Download Chromosome Structure

Chromosome Structure
and
DNA Sequence Organization
Timothy G. Standish, Ph. D.
Eukaryotes Have Large
Complex Geneomes
human genome is ≈ 3 x 109 bp
 3 x 109 bp x 0.34 nm/bp x 1 m/109 nm ≈
1m
 Because humans are diploid, each
nucleus contains 6 x 109 bp or ≈ 2 m of
DNA
 That is a lot to pack into a little nucleus!
 Eukaryotic DNA is highly packaged
 The
Eukaryotic DNA
Must be Packaged
 Eukaryotic
DNA exhibits many levels of
packaging
 The fundamental unit is the nucleosome,
DNA wound around histone proteins
 Nucleosomes arrange themselves
together to form higher and higher
levels of packaging.
Nucleosomes
 Nucleosome
- Nucle - kernel, some -
body
 The lowest DNA packaging level
 Can be thought of as like a length of
thread wound around a spool, the
thread representing DNA and the
spool being histone proteins
Nucleosome Structure
 Approximately 200 bp of DNA:
 Core DNA - 146 bp associated with
the histone octomer
 19 bases complete the two turns
around the histone octomer
 Linker DNA - 8 to 114 bp linking
nucleosomes together
 Four
The Histone Octomer
proteins: H2A, H2B, H3, and H4
 H3 and H4 are arginine rich and highly
conserved
 H2A and H2B are slightly enriched in lysine
 Both arginine and lysine are basic amino
acids making the histone proteins both basic
and positively charged
 The octomer is made of two copies of each
protein
The Histone Octomer
 A fifth
The Fifth Histone, H1
protein, H1, is part of the nucleosome,
but seems to be outside the octomer
 H1 varies between tissue and organisms and
seems to stick to the 19 bases attached to the
end of the core sequence
 Ausio (2000) discusses data showing that, at
least in fungi, survival is possible without H1
 Lack of H1 does not impact cell viability but
shortens the lifespan of the organism
 This raises the question of how H1 evolved in
single celled organisms
Packaging DNA
Histone
octomer
Histone proteins
B DNA Helix
2 nm
Packaging DNA
Histone
octomer
Histone proteins
B DNA Helix
2 nm
Packaging DNA
11 nm
Histone
octomer
Histone proteins
Nucleosome
B DNA Helix
2 nm
Packaging DNA
Histone H1
Packaging DNA
Histone H1
Packaging DNA
“Beads on
a string”
11 nm
30 nm
Tight helical
fiber
Looped
200 nm Domains
Protein scaffold
Packaging DNA
Nucleosomes
11 nm
30 nm
Tight helical fiber
Metaphase
Chromosome
700 nm
200 nm Looped Domains
2 nm
B DNA Helix
Protein scaffold
Highly Packaged DNA
Cannot be Expressed
The most highly packaged form of DNA is
“heterochromatin”
 Heterochromatin cannot be transcribed, therefore
expression of genes is prevented
 Constitutive heterochromatin - Permanently
unexpressed DNA e.g. satellite DNA
 Facultative heterochromatin - DNA that could be
expressed if it was not packaged

Junk DNA
 During
the late 1960s papers began to
appear that showed eukaryotic DNA
contained large amounts of repetitive
DNA that did not appear to code for
proteins (ie, Britten and Kohne, 1968).
 By the early 1970s, the term Junk DNA
had been coined to refer to this noncoding DNA (ie. Ohno, 1972).
Evidence
 Conservation
of protein (and DNA)
sequences is commonly interpreted to
indicate functionality
 Significant variation in non-coding DNA is
evident between relatively closely related
species and even within species (ie Zeyl and
Green, 1992).
 Mutation of some non-coding DNA does not
produce significant changes in phenotype
(Nei, 1987).
What is Junk DNA?
“Junk DNA” is DNA that does not code for
proteins, this is the definition that we will use.
 The meaning of “junk DNA” has become restricted
significantly in recent years as the functionality of
much of what was once considered junk has
become obvious. Most modern genetics texts
avoid the term. Even when junk DNA is
mentioned, it may be given significantly different
definitions. For example, Lodish et al. (1995)
called it “Extra DNA for which no function has
been found.”

Types of Junk DNA
 Nine different types of DNA were
listed as junk DNA by Nowak (1994)
 These nine types can be grouped into
three larger groups:
1Repetitive DNA sequences
2Untranslated parts of RNA transcripts
(pre-mRNA)
3Other non-coding sequences
Repetitive DNA
Repeated sequences seem too short to code for
proteins and are not known to be transcribed.
 Five major classes of repetitive DNA:

1 Satellites - Up to 105 tandem repeated short DNA
sequences, concentrated in heterochromatin at the ends
(Telomeres) and centers (centromeres) of chromosomes.
2 Minisatellites - Similar to satellites, but found in clusters
of fewer repeats, scattered throughout the genome
3 Microsatellites - Shorter still than minisatellites.
1 4 and 5 Short (300 bp) and Long (up to 7,000 bp)
Interspersed Elements (SINEs and LINEs) - Units of
DNA found distributed throughout the genome
Untranslated Parts of mRNA
 Not
all of the pre-mRNA transcribed from
DNA actually codes for the protein. These
non-coding parts are never translated.
 Three non-coding parts of eukaryotic mRNA:
1 5' untranslated region
2 Introns - Segments of DNA that are transcribed
into RNA, but are removed from the RNA
transcript before the RNA leaves the nucleus as
mRNA
3 3' untranslated region
A “Simple” Eukaryotic Gene
Transcription
Start Site
5’ Untranslated Region
Introns
5’
Exon 1 Int. 1
Promoter/
Control Region
Exon 2
3’ Untranslated Region
3’
Int. 2 Exon 3
Exons
RNA Transcript
Terminator
Sequence
Other Non-coding Sequences

Pseudogenes - DNA that resembles functional
genes, but is not known to produce functional
proteins. Two types:
1 Unprocessed pseudogenes
2 Processed pseudogenes

Heterogeneous Nuclear RNA - A mixture of
RNAs of varying lengths found in the nucleus.
Approximately 25 % of the hnRNA is pre-mRNA
that is being processed, the source and role of the
remainder is unknown.
Problems With Junk DNA
 Junk
DNA makes up a significant
portion of total genomic DNA in many
eukaryotes.
 97 % of human DNA is “junk”
 If this DNA is functionless, this
phenomenon presents interpretation
problems for both naturalism and
intelligent design theory.
The Problem for ID
It is hard to imagine a designer creating so
elegantly and efficiently at higher levels, but
leaving a lot of junk at the DNA level.
 This calls into question the intelligent design
argument that organisms are so complex and
efficient that they must be the result of design
rather than the result of random events.
 Darwinists have eagerly proclaimed junk DNA to
be molecular debris left behind in the genome as
organisms have changed over time - The pot
shards of evolution.

Straw Gods
 This
argument is based on assumptions
about the way the designer/God must be
 God is God and He can create in any way
He wants. If He wants to create
organisms with lots of unnecessary DNA,
then He can do that if He wants
 In other words, God can’t be defined, then
argued against on the basis of a faulty
definition
Darwinists Jumped on the Data



Dawkins (1993) and Orgel and Crick proposed that
successful genes are selfish in that they “care” only about
perpetuation of their own sequence. Thus repetitive DNA
represents successful selfish genes.
Brosius and Gould (1992) suggested nomenclature
assuming junk DNA was once functional DNA, currently
functionless, and is raw material for future functional genes.
Walter Gilbert and others (Gilbert and Glynias, 1993; Dorit
and Gilbert, 1991; Dorit et al., 1990) suggested exons are
the nuts and bolts of evolution while introns are the space
between them. Thus, to make a functional protein, standard
parts can be used, just as we use standard nuts, bolts and
other parts to make a bridge or bicycle
The Problem for Darwinists
Darwinism predicts at least some degree of
efficiency as natural selection should select against
less “fit” or efficient members of a population.
 Only the most efficient organisms would be
expected to survive in a selective environment.
The large amount of junk DNA in some
eukaryote’s genomes seems very inefficient.
 One would think that a trend would be evident in
organisms going from less to more efficient use of
DNA. In fact, if junk DNA really is junk, then the
trend is almost the opposite with the most primitive
organisms having the least junk DNA.

Changes in the Quantity of DNA
 The
amount of non-coding DNA can vary
significantly between closely related
organisms (ie salamanders) indicating that
changes in non-coding DNA is an easy
evolutionary step.
 If change is easy, why are those with more
than the average not less fit?
 If DNA is junk, it would be an added burden,
but the burden might not be significant, thus
change would be neutral in terms of fitness
Do Changes in Junk DNA
Quantity Impact Fitness?
Making DNA requires significant input of energy as
dNTPs, along with production of enzymes to
produce and maintain the DNA. Factor all that in to
the human average of 75 trillion cells with 6 x 109
bp/nucleus and the cost seems significant.
 Unneeded DNA presents a danger to the cell.

Mutations could resulted in the production of junk RNA
wasting resources and potentially interfering with production of
needed RNAs and consequently proteins.
 Junk proteins could be made that would waste cell resources at
best, or, at worst, may alter the activity of other proteins

Non-coding DNA has a
Significant Impact
 Sessions
and Larson (1987) showed that in
salamanders larger amounts of genomic DNA
correlates with slower development
 Meagher and Costich (1996) showed
significant negative correlation between junk
DNA content and calyx diameter in S. latifolia
 Petrov and Hartl (1998) have shown that, at
least in Drosophila species, functionless DNA
is rapidly lost
Evidence for Functionality in
Non-coding DNA




As early as 1981 (Shulman et al, 1981) statistical methods
were published for obtaining coding sequences out of the
morass of noncoding DNA.
More recently neural networks have been used to locate
protein coding regions (Uberbacher and Mural, 1991).
Searls (1992, 1997) suggested that DNA exhibits all the
characteristics of a language, including a grammar.
Mantegna et al (1994) applied a method for studying
languages (Zipf approach) to DNA sequences and suggested
“noncoding regions of DNA may carry biological
information.” (This has not gone unchallenged, see
Konopka and Martindale, 1995.)
Roles of Non-coding DNA
Expressed as RNA




Introns - May contain genes expressed independently of
the exons they fall between.
Many introns code for small nuclear RNAs (snoRNAs).
These accumulate in the nucleolus, and may play a role in
ribosome assembly. Thus the introns cut out of premRNA, may play a role in producing, or regulating
production of machinery to translate the mRNA’s code
3' Untranslated Regions - Play an important role in
regulating some genes (Wickens and Takayama, 1994).
Heterogeneous nuclear RNA - Only speculation is
possible, but with the discovery of ribozymes and RNAi it
is possible these RNAs are playing an important role
Roles of Non-coding DNA

Satellite DNA:
– Attachment sites of spindle fibers during cell division
– Telomeres protect the ends of chromosomes

Mini and Microsatellites - Defects are associated
with some types of cancer, Huntingtons disease
and fragile X disease
– May serve as sites for homologous recombination with
the Alu SINE
– A and T boxes resembling A-rich microsatellites are
found associated with the nuclear scaffold
– The AGAT minisatellite has a demonstrated function in
regulation
Conclusions
Less and less non-coding DNA looks like junk
 Some classes of non-coding DNA remain
problematic, particularly processed pseudogenes
 Discovery of important functions for non-coding
DNA calls into question any support the idea of
junk DNA provides Darwinism
 Proponents of ID must be cautious in accepting the
interpretation put on data by Darwinists
 Darwinists need to consider the predictions made
by their own theory before interpreting data to
discredit ID when the interpretation is equally
problematic in the context of natural selection

The Globin Gene Family
Globin genes code for the
a
b
protein portion of hemoglobin
 In adults, hemoglobin is made
Fe
up of an iron containing heme
molecule surrounded by 4
globin proteins: 2 a globins
b
a
and 2 b globins
 During development, different globin genes are
expressed which alter the oxygen affinity of
embryonic and fetal hemoglobin

Model For Evolution Of The
Globin Gene Family
Ancestral
Globin gene
Duplication
Mutation
a
b
Transposition
Chromosome 16
a
z
z
Embryo
b
Duplication and Mutation
e
g
Duplication and Mutation
Gg
a2 a1 yq
e
Ag
a
yz ya2 ya1
Fetus and
Adult
Embryo
Fetus
Chromosome 11
b
yb
d
b
Adult
Pseudo genes (y) resemble genes, but may lack introns and, along with other
differences typically have stop codons that come soon after the start codons.
Eukaryotic mRNA
5’ Untranslated Region
5’ G
Exon 1 Exon 2
3’ Untranslated Region
Exon 3
AAAAA
3’
Protein Coding Region
5’ Cap

RNA processing achieves three things:




3’ Poly A Tail
Removal of introns
Addition of a 5’ cap
Addition of a 3’ tail
This signals the mRNA is ready to move out of the
nucleus and may control its life span in the
cytoplasm
“Junk” DNA
It is common for only a small portion of a
eukaryotic cell’s DNA to code for proteins
 In humans, only about 3 % of DNA actually
codes for the about 100,000 proteins produced by
human cells
 Non-coding DNA was once called “junk” DNA
as it was thought to be the molecular debris left
over from the process of evolution
 We now know that much non-coding DNA is
involved in important functions like regulating
expression and maintaining the integrity of
chromosomes

Eukaryotes Have Large
Complex Geneomes
The human genome is about 3 x 109 base
pairs or ≈ 1 m of DNA
 That’s a lot more than a typical bacterial
genome
 E. coli has 4.3 x 106 bases in its genome
 Because humans are diploid, each nucleus
contains 6 x 109 base pairs or ≈ 2 m of DNA
 That is a lot to pack into a little nucleus!

 It
Only a Subset of Genes is
Expressed at any Given Time
takes lots of energy to express genes
 Thus it would be wasteful to express all
genes all the time
 By differential expression of genes, cells
can respond to changes in the environment
 Differential expression, allows cells to
specialize in multicelled organisms.
 Differential expression also allows
organisms to develop over time.
Eukaryotic DNA Must be
Packaged
Eukaryotic DNA exhibits many levels of
packaging
 The fundamental unit is the nucleosome,
DNA wound around histone proteins
 Nucleosomes arrange themselves together
to form higher and higher levels of
packaging.

Highly Packaged DNA Cannot
be Expressed
The most highly packaged form of DNA is
“heterochromatin”
 Heterochromatin cannot be transcribed,
therefore expression of genes is prevented
 Chromosome puffs on some insect
chomosomes illustrate where active gene
expression is going on

Logical Expression Control Points
Increasing cost
DNA packaging
 Transcription
 RNA processing
 mRNA Export
 mRNA masking/unmasking
and/or modification
 mRNA degradation
 Translation
 Protein modification
 Protein transport
 Protein degradation

The logical
place to
control
expression is
before the
gene is
transcribed
A “Simple” Eukaryotic Gene
Transcription
Start Site
5’
5’ Untranslated Region
Introns
Exon 1 Int. 1
Promoter/
Control Region
3’ Untranslated Region
Exon 2
3’
Int. 2 Exon 3
Exons
RNA Transcript
Terminator
Sequence
Enhancers
DNA
Many bases
5’
3’
Enhancer
5’
Promoter
TF
Transcribed Region
3’
TF
5’
TF TF RNA
RNA
Pol.
Pol.
5’
RNA
3’
Eukaryotic mRNA
5’ Untranslated Region
5’ G
Exon 1 Exon 2
3’ Untranslated Region
Exon 3
AAAAA
3’
Protein Coding Region
5’ Cap

RNA processing achieves three things:




3’ Poly A Tail
Removal of introns
Addition of a 5’ cap
Addition of a 3’ tail
This signals the mRNA is ready to move out of the
nucleus and may control its life span in the
cytoplasm