1. Basic concepts in molecular evolution
Gene: Sequence of DNA or RNA that
has a potentially functional transcript: protein-coding, tRNA, rRNA, snRNA, miRNA, ... (sometimes referred to as "productive gene"
serves as a regulatory or structural element: enhancer hub, centromeres, telomeres ... (sometimes refered to as "untranscribed gene" in literature)
Pseudogene: A non-functional DNA element with high degree of similarity to a functional (typically productive) gene
U.S. Dept of Energy Human Genome Program,
How would I know if a piece of DNA is functional or not?

2. Homologous genes: genes that share a common evolutionary origin
1. Orthologous genes
- descendants of an ancestral gene that was present
in the last common ancestor of two or more species
... so resulting from speciation event
eg. a-globin in mouse & a-globin in human
2. Paralogous genes
- arose by gene duplication within a lineage
eg. a-globin in mouse & b-globin in mouse
Memory aid: if similar genes are present in same genome, they must be paralogues
But that does not necessarily mean that similar genes in different organisms are orthologues.
eg. a-globin in mouse & b-globin in human are paralogues
… because a-globin and b-globin genes arose by duplication (long ago in ancestor of mouse and human)

3. “Typical” eukaryotic protein-coding gene
mRNA
coding sequence
Where is the promoter?
5’ UTR ?
3’ UTR ?
What regions will be present in the mature mRNA?
Is there an error in this figure?
Fig.1.4

4. Fig.1.4 Cis-acting element:
“RNA polymerase”
Promoter
“Splicing machinery”
AUG
UAA 5’ 3’
pre-mRNA
eg. cis-element for RNA stability in 3’ UTR
eg. RNA cis-element (5’ splice site)
mRNA 5’ 3’
AUG
UAA 5’ 3’
Regulatory small RNA (antisense)
Cis-acting element:
DNA (or RNA) sequences near a gene, that are important for its expression
Trans-acting factor:
protein (or RNA) that binds to cis-element to control gene expression
Fig.1.4

5. “Typical” bacterial gene organization
How many promoters are in the region shown in this figure? 2
How many proteins are encoded? 3
Operon = cluster of co-transcribed genes
Evolutionary advantages of operon organization?
- efficiency - co-ordination of gene expression
- economy - less space in genome
Fig.1.6

6. Typical prokaryotic gene: lacI in E. coli
----|----|----|----|----|----|----|----|----|----|----|----|----|----|----|----|----|----|
lacI CGAAGCGGCAUGCAUUUACGUUGACACCAUCGAAUGGCGCAAAACCUUUCGCGGUAUGGCAUGAUAGCGCCCGGAAGAGAGUCAAUUCAG
lacI GGUGGUGAAUGUGAAACCAGUAACGUUAUACGAUGUCGCAGAGUAUGCCGGUGUCUCUUAUCAGACCGUUUCCCGCGUGGUGAACCAGGC
......
lacI GCAGCUGGCACGACAGGUUUCCCGACUGGAAAGCGGGCAGUGAGCGCAACGCAAUUAAUGUGAGUUAGCUCACUCAUUAGGCACCCCAGG
----|----|----|----|----|----|----|----|----|----|---
lacI CUUUACACUUUAUGCUUCCGGCUCGUAUGUUGUGUGGAAUUGUGAGCGGAUAA
stop codon of the upstream mhpR
transcription start site SD
start codon
stop codon
ssu rRNA --GAUCACCUCCUUA 3'
mRNA GUGGUGGGA---- 5'
Frequent overlap between stop codon and start codon (of the downstream gene)
---UAAUG---, ---AUGA---

7. Gene family
an inclusive set of functionally diverged paralogous genes (Gene duplication is typically followed by subfunctionalization, neofunctionalization or degradation/deletion). It may include pseudogenes.
examples:
Human immunoglobulin genes: IgA, IgD, IgE, IgG, IgI
Alpha globin gene family
Beta globin gene family
Ubiquitin-specific protease gene family
Xuhua Xia

8. Human HBA on Chr 16
      
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9. Human HBB family at Chr 11
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10. Human HBB family at Chr 11  G   A 
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11. Protein-coding genes mRNA 5’ …. AUG GGA UUG CCC GCC …. 3’
5’ …. ATG GGA TTG CCC GCC …. 3’
“coding strand”
DNA
3’ .… TAC CCT AAC GGG CGG …. 5’
“template strand”
mRNA
5’ …. AUG GGA UUG CCC GCC …. 3’
DNA usually shown as single-stranded
with coding strand in 5’ to 3’ orientation
… so genetic code table can be used directly

12. Transcription and Translation
Gene Gene Gene 3
Polycistronic mRNA
RNA polymerase
Ribosome
GCC~tRNAGly
UCC~tRNAGly
Protein
UCC~tRNAGly
Initiation: Met-Gly-...
Elongation: Mn + M  Mn+1
UCC~tRNAGly
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13. Ribonucleotide concentration
rATP
1890
rCTP 53
rGTP
190
rUTP
130
Measured in the exponentially proliferating chick embryo fibroblasts, 2hrs, in moles 10-12 per 106 cells. The difference is expected to be more extreme in mitochondria.
NNA would seem to be a more efficient codon than NNC
XIA, X., Genetics 144:
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14. Standard Genetic Code Codon families have 1 – 6 members
Synonymous and nonsynonymous substitutions
0-fold, 2-fold, 3-fold, 4-fold degenerate sites
0-fold degenerate = non-degenerate
5’ …. AUG GGA UUG CCC CAC …. 3’
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15. Standard code 43 = 64 possible codons Codon families have 1 – 6
members
Initiation codon
When translating a nt sequence, always be sure to read it in the 5’ to 3’ direction !!
5’ …. AUG GGA UUG CCC CAC …. 3’
N-terminus…
Met
Gly
Leu
Pro
His
… C-terminus

16. Genetic code is not “universal”
Some mitochondria, a few bacteria, a few protists use a non-standard code
Table Vertebrate mitochondrial code
UGA = Trp (instead of stop codon)
AUA, AUG = Met
AGA, AGG = stop codons
Possible implications of different codes in nature?
“Defense” against foreign DNA invading the genome?
“standard”genetic code

17. AMINO ACIDS – Venn diagram showing properties
acidic:
Asp = GAU, GAC
Glu = GAA, GAG
Basic:
His, Lys, Arg
Fig. 1.9

18. Why study amino acid properties?
Protein properties often depends on the properties of their amino acids: Effect of mutation
Diagnosis, e.g., protein electrophoresis
Normal
 polypeptide (Hb-A): Val-His-Leu-Thr-Pro-Glu-Glu…… GAA
Sickel-cell
 polypeptide (Hb-S): Val-His-Leu-Thr-Pro-Val-Glu…… GUA

19. Amino acid substitutions:
(polarity, molecular volume...)
Grantham’s distance: F(V, P, C)
Miyata’s distance: F(V, P)
Amino acid substitutions:
Conservative
Ile
Leu
Radical
Table 4.7
Cys
Trp

20. Amino acid substitution matrices
----|----|----|----|----|----|----|----|----|----|----|----|--
S1 RWFFSTNHKDIGTLYLVFGAWAGMVGTALSLLIRAELSQPGALLGDDQIYNVIVTAHAFVMI
S2 RWLFSTNHKDIGTLYLLFGAWAGVLGTALSLLIRAELGQPGNLLGNDHIYNVIVTAHAFVMI
BLOSUM = BLOcks Substitution Matrix a substitution matrix used for sequence alignment of proteins (to score alignments between evolutionarily divergent protein sequences).
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21. - based on observed frequencies of amino acids
BLOSUM62 matrix
- based on observed frequencies of amino acids
replacing other amino acids during protein evolution,
particularly within conserved regions
Positive value for chemically similar substitutions
Leu to Ile = 2
Negative value for dissimilar
Cys to Trp = - 2
Large value for rare amino acids, & no change (diagonal) usually correlated with important function
Cys unchanged = 9

22. For the 61 sense codons, how many substitution mutations are possible?
43 = 64 possible codons
3 are stop codons
UAA, UAG, UGA
For the 61 sense codons, how many substitution mutations
are possible?
Change of 1st position of codon to each of 3 other nucleotides…
2nd position of codon… 3rd position of codon…
( ) x 61
= 549
How many lead to amino acid changes ? (Table 1.5)
(i.e. non-synonymous substitutions )

23. change in 2d position of codon always alters the amino acid encoded
Synonymous
Nonsynonymous
1st position
Note: This table summarizes outcome of nt sub at each site within codon, not the frequency of change seen in nature
2nd position
change in 2d position of codon always alters the amino acid encoded
3rd position
3rd position change often is “silent” (encoding same aa)

24. … but in nature, nt subs do not all occur with equal frequency:
From Table 1.3, can see that most types of nt substitutions result in amino acid changes…
… but in nature, nt subs do not all occur with equal frequency:
& synonymous subs occur much more frequently than non-synonymous ones
- that’s expected because most amino acids changes would be detrimental…
syn subs usually >> non-syn subs
Also some amino acids are more common than others in proteins
eg. Cys typically rare (but often has important function in protein folding)
For protein Y of 200 amino acids, approximately how many non-synonymous sites are expected in its mRNA?