Midterm 2

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 -one copy per haploid genome
• duplicated - genes with similar seq nearby, gene families

• 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

ribosomal RNA encoding gene

• simple seq DNA
• interspersed repeats (mobile elements)

• mobile elements -
• transposons
• LTR retrotransposons
• non-LTR retrotransposons (SINEs/LINEs)

• transposon - cut and paste
• retrotransposon - copy and paste

• 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

• (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- DNA and genes that make us up
• non-nuclear - genome in organelles, such as in mitochondria or chloroplasts
• (endosymbiont theory)

numbers, shape and size of chromosomes

• traditional - Giemsa
• modern - 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

metaphase

two sister chromatids joined at the centromere, which telomeres at the end of chromosomes

After replication, single chromosome becomes two chromosomes but the sister chromatids are held together at the middle until they are ready to let go

• interphase - highly extended structure (replication, transcription)
• metaphase- tightly packed chromosomes

• DNA double helix
• beads on a string
• packed nucleosomes
• loop structures
• condensed chromosome

• 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

• 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

• Change chromatin compaction
• inhibit binding of certain proteins
• Facilitate binding of certain proteins

• 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

heterochromatin will not be as close together or as compact because no oligomerization

region of chromosome that controls movement during cell division

protein structure at centromere that attaches the centromere to mitotic spindles

end of chromosome with repetitive sequence

• 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

• 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)

SMC protein complexes

• Heterochromatin = very condensed, so a lot of tri-methylation
• Euchromatin = much more open, more acetylation

• 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 you want to know what 2 regions of chromatin are close to each other so that you can localize chromosomes

• 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

• Changes C to T
• results in incorrect base pairing that needs to be repaired

• S-Phase
• because this is when homologous chromosomes are held together very closely

• 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

• 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

• 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

• 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

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correct sequence to what it was like before, restore the sequence perfectly

because it makes a lot of mistakes, DSB does not leave perfect blunts all the time

• 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

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 overcome mechanisms that limit growth (such as telomere shortening)

bind to promoter DNA and signal the RNA polymerase where to bind and start

transcription initiation factor because it is released after initiation

how long an enzyme can grab/hold onto something

has RNA helicase activity, disrupts the mRNA-DNA-RNA polymerase complex

• 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

it would be copied farther along in genome because it will not be able to catch up with the RNA polymerase

• 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

DNA that directs RNA polymerase to bind and begin transcription

Specific DNA in a bacteria that controls the transcription of an adjacent gene

• Both
• Negative - lac repressor
• Positive - CAP protein (when bound to DNA, it activates transcription, when not bound there is no transcription)

• * 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

• 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

signal from enhancer is what tells the cell to start trx after sigma factor and RNA polymerase bind to the promoter

has one recessive copy and one wild type copy, but the phenotype is still visible (ex: can see the wings curled)

• 1. have multiple inversions to prevent recombination
• 2. carry at least one recessive lethal mutation
• 3. carry at least one dominant marker

complementation test

ability to replace a missing function

• 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

causes errors in DNA replication

• 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

• 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

• Multiple inversions on balancing chromosomes
• single inversion- chromosomes still pair by twisting themselves in pretzel shape
• multiple - chromosomes don’t pair, therefore preventing recombination

not sex-linked, only need one copy of gene for it to show

equal probability for sons and daughters to be affected

All it takes is mother to be carrier

mother has to be carrier and father has to be affected

• important for initiation factor to bind
• where RNA polymerase complex assembles

close to promoter, important for recognition and involved in tethering - forming DNA platform for initiation factors to bind

control tissue specific and temporal expression

• 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)

because when you lose C’s, turns methylated C’s into T’s which is a problem

tell transcription factors where the promoter is

• 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

to open up DNA for transcription