Genetics test 3 Dr. Seibenhener

what most people think of when they hear the word chromosome?

metaphase chromosome

general chromosome infomation

humans have 22 pairs of autosomes and 1 pair of sex chromosomes (allosomes)

for a total of 23 pairs of chromosomes (46 chromosomes)

about 6 billion bp in every cell ~2 meters of DNA in a 10 nm nucleus

chromatin condensation

DNA

Chromatin

Metaphase Chromosome

what is chromatin?

DNA + protein (histones)

2 types of chromatin

euchromatin and heterochromatin

euchromatin (active)

lightly packed chromatin rich in gene concentration and is most often under active transcription

heterochromatin (silent)

tightly packed chromatin consisting mainly of the genetically inactive sequences

examples of heterochromatin

telomeres

centromeres

why are the genes in heterochromatin inactive?

it is bound so tightly that they cannot be accessed by transcription mechanisms

2 types of heterochromatin

1. Constitutive

2. Facultative

constitutive heterochromatin

Regions that are always heterochromatic

Permanently inactive with regard to transcription, as tightly packed as you can get

Usually contain highly repetitive sequences

Example: centromeres

Constitutive heterochromatin is located in

the centromeres of all chromosomes

facultative heterochromatin

"change"

Regions that can interconvert between euchromatin and heterochromatin (less tight to tight)

facultative heterochromatin example

heat shock proteins

-you don't need them all the time, they can remain in facultative until they are needed and then they change to become accessible

every cell in the body has the same

genetic information

models to explain association of DNA and proteins

1. folded fiber model

2. nucleosome model

Folded fiber model

A model of eukaryotic chromosome organization in which each sister chromatid consists of a single chromatin fiber composed of double-stranded DNA and proteins wound like a tightly coiled ball of yarn (DNA IS RANDOMLY PACKED)

-whole mounts of human WBC

-found few or no free fiber ends and concluded each chromatid must consist of a single fiber

-0.2 nm double helix of DNA

Type A fiber 1-10 nm

Type B fiber 20-25 nm (extensive type B folding forms a chromatid)

Nucleosome Model

Most commonly accepted model for DNA packaging

This model allows for more packaging than folded fiber, more organized

nucleosome

nucleosome

simplest packaging structure of all eukaryotic chromatin

classes of histones

core and linker

core histones

The four histone proteins (H2A, H2B, H3 and H4) that form the octameric core of a nucleosome

consists of approx. 120 amino acids each

highly conserved

in combination form the core particle

linker histone

The histone protein H1, which binds to the linker DNA adjacent to the nucleosome.

consist of approx. 200 amino acids

tissue specific expression and not highly conserved

loosely associated with core particle

each of the four core histones are __________, leading to the structure of an octamer

dimers

parts of the nucleosome

the octamer (8 histones)

the DNA

and the H1 linker

formation of the nucleosome

DNA 2 nm

nucleosomes 10 nm

soleniod, zig zag 30 nm

chromatin loops 300 nm

condensed chromatin loops 700 nm

chromosome 1400 nm

formation of the nucleosome: DNA

200 bp DNA

-146 bp wrap core

-54 bp link to the next nucleosome by linker DNA

reduce DNA length by ~7 times

linear arrangement

10 nm fiber: primary packaging of the chromatin

histone tails

strings of amino acids that protrude from the histone proteins in the nucleosome

are flexible

allows histone protein to grab onto the DNA

"beads on a string"

nucleosome

supercoiling of DNA- formation of the solenoid

H1 histone is responsible for packaging. it pulls it all together

Solenoid: helical coiling of 10 nm fibers consisting of 6 nucleosomes

-The 30 nm particle

Supercoiling- reduces the length of DNA by about 7 times

how does the histone H1 work?

it binds 2 distinct region:

1. linker DNA

2. portions of the 146 bp core

40x compaction, induces tighter DNA wrapping around histones, makes the solenoids come together

Alternate model of super coiling in DNA: the Zig Zag model

the DNA backbone (and solenoid) is not flexible enough to bend between nucleosomes; straight linker DNA connects opposite nucleosomes

The zig zag model takes the bends out, stacks a few solenoids on top of each other, allows more compaction because it puts DNA in the middle

Is still a 30 nm model

experimental results show that both the solenoid and zig zag topologies may both be present in the chromatin fiber

higher order coiling in the 300 nm fiber: chromatin loops

rosettes of chromatin loops are built around a scaffolding of topoisomerase 2

the loops have solenoids

have a compaction level of EUCHROMATIN (loose, genes are accessible, transcription can happen)

final condensation: the metaphase chromosome

DNA diameter about 700 nm

spiral scaffolding composed of topoisomerase 2 and about 15 non-histone proteins

compaction level of HETEROCHROMATIN

Chromatin Formation and the Cell Cycle

...

interphase

DNA is replicated in the S phase, DNA is accessible and lightly packed (G1, S, and G2 phase)

Early prophase

chromosome condenses, you cant access the genes

Late prophase

nuclear envelope breaks down, mitotic spindle assembles

Metaphase

mitotic spindle arranges chromosomes at cell's equator

anaphase

sister chromatids separate/torn apart

telophase

cell plate forms, cell division in cytokinesis; then we go back into G1 of interphase and it starts over

where the chromatin structures are during cell division

2 nm fiber-10 nm: not compacted, G1 phase

30 nm fiber- end of the S

300nm fiber- G2 phase

700 nm chromatin- early prophase

chromosome: in metaphase of mitosis

karyotype

picture of metaphase chromosomes in the nuclues of eukaryotic cells

let us looks at: chromosome size, position, and hetero vs euchromatin

we have 46

apes have 48

Down's Syndrome (Trisomy 21)

primary risk factor: mothers age

parents usually genetically normal

failure of chromosome 21 to separate in sperm or egg

Klinefelter's syndrome (XXY)

parents usually genetically normal

failure to separate the X chromosome during meiosis

true of false: large DNA molecules must be highly condensed to fit within the nucleus of a cell

true

stages of condensation

nucleosomes

solenoids

chromatin loops

condensed chromatin

chromatin folded around a protein scaffold

chromosome condensation is intimately associated with the

cell cycle

Pre-Darwian "great chain of being" thinking

Where human genome considered the most complex, but does the amount of DNA actually lead to complexity?

biased data set based on already sequenced "small" genomes

Junk DNA

discovery of repetitive DNA sequences

What defines complexity? (2)

1. number and types of cells

2. degree of cellular organization

C-value paradox

genome size does not correlate with organismal complexity

-excess (junk) DNA is present in the genome that does not seem to be essential for the development/evolutionary divergence of an organism

Gregory's Onion test

an onion

-diploid 2n=16

-haploid n=17 pg

-so, 17x 10^6 bp (5X MORE NON-CODING DNA)

humans

-diploid 2n=48

-haploid n=3.5 pg

-so, 3.5x 10^6 (LESS THAN AN ONION)

human vs onion: which one is more advanced?

amount of DNA: onion

number of genes: both the same

nutrition: onion

alternative splicing: humans

transcription factors: humans

genome size and cell volume

We are more complex because we have higher levels of control of our genome

Genome size and cell volume: plant cells typically larger than animal cells, DNA has a structural element in defining nucleus shape

Coding sequences in the human genome

2-5% of the human genome

about 20k protein coding genes

G-value paradox

the number of genes does not correlate with organism's complexity

C vs G value paradox

genome size vs number of genes

Types of DNA in the genome: 3 classes of nucleotide sequences

1. highly repetitive DNA sequence (HR)

2. moderately repetitive DNA sequence (MR)

3. single copy DNA sequence (unique)

highly repetitive DNA sequence (HR)

10 % of the human genome

mostly in heterochromatin regions around centromere/telomere

"non coding DNA regions"

function range: structural and organizational role to junk

example: alpha satellite DNA

moderately repetitive DNA sequence (MR)

30% of human genome

mostly in euchromatin

300 bp in size

10-10^6 copies per genome

includes "redundant" genes for histones, rRNA, and proteins

example: micro satellite DNA (repetitive sequences 2-5 bp) and interspersed repetitive DNA (transposable elements)

Microsatellites

variable number of tandem repeats typically occurring in non-coding regions of the genome

useful genetic markers as they tend to by highly polymorphic

used to sequence the human genome, markers for certain diseases, primary markers for DNA testing in forensics

can be di, tri, or tetra

occurs through a mutation process knows as "slippage recognition"

-repetitive sequence can cause slip forwards (skip info) or slip backwards (replication of old info)

single copy DNA sequence (unique)

1-5% human genome

most thoughout euchromatin

genes= "coding DNA regions"

about 20k genes

present at single or low copy number

HR+ MR+ unique

10 + 30 + 5= 45%

not 100%, the rest is junk DNA

where do we find the different types of DNA in the genome?

1. HR: in the heterochromatin, tightly coiled

-telomeres and centromeres

2. MR: scattered throughout euchromatin

-facultative heterochromatin

3. unique: euchromatin

-GENES

What is a gene?

the basic physical and functional unit of heredity

gene

sequence of unique nucleotides (genotype) that carry the genetic information which is to be expressed (phenotype)

the instruction manual for our bodies

every person has 2 copies of each gene

molecular level "gene"

DNA sequence that gives rise to an RNA molecule

the transcriptional unit

extends from the promoter to the terminator

On the DNA:

*regulatory sequence: site for the binding of regulatory proteins

1. promoter: signals beginning of transcription

2. transcribed region: part of this region contains the information that specifies an amino acid sequence

3. terminator: signals the end of transcription

is transcribed to mRNA

exon

coding sequences= phenotype

introns

intervening sequences= areas of genes that do not generally code for phenotype

flanking regions

5' untranslated region and 3' untranslated region

5' untranslated region (5' UTR)

mRNA that is directly upstream from the initiation code

-5' region untranslated forms a hairpin

3' untranslated region (3' UTR)

section of mRNA that immediately follows the translation termination codon

promoter

is a DNA sequence onto which the transcription machinery binds and initiates transcription

TATA box

highly conserved sequence in DNA serving as the binding site for transcription factor binding

core DNA sequence: 5'- TATAAA- 3'

on/off switch for transcription, where transcription factors bind

basal level of transcription (to produce one gene product)

Regulatory sequences

CAAT box

GC box (enhancer)

CAAT box

5'- GGCCAATCT- 3'

consensus sequence that occurs upstream by 60-100 bases to the initial transcription site; typically required for inducible genes to be produced in sufficient amounts

GC box

ENHANCER region of the DNA that can be bound with proteins (activators) to activate transcription of a gene or genes

"ramped up" level of transcription

termination/terminator

region of DNA where transcription ends

termination: the end of the genes

terminator: section of nucleic acid sequence that marks the end of a gene or operon in genomic DNA during transcription

helps identify the addition of the poly-A tail to the transcript

start site of transcription

+1

AUG codon codes for start

5 types of genes

1. solitary genes (unique)

2. duplicated genes

3. multigene families

4. psuedogenes

5. repeated genes

solitary gene (unique)

-a single copy of a gene (haploid situation); 2 copies in diploid

-comprises the bulk of euchromatin

duplicated genes

process by which a portion of a chromosome is duplicated in an additional copy of a gene

-results in a copy of the original gene called a paralog gene

-either of the 2 genes may mutate and change the original function of the gene

-usually occurs due to an error during meiosis

multigene families

-set of several similar genes, formed by duplication of a single original gene, and generally with similar biochemical functions

-most often located in similar regions of the chromosome

-most often used or synthesized at different times

psuedogenes

-dysfunctional relatives of genes that have lost their protein-coding ability

repeated genes

multiple copies of small genes clustered throughout the genome at specific chromosomal sites; present in a high copy number and in a tandem (head to tail) configuration

Central Dogma of Molecular Biology

DNA-transcription-RNA-translation-protein

1 gene (genotype) to 1 mRNA (intermediate) to 1 "protein" (phenotype)

but this isnt really accurate, more than one mRNA and protein being made

Transcription

DNA to mRNA

Where: in eukaryotes, cell nucleus

When: either in G1 or G2 (period when genes coding for cellular organelles are synthesized)

A. Not happening in mitosis because the genetic material it tight here (heterochromatin)

B. Not in S phase (replication) because different machinery

How: see basic rules below

basic rules of transcription

-results in an RNA compliment

A. Antiparallel

B. Unidirectional

-only one of the two DNA strands serves as a template strand

A. The strand being transcribed is the template strand because both strands cant be transcribed at the same time

-selective process

A. Replication does the entire DNA strand, transcription is controlled by transcription factors (which ones turn a gene on or off), where on= you are transcribing the gene

B. Histones: house keeping genes, actin- proteins we need all the time; always on

C. Heat shock proteins, kinases are tuned on/off based on cellular environment

-always in the 5' to 3' direction

A. Complimentary RNA will be read 3-5' (opposite direction)

steps of transcription

0. Pre-recognition

1. Recognition

a. Pre-initiation complex formation

2. Initiation

a. Binding of the RNA complex

3. Elongation

a. Movement of RNA pol 2 and formation of mRNA

4. Termination

a. Cleavage of new transcript

step 0. Pre-Recognition (DNA access)

1. DNA is packaged in the nucleus as Heterochromatin

2. DNA must convert to Euchromatin prior to Transcription so that the machinery can access the DNA

3. Part of the gene expression regulation

Transcription machinery accesses the DNA strand in the "active" euchromatin region

Transcription machinery is not able to access DNA strand in the "silent" heterochromatin region

Nucleosome (10 nm fiber) and acetylation

The core is very positive (basic) and DNA is negative, thus they attract

a. Decrease the positive charge of the histone core loosens the DNA strand; Its easier to loosen the core

b. Lys (K) acetylation

i. Histone acetyl transferase (HAT)

ii. Histone deacetylase (HDAC)

Histone acetyl transferase (HAT)

Transfers acetyl group to histone; this decreases the positive charge of the histone core, making it more negative (but is still positive)

Histone deacetylase (HDAC)

1) Takes away an acetyl group from a histone, more positive direction, DNA gets tighter

step 1. recognition: pre initiation complex formation

1. Recognition process: pre initiation complex formation

a. Formation of a large complex of proteins (PIC_ required for RNA pol 2 to bind

i. First step: TATA binding protein (TBP) binds promotor region (TATA box)

ii. General transcription factors (TF 2D) recruited by TBP

a) Form the pre-initiation complex

step 2. initiation

Initiation: binding of RNA pol complex

-other transcription factors and RNA pol 2 recruited to complex

-mediator complex: about 20 proteins (ATPase and helicase unwind DNA, about one turn of DNA unwinds and forms transcription bubble)

-template strand: binds to RNA pol 2 active site

-RNA pol 2 is unphsphorylated at its carboxyl end (CTD): is the on/off switch (phosphorylation turns it on)

step 3. elongation

Elongation: movement of the RNA pol 2 and formation of the mRNA

-RNA pol 2 is phosporylated at its carboxyl end (CTD)

-RNA pol2 traverses the template strand (3' to 5') and creates an RNA copy, but transcription occurs 5'- 3'

-exact copy of the coding strand (except the T and U, and there is ribose 5c sugar for RNA)

coding and template strand

coding 5-3'

template 3-5'

step 4. termination

termination: cleavage of new transcript

-two protein complexes carried by CTD recognize pol-A signal (AAUAAA)

1. CPSF (cleavage and polyadenylation specialty factor)

2. CSTF (cleavage stimulation factor)

-other proteins are recruited to carry out cleavage

-poly-A polymerase adds the poly-A tail, IS NOT TEMPLATE DEPENDENT

-final product is an mRNA

why do we need post-transcriptional regulations?

Transcription occurs in the nucleus. To get the mRNA to be translated, we must move it to the cytoplasm, which is a harmful environment on single stranded nucleotides. We have to protect the ends.