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Chromosome Structure and DNA Sequence Organization

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Title: Chromosome Structure and DNA Sequence Organization


1
Chromosome StructureandDNA Sequence Organization
  • Timothy G. Standish, Ph. D.

2
Eukaryotes Have Large Complex Genomes
  • The human genome is 3 x 109 bp
  • 3 x 109 bp x 0.34 nm/bp x 1 m/109 nm 1 m
  • 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

3
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.

4
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

5
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

6
The Histone Octomer
  • Four 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

7
The Histone Octomer
8
The Fifth Histone, H1
  • A fifth 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

9
Packaging DNA
B DNA Helix
10
Packaging DNA
T
G
A
Histone octomer
C
G C
TA
G C
C G
T A
A T
A T
C G
B DNA Helix
G C
T A
11
Packaging DNA
T
G
A
Histone octomer
C
G C
TA
G C
C G
T A
A T
A T
C G
B DNA Helix
G C
T A
12
Packaging DNA
13
Packaging DNA
Histone H1
14
Packaging DNA
Beads on a string
Looped Domains
Tight helical fiber
15
Packaging DNA
Metaphase Chromosome
16
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

17
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 (i.e., Britten and Kohne, 1968).
  • By the early 1970s, the term Junk DNA had been
    coined to refer to this non-coding DNA (i.e.,
    Ohno, 1972).

18
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 (i.e., Zeyl and
    Green, 1992).
  • Mutation of some non-coding DNA does not produce
    significant changes in phenotype (Nei, 1987).

19
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.

20
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
  • Repetitive DNA sequences
  • Untranslated parts of RNA transcripts (pre-mRNA)
  • Other non-coding sequences

21
Repetitive DNA
  • Repeated sequences seem too short to code for
    proteins and are not known to be transcribed.
  • Five major classes of repetitive DNA
  • Satellites - Up to 105 tandem repeated short DNA
    sequences, concentrated in heterochromatin at the
    ends (telomeres) and centers (centromeres) of
    chromosomes.
  • Minisatellites - Similar to satellites, but found
    in clusters of fewer repeats, scattered
    throughout the genome
  • Microsatellites - Shorter still than
    minisatellites.
  • 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

22
Untranslated Parts of mRNA
  • Not all of the pre-mRNA transcribed from DNA
    actually code for the protein. These non-coding
    parts are never translated.
  • Three non-coding parts of eukaryotic mRNA
  • 5' untranslated region
  • 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' untranslated region

23
A Simple Eukaryotic Gene
24
Other Non-coding Sequences
  • Pseudogenes - DNA that resembles functional
    genes, but is not known to produce functional
    proteins. Two types
  • Unprocessed pseudogenes
  • 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.

25
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.

26
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 potsherds
    of evolution.

27
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 cant be defined, then argued
    against on the basis of a faulty definition

28
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

29
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 eukaryotes
    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.

30
Changes in the Quantity of DNA
  • The amount of non-coding DNA can vary
    significantly between closely related organisms
    (i.e., 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

31
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
    into 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

32
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

33
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.)

34
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 pre-mRNA, may play a role
    in producing, or regulating production of
    machinery to translate the mRNAs 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

35
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

36
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

37
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38
The Globin Gene Family
  • Globin genes code for the protein portion of
    hemoglobin
  • In adults, hemoglobin is made up of an iron
    containing heme molecule surrounded by 4 globin
    proteins 2 a globins and 2 b globins
  • During development, different globin genes are
    expressed which alter the oxygen affinity of
    embryonic and fetal hemoglobin

39
Model For Evolution Of The Globin Gene Family
Pseudogenes (y) resemble genes, but may lack
introns and, along with other differences
typically have stop codons that come soon after
the start codons.
40
Eukaryotic mRNA
3 Untranslated Region
5 Untranslated Region
3
5
G
AAAAA
Exon 2
Exon 3
Exon 1
Protein Coding Region
3 Poly A Tail
5 Cap
  • RNA processing achieves three things
  • 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 lifespan in the
    cytoplasm

41
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42
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43
Junk DNA
  • It is common for only a small portion of a
    eukaryotic cells 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

44
Eukaryotes Have Large Complex Genomes
  • The human genome is about 3 x 109 base pairs or
    1 m of DNA
  • Thats 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!

45
Only a Subset of Genes is Expressed at any Given
Time
  • It 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.

46
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.

47
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

48
Logical Expression Control Points
  • 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
49
A Simple Eukaryotic Gene
Transcription Start Site
3 Untranslated Region
5 Untranslated Region
Introns
3
5
Int. 2
Int. 1
Exon 2
Exon 3
Exon 1
Terminator Sequence
Promoter/ Control Region
Exons
RNA Transcript
50
Enhancers
Many bases
TF
TF
TF
51
Eukaryotic mRNA
3 Untranslated Region
5 Untranslated Region
3
5
G
AAAAA
Exon 2
Exon 3
Exon 1
Protein Coding Region
3 Poly A Tail
5 Cap
  • RNA processing achieves three things
  • 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 lifespan in the
    cytoplasm
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