1. Organization of the eukaryotic genomes

2. Genome Size of genome? Nuclear / organelle genome
DNA: coding, non-coding, repetitive DNA
Complexity of genes
Transposable elements
Multigenes
Pseudogenes
Regulatory sequences for Transcription?
Density of genes?

3. Genome organization Prokaryotes Eukaryotes Most genome is coding
Small amount of non-coding is regulatory sequences
Eukaryotes
Most genome is non-coding (98%)
Regulatory sequences
Introns
Repetitive DNA

4. Prokaryote genomes Example: E. coli 89% coding 4,285 genes
122 structural RNA genes
Prophage remains
Insertion sequence (IS) elements
Horizontal transfers

5. Prokaryotic genome organization:
Haploid circular genomes ( Mbp, genes)
Operons: polycistronic transcription units
Environment-specific genes on plasmids and other types of mobile genetic elements
Usually asexual reproduction, great variety of recombination mechanisms
Transcription and translation take place in the same compartment

6. Eukaryotic genome Example: C. elegans 10 chromosomes 19,099 genes
Coding region – 27%
Average of 5 introns/gene
Both long and short duplications

7. Eukaryotic genome organization
Multiple genomes: nuclear, plastid: mitochondria, chloroplasts
Plastid genomes resemble prokaryotic genomes
Multiple linear chromosomes, total size 5-10,000 MB, 5000 to genes
Monocistronic transcription units
Discontinuous coding regions (introns and exons)

8. Eukaryotic genome organization (contd.)
Large amounts of non-coding DNA
Transcription and translation take place in different compartments
Variety of RNAs: Coding (mRNA, rRNA, tRNA), Non-coding (snRNA, snoRNA, microRNAs, etc).
Often diploid genomes and obligatory sexual reproduction
Standard mechanism of recombination: meiosis

9. Hierarchy of gene organization
Gene – single unit of genetic function
Operon – genes transcribed in single transcript
Regulon – genes controlled by same regulator
Modulon – genes modulated by same stimilus
Element – plasmid, phage, chromosome,
** order of ascending
complexity
Genome

10. Finding genes in eukaryotic DNA
Types of genes include
protein-coding genes
pseudogenes
functional RNA genes: tRNA, rRNA and others
--snoRNA small nucleolar RNA
--snRNA small nuclear RNA
--miRNA microRNA
There are several kinds of exons:
-- noncoding
-- initial coding exons
-- internal exons
-- terminal exons
-- some single-exon genes are intronless

11. Mitochondrial Genome Limited autonomy of mt genomes mt encoded nuclear
NADH dehydrogase 7 subunits >41 subunits
Succinate CoQ red 0 subunits 4 subunits
Cytochrome b/c comp 1 subunit 10 subunits
Cytochrome C oxidase 3 subunits 10 subunits
ATP synthase complex 2 subunits 14 subunits
tRNA components 22 tRNAs none
rRNA components 2 components none
Ribosomal proteins none ~80
Other mt proteins none mtDNA pol, RNA pol

12. Human Mitochondrial Genome
Small (16.5 kb) circular DNA
rRNA, tRNA and protein encoding genes (37)
1 gene/0.45 kb
Very few repeats
No introns
93% coding;
Genes are transcribed as multimeric transcripts
Recombination not evident
Maternal inheritance

13. What are the mitochondrial genes?
24 of 37genes are RNA coding
22 mt tRNA
2 mit ribosomal RNA (23S, 16S)
13 of 37 genes are protein coding
(synthethized on ribosomes inside mitochondria)
some subunits of respiratory complexes and oxidative phosphorylation enzymes

14. Two overlapping genes encoded by same strand of mt DNA (ATPase 8/ ATPase 6) (unique example)
Two independent AUG located in Frame-shift to each other,
second stop codon is derived from TA + A (from poly-A)

15. Mitochondrial codon table
22 tRNA
cover for
60 positions
via
third base
wobble
AUA=ile
UGA=stop

16. Human Nuclear Genome 3200 Mb 23 (XX) or 24 (XY) linear chromosomes
30,000 genes
1 gene/100kb
Introns in the most of the genes
1.5 % of DNA is coding
Genes are transcribed individually
Repetitive DNA sequences (45%)
Recombination at least once for each chrom.
Mendelian inheritance (X + auto), paternal (Y)

17. REPEATS!!!!

18. why eukaryotic genome sizes vary
C value paradox:
why eukaryotic genome sizes vary
The haploid genome size of eukaryotes (called the C value)
varies enormously.
Small genomes include:
Encephalotiozoon cuniculi (2.9 Mb)
A variety of fungi (10-40 Mb)
Takifugu rubripes (pufferfish) (365 Mb)(same number of genes as other fish or as the human genome, but 1/10th the size)
Human 3200 Mb
Large genomes include:
Pinus resinosa (Canadian red pine)(68 Gb)
Protopterus aethiopicus (Marbled lungfish)(140 Gb)
Amoeba dubia (amoeba)(690 Gb)

19. Genome sizes in nucleotide base pairs
plasmids
viruses
bacteria
fungi
plants
algae
insects
mollusks
bony fish
The size of the human
genome is ~ 3 X 109 bp;
almost all of its complexity
is in single-copy DNA.
The human genome is thought
to contain ~30,000 genes.
amphibians
reptiles
birds
mammals
104
105
106
107
108
109
1010
1011

20. why eukaryotic genome sizes vary
C value paradox:
why eukaryotic genome sizes vary
The range in C values does not correlate well with the complexity of the organism. This phenomenon is called the C value paradox.
Why?

21. Britten and Kohne (1968) identified repetitive DNA classes
Reassociation Kinetics = isolated genomic DNA, Shear, denature (melted), & measure the rates of DNA reassociation.

22. Repetitive DNA
Two types
Tandemly repetitive
Interspersed repetitive

23. Tandem repeats Tandem repeats:
Tandem repeats occur in DNA when a pattern of two or more nucleotides is repeated and the repetitions are adjacent to each other
Form different density band on density gradient centrifugation (from bulk DNA) -satellite
Example:
A-T-T-C-G-A-T-T-C-G-A-T-T-C-G
Tandem repeats:
Satellite DNA:
Microsatellite:
Minisatellite:

24. Satellite DNA Unit - 5-300 bp depending on species.
Repeat times.
Location - Generally heterochromatic.
Examples - Centromeric DNA, telomeric DNA. There are at least 10 distinct human types of satellite DNA.

25. Microsatellite DNA Unit - 2-4 bp (most 2).
Repeat - on the order of times.
Location - Generally euchromatic.
Examples - Most useful marker for population level studies..

26. Minisatellite DNA Unit - 15-400 bp (average about 20).
Repeat - Generally times ( bp long).
Location - Generally euchromatic.
Examples - DNA fingerprints. Tandemly repeated but often in dispersed clusters. Also called VNTR’s (variable number tandem repeats).

27. Tandemly Repetitive DNA Can Cause Diseases:
Fragile X Syndrome
“CGG” is repeated hundreds or even thousands of times creating a “fragile” site on the X chromosome.
It leads to mental retardation.
Huntington's Disease
“CAG” repeat causes a protein to have long stretches of the amino acid glutamine.
Leads to a neurological disorder that results in death

28. Interspersed Repetitive DNA
Interspersed repetitive DNA accounts for 25–40 % of mammalian DNA.
They are scattered randomly throughout the genome.
The units are 100 – 1000 base pairs long.
Copies are similar but not identical to each other.
Interspersed repetitive genes are not stably integrated in the genome; they move from place to place.
They can sometimes mess up good genes

29. Interspersed Repetitive DNA
These are:
Retrotransposons (class I transposable elements) (copy and paste), copy themselves to RNA and then back to DNA (using reverse transcriptase) to integrate into the genome.
Transposons (Class II TEs) (cut and paste) uses transposases to make makes a staggered sticky cut.

30. Interspersed Repetitive DNA
Retrotransposons are:
long terminal repeat (LTR) Any transposon flanked by Long Terminal Repeats. (also called retrovirus-like elements). None are active in humans, some are mobile in mice.
long interspersed nuclear elements (LINEs) encodes RT and
short interspersed nuclear elements (SINEs) uses RT from LINEs. example Alu made up of 350 base pairs long, recognized by the RE AluI (Non-autonomous)

31. Long interspersed nuclear elements (LINEs ) 20% of genome
RNA binding
also endonuclease
Internal
promoter
LINE1 – active
(Also many truncated inactive sequences)
Line2 – inactive
Line 3 – inactive
LINEs prefer AT-rich euchromatic bands
In everyone’s genome copies of LINE1
are still capable of transposing,
and may occasionally cause the disease by gene disruption

32. Mechanism of LINE repeat jumps
Full length LINE transcript is generated from 5’-UTR-based promoter
ORF1 and ORF2 translated into proteins that stay bound to LINE mRNA
ORF1/ORF2/mRNA complex moves back into the nucleus
5’ 3’
orf2
5’ 3’
orf1
Product of ORF2
cut ds DNA
5’ 3’
orf1
orf2 3’ 5’
Freed 3’ serves as a primer for
LINE reverse transcription from 3’ UTR
5’ ’
3’ ’

33. ORF2 and ORF1 function
ORF1 keeps ORF2 and LINE mRNA bound together and retracted into nucleus
ORF2 (endonuclease) cut dsDNA to provide free 3’ end as a primer to LINE 3’UTR
ORF2 (reverse transcriptase)
makes cDNA copy of LINE mRNA, which becomes integrated into chromosomal DNA
(as it bound to it by former 3’ freed end)
TTTT A is ORF1 cleavage site,
that is why integration prefers AT rich regions

34. Short interspersed nuclear elements (SINE) 13% of genome
Non-autonomous (no RT)
bp long;
No open reading frames (no start/stop codon)
Derived from tRNA (transcribed with
RNA pol III, leaving internal promoter)
Depend on LINE machinery for its movement

35. AluI - elements Derived from signal recognition particle 7SL
Internal promoter is active, but require appropriate flanking sequence for activation
Integrates in GC rich sequences
Only active SINE in the human genome

36. Diseases caused by Alu-integration
Neurofibromatosis (Shwann cell tumors),
haemophilia,
breast cancer,
Apert syndrome (distortions of the head and face and webbing of the hands and feet),
cholinesterase deficiency (congenital myasthenic syndrome)
complement deficiency (hereditary angioedema)
α-thalassaemia
Several types of cancer, including Ewing sarcoma, breast cancer, acute myelogenous leukaemia

37. Genes
About 30,000 genes, not a particularly large number compared to other species.
Gene density varies along the chromosomes: genes are mostly in euchromatin,
Most genes (90-95% probably) code for proteins. However, there are a significant number of RNA genes.

38. Gene families
A gene family is a group of genes that share important characteristics. These may be
Structural: have similar sequence of DNA building blocks (nucleotides). Their products (such as proteins) have a similar structure or function.
Functional: have proteins produced from these genes work together as a unit or participate in the same process

39. Gene families (structural)
Classical gene families (overall conservativeness) Histones, alpha and beta- globines
Gene families with large conservative domains (other parts could be low conservative) HLH/bZIP box transcription factors
Gene families with short conservative motifs e.g. DEAD box (Asp-Glu-Ala-Asp), WD (Trp- Asp) repeat

40. Gene families (functional)
1 Regulatory protein gene families
2 Immune system proteins
3 Motor proteins
4 Signal transducing proteins
5 Transporters
6 Unclassified families

41. Multigene families
Some genes are Transcribed (But Don't Make Proteins)
The entire family of genes probably evolved from a single ancestral gene.
Famous examples: rRNA, globin genes
Four different pieces of rRNA are used to make up a ribosome: 18S, 5.8S, 28S, and 5S.
It turns out that three of these rRNAs (18S, 5.8S, 28S, ) occur in the genome as a gene (on chrom 13, 14, 15, 21, 22) & transcribed together. (one 5S on chrom. 1)
The entire multigene family is repeated nearly 300 times in clusters on five different chromosomes!
It makes sense to have many repeats of this multigene family because each cell needs many ribosomes for protein synthesis

42. Multigene family: rRNA Genes
RNA polymerase I synthesizes 45S which matures into 28S, 18S and 5.8S rRNAs
RNA polymerase II synthesizes mRNAs and most snRNA and microRNAs.
RNA polymerase III synthesizes tRNAs, rRNA 5S and other small RNAs found in the nucleus and cytosol.

43. tRNA genes (497 nuclear genes + 324 putative pseudogenes)
Humans have fewer tRNA genes that the worm (584), but more than the fly (284);
Frog (Xenopus laevis) has thousands of tRNA genes;
Number of tRNA genes correlates with size of the oocytes;
In large oocytes lots of protein needs to be sythesized simultaneously.

44. Fascinating world of RNAs coding & non-coding

45. Non-coding RNAs tRNA & rRNA
4.5S & 7S RNA (Signal Recognition Particles)
snRNA – Pre-mRNA splicing
snoRNA – rRNA modification
siRNA – small interfering RNA
gRNA – guide RNA in RNA editing
Telomerase RNA – primer for telomeric DNA synthesis
tmRNA is a hybrid molecule, half tRNA, half mRNA
Xist: The X chromosome silencing is mediated by Xist – a 16,000 nt long ncRNA
shRNA (small heterochromatic RNAs ): expresses only one allele while other is silenced
LNA Locked Nucleic Acid
piRNA Piwi-interacting RNA

46. Protein-coding Genes Highly expressed genes usually have short introns
Genes vary greatly in size and organization.
Intron less: Some genes don’t have any introns. Most common example is the histone genes.
Some genes are quite huge: dystrophin (associated with Duchenne muscular dystrophy) is 2.4 Mbp and takes 16 hours to transcribe. More than 99% of this gene is intron (total of 79 introns).
Highly expressed genes usually have short introns
Most exons are short: 200 bp on average. Intron size varies widely, from tens to millions of base pairs.

47. Pseudogenes
Pseudogenes are defective copies of genes. They have lost their protein-coding ability
have stop codons in middle of gene
they lack promoters, or
truncated
just fragments of genes.
accumulation of multiple mutations
Processed pseudogenes copied from mRNA and incorporated into the chromosome but lack of protein-coding ability (no intron/ poly-A tail present/ no promoter)
Non-processed pseudogenes are the result of tandem gene duplication or transposable element movement. When a functional gene get duplicated, one copy isn’t necessary for life.

48. Processed pseudogenes

49. Complexity
Gene number
DNA amount

50. Why so small amount of genes we, humans, kings of nature, have?
Human 30,000 genes Drosophila – 13,000 Nematode – 19,000
Potential of proteome and transcriptome diversity is so great
that it is no need for increase of amount of genes

51. Solutions ? Solution 1 to the N-value paradox:
Many protein-encoding genes produce more than one protein product (e.g., by alternative splicing or by RNA editing).
Solution 2 to the N-value paradox:
We are counting the wrong things, we should count other genetic elements (e.g., small RNAs).
Solution 3 to the N-value paradox:
We should look at connectivity rather than at nodes.
These should be exciting and should stimulate the next generation of genomic investigation.

52. 09_25_Chromosome22.jpg
09_25_Chromosome22.jpg

53. Some more statistics Gene density 1/100 kb (vary widely);
Averagely 9 exons per gene
363 exons in titin (molecular spring for elasticity of muscle) gene
Many genes are intronsless
Largest intron is 800 kb (WWOX gene)
Smallest introns – 10 bp
Average 5’ UTR kb
Average 3’ UTR kb
Largest protein: titin: 38,138 aa

54. INTRONLESS GENES Interferon genes Histone genes
Many ribonuclease genes
Heat shock protein genes
Many G-protein coupled receptors
Some genes with HMG boxes
Various neurotransmitters receptors and hormone receptors

55. to the length of the gene
Smallest human genes
Percentages
describe
exon content
to the length of the gene

56. Typical human genes

57. Extra Large human genes

58. Presumable functions of human genes

59. Genes within genes Neurofibromatosis gene (NF1) intron 26 encode :
OGMP (oligodendrocyte myelin glycoprotein),
EVI2A and EVO2B, (homologues of ecotropic viral intergration sites in mouse)