fine structure of a gene

7/29/2019 fine structure of a gene

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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:


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


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GENE AND GENOME

genesize(kb)

numberof exons



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



factor VIII 186 26 375 7,100

CFTR 250 27 227 9,100

Dystrophin 2400 79 180 30,000


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



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



factor VIII 186 26 375 7,100

CFTR 250 27 227 9,100

Dystrophin 2400 79 180 30,000


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



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



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


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.


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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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.



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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



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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



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Level 2: 30-nm chromatin fiber

Histone H1 brings nucleosomes together

DNA is 40-fold more compact



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10-nm fiber 30-nm fiber

Level 2: 30-nm chromatin fiber



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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



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Level 4: Radial loop scaff old

Additional looping and gathering of loops

10000-fold more compact at metaphase



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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).



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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



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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.




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example Condensation of X chromosome in cells derived from females

Barr body

drumstick




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Euchromatin = the more

diffuse region of the interphasechromosome consisting of less-densed chromatin.

nucleus




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Modification of histones

(acetylation, methylation)

can change the chromatin

structure, and hence, the

level of gene expression.




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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)



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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)



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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



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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



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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.



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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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Documents

fine structure of a gene