chromosome structure and dna sequence organization timothy g. standish, ph. d

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Chromosome Chromosome Structure Structure and and DNA Sequence DNA Sequence Organization Organization Timothy G. Standish, Ph. D.

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Chromosome StructureChromosome Structureandand

DNA Sequence OrganizationDNA Sequence Organization

Timothy G. Standish, Ph. D.

Eukaryotes Have Large Eukaryotes Have Large Complex GeneomesComplex Geneomes

The human genome is ≈ 3 x 109 bp3 x 109 bp x 0.34 nm/bp x 1 m/109 nm ≈

1 mBecause 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

Eukaryotic DNA Eukaryotic DNA Must be PackagedMust 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.

NucleosomesNucleosomesNucleosome - Nucle - kernel, some -

bodyThe lowest DNA packaging levelCan be thought of as like a length of

thread wound around a spool, the thread representing DNA and the spool being histone proteins

Nucleosome StructureNucleosome StructureApproximately 200 bp of DNA:Core DNA - 146 bp associated with

the histone octomer19 bases complete the two turns

around the histone octomerLinker DNA - 8 to 114 bp linking

nucleosomes together

The Histone OctomerThe 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

The Histone OctomerThe Histone Octomer

The Fifth Histone, H1The 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

A TT AG CC

G

G C

TA

T

AG

C

C G

G C

T A

A T

Packaging DNAPackaging DNA

Histone proteins

Histoneoctomer

B DNA Helix 2 nm

A TT AG CC

G

G C

TA

T

AG

C

C G

G C

T A

A T

Packaging DNAPackaging DNA

Histone proteins

B DNA Helix

Histoneoctomer

2 nm

A TT AG CC

G

G C

TA

T

AG

C

C G

G C

T A

A T

Packaging DNAPackaging DNA

Histone proteins

Histoneoctomer

Nucleosome

11 nm

B DNA Helix 2 nm

Packaging DNAPackaging DNA

A TT AC G

C G

G C

T A

A T

Histone H1

Packaging DNAPackaging DNA

A TT AC G

C G

G C

T A

A T

Histone H1

Packaging DNAPackaging DNA

A TT AC G

C G

G C

T A

A T

Protein scaffold

11 nm“Beads on a string”

30 nm

Tight helical fiber

Looped Domains200 nm

Packaging DNAPackaging DNA

G

C

A

T

Protein scaffold

Metaphase Chromosome

700 nm

11 nm

30 nm200 nm

2 nm

Looped Domains

Nucleosomes

B DNA Helix

Tight helical fiber

Highly Packaged DNA Highly Packaged DNA Cannot be ExpressedCannot 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 DNAJunk DNADuring 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 non-coding DNA (ie. Ohno, 1972).

EvidenceEvidence 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?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 DNATypes of Junk DNANine 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 DNARepetitive 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 mRNAUntranslated 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 region2 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 GeneA “Simple” Eukaryotic GeneTranscription Start Site

Terminator Sequence

3’

Promoter/Control Region

5’

Introns

RNA Transcript

5’ Untranslated Region3’ Untranslated Region

Exon 2 Exon 3Int. 2Exon 1 Int. 1

Exons

Other Non-coding SequencesOther Non-coding Sequences Pseudogenes - DNA that resembles functional genes, but

is not known to produce functional proteins. Two types:1 Unprocessed pseudogenes2 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 DNAProblems With Junk DNAJunk 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 IDThe 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 GodsStraw GodsThis argument is based on assumptions

about the way the designer/God must beGod 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 DataDarwinists 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 DarwinistsThe 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 DNAChanges 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 Do Changes in Junk DNA Quantity Impact Fitness?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 Non-coding DNA has a Significant ImpactSignificant 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 Evidence for Functionality in Non-coding DNANon-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 Roles of Non-coding DNA Expressed as RNAExpressed 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 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 DNARoles 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

ConclusionsConclusions 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 FamilyThe 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 globins and 2 globins

During development, different globin genes are expressed which alter the oxygen affinity of embryonic and fetal hemoglobin

Fe

Model For Evolution Of The Model For Evolution Of The Globin Gene FamilyGlobin Gene Family

Ancestral

Globin geneDuplication

Duplication and Mutation

Chromosome 16 Chromosome 11

Transposition

Mutation

Duplication and Mutation

AdultEmbryo FetusEmbryo Fetus andAdult

Pseudo genes () resemble genes, but may lack introns and, along with other differences typically have stop codons that come soon after the start codons.

Eukaryotic mRNAEukaryotic mRNA

Protein Coding Region

3’ Untranslated Region5’ Untranslated Region

Exon 2 Exon 3Exon 1 AAAAAG 3’5’

3’ Poly A Tail5’ 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 life span in the cytoplasm

““Junk” DNAJunk” 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 Eukaryotes Have Large Complex GeneomesComplex 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!

Only a Subset of Genes is Only a Subset of Genes is Expressed at any Given TimeExpressed 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.

Eukaryotic DNA Must be Eukaryotic DNA Must be PackagedPackaged

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 Highly Packaged DNA Cannot be Expressedbe 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 PointsLogical 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

Increasing costIncreasing cost

The logical place to control

expression is before the

gene is transcribed

The logical place to control

expression is before the

gene is transcribed

A “Simple” Eukaryotic GeneA “Simple” Eukaryotic Gene

Terminator Sequence

Promoter/Control Region

Transcription Start Site

3’5’

RNA Transcript

Introns

Exon 2 Exon 3Int. 2Exon 1 Int. 1

3’ Untranslated Region5’ Untranslated Region

Exons

5’DNA

3’

EnhancersEnhancers

Enhancer Transcribed Region

3’5’ TF TFTF

3’5’ TF TFTF

5’RNA

RNAPol.

RNAPol.

Many bases

Promoter

Eukaryotic mRNAEukaryotic mRNA

Protein Coding Region

3’ Untranslated Region5’ Untranslated Region

Exon 2 Exon 3Exon 1 AAAAAG 3’5’

3’ Poly A Tail5’ 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 life span in the cytoplasm