1. Genome Organization & Protein Synthesis and Processing in Plants

2. Viral genomes
Viral genomes: ssRNA, dsRNA, ssDNA, dsDNA, linear or ciruclar
Viruses with RNA genomes:
Almost all plant viruses and some bacterial and animal viruses
Genomes are rather small (a few thousand nucleotides)
Viruses with DNA genomes (e.g. lambda = 48,502 bp):
Often a circular genome.
Replicative form of viral genomes
all ssRNA viruses produce dsRNA molecules
many linear DNA molecules become circular
Molecular weight and contour length:
duplex length per nucleotide = 3.4 Å
Mol. Weight per base pair = ~ 660

3. Procaryotic genomes Generally 1 circular chromosome (dsDNA)
Usually without introns
Relatively high gene density (~2500 genes per mm of E. coli DNA)
Contour length of E.coli genome: 1.7 mm
Often indigenous plasmids are present

4. Plasmids Extra chromosomal circular DNAs -lactamase ori
Found in bacteria, yeast and other fungi
Size varies form ~ 3,000 bp to 100,000 bp.
Replicate autonomously (origin of replication)
May contain resistance genes
May be transferred from one bacterium to another
May be transferred across kingdoms
Multicopy plasmids (~ up to 400 plasmids/per cell)
Low copy plasmids (1 –2 copies per cell)
Plasmids may be incompatible with each other
Are used as vectors that could carry a foreign gene of interest (e.g. insulin)
foreign gene

5. Eukaryotic genome Moderately repetitive
Functional (protein coding, tRNA coding)
Unknown function
SINEs (short interspersed elements) bp
100,000 copies
LINEs (long interspersed elements)
1-5 kb
10-10,000 copies

6. Eukaryotic genome Highly repetitive Minisatellites Microsatellites
Repeats of bp
1-5 kb long
Scattered throughout genome
Microsatellites
Repeats up to 13 bp
100s of kb long, 106 copies
Around centromere
Telomeres
Short repeats (6 bp)
250-1,000 at ends of chromosomes

7. Eucaryotic genomes Located on several chromosomes
Relatively low gene density (50 genes per mm of DNA in humans)
Contour length of DNA from a single human cell = 2 meters
Approximately 1011 cells = total length 2 x 1011 km
Distance between sun and earth (1.5 x 108 km)
Human chromosomes vary in length over a 25 fold range
Carry organelles genome as well

8. Mitochondrial genome (mtDNA)
Multiple identical circular chromosomes
Size ~15 Kb in animals
Size ~ 200 kb to 2,500 kb in plants
Over 95% of mitochondrial proteins are encoded in the nuclear genome.
Often A+T rich genomes.
Mt DNA is replicated before or during mitosis

9. Chloroplast genome (cpDNA)
Multiple circular molecules
Size ranges from 120 kb to 160 kb
Similar to mtDNA
Many chloroplast proteins are encoded in the nucleus (separate signal sequence)

10. “Cellular” Genomes Viruses Procaryotes Eucaryotes
Nucleus
Capsid
Plasmids
Viral genome
Bacterial
chromosome
Chromosomes
(Nuclear genome)
Mitochondrial
genome
Chloroplast
genome
Genome: all of an organism’s genes plus intergenic DNA
Intergenic DNA = DNA between genes

11. Estimated genome sizes
mammals
plants
fungi
bacteria (>100)
mitochondria (~ 100)
viruses (1024)
1e1 1e2 1e e4 1e5 1e6 1e7 1e8 1e9 1e10 1e11 1e12
Size in nucleotides. Number in ( ) = completely sequenced genomes

12. Size of genomes Epstein-Barr virus 0.172 x 106 E. coli 4.6 x 106
S. cerevisiae
12.1 x 106
C. elegans
95.5 x 106
A. thaliana
117 x 106
D. melanogaster
180 x 106
H. sapiens
3200 x 106

13. Chromosome organization
Eucaryotic chromosome
Telomere
Centromere
Telomere
p-arm
q-arm
Centromere:
DNA sequence that serve as an attachment for protein during mitosis.
In yeast these sequences (~ 130 nts) are very A+T rich.
In higher eucaryotes centromers are much longer and contain
“satellite DNA”
Telomeres:
At the end of chromosomes; help stabilize the chromosome
In yeast telomeres are ~ 100 bp long (imperfect repeats)
Repeats are added by a specific telomerase
5’ – (TxGy)n
3’ – (AxCy)n
x and y = 1 - 4
n = 20 to 100; (1500 in mammals)

14. Gene classification intergenic region non-coding genes coding genes
Chromosome
(simplified)
Messenger RNA
Structural RNA
Proteins
transfer
RNA
ribosomal
RNA
other
RNA
Structural proteins
Enzymes

15. What is a gene ? Definitions
Classical definition: Portion of a DNA that determines a single character (phenotype)
One gene – one enzyme (Beadle & Tatum 1940): “Every gene encodes the information for one enzyme”
One gene – one protein: “One gene contains information for one protein (structural proteins included) one gene – one polypeptide
Current definition: A piece of DNA (or in some cases RNA) that contains the primary sequence to produce a functional biological gene product (RNA, protein).

16. Coding region
Nucleotides (open reading frame) encoding the amino acid sequence of a protein
The molecular definition of gene includes more than just the coding region

17. Noncoding regions Regulatory regions Introns
RNA polymerase binding site
Transcription factor binding sites
Introns
Polyadenylation [poly(A)] sites

18. Gene Molecular definition:
Entire nucleic acid sequence necessary for the synthesis of a functional polypeptide (protein chain) or functional RNA

19. Anatomy of a gene ORF. From start (ATG) to stop (TGA, TAA, TAG)
Upstream region with binding site. (e.g. TATA box).
Poly-a ‘tail’
Splices. Bounded by AG and GT splice signals.

20. Bacterial genes Most do not have introns
Many are organized in operons: contiguous genes, transcribed as a single polycistronic mRNA, that encode proteins with related functions
Polycistronic mRNA encodes several proteins

21. Bacterial operon
What would be the effect of a mutation in the control region (a) compared to a mutation in a structural gene (b)?

22. Eucaryotic genes Hemoglobin beta subunit gene Splicing
Exon 1
90 bp
Intron A
131 bp
Exon 2
222 bp
Intron B
851 bp
Exon 3
126 bp
Splicing
Introns: intervening sequences within a gene that are not translated
into a protein sequence. Collagen has 50 introns.
Exons: sequences within a gene that encode protein sequences
Splicing: Removal of introns from the mRNA molecule.

23. Regulatory mechanisms
‘organize expression of genes’ (function calls)
Promoter region (binding site), usually near coding region
Binding can block (inhibit) expression
Computational challenges
Identify binding sites
Correlate sequence to expression

24. Eukaryotic genes Most have introns
Produce monocistronic mRNA: only one encoded protein
Large

25. Alternative splicing Splicing is the removal of introns
mRNA from some genes can be spliced into two or more different mRNAs

26. “Nonfunctional” DNA Higher eukaryotes have a lot of noncoding DNA
80 kb
Higher eukaryotes have a lot of noncoding DNA
Some has no known structural or regulatory function (no genes)

27. Types of eukaryotic DNA

28. Duplicated genes Encode closely related (homologous) proteins
Clustered together in genome
Formed by duplication of an ancestral gene followed by mutation
Five functional genes and two pseudogenes

29. Pseudogenes Nonfunctional copies of genes
Formed by duplication of ancestral gene, or reverse transcription (and integration)
Not expressed due to mutations that produce a stop codon (nonsense or frameshift) or prevent mRNA processing, or due to lack of regulatory sequences

30. Repetitive DNA Moderately repeated DNA Simple-sequence DNA
Tandemly repeated rRNA, tRNA and histone genes (gene products needed in high amounts)
Large duplicated gene families
Mobile DNA
Simple-sequence DNA
Tandemly repeated short sequences
Found in centromeres and telomeres (and others)
Used in DNA fingerprinting to identify individuals

31. Types of DNA repeats Perfect repeats vs degenerate repeats
Tandem repeats (e.g. satellite DNA)
5’-CATGTGCTGAAGGCTATGTGCTGCGACG- 3’
3’-GTACACGACTTCCGATACACGACGCTGC- 5’
Inverted repeats (e.g. in transposons)
Loop
5’-CATGTGCTGAAGGCTCAGCACATCGACG- 3’
3’-GTACACGACTTCCGAGTCGTGTAGCTGC- 5’
Stem
Form stem-loop structures
Palindroms = adjacent inverted repeats
(e.g. restriction sites)
Form hairpin structures
Hairpin

32. Moderately repetitive
Repetitive sequences
Satellite DNA
Chromosomal DNA
Caesium chloride
density gradient
Repeats in the mouse genome
Type
No. of Repeats
Size
Percent of genome
Highly repetitive
> 1 Mill
< 10 bp
10 %
Moderately repetitive
> 1000
~ ~300 bp
20 %

33. DNA repeats and forensics
AluSTXa
Gender determination
Standard technique: PCR amplification of the amelogenin locus
(Males = XY => bp)
AluSTXa Alu insertion on X
AluSTYa Alu insertion on Y
M F Suspect
878 bp
556 bp
AluSTYa
X-Y homologous regions
AluSTYa
M F Suspect X
528 bp
199 bp Y
Alu sequence

34. Mobile DNA Move within genomes
Most of moderately repeated DNA sequences found throughout higher eukaryotic genomes
L1 LINE is ~5% of human DNA (~50,000 copies)
Alu is ~5% of human DNA (>500,000 copies)
Some encode enzymes that catalyze movement

35. Transposition Movement of mobile DNA
Involves copying of mobile DNA element and insertion into new site in genome

36. Why? Molecular parasite: “selfish DNA”
Probably have significant effect on evolution by facilitating gene duplication, which provides the fuel for evolution, and exon shuffling

37. RNA or DNA intermediate
Transposon moves using DNA intermediate
Retrotransposon moves using RNA intermediate

38. Types of mobile DNA elements

39. LTR (long terminal repeat)
Flank viral retrotransposons and retroviruses
Contain regulatory sequences
Transcription start site and poly (A) site

41. LINES and SINES Non-viral retro-transposons
RNA intermediate
Lack LTR
LINES (long interspersed elements)
~6000 to 7000 base pairs
L1 LINE (~5% of human DNA)
Encode enzymes that catalyze movement
SINES (short interspersed elements)
~300 base pairs
Alu (~5% of human DNA)

42. Proteins Most protein sequences (today) are inferred
What’s wrong with this?
Proteins (and nucleic acids) are modified
‘mature’ Rna
Computational challenges
Identify (possible) aspects of molecular life cycle
Identify protein-protein and protein-nucleic acid interactions

43. Genetic variation
Variable number tandem repeats (minisatellites) bp. Forensic applications.
Short tandem repeat polymorphisms (microsatellites). 2-5 bp, consecutive copies.
Single nucleotide polymorphisms

44. Single nucleotide polymorphisms
1/2000 bp.
Types
Silent
Truncating
Shifting
Significance: much of individual variation.
Challenge: correlation to disease

45. Yeast genome 4.6 x 106 bp. One chromosome. Published 1997.
4,285 protein-coding genes
122 structural RNA genes
Repeats. Regulatory elements. Transposons.
Lateral transfers.

46. Yeast protein functions
Regulatory 45
1.05%
Cell structure
182
4.24
Transposons,etc 87
2.03
Transport & binding
281
6.55
Putative transport
146
3.40
Replication, repair
115
2.68
Transcription 55
1.28
Translation
Enzymes
251
5.85
Unknown
1632
38.06