Exam 3 Flashcards

Question

What are some examples of mitochondrial DNA depletion syndromes? How do they differ?

Answer 1

* Alpers Syndrome * Toddler form of hepatic mtDNA depletion syndrome * Characterized by diffuse cerebral degeneration, psychomotor retardation, intractable epilepsy, and liver failure * Progressive external ophthalmoplegia (PEO) * Characterized by chronic, progressive, bilateral, and usually symmetric ocular motility deficit and ptosis * Accompanied by myasthenia gravis * Ataxia-neuropathy syndrome * Similar to PEO but neuromuscular defects preceed ocular ptosis

Answer 2

* Telomerase adds more T and G residues to the TG primer of DNA * This repositions the internal template RNA and allows the addition of more T and G residues * The complementary stand is synthesized by cellular DNA polymerases * The single-stranded tail synthesized by telomerase is folded back and paired with its complement in the duplex portion of the telomere * Telomere is bound by several telomere-binding proteins (TRF1, TRF2)

Answer 3

* Each mitochondrion contains 2-10 copies of the ~17 kb genome, which are not evenly distributed during cellular division * Many proteins required for mitochondrial function are nuclear encoded, including the DNA Polymerase Y used in mtDNA replication * Heteroplasmy - one cell contains multiple types of mtDNA with varying levels of each * Can cause complicated disease patterns when deleterious mutations are present

Answer 4

* Genetic, neurodevelopmental disorder due to defects on the X chromosome * Only affects females * Second only to Down syndrome for genetic causes of mental retardation in females * Effects the way DNA is packaged in a cell * Molecular cause is defects in the methyl-CpG-binding protein 2 involved in chromatin conformation

Answer 5

* Irregularly-shaped region within the cell of prokaryotes which has nuclear material without a nuclear membrane

Answer 6

* Base pair rule * A = T/U * C = G

Answer 7

* DNA backbone is alternating phosphate and pentose residues * Backbone is hydrophilic - phosphate groups are completely ionized * Bases are hydrophobic

Answer 8

A combination of Van de Waals and dipole-dipole interactions between bases

Answer 9

* Base stacking * Not base specific * Includes various Van de Waals forces such as dipole interactions and London dispersion forces * Hydrogen bonds between bases * Weaker than base stacking * Specific for bases

Answer 10

* Van der Waals forces * Specific intermolecular interactions observed in liquids and solids * Electrostatic in nature, arising from interactions of positively and negatively charged species * Dipole-dipole * Intermolecular force between two molecules that have net dipole moments - asymmetrical charge distributions where polar molecules develop partial positive and partial negative charges * Type of Van der Waals force * London dispersion * Interaction between instantaneous dipole-induced dipole * All electrons come to one side of the atom, making it an instantaneous dipole that repels electrons of neighboring atoms * Type of Van der Waals force

Answer 11

* 10 base pairs (34 A, 3.4 nm) per turn of the helix * Later measurements revealed 10.5 base pairs (36 A, 3.6 nm) per turn * Major and minor grooves * Antiparallel * Bases are perpendicular to the backbone * DNA double helix is held together by base-stacking interactions and hydrogen bonding

Answer 12

* A form * Right handed helix * 26 A diameter * Anti glycosyl bond conformation * Found in dehydrated DNA such as during crystallization * Found in DNA-RNA hybrids or RNA-RNA double stranded regions * B form * Right handed helix * 20 A diameter * Anti glycosyl bond conformation * Z form * Left handed helix * 18 A diameter * Anti glycosyl bond conformation for pyrimidines, syn for purines * May be important in gene regulation * Deviations in structure do not effect complementarity, antiparallelity, A/T and G/C pairing

Answer 13

* Palindromes - sequences of double stranded nucleic acids with twofold symmetry. * In order to superimpose one region on the other, it much be rotated 180 degrees around both axises * Mirror - symmetric sequences within each strand itself

Answer 14

* Palindromic sequences can form alternative structures with intrastrand base pairing * Hairpin - when only a single strand is involved * Cruciform - when both strands of a duplex DNA/RNA are involved

Answer 15

* 3' untranslated regions of mRNAs * Best known to regulate mRNA-based processes, such as mRNA localization, mRNA stability, and translation * Mediated by hairpins and cruciform structures by providing a "handle" for proteins to carry the mRNA with

Answer 16

* G = U base pair only when presynthesized strands of RNA fold up or anneal with each other * No RNA polymerases insert a U opposite to a G or vise versa * A-form DNA is common in RNA secondary structure

Answer 17

* Denaturation * Disruption of the non-covalent interactions which hold the DNA duplex together * Can be caused by heat, extreme pHs, urea, other denaturing agents * DNA begins to complete denature at 80⁰ C * Annealing * Restoring partially denatured DNA * Can hold happen if DNA is not completely denatured

Answer 18

* Upon denaturation, DNA solutions become less viscous and absorb more UV light * Used as a standard to measure purity of DNA in preparations

Answer 19

* Different regions of DNA will have different melting temperatures (T_m) based on their relative amounts of GC base pairs * Result of 3 hydrogen bonds vs 2 * RNA double helices are more stable than DNA * RNA:DNA double helices are intermediate

Answer 20

* DNA * "Beads on a string" chromatin * 30 nm fiber * 1 loop (75,000 bp) * 1 rosette (6 loops) * 1 coil (30 rosettes) * 2 chromatids (10 coils each)

Answer 21

Topoisomerase II

Answer 22

* Humans have 2 versions of each chromosome, one derived from the maternal and one from the paternal source (diploid) * Each member of the chromosomal pair is called a homologous chromosome (same genes but possibly different alleles) * After DNA replication, each chromosome has an identical copy, joined by a centromere and referred to as sister chromatids (identical copies of a single chromosome)

Answer 23

* Positive - overwound DNA * Negative - underwound DNA

Answer 24

Topoisomerases relax positive supercoiling stress and maintain negative supercoiling by breaking DNA then resealing the breaks

Answer 25

* Form of gene regulation * Acts to sterically inhibit interaction with the DNA molecule * Acts by inhibiting the binding of trans-acting factors, typically repressing transcription * Sometimes methylation affects the bind of repressive factors and activates transcription instead * Occurs in promoter regions * Usually silences genes, but may also activate them

Answer 26

DNMT (DNA methyltransferase)

Answer 27

* Heritable changes in gene expression that occur without a change in DNA sequence * Permanent or reversible * Heritable environmental induction

Answer 28

* DNA methylation * DNA methyltransferases, DNMT * Histone modification * Histone deacetylases (HDAC) * Histone acetylases * Histone methyltransferases * Methyl-binding domain protein, MECP2 * Phosphorylation * RNA-mediated epigenetic regulation * Small RNAs * Long non-coding RNAs, lncRNA

Answer 29

**Normal functions** * Correct organization of chromatin * Controls active and inactive states of embryonic and somatic cells * Specific DNA methylation and histone modifications * Controls gene and tissue specific epigenetic patterns * Silencing repetitive elements * Ensures chromatin order and proper gene expression patterns are maintained * Genomic imprinting * Essential for development * X chromosome inactivation * Balances gene expression between males and females

Answer 30

* DNA hypermethylation * Results in chromatin condensation and silencing of tumor suppressors and other genes * DNA hypomethylation * Activates oncogenes * Results in chromosomal instability * Activates transposons * Mutations at methylated cytosines * Results in inappropriate gene expression * Imprinting defects * Results in loss of parental identity

Answer 31

* Generally occurs when a cytosine is followed by a guanine (CpG dinucleotide) * Outside of genes, 5-methylcytosine is typically associated with the exclusion of transcription factors (silencing gene expression) * May increase/activate gene expression, but less common

Answer 32

* Global DNA demethylation in the developing zygote and primordial germ cells of the embryo * Locus-specific in some somatic cells * TET family of proteins- act as enzymes in the demethylation reaction

Answer 33

* Methylation of CpG sites within INS promoter suppressed insulin promoter-driven reporter gene activity by almost 90% * Specific methylation of CpG site in the CRE in the promoter alone suppressed insulin promoter activity by 50%

Answer 34

* **Histone modifications** - covalent modifications to histone proteins associated to how tightly the nucleosomes are packed together * **Nucleosome** - complex of chromosome and associated histone proteins * **Heterochromatin** - regions of tightly packed nucleosomes * **Euchromatin** - less tightly packed, allowing binding of RNA polymerase and regulatory proteins controlling gene expression * **Enzymes** * Histone deacetylases, HDAC * Histone acetylases * Histone methyltransferases

Answer 35

* Parent histones are randomly spread among daughter chromosomes during DNA replication * Histone H3 Lysine 9 methyltransferase (H3K9 HMT) binds to nucleosomes with "methyl marks" and methylates the remaining unmarked nucleosomes

Answer 36

* Small RNAs recruit proteins (Argonaute, AGO, PIWI) to transcript through base-pairing interactions to recruit modifying enzymes * Long RNAs act as scaffolds for RNA-binding proteins to recruit chromatin-modifying complexes

Answer 37

Functions: * Heterochromatin maintenance * Developmental controls * Tissue specific expression controls

Answer 38

* X chromosome inactivation * Placental - random X inactivation * Marsupials - imprinted X inactivation * Imprinting * Parent of origin dependent monoallelic DNA methylation

Answer 39

1. The X-inactivation center (XIC) on two X chromosomes sense each other and pair 2. Originally, both chromosomes express Xist. However, one (random) chromosome stops expression of Xist. 3. Xist is transcribed on one, and Tsix on the other 4. Xist RNA coats the chromosome from which it is transcribed. 5. Xist recruits histone modifications and DNA methylations 6. Changes in chromatin in the Xist-coated X chromosome inactive most transcription from the chromosome * Xist - non coding RNA unique to placental mammals * Tsix - antisense gene of Xist that blocks Xist transcription

Answer 40

* The allele for fur color is on the X chromosome * After cell division and X chromosome inactivation, some cells have an active X chromosome with orange fur and some have an active X chromosome with black fur * These two cell populations (orange and black) are randomly distributed throughout the cat's fur

Answer 41

Only one copy of a gene in an individual (either from their mother or their father) is expressed. The other is suppressed by epigenetic tags on the DNA.

Answer 42

* In mice, the Igf2 is expressed in the paternal allele (but not in the maternal allele) * In mice, Igf2r is expressed in the maternal allele (not paternal). * In humans, only the Igf2 is silenced in the maternal allele

Answer 43

* Two regulatory regions are important for their parental allele-specific expression: a differentially methylated domain (DMD) upstream of H19 and a set of tissue-specific enhancers downstream of H19. * The enhancers specifically activate Igf2 on the paternal chromosome and H19 on the maternal chromosome. * A methylated DMD on the paternal chromosome inactivates adjacent H19 * An unmethylated DMD on the maternal chromosome insulates Igf2 from the enhancers.

Answer 44

* Genetic mutation in the gene encoding for Methyl CpG Binding Protein (MECP2), which is part of the gene silencing complex * MECP is located on X chromosome * Lethal in males (who only have one X) * Severely affects neurological development * Impaired communicaiton and motor skills * Early onset (1 y/o) * Generally de novo mutation *

Answer 45

* Polymorphism in MTHFR (methylenetetrahydrofolate reductase) * 677CT polymorphism (alters patterns of DNA methylation

Answer 46

* **Imprinting disorders** * Since imprinted genes are functionally haploid, loss of function mutations can lead to abnormalities * **Angelman syndrome** - maternal LOF, chromosome 15 * **Prader-Willi syndrome** - paternal LOF, chromosome 15 * **Beckwith-Weidemann syndrome** - Activation of Igf2 on maternal chromosome 11

Answer 47

* Defects in histone modifications * Coffin-Lowry syndrome * Defects of methylation * Fragile X syndrome

Answer 48

* If pericentromeric heterochromatin is _hypomethylated_ --> mitotic recombination, genomic instability * If the CpG island is _hypermethylated_ --> transcriptional repression, loss of TSG (tumor suppression gene) expression * Both cases lead to cancer

Answer 49

* Bisulfite Sequencing for DNA methylation 1. An allele goes through bisulfide treatment, alkylation, and spontaneous denaturation. 2. Unmethylated cytosines are turned into uracil, while the methylated cytosines are kept intact. 3. The allele goes through non-methylation specific PCR and methylation-specific PCR. 4. Differentiation of bisulfite-generated polymorphisms: Original allele and new allele (with uracil) are compared to find the methylation pattern

Answer 50

* Negative (-): Suppressor * DNA methyltransferases (DNMT) * Histone deacetylases (HDAC) * Histone methyltransferases * Positive (+): Expressor * Histone acetylases

Answer 51

* Repair involving excision of lesion followed by replacement * Mismatch repair * Nucleotide excision repair * Base excision repair * Direct repair * Multiple types, all dedicated to repairing specific lesions * Double-stranded break repair * Nonhomologous end-joining * Homologous recombination repair

Answer 52

1. After DNA replication, for a short period of time, the template strand is methylated but the new strand is not * This is how the polymerase (or other proofreaders) recognize which strand has the mistake 2. MutL-MutS complex forms at the site of mismatch and moves along the DNA to the MutH (bound at the 5'GATC sequence) 3. MutH cleaves the unmethylated strand on the 5' side of the G in the GATC sequence 4. The combined action of DNA helicase II, SSB, and one (of four) exonucleases removes a segment from the new strand between the MutH cleavage site and a point just beyond the mismatch. 5. The resulting gap is filled in by DNA polymerase III, and the nick is sealed by DNA ligase 6. The new strand is methylated by dam methylase

Answer 53

MLH1, MSH2, MSH6, PMS2

Answer 54

Increases fidelity of DNA replication by 10²-10³

Answer 55

* Adenine --> Hypoxanthine * Guanine --> Xanthine * Cytosine --> Uracil * 5-methyl cytosine --> thymine * Thymine --> no deamination * Methylated C is a common site for mutation * Deaminated base is removed by DNA glycosylase * Exception: Deamination of methylated C at CG dinucleotide generates T that is inefficiently removed

Answer 56

* Creates an abasic site * Higher rates for purines than for pyrimidines * Much slower in ribonucleotides and RNA

Answer 57

1. DNA glycosylase recognizes a damaged base and cleaves between the base and deoxyribose in the backbone 2. An AP endonuclease cleaves the phosphodiester backbone near the AP site 3. DNA polymerase I initiates repair synthesis from the free 3' hydroxyl at the nick, removing and replacing a portion of the damaged strand * 5' --> 3' exonuclease activity 4. The nick remaining after DNA polymerase I has dissociated is selaed by DNA ligase * DNA glycosylases are specific for each type of damage * Responsible for repair of deamination of cytosine * Can distinguish thymine from deaminated cytosine * Most bacteria have 1 uracil DNA glycosylase, while humans have 4 * Mode of repair for particularly common lesions * Repairs altered bases resulting from spontaneous changes and environmental insults

Answer 58

* Methyltransferases - add methyl groups * Ten-eleven translocation (TET) enzymes - change the methyl groups

Answer 59

* Catalyzes the transfer of methyl groups to specific CpG structures in DNA * Process is called DNA methylation * Encoded in humans by the DNMT3A gene

Answer 60

G/T mismatch-specific thymine DNA glycosylase

Answer 61

Suggests that TET enzymes are responsible for maintaining low levels of DNA methylation in stem cells

Answer 62

* Autosomal recessive disorder * Extreme light sensitivity * Neurological defects * Seven different enzymes in NER * 6x more common in Jananese

Answer 63

1. An excinuclease binds to DNA at the site of a bulky lesion and cleaves the damaged DNA strand on either side of the lesion 2. The DNA segment (13 nucleotides or 29 nucleotides) is removed with the aid of helicase 3. The gap is filled in by DNA polymerase (I or e) 4. The remaining nick is sealed with DNA ligase * Used to repair DNA lesions that cause large distortions in helical structure * Repairs benso[a]pyrene-guanine (cigarette smoke damage) * Repairs polycyclic aromatic hydrocarbon damage * Excinucleases are unique endonucleases that cut in two different patterns

Answer 64

**What is it?** * Particularly notable for its high mutagenicity, pairing with T during DNA replication * O6-mG lesions block the replicative DNA polymerases Pol(a) and Pol(S) * Formed in DNA by alkylation of the oxygen atom of guanine, most often by N-nitroso compounds (NOC) and sometimes due to methylation by other compounds such as endogenous S-adenosyl methionine **How is it repaired?** * Methyltransferase is used to turn O6-mG --> Guanine * Direct repair mechanism * After repair, inactive enzyme is degraded * Repair does not involve polymerase

Answer 65

* NOC are found in some foods (bacon, sausages, cheese) and tobacco smoke * Formed in the gastrointestinal tract, especially after consumption of red meat. * Endogenous nitric oxide levels were found to be enhanced under chronic inflammatory conditions, and this could favor NOC formation in the large intestine

Answer 66

* Differs from other repair mechanisms in that the damage is directly repaired, rather than excising the damge and replacing the nucleotides * Enzymes are dedicated to repairing specific DNA damage * The enzyme catalyzes a single reaction, then is inactived and degraded * Repair does not involve DNA polymerase

Answer 67

* Methylation of guanine --> O⁶-methylguanine * O6-mG pairs with thymine, rather than cytosine * If not repaired, G-C will turn into A-T after replication * The methylation interferes with hydrogen bonding and is thus mutational

Answer 68

1. A blue light photon (300-500 nm) is absorbed by MTHFpolyGlu 2. The excitation energy passes to FADH- in the active site 3. The excited flavin (FADH-) donates an electron to the pyrimidine dimer to generate an unstable dimer radical 4. Electron rearrangement restores monomeric pyrimidines 5. Electron is transferred back to the flavin radical to regenerate FADH- * Does not happen in placental mammals (NER used instead)

Answer 69

* AlkB is an alpha-ketoglutarate-dependent hydroxylase (a superfamily of non-haem iron containing proteins) * Specifically, AlkB removes the alkylation damage to ssDNA caused by SN2 type of chemical agents * Removes methyl groups from 1-methyl adenines and 3-methyl cytosines * AlkB oxidatively demethylates the DNA substrate * Accompanied with the release of CO2, succinate and formaldehyde

Answer 70

* Caused by: * High-energy radiation * Oxidative free radicals * Errors during replication * Recombinational DNA repair * Homologous genetic recombination as a DNA repair process that is typically directed at the reconstruction of stalled or collapsed replication forks * Nonhomologous end joining (NHEJ) * An alternative process for double-stranded break repair that does not entail recombination

Answer 71

* DNA damage encountered during replication can cause the collapse of the replication fork * This halts replication. B/c of this, there is no other strand to assist in the repair * Error-prone repair (when lesions are numerous) * Involves a novel DNA polymerase V (encoded by umuC and umuD genes) that can replicate, albeit inaccurately, over many types of lesions * Mutations often result

Answer 72

* Both involved in homologous recombination repair * Mutations account for 20-25% of hereditary breast cancers, and 5-10% of all breast cancers * Women with abnormal BRCA1/BRCA2 gene --> 80% more likely to develop breast cancer by age 90 * Risk of ovarian cancer * 55% for women with BRCA1 mutation * 25% for woman with BRCA2 mutation

Answer 73

* When the replication fork encounters a damaged site in the template strand, it collapses. 1. 5'-ending strand is degraded 2. 3'-single stranded extenstion is bound by a recombinase that pairs with a complementary sequence in the intact DNA duplex 3. This creates a branched DNA structure, where 3 DNAs come together 4. Branched migration - moves the DNA branch 5. This creates a Holliday intermediate (X-like crossover structure) 6. A special class of nucleases cleaves the Holliday intermediate 7. Ligation restores the replication fork

Answer 74

* DNA sequences are rearranged in recombination reactions, usually in processes tightly coordinated with DNA replication or repair * **Homologous recombination** * Involves genetic exchanges between any two DNAs that share an extended region of nearly identical sequences * **Site-specific recombination** * Involves genetic exchanges only at a particular DNA sequence * **DNA transposition** * Involves a short segment of DNA with the capacity to move from one location in a chromosome to another ("jumping genes")

Answer 75

* Initiated by double-stranded breaks * Forms Holliday intermediates * Three functions: * DNA repair especially at stalled replication forks * Assists in segregation of chromosomes * Enhancement of genetic diveristy * Crossing over: * Produces an exchange of genetic material * Chiasmata - points of joining between two pairs of homologous chromatids * Chiasmata are the physcial manifestation of prior homologous recombination (crossing-over) events

Answer 76

* Forms during crossing over (a natural genetic process that occurs between homologous chromosomes and leads to switching genetic material) * Cleavage of Holliday intermediate by specialized nucleases can generate strands than have recombined * Recombination increases genetic variation in a population * Equal probabilities of recombination products and non-recombined products

Answer 77

* RuvA (associated stick proteins) is part of the RuvABC complex of three proteins that mediate branch migration and resolve the Holliday junction created during homologous recombination in bacteria.

Answer 78

* Results in insertion or duplication in one strand, and deletions in the other * Contributing factors to unequal crossing over: * Short, repetitive sequences * Similar regions in different genes such as in the beta and delta globulin genes

Answer 79

* Results in the conversion of one allele to the other allele * Situation in which the products of meiosis from an AA' individual are 3A and 1A' (or 1A and 3A') - not 2A and 2A' * Heteroduplex DNA is formed at the sites of homologous recombination between maternal and paternal chromosomes. * If the maternal and paternal DNA sequences are slightly different, the heteroduplex region will include some mismatched base pairs, which may then be corrected by the DNA mismatch repair machinery. * Such repair can “erase” nucleotide sequences on either the paternal or the maternal strand. * The consequence of this mismatch repair is gene conversion, detected as a deviation from the segregation of equal copies of maternal and paternal alleles that normally occurs in meiosis. * GC base pair bias - more retained than AT base pairs **Difference from crossing over** * Crossing over/recombination - neither paternal nor maternal DNA sequence segments are lost * Gene conversion - paternal or maternal sequence is deleted and replaced with a duplicate of the other

Answer 80

* Occurs only at specific target sequences * Found in virtually all cells * Functions in DNA integration and regulation of gene expression * Outcome depends on the location and orientation of the recombination sites in a dsDNA molecule * Recombination sites with opposite orientation in the same DNA molecule --> **inversion** * Recombination sites with the same orientation, either on one DNA molecule (producing a **deletion**), or on two DNA molecules (producing an **insertion**)

Answer 81

* Prophage formation allows the phage to go dormant and replicate passively with the chromosomal DNA * Region of complete homology is only 15 bp

Answer 82

* Attachment site on the λ phage DNA (attP) shares only 15 bp of complete homology with the bacterial site (attB) in the region of the crossover. * Reaction generates two new attachment sites (attR and attL) flanking the integrated phage DNA. * The recombinase is the λ integrase (or INT protein). * Integration and excision use different attachment sites and different auxiliary proteins. * Excision uses the proteins XIS, encoded by the bacteriophage, and FIS, encoded by the bacterium. * Both reactions require the protein IHF (integration host factor), encoded by the bacterium.

Answer 83

* In almost all cells, transposons use recombination to move within/between chromosomes * **Replicative transpositions** - leave a copy behind * **Direct transposition** - does not leave a copy behind 1. DNA is cleaved on each side of the transposon. 2. The liberated 3′‐hydroxyl groups at the ends of the transposon act as nucleophiles in a direct attack on phosphodiester bonds in the target DNA. 3. The target phosphodiester bonds are staggered in the two DNA strands. 4. The transposon is now linked to the target DNA. 5. In direct transposition, replication fills in gaps at each end to complete the process. * The cleaved host DNA left behind is either: repaired by DNA end joining or degraded. * Degradation can be lethal to an organism. 6. In replicative transposition, the entire transposon is replicated to create a cointegrate intermediate. * The cointegrate is often resolved later, with the aid of a separate site‐specific recombination system.

Answer 84

* The most abundant repeats are the Alu repeats of short interspersed repetitive elements (SINEs). * Alu elements are one of the most successful SINEs found in any organism. * Approximately 300 base pairs in length, making the retrotransposition competent RNA using a bipartite internal RNA polymerase III promoter. * The retrotransposition rate of Alu elements is estimated to be one in 21 births, which is ten times higher than that of LINE‐1. * Most L1s are inactive due to point mutations, rearrangements, or truncations.

Answer 85

* **Class I**: "copy and paste", retrotransposons * DNA transcribed into RNA, then reverse transcriptase is used to make a DNA copy of the RNA to insert in a new location * LTR retrotransposons * Non-LTR retrotransposons * LINEs * SINEs * **Class II**: "cut and paste" * Consists of DNA that moves directly from place to place * DNA transposons

Answer 86

* Inverted region (~50 bp) * Protein coding region (transposase) * Target-site direct repeat (5-11 bp)

Answer 87

1. Transposase makes blunt-ended cuts in donor DNA (IS10) at inverted regions, and staggered cuts in target DNA 2. Transposase ligates IS10 to 5' single stranded ends of target DNA 3. Cellular DNA polymerase extends 3' cut ends, and ligase joins extended 3' ends to the IS10 5' ends

Answer 88

1. Fusion * Retrovirus enters host cell via fusion 2. Reverse transcription * Enzyme capable of synthesizing DNA using RNA as a template * Viral DNA made from genomic ssRNA from nucleocapsid (virus) 3. Transport to nucleus and integration * Viral DNA transported to nucleus of host cell * Provirus 4. Transcription * Retroviral proteins are transcribed 5. Budding * Viral genomic ssRNA is encapsulated and leaves the host cell

Answer 89

1. RNA polymerase II makes a primary transcript from the integrated retroviral DNA * Start site - before "R" in LTR region * Poly(A) site - after "R" in second LTR region * Coding region in between the two LTRs * Host cell DNA is on either side of the LTRs 2. RNA-processing enzymes (Poly(A) polymerase) converts the primary transcript to retroviral RNA genome

Answer 90

1. tRNA is extended to form a DNA copy of 5' end of genomic RNA 2. The RNA of DNA-RNA hybrid is digested 3. DNA is hybridized with the remaining RNA R sequence (on the 3' end) 4. DNA strand extended from 3' end 5. Most of the hybrid RNA is digested 6. 3' end of the second DNA strand is synthesized 7. tRNA in the DNA-RNA hybrid is digested 8. Second DNA strand jumps to the 5' end 9. Second strand is extended from the 3' end

Answer 91

Experiment 1: * A yeast Ty element + Galactose(Gal)-sensitive promoter is engineered in plasmid vectors * Yeast is placed in media with galactose * Results: * Ty mRNA synthesis is increased * Transposition of Ty elements is increased Experiment 2 * A yeast Ty element + Gal-sensitive promotor + unrelated intron is engineered in plasmid vectors * Yeast is placed is media with galactose * Results: * Ty mRNAs lack intron * Transposed Ty elements lack intron

Answer 92

* Prior to start: * LINE ORF1/ORF2 are transcribed into LINE RNAs * In cytoplasm, LINE RNAs are translated into proteins * LINE RNAs with bound LINE proteins are transported back into the nucleus 1. Nicking * ORF2 makes cuts in the AT-rich regions 2. Priming of reverse transcription by chromosomal DNA * Poly-A tails act as a primer for synthesis of new LINE DNA using LINE RNA as the template 3. Reverse transcription of LINE RNA by ORF2 4. LINE RNA and LINE DNA are integrated into the chromosomal DNA at ORF2 5. Copying of chromosomal DNA by ORF2 * ORF2 continues past end of LINE RNA and uses target DNA as template 6. Insertion completed by cellular enzymes * Cell's DNA polymerase extends 3' end and digests away the LINE RNA at the same time 7. New LINE ligated into place

Answer 93

* AT-rich region * ORF1 - RNA binding protein * ORF2 - reverse transcriptase + endonuclease * AT-rich region * Target site direct repeat

Answer 94

* LTR (250-600 bp) * Protein coding region * LTR * Target site direct repeat (5-10 bp)

Answer 95

* TSD - target site domain * Alu element * A+B = RNA pol III promoter * A₅TACA₅ * AA repeat region * TSD * Alu 1 recognition site: * 5' AG CT3 3' * 3' TC GA 5' * Needs L1 proteins

Answer 96

* By recombination between interspersed repeats * Double crossover between Alu elements * By transposition - DNA transposons * Excise transposase from gene 1, insert into second gene * By transposition - LINEs * LINE is transcribed and polyadenylated at end of downstream exon * ORF 2 reverse transcription and insertion into new gene * By duplication of DNA sequence at target site * Transposase makes staggered cuts in the target site * Transposon is inserted at the site of the cuts * Replication fills in gaps by duplicating the sequence flanking the transposon

Answer 97

* Codeine is transformed to morphine by **CYP 2D6** * Gene deletion - no 2D6 - poor metabolizers - codeine does not turn into morphine - no pain relief * Gene duplication - multiple 2D6's - ultra metabolizer - codeine is transformed to morphine at a very high/fast rate - overdose

Answer 98

* Nuclear envelope * Continuous with ER * Separates nuclear content from cytoplasm * Nuclear lamina * Cytoskeletal support * Nuclear pores * Controls traffic between nucleus and cytoplasm * Nucleolus * Site of rRNA transcription and processing * Chromatin * Genetic material (DNA and associated proteins)

Answer 99

* Most prominent nuclear body and ribosome production factory * Site of rRNA transcription and processing Structure * Fibrillar center * Location of rRNA genes * Dense fibrillar component * Active in transcription of rRNA * Granular component * Site of ribosome assembly * Associates with chromosome regions that contain genes for the 5.8S, 18S, and 28S rRNAs

Answer 100

1. Pre-rRNA genes are arranged in long, tandem arrays separated by non-transcribed spacer regions 2. Transcribed by RNA polymerase I * Transcribed as 45S ribosomal precursor 3. Primary transcript undergoes series of cleavages and modifications 4. Final products: * 5.8S and 28S rRNA - used by 60S ribosomal subunit * 18S rRNA - used by 40S ribosomal subunit 5. Early assembly of subunits happen in the nucleolus 6. Intermediate-late assembly is in the nucleoplasm 7. Mature subunits are transported to the cytoplasm (40S, 60S subunits) * 55 rRNA is transcribed elsewhere by RNA polymerase III

Answer 101

* Meswork underlying inner nuclear membrane * Functions: * Provides structural and functional support to the nucleus * Binds chromatin and other nuclear proteins * Regulated by phosphorylation * Lamin tetramer (part of lamin filament) --> phosphorylated lamin dimers * Enzyme: MPF

Answer 102

* Made of lamins - class of intermediate filaments * Three lamin genes: A/C, B1, B2 * Lamin A, C proteins * Products of single gene * Generated by alternative splicing of mRNA * Identical except for 133 amino acids present at C-terminus of lamin A * Expressed in most (but not all) cells * Expression occurs at different times during development * Lamin B1, B2 proteins * Each encoded by one gene * Expressed in _all_ cells * Modified by addition of isoprenyl group, which attached protein to inner nuclear membrane * Binds inner nuclear membrane proteins, such as emerin and lamin B * LINC complex * Connects the nuclear lamina with cytoskeleton protein (actin, IFs, microtubules) * Plays a role in nuclear migration, nuclear integrity, and chromosome movement during cell division

Answer 103

1. "Sticky" farnesyl is added to the lamin A protein, which helps it to reach the nuclear lamina * Farnesylation of a Cys residue * Farnesyl pyrophosphate --> Farnesylated Ras protein * Enzyme: farnesyl transferase 2. Farnesyl is cut off, allowing lamin A to be incorporated in the nuclear lamina

Answer 104

1. Activation - H3, H4, H2A, H2B 2. Repression - n/a 3. Activation - H3, H2A, H2B 4. Activation - H3, H4 5. * Activation: H3 Me3 promoter, H3 Me1 enhancer * Elongation: H3 transcribed * Repression: H3, H4 6. * Activation - H2B * Repression - H2A

Answer 105

Activation: * Acetylated lysine * H3, H4, H2A, H2B * Phosphorylated serine/threonine * H3, H2A, H2B * Methylated arginine * H3, H4 * Methylated lysine * H3 Me 3 promoter * H3 Me1 enhancer * Ubiquitinylated lysine * H2B

Answer 106

Repression: * Hypoacetylated lysine * No site listed * Methylated lysine * H3, H4 * Ubiquitinylated lysine * H2A

Answer 107

Elongation: * Methylated lysine * H3 in transcribed region

Answer 108

* Large complex that controls traffic between nucleus and cytoplasm * Small molecules - diffuse freely * Large molecules (protein, RNA) - transported through nuclear pore complexes (NPC) * Made of nucleoporins * Forms basket/net structure

Answer 109

* Structural * Y-complex * Forms basic structural scaffold * FG-nucleoporins * Lines central channel * Forms semi-permeable channel to control transport * named because of presence of hydrophobic FG-repeats (phenylalanine-glycine)

Answer 110

**Nuclear localization signal (NLS)** * Basic amino acid squence (Lys-Arg) * May also have Lys-rich sequence, without Arg * Binds to transport protein _importin_ so that cargo can be carried _into_ nucleus * Import can be regulated by blocking and unblocking NLS **Nuclear export signal (NES)** * Sequence rich in leucine (Leu) * Binds to transport protein _exportin_ so that cargo can be carried _out_ of the nucleus

Answer 111

* Striated muscle disease * Associated with the nucleus * Compared to normal (oval shaped) cell, EDMD2 cell looks like an elongated oval with a nipple and uneven color * Defects in emerin and/or lamin A/C * Muscle weakness in chest, upper arms, calves and feet

Answer 112

* Associated with the nucleus * Compared to normal (round) cell nucleus, HGP mutated nucleus looks shriveled and not round * Disorganization of heterochromatin * Defective repair of DNA damage and increased genomic instability * Premature aging * Defect in lamin A/C * Single base mutation at LMNA gene activates cryptic mRNA splice site * Lamin protein is shorter than normal * Farnesyl group stays attached

Answer 113

1. NLS on cargo protein is recognized and bound by importin in the cytoplasm 2. Importin interactions with FG-nucleoporins and translocates through the NPC 3. Inside the nucleus, importin binds to Ran-GTP 4. This causes a conformational change in importin, which displaces the NLS and releases the cargo protein into the nucleus 5. The importin-Ran-GTP complex is transported to the cytoplasm 6. GAP (Ran GTPase activating protein) changes Ran-GTP to Ran-GDP 7. Ran releases importin and diffuses through the pores, back into the nucleus

Answer 114

1. The heterotrimer complex (NXF1/NXT1) binds to cargo mRNA-protein complexes (mRNPs) 2. mRNP-NXF1-NXT1 complex interacts with FG-nucleoporins and translocates through the NPC into the cytoplasm 3. RNA helicase (Dbp5) removes NXF1 and NXT1 from the complex * ATPase * Hydrolysis of ATP generates the energy to remove the NX's 4. NXF1 and NXT1 are transported back into the nucleus via Ran-dependent import, while mRNP stays in cytoplasm

Answer 115

1. NES on the cargo protein is recognized and bound by exportin and Ran-GTP in the nucleus 2. Exportin interacts with FG-nucleoporins and translocates through the NPC into the cytoplasm 3. GAP (Ran GTPase activating protein) changes Ran-GTP to Ran-GDP 4. This causes a transformational change in exportin, which causes the release of the cargo protein into the cytoplasm 5. Exportin and Ran-GDP are transported back into the nucleus

Answer 116

1. Guanine nucleotide binding protein (binds either GTP or GDP) 2. Exchanges GDP --> GTP in Ran, in nucleus 3. Facilitates hydrolysis of Ran-GTP in cytoplasm 4. Binds to protein to import into nucleus 5. Binds to protein to export out of nucleus 6. (Out) Goes from cytoplasm to nucleus 7. (In) Goes from nucleus to cytoplasm

Answer 117

* Important for regulation in both prokaryotes and eukaryotes * Arise from larger precursors that are transcribed from non-protein-coding DNA * Types: * siRNA - short interfering RNA * miRNA - micro RNA * piRNA - Piwi associated RNA * Functions: * Transcription * Gene silencing * DNA replication * RNA processing, modification, stability * mRNA translation * Protein stability and translocation

Answer 118

Structure: * 21-23 nt dsRNA duplexes * Symmetric 2-3 nt 3' overhangs * 5'-phosphate and 3'-hydroxyl groups * Formed by RNAase III enzyme (Dicer) * dsRNA-specific endoribonuclease that cleaves long dsRNA into short fragments Generation: * Bidirectional transcription * Transcription through inverted repeats * Transcription of aberrant RNAs triggers dsRNA synthesis by RNA-dependent RNA polymerase

Answer 119

1. Coded for by genomic sequences and transcribed by RNA pol II 2. Primary miRNA transcripts are recognized by the microprocessor complex, comprised of Drosha (dsRNA-specific RNase) and DGCR8 (dsRNA binding protein) 3. Microprocessor digests the primary miRNA in the nuclease to release pre-miRNA hairpins * Cleaved at the base of the stem loop, releasing 60-70 nt pre-miRNA that has a 2 nt 3' overhang 4. Exportin-5 transports pre-miRNA from the nucleus to the cytoplasm 5. In the cytoplasm, pre-miRNAs are recognized by the RISC containing Dicer-TRBP-Ago * TRBP - dsRNA binding protein 6. Dicer (RNase III enzyme) cleaves ~ 22 nt from the Drosha cleavage site * Creates duplex miRNA with 2 nt 3' overhang 7. The duplex RNA strands are separated (without using ATP). One miRNA remains associated with Ago2 in the RISC complex (guide strand). 8. The RISC complex recognizes the target mRNA (based on complementarity between the guide miRNA and the mRNA transcript). 9. Ago2 cleaves the target mRNA for posttranscriptional gene silencing. 10. The cleaved product is released, enabling RSIC to catalyze the destruction of another target RNA

Answer 120

* Region of si/miRNAs that mediates target binding * 7 nucleotides long, at positions 2-8 from 5' end

Answer 121

* RNA-inducing silencing complex * Contains Argonaute family member, Dicer and dsRNA binding protein * RNAs require 5' phosphate to enter RISC * Those lacking the phoshate are rapidly phosphorylated by endogenous kinase * Results of RISC function: * mRNA cleavage * Protein synthesis block * Transcriptional gene silencing

Answer 122

* Part of RISC * Contains central PAZ domain and carboxyl terminal PIWI domain * PIWI domain --> binds to small RNAs at their 5' end * Slicer - endonuclease that cleaves target RNA in this domain * PAZ domain --> binds to 3' end of both target and small RNAs

Answer 123

1. Transcription (bidirectional, inverted repeat or aberrant transcription) 2. Dicer cleaves long dsRNA into shorter fragments 3. 1 strand of the siRNA duplex is associated with the AGO2 in the RISC complex (guide strand) 4. AGO2 recognizes target mRNA b/c of the guide strand. mRNA is sliced and released

Answer 124

Method 1: 1. Guides strand (antisense strand) guides RISC to its homologous target mRNA and binds the mRNA 2. RISC does an endonucleolytic cleavage at a single site in the center of the duplex region between the guide siRNA and target mRNA (10 nt from 5' end of siRNA) 3. This single site cleavage causes the rest of the mRNA to degrade Method 2: * Guides nuclear events, including histone and DNA methylation, resulting in transcriptional silencing * Direct formation of heterochromatin * Associates with RITS complex * Separated siRNA strands guide RITS complex to homologous gene * RITS complex includes proteins that induce chromatin condensation and methylation of histone H3

Answer 125

* Environmental and genetic factors * * Proteins can block RISC * Signals can alleviate repression * Proteins may prevent RISC from binding to mRNA

Answer 126

* Expression of miRNAs is significantly lower in tumors * Interfering with production of miRNAs can increase cell transformation and tumor development * let-7 miRNA blocks the expression of c-Myc and RasK oncogene proteins * During breast cancer metastais, some miRNAs are lost * miR-126 and miR-335 are lost in the majority of primary breast tumors from patients who relapse

Answer 127

Mice: * miR-1 expressed in developing heart muscle cells * Targets genes involved in differentiation of cells * Major target - Hand2 (transcription factor that promotes heart muscle cell expansion) * Negatively regulates heart muscle cell proliferation *Drosophilia* bantam * Represses apoptosis and promotes cell proliferation in developing fly * Inhibits translation of mRNA for *Hid* (key activator of apoptosis)

Answer 128

* *C. elegans* has 4 larval stages (L1-L4) * Lin-14 initiates L1 and inhibits L2 * Hbl-1 initates L2 and inhibits L3 * Lin-4 down regulates Lin-14, so Hbl-1 levels can increase to move cells to L2 * Loss of Hbl-1 initiates L3 * mi-48, mi-241 and mi-84 down regulate Hbl-1 so cells can move to L3 Proteins: * Lin-14, Hbl-1 miRNAs * Lin-4, mi-48, mi-241, mi-84

Answer 129

Interchangeable: * If animal miRNA encounters target with complete complementarity, it can enter cleavage pathway * siRNA can repress translation without cleavage if degree of complementarity to target is reduced Distinct: * Distinguished by biogenesis pathways and mechanism for regulating target genes * miRNA are processed from small hairpins embedded in longer primary transcripts * Common feature of targets is complementarity to 6-7 b of miRNA * siRNAs are processed from long dsRNAs * Targets are highly homologous to the siRNA precursor * Cannot be distinguished by chemical composition or function

Answer 130

* miRNA binds to sites that have partial sequence complementarity in 3' untranslated region of target mRNAs * Repress translation and inhibit protein synthesis * Inhibition requires binding of 2 or more RISC complexes to target mRNA * Virtually all of the miRNAs that have been studied in animals reduce steady state protein levels for the targeted gene(s) without impacting the corresponding levels of mRNA * Some exhibit temporal or tissue-specific patterns of gene expression * Multiple miRNAs may target same transcript

Answer 131

1. C. elegans (CE) - developmental timing 2. Ce - developmental timing 3. Ce - neuronal cell fate 4. Ce - neuronal cell fate 5. D. melanogaster (Dm) - cell death, proliferation 6. Dm - cell death, fat storage 7. M. musculus (Mm) - haematopoietic cell fate

Answer 132

1. Bacteria infected by virus cleaves viral DNA into short segments and adds these segments to CRISPR array 2. CRISPR array is transcribed - contains both CRISPR and viral sequences 3. CRISPR RNA is cleaved into guide RNAs (gRNAs) by nuclease complexed with trans-activating CRISPR RNA (tracrRNA) 4. Inactive Cas9 nuclease binds to gRNA and tracrRNA and becomes activated 5. gRNA + tracrRNA + Cas proteins all bind to specific viral DNA and cleave it

Answer 133

1. Foreign DNA from a virus or plasmid invades a cell 2. DNA fragments from the invading DNA are incorporated into the CRISPR locus as spacers 3. The cell transcribes pre-crRNA with the repeat/spacer group (invading DNA) 4. tracrRNA base pairs with CRISPR repeat sequences on the pre-crRNA. 5. RNase III, Csn 1 and other CRISPR proteins modify pre-crRNA/tracrRNA duplex to form a guide RNA 6. The inactive Cas9 protein binds to the gRNA and becomes activated 7. The activated Cas9/gRNA binds with the invading target DNA, downstream (3') of the PAM (protospacer adjacent motif) 8. Cas9 cleaves the target DNA upstream (5') of PAM

Answer 134

1. Normal or diseased donor gives cells that ar reprogrammed to induced pluripotent stem cells 2. iPSCs are genomically edited (with CRISPR, ZFN, TALEN, etc) 3. Edited iPSCs can be used in disease modeling, precision therapy and drug screening

Answer 135

* CRISPR can be used as a "Gene Drive" (modification of a genome leading to a version of a gene becoming dominant in a population) * CRISPR complexes on the Y-chromosome shred the X chromosome in males * Without the X sex chromosome, males produce only Y chromosome sperm * Over time, only males are left in the population, and the population collapses

Answer 136

* Gene knock-out * Gene repression * Gene activation * Genome-wide screening * Imaging genomic loci * Purification of genomic loci * By co-expressing gRNA specific to gene being targeted and Cas9 nuclease, one can delete or modify any gene sequence, if: * The sequence is unique compared to the rest of the genome * The target is present immediately upstream of a PAM (usually PAM is the sequence NGG)

Answer 137

1. Promote/activate gRNA and Cas9 expression * Complex should target specific DNA sequence 2. Cas9 cleaves dsDNA at the target sequence, creating a double-stranded break 3. The double stranded break can be repaired via nonhomologous end joining * Disrupts the reading frame * Leads to an induced, premature stop codon 4. Or, it can be repaied by homology-directed repair (HDR) * Homologous recombination * Specific change introduced to the genomic DNA * Cas9 can also cause a single strand break. Single stranded nicks improve recombination frequency

Answer 138

* Has two endonuclease acitivity domains * RuvC domain * HNH domain * Mutants: * Nickase HNH+ * Nickase RuvC+

Answer 139

* dCas9 (Cas9 with inactive nuclease) goes through transcription and translation * dCas9 binds with synthetic gRNA * Complex binds to target sequence on a gene in the promoter region * Complex interferes with gene transcription

Answer 140

* dCas9 (Cas with inactive nuclease) with VP16 domain goes through transcription and translation * dCas9 and VP16 fuse, and then combine with the synthetic gRNA to form a large complex * Complex binds to target sequence at the promoter region on a gene, and activate the promoter (which activates gene transcription)

Exam 3 Flashcards

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