DNA and the nucleus

What did Griffith and Avery show?

that DNA is the genetic material of bacteria

Who discovered a substance that was now know as DNA?

Johann Friedrich Miescher

Walther Flemming

first observed chromosomes while studying dividing cells under the microscope

Griffith experiment

studied a pathogenic bacterial strain that caused pneumonia in animals using an A-strain, which caused a fatal infection and R-strain which was unable to do so

Genetic transformation in the griffith experiment

when dead S-strain and living R strain were mixed together and used to infect mice, the mice died; griffith found live S in the dead mice and concluded that the R strain had been converted to S-strain

kilobase (kb)

multipkes of 1000 base pairs

Alpha form RNA

favored helix of dsRNA, not a significant form of DNA

directionality of A-form RNA

right handed with a wider minor groove

B form DNA

right handed helix with clear minor and major grooves

the vast majority of DNA is in which form?

B-form

Z-form DNA

disordered, left handed helix

positive supercoiling

twisted in the same direction as coiling

negative supercoiling

coiling in the opposite direction that it is already coiled

Supercoiling occurs in ds or ss DNA?

both, but is more easily studied in circular DNA

topoisomerases

induce and relax super coils

type 1 topoisomerase

introduce transient single strand breaks in DNA

type 2 topoisomerase

introduce double strand breaks

what kind of topoisomerase is DNA gyrase?

type 2 topoisomerase

How are DNA strands bound to each other?

by relatively weak noncovalent bonds

How is strand separation induced?

by raising temperature or pH

How can strand separation be monitored?

light aborption

All DNA absorbs light with a maximum around

260 nm

as strands separate, absorbance ____

increases rapidly

Tm

DNA melting temperature; temperature at which half the absorbance change is reached

how is renaturation achieved?

by lowering temperature to permit hydrogen bonds to reform

nucleic acid hybridization

nucleic acids are identified based on sequence

a probe

purified single stranded DNA with a sequence complementary to the sequence one is trying to detect

FLourescent in Situ Hydribization (FISH)

Flourescent ssDNA probe will anneal to complementary target sequences in sample DNA

What does FISH allow you to find?

specific regions of the genome in a cell of identify specific chromosomes

when bound to proteins DNA is converted to…

chromatin

nucleosome

basic unit of chromatin

histones

a group of small basic proteins with high lysine and arginine content

Basic proteins are ___ charged and attach to __ charged DNA

positive; negative

name the five main types of histones

1. H1

2. H2A

3. H2B

4. H3

5. H4

chromatin contains about equal numbers of the five types of histones except…

H1 which is half as abundant than the others

evidence for nucleosomes

electrophoresis shows a distinctive pattern of DNA fragments in repeating 200-bp intervals, which does not hapen when DNA alone is digested

nucleosomes are packed together to form..

chromatin fibers and chromosomes

constitutive heterochromatin

heterochromatin that is permanently compacted

constitutive heterochromatin function

structural function within chromosomes

2 important types of constitutive heterochromatin

1. centromeres

2. telomeres

What color do centromeres stain?

pink

what colors do telomeres stain?

yellow

centromeres

highly repetative internal sequence, bound by a complex of proteins

Are centromeres conserved?

no

Are telomeres conserved across vertebraes?

yes (TTAGGG)

How many centromeres per chromosome?

one

How many telomeres per chromosome?

two

centromere function

maintain sister chromatid cohesion and provide attachment sites for microtubules during mitosis/meiosis

telomere fucntion

protect ends of linear chromosomes from shortening during replication

Who discovered repeat DNA sequences and how?

Roy Britten and David Kohne, by breaking down, denaturing, and renaturing DNA

rate of renaturation depends on

concentration of each kind of DNA sequence

Cot

concentration of DNA x time

the more rapidly annealing sequences of the clad DNA contain

repeated DNA sequences that are present in multiple copies

two categories of repeated DNA

tandemly repeated DNA and interspersed repeated DNA

simple sequence repeated DNA

tandem repeats that are less than 10 bases per repeat

simple-sequence repeaets were originally called

satellite DNA

Transposable elements (transposons)

interspered repeat DNA that can move around the genome and leave copies of themselves behind

Roughly ___% of the human genome consists of transposable elements

50

most abundant transposable elements

LINES (long interspersed nuclear elements)

LINES (long interspersed nuclear elements )

6000-8000bp long and contain genes requried for their own mobilization

SINES

short interspersed nuclear elements; less than 500 bp

what do SINES rely on for movement?

enzymes

most common SINES in humans

ALU sequences, which account. for 10% of the human genome

Tandemly Repeated DNA

multiple copies arranged next to each other in a row

Tandemly repeated DNA accounts for ___% of the human genome

10-15%

Interspersed repeated DNA

unique sequences that are found individually but in multiple places in the genome

are tandem repeats or interspersed repeats longer>

Interspersed repeats

interspersed repeats account for __% of the human genome

25-50%

Which type of DNA makes up the smallest portion of the human genome?

exons; about 1.5%

Eukaryotes package some of their DNA in which two places

mitochondria and chloroplasts

Mitochondira and chloroplasts have their own ____ which are devoid of ___and usually ____ shaped

chromosomes; histones; circularlly

the nucleus

the site within the eukaryotic cell where the chromosomes are lcoalized and replicated and the DNA they contain is transcribed

The NPS (nuclear pore complex) is built from….

about 30 different proteins called nucleoporins

the central granule

is called the transported. and is likely involved in moving molecules across the nuclear envelope

the NPC is ____ symmetrical

strikingly

How do molecules enter and exit the nucleus?

through nuclear pores

how do small particles freely move through the NPC?

through tiny aqueous diffusion channels

Nuclear localization signals (NLS) function

enable large proteins to be recognized and transported by the NPC

Nuclear localization signals (NLS)

8-30 amino acids long and often contains proline + the basic amino acids lysine and arginine

large T antigen

one of the first NLS to be characterized, a protein made bu simian virus 40 (SV40)

how is RNA export mediated in the nucleus?

by adaptor proteins that bind to the RNA

Nuclear export signals NES

sequences in adaptor proteins that target the proteins and bind RNAs for export

exportins

recognize NES and mediate transport of hte complexes out of the nucleus