartic acid)
- * DNA-binding protein
- * In the absence of phosphorylation of aspartic acid, regulatory domain prevents DNA binding domain from binding to DNA
Regulation of glutamine at low levels
- Gln leaves, sensor domain changes conformation
- HKTD is free to do its kinase activity aka phosphorylate
- phosphorylates its own histodine
- after it autophosphorylates itself it transmits phosphorylation to the aspartic acid in ntrC
- which releases DNA binding protein, free to bind to DNA
How is prokaryotic enhancer involved in starting transcription?
signal from enhancer is what tells the cell to start trx after sigma factor and RNA polymerase bind to the promoter
What is a dominant visible allele?
has one recessive copy and one wild type copy, but the phenotype is still visible (ex: can see the wings curled)
3 properties of balancer chromosomes
- 1. have multiple inversions to prevent recombination
- 2. carry at least one recessive lethal mutation
- 3. carry at least one dominant marker
Test used to figure out if two mutations lie on the same gene?
What is complementation?
ability to replace a missing function
Describe process of complementation test
- cross together two mutations that produce the same phenotype
- - if the phenotype is NOT seen, the mutations are NOT in the same gene
- - if the phenotype IS seen, the mutations ARE on the same gene
What does EMS do?
causes errors in DNA replication
How to screen for recessive mutations?
- Ecpose males to EMS, carry mutations in their sperm cells
- * Cross them to wild type females
- * Collect male progeny from this cross
- * High probablity that males collected have some genes that are mutated
- * Each male has a different mutation because they each derive from a different sperm cell
- * Important to collect a lot of different mutants
- * Cross each mutant back to wild type female
- * Still only expect to see wild types because they are heterozygotes
- * Allow the progeny flies to mate with each other
- * recessive mutations appear in second generation
Why is crossover a problem when balancing mutations to keep stable stocks?
- If you have crossover during meiosis 1, then what you can create are recombinant chromosomes
- * One chromosome will have both lethal alleles and the other will have both wild type alleles
- * The wild type allele will take over the stock, no more balance
- * Recessive alleles make make the fly weak as heterozygotes
Solution for recombination while trying to make stable stocks?
- Multiple inversions on balancing chromosomes
- single inversion- chromosomes still pair by twisting themselves in pretzel shape
- multiple - chromosomes don’t pair, therefore preventing recombination
What does it mean if a gene is autosomal dominant?
not sex-linked, only need one copy of gene for it to show
What does autosomal recessive imply?
equal probability for sons and daughters to be affected
Why do males have higher probability of acquiring x-linked diseases?
All it takes is mother to be carrier
How can females be affected by x-linked diseases?
mother has to be carrier and father has to be affected
Role of TATA box?
- important for initiation factor to bind
- where RNA polymerase complex assembles
What do promoter-proximal elements do?
close to promoter, important for recognition and involved in tethering - forming DNA platform for initiation factors to bind
What do enhancers do?
control tissue specific and temporal expression
2 types of mammalian promoters
- 1. with a TATA box
- 2. CpG island promoter gene - transcribed at a lower level, contain proximal promoter elements, may contain seq resembling TATA box, less precise transcription start sites (may be more than one start site), support transcription in both directions (divergent transcription)
Why may CpG methylation be mutagenic?
because when you lose C’s, turns methylated C’s into T’s which is a problem
What do basal promoter elements do?
tell transcription factors where the promoter is
What does a mediator do? How?
- transcription coactivator that mediates interactions between enhancers and polymerase and promoter
- * Makes a loop between enhancer and promoter
- * Does this by having a lot of proteins that can physically interact and recruit
Role of co-activators in transcription?
to open up DNA for transcription
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