Exam 3 - PCB 3134 (ch 16)

DNA discovery

friedrich miescher (1869)

Chromosome discovery

flemming, a few years after DNA discovery

Genes and protein

1910-1940s protein was believed to be more complex than DNA, thus genes were made of protein

Griffith and avery

discovered 2 forms of bacterium

• S-strain (led to death of mice)

• R-strain

genetic transformation (griffith)

when S-strain was mixed with R-strain, mice died

S-strain was found alive in dead mice

conclusion: R-strain was transformed to s-strain

avery experiment

worked further on griffiths experiment, used enzymes

• the only enzyme in which mice survived while being applied was DNAase

• DNAase destroyed DNA of S-strain

DNA structure

antiparallel

double stranded deoxyribonucleotide

3’-5’

complementary sequences

complementary sequences

purine with pyrimidine

G—C —→ 3 hydrogen bonds

A—T —→ 2 hydrogen bonds

one DNA strand can be used as

a template for replication

DNA is twisted around

major and minor grooves

bond joining 5’ carbon to 3’ carbon

phosphodiester bonds

DNA length measurement

measured in base pairs, specifically kilobases (kB) 1000

Nucleic acid variants

• A-form RNA

• B-form RNA

• Z-form RNA

A-form RNA

• right handed

• helix is shorter and wider

• wider minor groove

• narrow major groove

B-form RNA

most common

• right handed

• wide major groove

• narrow minor groove

Z-form RNA

• left handed

• disordered

• narrow zig zag order

DNA twisting

can twist upon itself to supercoil

positive supercoil

same direction

negative supercoil

opposite direction

what DNA can supercoil

circular and linear, mainly linear

two states

supercoiled and relaxed, can go back and forth

supercoiling purpose

make chromosomal DNA compact

topoisomaerase

induce and relax supercoils

types of topoisomerase

• type 1 isomerase

• type 2 isomaerase

type 1 isomerase

introduces single stranded DNA breaks

type 2 isomerase

introduces double stranded DNA breaks

ex: DNA gyrase

DNA strands bound by

weak noncovalent bonds

denaturation

strands separating -melting

denaturation caused by

temp or pH increase

denaturation observational method

light absorption

DNA absorbs light at

260 nm, strand separation causes increase in absorption

DNA melting temp T_m

temp at ½ absorbance change is the T_m

T_m depends on

nucleotides

DNA renaturation

double helix reformation (reannealing)

renaturation caused by

lowering temp ; allowing hydrogen bonds to reform

Nucleic acid hybridization

Nucleic acids identified by sequences

Renaturation process

denatured DNA + ssDNA (probe) —> complementary

FISH

probe binds to complementary sequence

visual of region to identify chromosomes

fluorescent ssDNA

probe anneals to complementary sequence

DNA - eukaryotes

more per cell and interacts with proteins

DNA bound to protein

DNA is converted into chromatin

unit of chromatin/ chromosomes

nucleosomes

nucleosomes

DNA wrapped in histone proteins

nucleosome bead

8 Histones + 146 bp

histones

small basic proteins with high lysine and arginine content

5 histone types

H1, H2A, H2B, H3, and H4

amount of histones

equal amounts except for H1, it has ½

DNA and protein charges

DNA (-) and protein (+)

technique used to observe DNA degradation

gel electrophoresis

nucleosome evidence

1. chromatin exposed to nuclease

2. nuclease degrades DNA from protein

3. analyze dna by electrophoresis

electrophoresis

observe distinct patterns (200 bp)

Heterochromatin role

structural role in chromosomes

constitutive heterochromatin

permanently compacted

constitutive heterochromatin - types

centromeres and telomeres

centromere color

pink

telomere color

yellow

centromere characteristics

• internal chromosomes

• 1 per chromosome

• bound by proteins

• maintains sister chromatid cohesion

• sites for MT

• highly repetitive

telomere characterisitcs

• two per chromosome

• protect ends of chromosomes from shortening

• highly repetitive

discovery of repeated sequences

Roy britten and david kohne

experimental design

DNA fragments —→ denatured —→ renatured

Observation - roy experiment

rate of renaturation depending on conc of DNA sequence and kind

• more repeated DNA = faster reanneling

mammals-bacteria repeated DNA

mammals have more repeated DNA

two types of repeated DNA

• tandemly repeated DNA

• interspersed DNA

tandemly repeated DNA

simple sequence repeats <10 bases/ repeat

satellite DNA

tandem repeats (1000+) at one site

interspersed DNA types

transposable elements —→ transposons

transposons

can move around genome and leave copies of themselves

transposons examples

LINES and SINES

LINES

6000-800 bp, contain genes for mobilization

SINES

short interspersed nuclear elements

< 500 bp, rely on enzymes

tandemly repeats

multiple copies arranged in a row

1-2000 bp (most times <10)

10-15% of genome

Interspersed repeats

unique sequence found in multiple parts of genome

25-50% of genome

genome makeup

largest amount to least

• interspersed DNA

• Introns

• tandemly repeated DNA

• noncoding DNA

• alu elements

• extrons

mitochondria and chloroplast chromosomes

each have their own, in circular forms from histones

human mitochondria

• uses 5% of RNA

• 16,569 BP, 37 genes

nucleus

site for chromosome localization and replication

DNA transcription

nuclear pore complex

30 different proteins —→ nucleoporins

symmetrical

NPC - central granule

transporter —→ moves molecules across envelope

molecules entering NPC

• mRNA

• tRNA

• rRNA

molecules exiting NPC

proteins

what allows the entrance/ exit

aqueous diffusion channels

transport of large proteins (NPC)

can’t diffuse, active transport requiresd

Nuclear localization signals (NLS)

enables recognition of protein and transport by NPC

NLS composition

8-30 amino acid (pro-lys-arg)

NLS example

large T antigen, made by SV40

NLS does what

cause nuclear localization of protein

pyruvate kinase + NLS

RNA export

mediated by adaptor proteins

adaptor proteins contain what

nuclear export signals (NES)

NES function

target protein and RNAs for export

NES sequence recognized by

exportins —→ mediate transport out of cell

supercoiling

over/underwinding of DNA

probes

synthetic; DNA sequences that hybridize to target sequences

histone core

2 copies of H2A, H2B, H3, H4 + 1 copy of H1 (linker)

nuclease digestion experiment

partial digestion of chromatin reveals repeating 200 bp

—→ nucleosomes protect DNA from digestion

heterochromatin

always condensed

• telomeres/ centromeres

• non-coding, repetitive DNA

telomeres

5-15 kb, repeated TTAGGG

why does repetitive DNA reanneal faster

because it has more pairing options

nucleus is surrounded by

nuclear envelope