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SI BC 511SI BC 511: STRUCTURE: STRUCTURE AND BEHAVI ORAND BEHAVI OR
OF GENE AND CHROMOSOMEOF GENE AND CHROMOSOME
Chatchawan Srisawat M.D., Ph.D.
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nucleotide
deoxyribose
phosphate group
nitrogenous bases
adenine Aguanine Gcytosine Cthymine T
polydeoxyribonucleotide
DNA STRUCTURE
almost always found in a double-stranded form (via hydrogen
bonds between bases).
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Complementary base pairing: A - T , G - C
A T
G C
5
5 3
3
DNA STRUCTURE
Antiparallel strands of DNA
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DNA STRUCTURE
t he B-conformat ion- thetypical Watson-Crick double helix
(physiological form).
the Z-conformation is formed as
a result of a certain base-order;
left handed helix.
the A-conformation occurs when
DNA is dehydrated
Various conform at ions of DNA
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Major and minor grooves are important for DNA-protein interactions
DNA STRUCTURE
Rasmol
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DNA STRUCTURE
Major and minor grooves are important for DNA-protein interactions.
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DNA STRUCTURE
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GENE
GENE: a specific sequence of nucleotides in DNA or RNA that controlsthe transmission and expression of one or more traits by specifying the
structure of a protein or RNA
GENE = THE BASI C UNI T OF HEREDI TY
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Prokaryot ic gene
Eukaryotic gene
GENE
GENE: a specific sequence of nucleotides in DNA or RNA that controlsthe transmission and expression of one or more traits by specifying the
structure of a protein or RNA
Coding region encodes the amino acid sequence of a polypeptide.
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GENE EXPRESSI ON
Typical human genes include:
Regulatory sequences - promoter, enhancer, silencer
Coding regions (coded for protein)- Exons
Non-coding regions (interspersed between exons)- In t rons,
5 and 3 untranslated regions (UTR)
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GENE EXPRESSI ON
Question
I s the sequence of all exons in mRNA coded for a polypept ide ?
Exon: a segment of a gene that is represented in the mature RNA product. Individualexons may contain coding DNA/or non-coding DNA (untranslated sequences)
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The term genomerefers to the complete complement of DNA for a
given species.
GENE AND GENOME
Organism Genom e Size Est im ated
Bases Genes
Human (Homo sapiens) 3 billion 25,000
Laboratory mouse (M. musculus) 2.6 billion 30,000Mustard weed (A. thaliana) 100 million 25,000
Roundworm (C. elegans) 97 million 19,000
Fruit fly (D. melanogaster) 137 million 13,000
Yeast (S. cerevisiae) 12.1 million 6,000
Bacterium (E. coli) 4.6 million 3,200
Human immunodeficiency virus (HIV) 9700 9
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GENE AND GENOME
closed circular double stranded DNA consisting of 16,569 bp
encodes 37 genes: 2 rRNAs, 22 tRNAs, 13 protein subunits in
respiratory chain complexes (I, III, IV, V)
~20000-25000
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GENE AND GENOME
Coding DNA
- represents only ~ 3% of the genome
- encodes the amino acid sequence of a polypeptide, or afunctional mature RNA*
* some gene products are RNA (estimated 3000-4000 genes out of 25000 total genes).
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GENE AND GENOME
Gene number:
General facts about human genomeGeneral facts about human genome
37 genes (mitochondrial genome)
~ 20,000 25,000 genes (nuclear genome)
Gene densit y:
One gene per 0.45 kb (mitochondrial genome)
One gene per 40-45 kb (nuclear genome)
averages of about
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GENE AND GENOME
Average 10-15 kb, but enormous variationGene size:
General facts about human genomeGeneral facts about human genome
genesize(kb)
numberof exons
Average exonsize (bp)
Average intronsize (bp)
Histone H4
tRNA
0.4
0.1
1
2
300
50
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20insulin 1.4 3 155 480
-globin 1.6 3 150 490
Class I HLA 3.5 8 187 260
serum albumin 18 14 137 1,100type VII collagen 31 118 77 90
complement C3 41 29 122 900
factor VIII 186 26 375 7,100
CFTR 250 27 227 9,100
Dystrophin 2400 79 180 30,000
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GENE AND GENOME
genesize(kb)
numberof exons
Average exonsize (bp)
Average intronsize (bp)
Histone H4
tRNA
0.4
0.1
1
2
300
50
-
20insulin 1.4 3 155 480
-globin 1.6 3 150 490
Class I HLA 3.5 8 187 260
serum albumin 18 14 137 1,100type VII collagen 31 118 77 90
complement C3 41 29 122 900
factor VIII 186 26 375 7,100
CFTR 250 27 227 9,100
Dystrophin 2400 79 180 30,000
General facts about human genomeGeneral facts about human genome
Exon number: Generally correlated with gene size (but showswide variation)
The human genome contains about 12% single exonic genes (Sakharkar et al. 2004).
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GENE AND GENOME
genesize(kb)
numberof exons
Average exonsize (bp)
Average intronsize (bp)
Histone H4
tRNA
0.4
0.1
1
2
300
50
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20insulin 1.4 3 155 480
-globin 1.6 3 150 490
Class I HLA 3.5 8 187 260
serum albumin 18 14 137 1,100type VII collagen 31 118 77 90
complement C3 41 29 122 900
factor VIII 186 26 375 7,100
CFTR 250 27 227 9,100
Dystrophin 2400 79 180 30,000
General facts about human genomeGeneral facts about human genome
Exon size: On average, 200 bp (comparatively little length variation)
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I n t ron size: Enormous variation (strong correlation with gene size)
GENE AND GENOME
genesize(kb)
numberof exons
Average exonsize (bp)
Average intronsize (bp)
Histone H4
tRNA
0.4
0.1
1
2
300
50
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20insulin 1.4 3 155 480
-globin 1.6 3 150 490
Class I HLA 3.5 8 187 260
serum albumin 18 14 137 1,100type VII collagen 31 118 77 90
complement C3 41 29 122 900
factor VIII 186 26 375 7,100
CFTR 250 27 227 9,100
Dystrophin 2400 79 180 30,000
General facts about human genomeGeneral facts about human genome
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GENE AND GENOME
General facts about human genomeGeneral facts about human genome
Gene orientat ion:
5533
head-to-tail
head-to-head or tail-to-tail
5533
5533
overlap
5533
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GENE AND GENOME
- Some human genes can be found within other genes.
e.g. most small nucleolar RNA (snoRNA) genes are located withinribosome-associated proteins or nucleolar proteins.
General facts about human genomeGeneral facts about human genome
nested gene
Gene orientat ion:
- About 6% of human genes reside in introns of other genes.
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GENE AND GENOME
Pseudogene: a DNA sequence which shows a high degree of sequence
homology to a nonalleic functional gene but which is itself nonfunctional.
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GENE AND GENOME
nonprocessed pseudogene: a gene that has been inactivated(non-functional) because its nucleotide sequence has been changed
by mutation.
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GENE AND GENOME
processed pseudogene: non-functional due to lack of introns
and control region
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GENE AND GENOME
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GENE AND GENOME
Gene fragments: likely to haveoriginated from unequal crossoveror sister chromatid exchange
GENE AND GENOME
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GENE AND GENOME
Tandemly repeat s TTAGGG TTAGGG TTAGGG TTAGGG
I nterspersed repeat s TACTCTACG
TACTCTACG
GENE AND GENOME
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GENE AND GENOME
Tandemly repeat noncoding DNATandemly repeat noncoding DNA
1. Satellit e DNAs Blocks often from 100000 bp to several Mb in length
major chromosomal location: centromeres
function: not clear, might be important for centromere functionsize of repeats: 5 to 171 bp
2. Minisatell i t e DNAs Blocks often within 100 - 20000 bp range
major chromosomal location: at or close to telomeres
function: recombination hot spot?
size of repeats: 6 to 64 bp2.1 telomeric family
2.2 hypervariable family- number of repeats increases or
decreases between generations (highly polymorphic) -->
used as markers in DNA fingerprint application
3 classes
GENE AND GENOME
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GENE AND GENOME
Tandemly repeat noncoding DNATandemly repeat noncoding DNA
3. Microsatell i t e DNAs Blocks often less than 150 bp
major chromosomal location: dispersed throughout all chromosomes
function: not well understoodsize of repeats: 1 to 4 bp
CA 0.5 % of nuclear genomeCT 0.2 % of nuclear genome
Runs of A or T 0.3 % of nuclear genome
Tri- or tetranucleotride repeats - rare
3 classes
GENE AND GENOME
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GENE AND GENOME
Chr om osom al locat ion of m aj or repet it ive DNA classes
GENE AND GENOME
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GENE AND GENOME
Satellite
Minisattelite Microsatelite
GENE AND GENOME
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I nterspersed repet i t ive noncoding DNAI nterspersed repet i t ive noncoding DNA
GENE AND GENOME
Class family sizenumber ofcopies
% of genome
SINE aluMIR
~0.3kb~0.13kb
~1,000,000~400,000
~7%~1.7%
LINE LINE-1 (Kpn) 6.1kb (butmost aretruncated)
~300,000 ~5%
Others various ~0.4kb ~800,000 ~10%
SI NE = Short interspersed element LINE = Long interspersed element
Alu repeats are very common (once every 3 kb).
The function of Alu is unknown (speculated to promote unequalrecombination, which may be evolutionarily advantageous in promoting gene
duplication??).
GENE AND GENOME
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GENE AND GENOME
Location of repetitive DNAs in human retinoblastoma susceptibility gene
GENE AND GENOME
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GENE AND GENOME
Satellite Minisattelite
Microsatelite
LINE SINE
Transposon
GENE AND GENOME
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GENE AND GENOME
Genes and gene-relatedsequences1,200 Mb
Ext ragenic DNA2,000 Mb
Non-coding DNA
1200 Mb
I nt erspersedRepeats1,400 Mb
Otherintergenicregions
600 Mb
Nuclear genome (~ 3,000 Mb)
CodingDNA
48 Mb
LI NEs640 Mb
LTR250 Mb
SI NEs420 Mb
DNA t ransposons90 Mb
Others510 Mb
Microsatellites90 Mb
PseudogenesGene
fragments Introns,UTRs
GENE AND GENOME
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GENE AND GENOME
3% of the human genome are actually coded for proteins
A lot of the genome is junk why so much?
Pelagibacter ubique, one of the smallest self-replicating cells known
(almost no junk DNA in its genome)
CHROMOSOME STRUCTURE
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CHROMOSOME STRUCTURE
From gene to chromosome
The human genome contains 3 x 109 bp. If the DNA of all 46
chromosomes from one cell was linked together, it would measure
one meter in length.
However, in human as well as other eukaryotes, genomic DNA
can be highly folded, constrained, and compacted by histoneand non-histone proteins into chromatin and chromosome.
CHROMOSOME STRUCTURE
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CHROMOSOME STRUCTURE
Eukaryotic DNA is associated with
histone proteins.
histone
DNA
Histones are small (102 to 135 amino
acids) proteins that contain a very highproportion of positively charged amino
acids such as lysine and arginine.
Thus, they have high affinity for DNA
(negatively charged molecules).
Level 1: Nucleosome - the most fundamental unit of packaging
CHROMOSOME STRUCTURE
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Level 1: Nucleosome - the most fundamental unit of packaging
Nucleosome core part icle is consisted of a histone core octamer (twosubunits of H2A, H2B, H3, and H4) and 146 bp of DNA wrapped 1.75 turns
around the core.
Nucleosome (200 bp) :
Nucleosome core particle (146 bp)
+ linker DNA
CHROMOSOME STRUCTURE
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Level 2: 30-nm chromatin fiber
Histone H1 brings nucleosomes together
DNA is 40-fold more compact
CHROMOSOME STRUCTURE
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10-nm fiber 30-nm fiber
Level 2: 30-nm chromatin fiber
CHROMOSOME STRUCTURE
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Level 3: Radial loop scaff old
Scaffold proteins loop the 30-nm fiber
Specific, repeated DNA sequencesinteract with the scaffold proteins
CHROMOSOME STRUCTURE
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Level 4: Radial loop scaff old
Additional looping and gathering of loops
10000-fold more compact at metaphase
CHROMOSOME STRUCTURE
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Roles of chromat in st ructure on cel lu lar funct ionsRoles of chromat in st ructure on cel lu lar funct ions
packing long DNA into compact chromosomes during cell division.
controlling of gene expression by altering chromatin structures
- Packaging of DNA into chromatin and chromosome efficiently
compact it in the nucleus (~10000-fold more compact).
CHROMOSOME STRUCTURE
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The packaging of DNA into nucleosomes is generally regarded as ablock to transcription, presumably because the nucleosome interferes
with binding of activators.
Affinity of transcription factor for its binding site on DNA is decreased
when the DNA is reconstituted into nucleosomes.
transcription factor
transcriptional element
transcription transcription
Cont rol ling of gene expression by al tering chromat in st ructuresCont roll ing of gene expression by al tering chromat in st ructures
CHROMOSOME STRUCTURE
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nucleus
Heterochromatin = a
portion of the chromatin in
the interphase which remains
relatively compacted and istranscriptionally inactive.
Probably consists of closely
packed region of 30-nm
chromatin fiber.
Cont rol ling of gene expression by al tering chromat in st ructuresCont roll ing of gene expression by al tering chromat in st ructures
CHROMOSOME STRUCTURE
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example Condensation of X chromosome in cells derived from females
Barr body
drumstick
Cont rol ling of gene expression by al tering chromat in st ructuresCont roll ing of gene expression by al tering chromat in st ructures
CHROMOSOME STRUCTURE
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Euchromatin = the more
diffuse region of the interphasechromosome consisting of less-densed chromatin.
nucleus
Cont rol ling of gene expression by al tering chromat in st ructuresCont roll ing of gene expression by al tering chromat in st ructures
CHROMOSOME STRUCTURE
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Modification of histones
(acetylation, methylation)
can change the chromatin
structure, and hence, the
level of gene expression.
Cont rol ling of gene expression by al tering chromat in st ructuresCont roll ing of gene expression by al tering chromat in st ructures
CHROMOSOME STRUCTURE
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Centromere is required to
attach to spindle at mitosis,
so chromosomes segregateinto new cells
Telomeres protect the endsof chromosomes
Replicat ion origins are
where DNA replication starts
I mpor tant features of chromosome
FEATURES OF CHROMOSOME
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CENTROMERECENTROMERE
Hold sister chromatid together
Bind spindle fiber, allowingsegregation
In mammals, it consists of
blocks ofsatel l it e DNA.
Tightly condensed chromatinstructure (heterochromatin)
FEATURES OF CHROMOSOME
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TELOMERETELOMERE
protect the ends of chromosomes from degradationand loss of DNA sequence
consists of 10-15 kb TTAGGG sequence (telomericfami ly of minisatel li te DNAs)
FEATURES OF CHROMOSOME
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Gap
Shortening of theDNA ends
(telomeres) witheach replication
Shortening of theDNA ends
(telomeres) witheach replication
5 33 5
5 33 5
35
53
53
35
5
335
5 33 5
primerprimerprimer
FEATURES OF CHROMOSOME
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Excessive shortening of telomeres may involve genes
and disrupt the coding regions.
Aging and cell death
somat ic cells: e.g. skin cells (keratinocytes), fibroblasts, etc
FEATURES OF CHROMOSOME
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In germ line, telomerase is expressed to maintain the length of telomere.
Abnormal expression may be found in neoplastic cells.Abnormal expression may be found in neoplastic cells.
FEATURES OF CHROMOSOME
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REPLI CATI ON ORI GI NREPLI CATI ON ORI GI N
Sequences recognized by initiator protein
Mulitple origins needed to replicate chromosome efficiently
Sites of DNA replication initiation
Yeasts autonomously replicating sequence (ARS)
HUMAN ARTI FI CI AL CHROMOSOME
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An ideal vehicle for gene delivery
Large insert capacity
Predictable gene
expression (endogenous
machinery)
Stable inheritance withoutintegration
Non-immunogenic
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