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KCUMB Molecular Biology > EXAM I Ferguson > Flashcards

EXAM I Ferguson Flashcards

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1
Q

What two bonds are important in the formation of DNA?

A

Glycosidic - links sugar to base

Phosphodiester - links bases

10bp/turn

symmetric

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2
Q

How many nts are distributed over how many chromosomes?

A

3.2x10^9 nts over 24 different chromosomes

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3
Q

What are banding patterns, karyotyping, and whole chromosome painting used for?

A

The identification and detection of chromosome numbers

Banding pattern - staining & light microscopy

Karyotyping - detect abnormalities, determine loss or gain of chromosomes

Whole Chrom. Painting

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4
Q

3 requirements to pass genetic information to daughter cells

A

OriR

Centromere - kinetochore attachment

Telomere

(ENTIRE genome is replicated; DNA serves as its own template)

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5
Q

What are the common interactions between DNA and histone proteins?

A

H.H.S. - hungry, hungry, salty!

Hydrophobic interactions

H-bonds

Salt linkages - Arginine & Lysine

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6
Q

What are the roles of arginine and lysine during the packing of DNA with histones?

A

They are (+) charges which neutralizes the (-) charge of the DNA backbone, allowing the interactions (hydrophobic, H-bonds, salt linkages) to occur

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7
Q

What does chromatin remodeling allow for? What enzyme is used? And does this process require energy?

A

Looser arrangment of histones around DNA to allow greater access for proteins

Helicase requires ATP - cuts linker DNA into fragments

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8
Q

Define epigenetics and provide some examples

A

The study of changes in organisms caused by gene expression modifications RATHER than alteration of the genetic code itself

i.e. DNA methylation, Maternal Imprinting, Histone modifications, chromatin structures

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9
Q

Evidence that histones are more than just structural (they’re also functional; 3 ways)

A

Highly conserved

DNA contains equal amounts of histone and nonhistone protein

Heterochromatin is used for gene silencing & passed on to daughter cells = Drosophila experiment (zone of inactivation) = position effect

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10
Q

Which amino acid(s) are important in histone modification? What enzymes add and remove it?

A

Lysine

Methylation of lysine via methyl transferase, removed by methyl demethylase

Acetylation of lysine via histone acetyl transferase (HATs), removed by histone deacetylase complexes (HDACs)

Serine = phosphorylated

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11
Q

What is the function of the code-reader complex for histone codes? List its components, what are some examples of the consequences of the histone codes? Function of code reader enzymes?

A

Binds histone codes on the N-terminus histone tail via covalent modifications which determines DNA packaging in nucelosomes

Components: protein modules (that bind specific histone modifications on nucleosomes), scaffold protein

Displays catalytic activity

Ex: gene silencing, gene expression, etc.

Code reader enzymes spread the marker over chromosome for continued modification

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12
Q

Function of barrier proteins during nucleosome/chromatin modification; example is HS4 region

T/F - remodeling is dependent upon ATP

A

Barrier sequences (i.e. proteins or enzymes) halt the spread of chromatin modification

HS4 region protect beta-globin locus from silencing, containing a cluster of histone acetylase binding sites

True - ATP-dependent chromatin remodeling protein - assists with condensation/decondensation

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13
Q

Explain the role of centromeric heterochromatin (H3) in chromatin modifications

Which set of histones are always passed on to daughter cells in forming the centromere?

A
  • H3 histone = CENP-A that forms kinetochore (required for the attachment of mitotic spindle)

Centromere sequences contain non-required alpha satellite DNA, also found at non-centromeric positions

New centromeres can spontaneously form on fragmented DNA

  • H3-H4 tetramer directly inherited
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14
Q

What type of chromatin is rich in gene expression? Where are heterochromatin located during gene expression?

A

Euchromatin

Heterochromatin = towards or inside the nucleus

Active chromatin form loops

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15
Q

What is the role of condensins? T/F ATP required?

A

Used during condensation (disentangelment of sister chromatids to allow separation for cell division), which aids the compaction via ATP hydrolysis to coil DNA into chromatids

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16
Q

How do genomic changes occur (genome evolution)

A

Via mistakes during DNA replication: (rare)

I (goto) DownTown, SD

Inversions, Translocations, Deletions, Substitutions, Duplication

Base pair substitutions

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17
Q

How are phylogenetic trees used for comparative genomics?

A

Able to compare genomes of contemporary organisms; they show differences between humans and apes

Able to determine sequence conservation

One reason for sequence conservation could be that eliminating mutations could interfere with important genetic functions = purifying selection

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18
Q

What is the purpose of sequence comparisons in genome evolution?

A

Helps to provide inside into purpose or function

5% human genome conserved, but 1.5% codes for proteins

Therefore, the human genome is highly conserved

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19
Q

Describe the importance of gene duplication in regards to the globin chain

A

Duplication and Divergence

Started with a single-chain globin that binds 1 oxygen molecule, evolution allowed for a 2nd globin chain by gene duplication followed by mutation, resulting in 4 globin chains binding 4 oxygens

Mutation gave rise beta and alpha genes; translocation = moved alpha to separate chrom.; further duplication & mutation = more specialized beta molecules (fetal, adult)

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20
Q

Why do germ cells and somatic cells have a high fidelity rate?

A

Germ-cells = to maintain the species

Somatic cells = to avoid uncontrolled proliferation/cancer

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21
Q

What are the key players in DNA replication?

A

DNA Polymerase

DNA Primase

Helicase

OriR (A-T rich)

ssDNA Binding Protein

Topoisomerase

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22
Q

What is the function of mismatch repair during DNA replication? What players are involved?

A

Corrects the errors that DNA proofreading (via 3’-5’ exonuclease) does not fix

MutS - sticks and binds to mismatch

MutL - looks for errors and triggers degradation of nicked strand

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23
Q

What are the proteins at the replication fork in DNA replication? ATP required?

A
  • Sliding Clamp + Clamp loader - keeps DNA polymerase on DNA when moving and is removed once it encounters a double strand
  • Assembled via ATP hydrolysis
  • Stays on continuously in leading strand, new clamps are assembled on lagging strand at each Okazaki fragment
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24
Q

At what rate does helicase unwind DNA?

A

1000 nt pairs/sec

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25
Q

What is the role of helicase loading proteins (Cdc6 and Cdt1) and Cdks during DNA replication regulation?

A

Helicase loading proteins bind to ORC

Cdks removed helicase loading proteins, activating helicase and unwinding DNA and loading DNA polymerase

= S phase

26
Q

How are histones disassembled (broken up into) during replication and how are they reassembled afterwards?

A

Disassembled = destablized into H3-H4 tetramer distributed randomly to daughter duplexes and 2 H2A-H2B dimers (1/2 old; 1/2 new) with the help of histone chaperones (chromatin assembly factors) directed by sliding clamp PCNA

Daughter strands contain a hybrid of parental and new histones = epigenetic inheritance

27
Q

Explain the role of telomerase in replicating chromosome ends; what is the special sequence? Define T-loops

A

On the lagging strand, there is not enough space for primase to place ~10nts at the end, telomerase replenishes by elongating parental strand in 5’-3’ direction using an RNA template and DNA polymerase continues replicating lagging strand

Ensures that 3’ end is longer leaving a protruding SS end that loops back and tucks into repeat = T-loops protects ends

Shelterin - protective cap

28
Q

What is caused by dyskeratosis congenita in telomeres?

A

Mutant telomerase RNA gene

Prematurely shortened telomeres; chromosome ends aren’t stablized and secured (T-loops)

29
Q

What are the two types of DNA damage and what are the results if not repaired?

A

Depurination - Removing a purine (A/G) base via hydrolyzing glycosyl linkages leaving a free OH group = can result in deletion

Deamination - switching out bases resulting in base-pair substitutions; i.e. UV radiation makes covalent bonds b/w adjacent pyrimidines (T-T, C-T) = pyrimidine dimers

30
Q

What are the 4 types of DNA repair pathways?

A

Boys Never Take (you on) Dates

Base excision repair

Nucleotide excision repair

Trancription-coupled repair

Double-stranded break repair (homologous vs. non)

31
Q

Describe the mechanism of base excision repair; what are the key players

A

DNA glycosylases (unique for each base; i.e. uracil specific) scans and cleaves glycosyl bond connecting base w/ sugar

AP endonuclease and phosphodiesterase cut phosphodiester backbone removing the base

AP endonuclease directly repairs depurinations

32
Q

Describe the mechanism of Nt excision repair; what makes it different from base excision repair?

A

The way damage is removed is different; phosphodiester bond is cleaved on both sides

Bulky lesions are removed i.e. chemically-induced, thymine dimers

Multienzyme complex scans double helix; Gap is repaired by DNA polymerase and ligase

33
Q

What is the mechanism of transcription-coupled repair? What disease is associated with this?

A

Active RNA polymerase is linked with DNA repair = occurs simultaneously = urgent repair systems

RNA stays along, waits, and continues transcription by fixing the repair first

Cockayne’s Syndrome - defect in the coupled-repair

RNA polymerase is permanently stalled at sites of damage

34
Q

Why are methylated cytosines problematic (DNA repair)

A

Deamination of methyl-C produces T mismatched with G which is recognized by a DNA glycosylase which is relatively ineffective

Occurs at some CpG sequences and is associated with inactive genes

35
Q

Which DNA repair mechanism is most prominent in humans?

A

Non-homologous double strand break repair

Broken ends are joined and sealed via DNA ligation with a few nts being lost

36
Q

Define the ATM protein that’s involved in DNA repair

A

Kinase that generates intracellular signals that alert the cell to DNA damage and upregulates expression of DNA repair genes

Mutation = AT; Ataxia telengiectasia - neurodegeneration, predisposition to cancer, etc.

37
Q

List two ways in which double stranded breaks can occur

A

Homologous recombination

Nonhomologous end joining - Broken ends are joined and sealed via DNA ligation with a few nts being lost

38
Q

List the steps in repairing ds stranded breaks via homologous recombination (HR) (can also repair broken or stalled replication forks)

A
  1. 5’ ends are degraded by 5’ exonuclease, leaving a 3’ overhang
  2. 3’ end invades homologous ds template and primes repair DNA synthesis = strand invasion
  3. Back home; The newly synthesized 3’ end of invading strand anneals w/ original damaged DNA via complementary base pairing
  4. gaps filled and ligated
39
Q

Importance of RecA protein in single strand DNA pairing?

A

RecA directs the invading single strand to search for homologous sequence to invade and form a heteroduplex DNA = synapsis reaction

40
Q

List the major differences between homologous recombination and non-homologous end joining ds break repair processes

A

Homologous Recombination:

  • Template = daughter DNA duplex
  • No loss or alteration of DNA
  • Can repair other types of DNA damage, conserved

Non-Homologous End-Joining

  • No template required
  • Mutation created at repair site (due to overhang; flush ends being chewed by exonuclease)
  • Translocations can occur
41
Q

What two events occur that cause homolognous recombination?

A

Crossing over

Gene conversion

Occurs after meiosis and duplication on the diploid cell

42
Q

When does meiotic recombination occur and what does it begin with? List the basic steps

A

Maternal and paternal homologous chromosomes are paired

Begins with a double stranded break

Strand invasion

Double Holliday junction

Resolution

43
Q

Define Holliday Junction

A

DNA intermediate with 4 DNA strands from two different helices

Transient structures

Cleaved by Endonuclease (RuvC); 2 outcomes:

Crossing Over = rare

Gene Conversion = common

44
Q

What occurs in each scenario of the Holliday Junction?

A

Left - cut at BOTH HJ (both crosses); strands separate with minimal exchange of sequences

Right - cut in opposite directions; each chromosome are swapped = crossover event

45
Q

What occurs during branch migration? Does this mechanism require energy? What type of DNA does a recombination event create?

A

Strand invasion occurs and one strand creates a branch point and moves along the heteroduplex

Requires ATP

Recombination results in heteroduplex DNA

46
Q

What rare event doesn’t allow for equal exchange of genetic material between maternal and paternal genes? When does this occur

A

Gene Conversion

Occurs during homologous recombination during DNA synthesis

Due to mismatch repair in heteroduplex DNA regions

47
Q

What are the 3 types of transpositional recombination? List the type of Conservative site-specific recombination?

A

DNA-only transposons

Retroviral-like Retrotransposons

Nonretrovial Retrotransposons

Bacteriophage lambda

48
Q

Define transposon; what enzyme does it encode?

A

Specialized DNA segments that move from one position in the genome to another

Can provide antibiotic resistance in bacteria

Encodes Transposase that allows insertion into a target DNA site

49
Q

What can occur from meiotic errors?

A

Nondisjunction - homologs fail to separate; common during egg development at Meiosis I (most common abnormality)

Aneuploid - abnormal chromosome number

Euploid - normal chromosomes!!! :))

50
Q

Define conservative site specific recombination; what are the three outcomes and what causes them to occur; how does this differ from transposition? Which microbe uses this?

A

Mediates rearrangements of other types of mobile DNA elements (other than transposons); Bacteriophage lambda; can be used to turn genes on/off = Cre-lox recombination

DNA integration, DNA excision, DNA inversion

EACH DNA contains special recognition sites for recombinase, no intermediates

Sites are same oritentation = integrated or excised

Inverted sites = DNA inversion

51
Q

Define cohesin used in meiosis I, when do chromosomes replicate?

A

Cohesin - holds two sister chromatids together during S phase

Replication uccurs during S phase

Plays important role in segregating homologs during meiosis I, in meiosis II, sister chromatids are separted and cohesin breaks down

52
Q

Define 3 terms that occur during Prophase I

Bivalent

Chiasma

Synaptonemal complex

A

Bivalent - 4 chromatin structure containing replicated maternal and replicated paternal chromosomes (2 chromosomes, 4 chromatids)

Chiasma - crossing over

Synaptonemal complex - where homologs are joined via small region of homology = pseudoautosomal region

53
Q

What are the 5 phases of prophase? What occurs at each step

A

Little Zebras Pack Dancing Dicks

Leptotene - homologs begin to condense/pair

Zygotene - synaptonemal complex forms via transverse filaments

Pachytene - crossing-over

Diplotene - breaking down of synaptonemal complex

Diakinesis - max condensation, homologs separate, transition into metaphase

54
Q
A
55
Q

Where in the body is the sex chromosome determined during primordial germ cell development?

What gene is important for testis development?

A

Genital ridge

  • PGCs proliferate via mitosis then meiosis to differentiate into mature haploid gametes
  • SRY gene - cells diff into Sertoli cells, Sox9 gene. Sertoli cells secrete anti-Mullerian hormone and induce Leydig cel diff.
56
Q

Where does spermatogenesis occur?

A

Seminiferous tubules - maturation moves them closer to the lumen

57
Q

What are the requirements of fertilization?

A

Albumin - helps extract cholesterol from membrane, increasing ability to fuse w/ acrosome membrane

Ca2+, HCO3- - initiation of capacitation-associated changes

58
Q

List the basic steps of fertilization

A
  1. Sperm binds to zona pellucida
  2. Acrosomal rxn
  3. Penetration thru ZP
  4. Fusion of plasma membranes
  5. Sperm contents enter egg cytoplasm; cortical rxn occurs & meiosis resumes (high [Ca2+])
59
Q

Describe the cortical rxn

A

The release of enzymes via cortical granules from the egg to change ZP to block polyspermy

Inactivation of ZP3 so it doesn’t bind to sperm or induce acrosome rxn

60
Q
A

KCUMB Molecular Biology (6 decks)

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