SIBC 511: STRUCTURE AND BEHAVIOR OF GENE AND CHROMOSOME

Chatchawan Srisawat M.D., Ph.D.

DNA STRUCTURE
nucleotide deoxyribose phosphate group nitrogenous bases adenine guanine cytosine thymine A G C T

polydeoxyribonucleotide almost always found in a double-stranded form (via hydrogen bonds between bases).

DNA STRUCTURE
3 5

G
Antiparallel strands of DNA Complementary base pairing: A - T , G - C

DNA STRUCTURE

Various conformations of DNA the B-conformation- the typical 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

DNA STRUCTURE

Major and minor grooves are important for DNA-protein interactions

Rasmol

DNA STRUCTURE

Major and minor grooves are important for DNA-protein interactions.

DNA STRUCTURE

GENE GENE: a specific sequence of nucleotides in DNA or RNA that controls

the transmission and expression of one or more traits by specifying the structure of a protein or RNA

GENE = THE BASIC UNIT OF HEREDITY

GENE GENE: a specific sequence of nucleotides in DNA or RNA that controls

the transmission and expression of one or more traits by specifying the structure of a protein or RNA

Prokaryotic gene

Eukaryotic gene

Coding region encodes the amino acid sequence of a polypeptide.

GENE EXPRESSION

Typical human genes include: Regulatory sequences - promoter, enhancer, silencer Coding regions (coded for protein)- Exons Non-coding regions (interspersed between exons)- Introns, 5 and 3 untranslated regions (UTR)

GENE EXPRESSION

Question Is the sequence of all exons in mRNA coded for a polypeptide ?

Exon: a segment of a gene that is represented in the mature RNA product. Individual exons may contain coding DNA/or non-coding DNA (untranslated sequences)

GENE AND GENOME

The term genome refers to the complete complement of DNA for a given species.
Organism Human (Homo sapiens) Laboratory mouse (M. musculus) Mustard weed (A. thaliana) Roundworm (C. elegans) Fruit fly (D. melanogaster) Yeast (S. cerevisiae) Bacterium (E. coli) Human immunodeficiency virus (HIV) Genome Size Estimated Bases 3 billion 2.6 billion 100 million 97 million 137 million 12.1 million 4.6 million 9700 Genes 25,000 30,000 25,000 19,000 13,000 6,000 3,200 9

GENE AND GENOME

~20000-25000

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)

GENE AND GENOME

Coding DNA - encodes the amino acid sequence of a polypeptide, or a functional mature RNA* - represents only ~ 3% of the genome
* some gene products are RNA (estimated 3000-4000 genes out of 25000 total genes).

GENE AND GENOME

General facts about human genome General facts about human genome Gene number: 37 genes (mitochondrial genome) ~ 20,000 25,000 genes (nuclear genome)

Gene density:

averages of about

One gene per 0.45 kb (mitochondrial genome) One gene per 40-45 kb (nuclear genome)

GENE AND GENOME

General facts about human genome General facts about human genome Gene size: Average 10-15 kb, but enormous variation

gene Histone H4 tRNA insulin -globin Class I HLA serum albumin type VII collagen complement C3 factor VIII CFTR Dystrophin

size (kb) 0.4 0.1 1.4 1.6 3.5 18 31 41 186 250 2400

number of exons 1 2 3 3 8 14 118 29 26 27 79

Average exon size (bp) 300 50 155 150 187 137 77 122 375 227 180

Average intron size (bp) 20 480 490 260 1,100 90 900 7,100 9,100 30,000

GENE AND GENOME

General facts about human genome General facts about human genome Exon number: Generally correlated with gene size (but shows wide variation)
size (kb) 0.4 0.1 1.4 1.6 3.5 18 31 41 186 250 2400 number of exons 1 2 3 3 8 14 118 29 26 27 79 Average exon size (bp) 300 50 155 150 187 137 77 122 375 227 180 Average intron size (bp) 20 480 490 260 1,100 90 900 7,100 9,100 30,000

The human genome contains about 12% single exonic genes (Sakharkar et al. 2004).

gene Histone H4 tRNA insulin -globin Class I HLA serum albumin type VII collagen complement C3 factor VIII CFTR Dystrophin

GENE AND GENOME

General facts about human genome General facts about human genome Exon size: On average, 200 bp (comparatively little length variation)

gene Histone H4 tRNA insulin -globin Class I HLA serum albumin type VII collagen complement C3 factor VIII CFTR Dystrophin

size (kb) 0.4 0.1 1.4 1.6 3.5 18 31 41 186 250 2400

number of exons 1 2 3 3 8 14 118 29 26 27 79

Average exon size (bp) 300 50 155 150 187 137 77 122 375 227 180

Average intron size (bp) 20 480 490 260 1,100 90 900 7,100 9,100 30,000

GENE AND GENOME

General facts about human genome General facts about human genome Intron size: Enormous variation (strong correlation with gene size)

gene Histone H4 tRNA insulin -globin Class I HLA serum albumin type VII collagen complement C3 factor VIII CFTR Dystrophin

size (kb) 0.4 0.1 1.4 1.6 3.5 18 31 41 186 250 2400

number of exons 1 2 3 3 8 14 118 29 26 27 79

Average exon size (bp) 300 50 155 150 187 137 77 122 375 227 180

Average intron size (bp) 20 480 490 260 1,100 90 900 7,100 9,100 30,000

GENE AND GENOME

General facts about human genome General facts about human genome Gene orientation: head-to-tail
5 3 3 5

head-to-head or tail-to-tail
5 3 5 3 3 5 3 5

overlap
5 3 3 5

GENE AND GENOME

General facts about human genome General facts about human genome Gene orientation: nested gene - Some human genes can be found within other genes.
e.g. most small nucleolar RNA (snoRNA) genes are located within ribosome-associated proteins or nucleolar proteins.

- About 6% of human genes reside in introns of other genes.

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.

GENE AND GENOME

nonprocessed pseudogene: a gene that has been inactivated (non-functional) because its nucleotide sequence has been changed by mutation.

GENE AND GENOME

processed pseudogene: non-functional due to lack of introns and control region

GENE AND GENOME

GENE AND GENOME

Gene fragments: likely to have originated from unequal crossover or sister chromatid exchange

GENE AND GENOME

Tandemly repeats Interspersed repeats

TTAGGG TTAGGG TTAGGG TTAGGG

TACTCTACG TACTCTACG

GENE AND GENOME

Tandemly repeat noncoding DNA Tandemly repeat noncoding DNA 3 classes

Blocks often from 100000 bp to several Mb in length 1. Satellite DNAs major chromosomal location: centromeres function: not clear, might be important for centromere function size of repeats: 5 to 171 bp

2. Minisatellite 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 bp 2.1 telomeric family 2.2 hypervariable family- number of repeats increases or decreases between generations (highly polymorphic) --> used as markers in DNA fingerprint application

GENE AND GENOME

Tandemly repeat noncoding DNA Tandemly repeat noncoding DNA 3 classes

3. Microsatellite DNAs Blocks often less than 150 bp major chromosomal location: dispersed throughout all chromosomes function: not well understood size of repeats: 1 to 4 bp Runs of A or T CA CT
0.3 % of nuclear genome 0.5 % of nuclear genome 0.2 % of nuclear genome

Tri- or tetranucleotride repeats - rare

GENE AND GENOME

Chromosomal location of major repetitive DNA classes

GENE AND GENOME

Satellite Minisattelite Microsatelite

GENE AND GENOME

Interspersed repetitive noncoding DNA Interspersed repetitive noncoding DNA
Class SINE LINE family size number of copies ~1,000,000 ~400,000 ~300,000 % of genome ~7% ~1.7% ~5%

Others

alu ~0.3kb MIR ~0.13kb LINE-1 (Kpn) 6.1kb (but most are truncated) various ~0.4kb

~800,000

~10%

SINE = 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 unequal recombination, which may be evolutionarily advantageous in promoting gene duplication??).

GENE AND GENOME

Location of repetitive DNAs in human retinoblastoma susceptibility gene

GENE AND GENOME

Satellite Minisattelite Microsatelite

LINE SINE Transposon

GENE AND GENOME Nuclear genome

Genes and gene-related sequences 1,200 Mb Coding DNA 48 Mb (~3,000 Mb)

Extragenic DNA
2,000 Mb

Interspersed Repeats Non-coding DNA

1200 Mb LINEs 640 Mb SINEs 420 Mb Introns, UTRs DNA transposons 90 Mb LTR 250 Mb 1,400 Mb

Other intergenic regions

600 Mb

Pseudogenes

Gene fragments

Microsatellites 90 Mb Others 510 Mb

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
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 histone and non-histone proteins into chromatin and chromosome.

CHROMOSOME STRUCTURE
Level 1: Nucleosome - the most fundamental unit of packaging

DNA

Eukaryotic DNA is associated with histone proteins. Histones are small (102 to 135 amino acids) proteins that contain a very high proportion of positively charged amino acids such as lysine and arginine. Thus, they have high affinity for DNA (negatively charged molecules).

histone

CHROMOSOME STRUCTURE
Level 1: Nucleosome - the most fundamental unit of packaging

Nucleosome (200 bp):

Nucleosome core particle (146 bp) + linker DNA

Nucleosome core particle is consisted of a histone core octamer (two subunits of H2A, H2B, H3, and H4) and 146 bp of DNA wrapped 1.75 turns around the core.

CHROMOSOME STRUCTURE
Level 2: 30-nm chromatin fiber

Histone H1 brings nucleosomes together DNA is 40-fold more compact

CHROMOSOME STRUCTURE
Level 2: 30-nm chromatin fiber
10-nm fiber 30-nm fiber

CHROMOSOME STRUCTURE
Level 3: Radial loop scaffold

Scaffold proteins loop the 30-nm fiber Specific, repeated DNA sequences interact with the scaffold proteins

CHROMOSOME STRUCTURE
Level 4: Radial loop scaffold

Additional looping and gathering of loops 10000-fold more compact at metaphase

CHROMOSOME STRUCTURE
Roles of chromatin structure on cellular functions Roles of chromatin structure on cellular functions packing long DNA into compact chromosomes during cell division. - Packaging of DNA into chromatin and chromosome efficiently compact it in the nucleus (~10000-fold more compact).

controlling of gene expression by altering chromatin structures

CHROMOSOME STRUCTURE
Controlling of gene expression by altering chromatin structures Controlling of gene expression by altering chromatin structures Affinity of transcription factor for its binding site on DNA is decreased when the DNA is reconstituted into nucleosomes. The packaging of DNA into nucleosomes is generally regarded as a block to transcription, presumably because the nucleosome interferes with binding of activators.

transcription factor

transcriptional element

transcription

transcription

CHROMOSOME STRUCTURE
Controlling of gene expression by altering chromatin structures Controlling of gene expression by altering chromatin structures Heterochromatin = a portion of the chromatin in the interphase which remains relatively compacted and is transcriptionally inactive. Probably consists of closely packed region of 30-nm chromatin fiber. nucleus

CHROMOSOME STRUCTURE
Controlling of gene expression by altering chromatin structures Controlling of gene expression by altering chromatin structures

drumstick

Barr body
example
Condensation of X chromosome in cells derived from females

CHROMOSOME STRUCTURE
Controlling of gene expression by altering chromatin structures Controlling of gene expression by altering chromatin structures

Euchromatin = the more diffuse region of the interphase chromosome consisting of lessdensed chromatin. nucleus

CHROMOSOME STRUCTURE
Controlling of gene expression by altering chromatin structures Controlling of gene expression by altering chromatin structures

Modification of histones (acetylation, methylation) can change the chromatin structure, and hence, the level of gene expression.

CHROMOSOME STRUCTURE
Important features of chromosome

Centromere is required to attach to spindle at mitosis, so chromosomes segregate into new cells Telomeres protect the ends of chromosomes Replication origins are where DNA replication starts

FEATURES OF CHROMOSOME
CENTROMERE CENTROMERE In mammals, it consists of blocks of satellite DNA. Tightly condensed chromatin structure (heterochromatin) Hold sister chromatid together Bind spindle fiber, allowing segregation

FEATURES OF CHROMOSOME
TELOMERE TELOMERE

consists of 10-15 kb TTAGGG sequence (telomeric family of minisatellite DNAs) protect the ends of chromosomes from degradation and loss of DNA sequence

FEATURES OF CHROMOSOME

3 5 3 5 5 3 3 5 5 3 3 5 5 3
primer primer primer

5 3 5 3 3 5

Gap

5 3 3 5

Shortening of the Shortening of the DNA ends DNA ends (telomeres) with (telomeres) with each replication each replication

FEATURES OF CHROMOSOME

Excessive shortening of telomeres may involve genes and disrupt the coding regions.

Aging and cell death

somatic cells: e.g. skin cells (keratinocytes), fibroblasts, etc

FEATURES OF CHROMOSOME
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
REPLICATION ORIGIN REPLICATION ORIGIN

Yeasts autonomously replicating sequence (ARS)

Sites of DNA replication initiation Sequences recognized by initiator protein Mulitple origins needed to replicate chromosome efficiently

HUMAN ARTIFICIAL CHROMOSOME

Large insert capacity Predictable gene expression (endogenous machinery) Stable inheritance without integration Non-immunogenic

An ideal vehicle for gene delivery