Genetics Exam 3-1

Front 1

Nucleic acids made of...

Back 1

NUCLEOTIDES = 5 carbon sugar + phosphate + nitrogenous base

Front 2

RNA vs DNA structure

Back 2

RNA contains ribose in ribonucleotides (NTPs - OH)

DNA contains deoxyribose in deoxyribonucleotides (dNTPs - H)

Front 3

Purines vs Pyrimidines

2 vs 3 H bonds

Back 3

Purines = 2 rings. Adenine, Guanine

Pyrimidines = 1 ring. Thymine, Cytosine.

3 bonds = Guanine, Cytosine

2 bonds = Thymine, Adenine.

Front 4

Phosphodiester bonds

5' vs 3' end

Back 4

Bonds between hydroxyl and phosphate. Makes sugar phosphate backbone.

5' end = start = phosphate

3' end = end = hydroxyl

Front 5

3 forms of DNA, special form

Back 5

B-DNA = common

Z-DNA = special, counter clockwise

A-DNA = theoretical

ssDNA in viruses

Front 6

B-DNA structure

Back 6

Alpha helix, clockwise, 10bp per rotation, .34nm apart, 1 rotaiton = 3.4 nm.

Minor groove where base pairing occurs

Major groove = large gap.

Front 7

DNA special structures (2)

Back 7

Stem structure

Stem loop

Formed due to inverted repeats

Front 8

DNA methylation

Back 8

Addition of methyl group in DNA

Usually C in CpG sequence

Methylated = gene off, silenced.

Occurs near 5' ends of genes in promoter region

Front 9

2 ways methylation of promoter region work

Back 9

Prevents binding of transcription factors

Acts as binding site for proteins involved in condensing chromatin

Front 10

Maintenance methyltransferase

Back 10

DNA methylation patterns can be faithfully inherited - adds methyl group to opposite methyl C-G

Front 11

Supercoiling in DNA

Back 11

Lowest energy state = 10bp per turn

Supercoiled is subjected to over/underwinding (positive/negative supercoiling)

MOST DNA is NEGATIVELY supercoiled (easier to unwind, takes up less space)

Front 12

Topoisomerase

Back 12

adds or removes rotations to unwind/supercoil DNA

Front 13

How does bacterial dna fit

Back 13

Supercoiled, complexted to proteins (NOT hisotones)

Front 14

Forms of chromatin (DNA + Protein - histone/nonhistone)

Back 14

Meiosis/mitosis = most condensed, chromosome

Interphase = DNA msut be partially unwound, accesible to enzymes, chromatin

Euchromatin = undergoes condensation, decondensation during cell cycle

Heterochromatin = stays condensed, does not unwind.

Front 15

Forms of chromatin

Back 15

Euchromatin:

10nm fiber beads on a string

30nm fiber (usually unwound to 10nm)

Actively expressed.

Heterochromatin: transcriptionally inactive, more compactly folded forms of chromatin.

Includes: centromere, telomere, methylated DNA, methylated histones

Front 16

Polytene chromosomes

Back 16

giant chromosomes in some tissues in drosophila, many rounds of replication occur without cell division. Chromosome puffs = actively transcribed regions

Front 17

Changes in chromatin structure: DNAse 1 sensitivity.

Back 17

DNAase1 digests DNA when not bound to protein (histones)

DNAase1 sensitivity is correlated to active gene transcription - chromatin decondenses, RNA polymerase can bind to DNA now that it's less tightly packed.

SEE SLIDE 45

Front 18

Chromatin structure (2)

Back 18

Chromatin = DNA + Proteins (Histone/nonhistone)

DNA + core of 8 histones = nucleosome

Histones are + charged

DNA are - charged, bind to each other

DNA wrapped around histones (H2A, H2B, H3, H4)

Front 19

10 nm fiber

Back 19

Beads on a string

Nucleosomes with linker regions (attach to H1 Histone "clip").

Nonhistone proteins on linker region

Front 20

How can changes in histones/proteins alter gene activity

Back 20

1. Histone code: Histones tagged by methyl, acetyl, phosphate groups

2. Modification of histones acts as signal to other proteins for modifying chromatin STRUCTURE (wind/unwind)

OR

May provide BINDING SITE for proteins that activate/repress transcription

Front 21

Histone acetylation (which histones, by what, how)

Back 21

H3 and H4

HAT acetylates, activates

HDAC deacetylates, deactivates

Chromatin remodeling complex (SWISNF) attaches to acetylated histones, slides nucleosomes, expose dna --> activation

Front 22

Define: epigenetics. Name 3 types

Back 22

Stable changes in chromatin structure/gene expression retained through cell division (passed on to future generations of cells and future generations of organisms)

DNA methylation: OFF

Histone methylation: OFF

Histone acetylation: ON

Front 23

Genomic imprinting

Back 23

Expression depends on whether gene inherited from mother or father

Imprinting caused by epigenetic changes in chromatin structure.

Front 24

Igf2

Back 24

In mice and human: only paternal copy expressed. Maternal is METHYLATED.

Promotes feta and placental growth.

If paternal copy deleted--> small placenta, low birth weight offspring

Front 25

H19

Back 25

mehtylated in sperm, unmethylated in egg. Only maternal expressed.

Paternal demethylated in germ cell, new pattern established before gametes made

Front 26

3 types of sequences needed in chromosomes

Back 26

1. ORI: origin of replication

2. Centromere

3. Telomere

Front 27

Centromere

Back 27

Binding sites for kinetochore (protein disk) where kinetochore microtubules (spindle fibers) attach.

Large, 100K base pairs. Mainly heterochromatin. Short tandemly repeated sequence (nonspecific)

Not defined by DNA sequence but epigenetic changes (CenH3 replaces H3, promotes formation of kinetochore, attachment of spindle fibers).

Front 28

Telomeres

Back 28

Repeated sequence at ends of chromosome (unique to species, but similar pattern: TT towards middle, GG towards end)

3' overhang involved in replicating DNA. Folds over to protect ends of DNA.

Form loops to prvenet DNA breakdown

Length of telomeres change with age

Front 29

Telomerase

Back 29

Adds extra repeats to template (long strand) so DNA synthesis can occur on shorter strand.

Front 30

Genome size

Back 30

Huge variation in C (DNA content per cell) in organisms.

Mostly noncoding spacer DNA

Front 31

3 types of DNA sequences in Eukaryotes

Back 31

1. Unique sequence DNA: found 1-few times in DNA. Some code for protein, many have unknown function

Single copy genes: 25-80% of protein coding genes

Gene families: several similar but not identical genes (ex: globins)

2. Moderately repetitive DNA

3. Highly repetitive DNA

Front 32

Moderately repetitive DNA

Back 32

150-300 bp repeated thousands of times.

Functional sequences: rRNA genes, tRNA genes (need many of each).

No known function: tandem repeats, interspersed repeats (SINES, LINES)

Front 33

SINES

Back 33

Short interspersed elements. Alu sequence. 200 bp long. repeated a million times (11% of human genome).

Front 34

LINES

Back 34

long interspersed elements. 1000s of bp long. LINE1 = 17% of human genome.

Front 35

Highly repetitive DNA

Back 35

Short sequences (<10bp) repeated millions of times

Clustered in certain region of chromosome (centromere, telomere)

Satellite DNA: different GC content than rest of DNA

RARELY TRANSCRIBED into RNA.

Front 36

Transposable elements

Back 36

Mobile, jumping dna sequences

Abundant, 45% of DNA

Can insert in many locations

Can cause mutations: insertional inactivation (transposon jumps in, inactivates).

Promotes DNA rearrangements.

Front 37

General features of transposable elements (2)

Back 37

Short flanking direct repeats (3-12 bp): not part of transposon, created with it jumps in

Terminal inverted repeats: (9-40 bp), inverted and complementary. Binding site for transposase enzyme

Front 38

Common features of transposition

Back 38

Target sequence = sequence recognized by transposon

Staggered breaks made in target DNA

DNA replicated to fill in gaps, generates direct repeats

Front 39

Types of transposable elements

Back 39

DNA transposons: class II. Transpose as DNA. Replicative or nonreplicative. In bacteria and eukaryotes.

Retrotransposons: Class I. Transpose through RNA intermediate. Nonreplicative. Only in eukaryotes (common).

Front 40

Transposition causes

Back 40

Mutations.

Can disrupt gene and make it nonfunctional = insertional inactivation (50% in drosophila).

If inserted into promoter, can affect level of expression.

Can cause DNA rearrangements = deletions, inversions, duplications. (slides 86-88)

Front 41

Regulation of transposition (4)

Back 41

Many organisms need to limit tranposition.

1. Methylating DNA in regions where transposition common

2. Alteration of chromatin structure to prevent transcription of transposons.

3. Prevent translation of transposase (enzyme needed to jump) RNA by RNA interference

4. Repressor proteins

Front 42

Insertion sequences

Back 42

Simplest type, bacteria and plasmids

Contain:

Inverted repeats

Transposase gene

Generate flanking direct repeats

Front 43

Composite transposons

Back 43

Segment of DNA flanked by 2 copies of insertion sequence

Contains terminal inverted repeats + transposase gene

DNA may have MANY genes, including antibiotic resistance.

Makes flanking direct repeats.

Front 44

Noncomposite transposons

Back 44

No insertion sequences

Terminal inverted repeats

Transposase

Many other genes

Generate flanking direct repeats

Front 45

Transposable elements in eukaryotes (2)

Back 45

Transposable elements (P, Ac/Ds): Terminal inverted repeats, transposase gene, flanking direct repeats, DNA

Retrotransposons (Ty, Copia, Alu): Terminal direct repeas, several genes (reverse transcriptase) required, flanking direct repeats, RNA intermediate.

Front 46

Ty in Yeast

Back 46

Example of retrotransposon

Terminal direct repeats (delta sequences - contain promoters for Ty genes, stimulate transcription of downstream genes), retrovirus similarity

Front 47

Ac/Ds in maise

Back 47

Variegated corn kernels caused by unstable mutation (transposons).

Chromosome breakage occured at Ds (dissociation) gene only if Ac (activator) present

Front 48

Ac elements in maize

Back 48

DNA transposable element = inverted terminal repeats

Makes flanking direct repeats

Contains transposase gene - can move by itself

Front 49

Ds element in maize

Back 49

Same as Ac but with missing transposase parts

Needs Ac present to make transposase and jump

Front 50

Maize geno/phenotype

Back 50

cc, no transposition = white

Cc, no transposition = purple

Cc -> Ctc = white

Ctc -> Cc = purple

later transposition OUT = smaller purple specks.

Front 51

Transposable elements in humans, 2 examples

Back 51

45% of human genome (mostly inactive)

Alu

SINES

Front 52

DNA replication

Back 52

1. Semiconservative

2. Bidirectional from ORI sites

3. During S phase

Front 53

3 types of DNA replication

Back 53

Theta - organisms with circular DNA, 1 ORI

Rolling circle - viruses, plasmid, 1 ORI

Linear eukaryotic - many ORI, linear DNA

Front 54

Requirements for DNA Replication (REVIEW FUNCTIONS - pg 22)

Back 54

Substrate = dNTPs

Template = ssDNA

Primer

Enzymes:

Initiator protein

helicase

Topoisomerase = DNA Gyrase

Single stranded DNA Binding proteins: SSB

Primase

DNA Polymerase III

DNA Polymerase I

DNA Ligase

Front 55

Differences between prokaryotic and eukaryotic replication

Back 55

Eukaryotes have much more DNA, so multiple ORI

Licensing of DNA replication: Replication licensing factor binds to ORI, replication enzymes bind to each licensed ORI. After replication forks move away, licensing factor is removed so DNA only replicated once from that ORI per cell cycle.

Front 56

New strand built ___ to ___ end

exonuclease activity

taq polymerase

Back 56

5' to 3'

Proofreading. Also when DNA polymerase I removes RNA primers. 3' to 5'

special thermostable polymerase in PCR

Front 57

DNA Polymerase III

DNA Polymerase I

DNA ligase

Back 57

Adds dNTPs 5'-->3' on both leading/lagging strand, proofreads

Removes RNA primers, adds DNA

makes last phosphodiester bond to connect DNA.

Front 58

Okazaki fragments

dNTPS

Back 58

pieces made by lagging strand

When bond brokent to realease nucleotide, energy is also provided