genetics is a study of heredity, by studying microbial genetics, which is the most basic, one can extrapolate it to complex genetic studies of complex biological systems. effect of mutagens on genes is eye opening

2.  Genetics :- Science of heredity study of what genes are, how they
carry info, how they are replicated, and how expression of the
info determines characteristics of the organism.
 Genome :- All genetic info in a cell
 Chromosome :- organized unit of genome
 Genes = segments of DNA that code for functional products
(rRNA, tRNA or protein)
 Genomics :- Field of genetics involved in sequencing and
molecular characterization of genomes
 Many organisms sequences known: e.g. E.coli = ~3-4 thousand
genes; Yeast= ~5-6 thousand genes; Human= ~30 thousand
genes

3. DNA
 It is a very large molecule made up of a long chain of sub-unitsThe
sub-units are called nucleotides
 Each nucleotide is made up of a sugar called deoxyribose a
phosphate group -PO4 and an organic base
Ribose & deoxyribose
 Ribose is a sugar, like glucose, but with only five carbon atoms in
its molecule
 Deoxyribose is almost the same but lacks one oxygen atom Both
molecules may be represented by the symbol

4. The Most Common Organic Bases:
Adenine (A)
Thymine (T)
Cytosine (C)
Guanine (G)

5. adenine
deoxyribose
PO4
Nucleotides= The phosphate+ The deoxyribose+ One of the bases

6.  A molecule of DNA is
formed by millions of
nucleotides joined together
in a long chain.
 In fact, the DNA usually
consists of a double strand
of nucleotides.
 The sugar-phosphate chains
are on the outside and the
strands are held together by
chemical bonds between the
bases.
PO4
PO4
PO4
PO4
sugar-phosphate
backbone
+ bases
Joined Nucleotides

7. PO4
PO4
PO4
thymine
PO4
PO4
PO4
PO4
adenine
cytosine
PO4
guanine
Bonding

8. Genes
 Genes are like committee members; all of the genes (the
genotype) sit down together at a table and decide what the
organism is going to look like (the phenotype)

9. Genotype
 The inherited instructions an organism carries within its genetic
code
 Each gene has an opinion (an allele).
 When all of the genes are together at the table, they state what
their opinion is (what allele they are).
 Allele:
An alternate form of a gene
 Alleles as different flavors of genes

10. A
a
C
b
c
B
Genotype

11. Dominant Alleles
 Genes have different personalities
 Some genes are loud and bossy. They are always convinced that
their ideas are the best, and they have to express them to
everyone!
 We say that these genes are dominant alleles
 Dominant alleles are written with capital letters.

12. Recessive Alleles
 Some genes are very shy. They stare at their feet and just
mumble something whenever they are asked a question. They
might have some cool ideas, but they are afraid that they’ll be
laughed at if they tell the dominant genes about them, so they
keep quiet.
 We say that these genes are recessive alleles
 Recessive alleles are written with lower-case letters

13. Phenotype
 Any observable trait or characteristic of an organism
 When the committee is deciding on what the animal will
look like (phenotype), the genes split up into little sub-
committees for each trait.
 A subcommittee consists of only
 the copy of the gene from
 Mom and the copy
 of the gene from Dad.

14. Phenotype
• These two genes might discover that they
are identical and they both agree that the
exact same thing should be done.
• This means the two alleles are
homozygous
a a
We will express a
A A
We will express A

15. Phenotype
• These two genes might discover that they are different
alleles. Each gene has a different idea about how the trait
should turn out.
• This means the two alleles are heterozygous
• In this case:
– The recessive allele can’t get a word in edgewise!
– The dominant gene won't listen to anything that recessive
has to say.
– When the decisions are made about what the animal will
look like, you see only the dominant gene's ideas.
A a
We will express A

16. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Model for the formation of a replication bubble at a replication origin in
E. coli and the initiation of the new DNA strand
DNA Replication

17. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Model for the events occurring around a single replication fork of
the E. coli chromosome

18. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

19. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Model for the “replication machine,” or replisome, the complex of key
replication proteins, with the DNA at the replication fork

20. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Bidirectional replication of circular DNA molecules

21. Replication of circular DNA and the supercoiling problem
 Some circular chromosomes (e.g., E. coli) are circular
throughout replication, creating a theta-like (θ) shape. As the
strands separate on one side of the circle, positive supercoils
form elsewhere in the molecule. Replication fork moves about
500 nt/ second, so at 10 bp/turn, replication fork rotates at
3,000 rpm.
 Topoisomerases relieve the supercoils, allowing the DNA
strands to continue separating as the replication forks advance.

22. DNA replication accurate: DNA polymerase has proofreading ability
to insure proper base pairing before backbone is chemically bonded
Error rate = ~1 in 109 bases error = mutation
Gene Expression:
• RNA and protein synthesis
• DNA replication only occurs in cells that are dividing
• Gene expression occurs in all cells all the time: cells are
constructed of protein and require enzymes to function
DNA --------------> RNA --------------> Protein
transcription translation

23. Gene Expression - Transcription
When a protein is needed by a cell, the genetic code for that protein
must be read from the DNA and processed.
A two step process:
1. Transcription = synthesis of a single-stranded RNA molecule
using the DNA template (1 strand of DNA is transcribed).
2. Translation = conversion of a messenger RNA sequence into the
amino acid sequence of a polypeptide (i.e., protein synthesis)
 Both processes occur throughout the cell cycle. Transcription
occurs in the nucleus, whereas translation occurs in the
cytoplasm.

24. DNA
molecule
Gene 1
Gene 2
Gene 3
DNA strand
(template)
3
TRANSCRIPTION
Codon
mRNA
TRANSLATION
Protein
Amino acid
35
5DNA
RNA
Protein

25. Five different types of RNA, each encoded by different genes:
1. mRNA Messenger RNA, encodes the amino acid sequence of a
polypeptide.
2. tRNA Transfer RNA, transports amino acids to ribosomes during
translation.
3. rRNA Ribosomal RNA, forms complexes called ribosomes with
protein, the structure on which mRNA is translated.
4. snRNA
5. miRNA/siRNA
Small nuclear RNA, forms complexes with proteins used in
eukaryotic RNA processing (e.g., exon splicing and intron
removal).
Micro RNA/small interfering RNA, short ~22 nt RNA
sequences that bind to 3’ UTR target mRNAs and result in
gene silencing.

26. Transcription: How is an RNA strand synthesized?
1. Regulated by gene regulatory elements within each gene.
2. DNA unwinds next to a gene.
3. RNA is transcribed 5’ to 3’ from the template (3’ to 5’).
4. Similar to DNA synthesis, except:
 NTPs instead of dNTPs (no deoxy-)
 No primer and No proofreading
 Adds Uracil (U) instead of thymine (T)
 RNA polymerase

27. Three Steps to Transcription:
1. Initiation
2. Elongation
3. Termination
 Occur in both prokaryotes and eukaryotes.
 Elongation is conserved in prokaryotes and eukaryotes.
 Initiation and termination proceed differently.

28. Step 1-Initiation, E. coli model:
Each gene has three regions:
1. 5’ Promoter, attracts RNA polymerase
-10 bp 5’-TATAAT-3’
-35 bp 5’-TTGACA-3’
2. Transcribed sequence (transcript) or RNA coding sequence
3. 3’ Terminator, signals the stop point

29. Step 1-Initiation, E. coli model:
1. RNA polymerase combines with sigma factor (a polypeptide) to create RNA
polymerase holoenzyme
 Recognizes promoters and initiates transcription.
 Sigma factor required for efficient binding and transcription.
 Different sigma factors recognize different promoter sequences.
2. RNA polymerase holoenzyme binds promoters and untwists DNA
 Binds loosely to the -35 promoter (DNA is d.s.)
 Binds tightly to the -10 promoter and untwists
3. Different types and levels of sigma factors influence the level and dynamics of
gene expression (how much and efficiency).

31. Step 2-Elongation, E. coli model:
1. After 8-9 bp of RNA synthesis occurs, sigma factor is released and
recycled for other reactions.
2. RNA polymerase completes the transcription at 30-50 bp/second.
3. DNA untwists rapidly, and re-anneals behind the enzyme.
4. Part of the new RNA strand is hybrid DNA-RNA, but most RNA is
displaced as the helix reforms.

33. Step 3-Termination, E. coli model:
Two types of terminator sequences occur in prokaryotes:
1. Type I (-independent)
Palindromic, inverse repeat forms a hairpin loop and is believed to
physically destabilize the DNA-RNA hybrid.
2. Type II (-dependent)
Involves  factor proteins that break the hydrogen bonds between the
template DNA and RNA.

34. Mechanism of Translation
Ribosomes
• Bind messenger (mRNA) - Attract transfer RNA (tRNA) to
mRNA - tRNA covalently linked to specific amino acid (aa-
tRNA)
• Complementary basepairs form between mRNA and aa-tRNA
(codon-anticodon interactions)
• Enzyme in ribosome catalyzes peptide bond between amino acids
> polypeptide chain grows

35. Amino acid
attachment site
Hydrogen
bonds
3
5
Two-dimensional structure
Anticodon
Amino acid
attachment site
3
5
Hydrogen
bonds
Anticodon Anticodon
Symbol used in this bookThree-dimensional structure
3 5
tRNA structure
~ 80 nt long
Three different schematics
In what ways do they
convey the same
and different information?

36. Polypeptide
tRNA with
amino acid
attached
Ribosome
tRNA
Anticodon
35
mRNA
Amino
acids
Codons

37. Accurate translation requires two steps
 A correct match between trna and an amino acid catalyzed by
aminoacyl-trna synthetase
 A correct match between the trna anticodon and an mrna
codon

38. Amino acid Aminoacyl-tRNA
synthetase (enzyme)
Pyrophosphate
Phosphates
tRNA
AMP
Aminoacyl tRNA
(an “activated
amino acid”)

39. P site (Peptidyl-tRNA
binding site)
E site
(Exit site)
mRNA
binding site
A site (Aminoacyl-
tRNA binding site)
Large
subunit
Small
subunit
Schematic model showing binding sites on ribosome
E P A

40. Amino end
mRNA
5
3
Growing polypeptide
Next amino acid
to be added to
polypeptide chain
tRNA
Codons
Schematic model with mRNA and tRNA
E
Ribosome translates 5’ to 3’ on mRNA.
Polypeptide chain grows amino end first, carboxyl end last.

41. Building a Polypeptide
• The three stages of translation:
– Initiation
– Elongation
– Termination
All three stages require protein translation factors

42. Ribosome Association and Initiation of Translation
 Small ribosomal subunit binds mRNA and special initiator
tRNA (met-tRNAi) (carries the amino acid methionine)
 Small subunit scans along the mRNA until first start codon
(AUG).
 Initiation factors bring in large subunit initiator tRNA occupies
the P site.

43. Second mRNA base
ThirdmRNAbase(3end)
Genetic Code Codon Table
Memorize
Start
Codon

44. GTPInitiator tRNA
mRNA
5
3
mRNA binding site
Small
ribosomal
subunit
Start codon
P site
5 3
Translation initiation complex
E A
Large
ribosomal
subunit
GDP

45. Elongation of the Polypeptide Chain
Amino acids are added one by one to the preceding amino acid
Elongation factors facilitate :
1. codon recognition
2. peptide bond formation
3. translocation

46. Ribosome ready for
next aminoacyl tRNA
mRNA
5
Amino end
of polypeptide
E
P
site
A
site
3
2
2 GDP
E
P A
GTP
GTP
GDP
E
P A
E
P A
1. Recognition
2. Peptide bond
formation
3. Translocation

47. Termination of Translation
- Occurs when stop codon in mRNA reaches A site of ribosome
- A site accepts protein called release factor
-Release factor causes addition of water molecule instead of amino
acid
- Polypeptide released, ribosomal subunits dissociate and fall off
mRNA

48. Second mRNA base
ThirdmRNAbase(3end)
Genetic Code Codon Table
Memorize
Stop
Codons

49. 3
The release factor hydrolyzes the
bond between the tRNA in the
P site and the last amino acid of the
polypeptide chain. The polypeptide
is thus freed from the ribosome.
The two ribosomal subunits
and the other components
of the assembly dissociate.
Release
factor
Stop codon
(UAG, UAA, or UGA)
5
3
5
3
5
Free
polypeptide
When a ribosome reaches a stop
codon on mRNA, the A site of the
ribosome accepts a protein called
a release factor instead of tRNA.

50. The Genetic Code
• Genetic information is encoded as a sequence of nonoverlapping base triplets, or
codons
• The gene determines the sequence of bases along the length of an mRNA molecule
DNA
molecule
Gene 1
Gene 2
Gene 3
DNA strand
(template)
TRANSCRIPTION
mRNA
Protein
TRANSLATION
Amino acid
A C C A A A C C G A G T
U G G U U U G G C U C A
Trp Phe Gly Ser
Codon
3 5
35

51. The Genetic Code
• Codons: 3 base code for the production of a specific amino
acid, sequence of three of the four different nucleotides
• Since there are 4 bases and 3 positions in each codon, there are
4 x 4 x 4 = 64 possible codons
• 64 codons but only 20 amino acids, therefore most have more
than 1 codon
• 3 of the 64 codons are used as STOP signals; they are found at
the end of every gene and mark the end of the protein
• One codon is used as a START signal: it is at the start of every
protein
• Universal: in all living organisms

52. The Genetic Code
• A codon in messenger RNA is either translated into an amino
acid or serves as a translational start/stop signal
Second mRNA base
U C A G
U
C
A
G
UUU
UUC
UUA
UUG
CUU
CUC
CUA
CUG
AUU
AUC
AUA
AUG
GUU
GUC
GUA
GUG
Met or
start
Phe
Leu
Leu
lle
Val
UCU
UCC
UCA
UCG
CCU
CCC
CCA
CCG
ACU
ACC
ACA
ACG
GCU
GCC
GCA
GCG
Ser
Pro
Thr
Ala
UAU
UAC
UGU
UGC
Tyr Cys
CAU
CAC
CAA
CAG
CGU
CGC
CGA
CGG
AAU
AAC
AAA
AAG
AGU
AGC
AGA
AGG
GAU
GAC
GAA
GAG
GGU
GGC
GGA
GGG
UGG
UAA
UAG Stop
Stop UGA Stop
Trp
His
Gln
Asn
Lys
Asp
Arg
Ser
Arg
Gly
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
FirstmRNAbase(5end)
ThirdmRNAbase(3end)
Glu

53. Regulation of Bacterial Gene Expression
-protein synthesis metabolically expensive: cells only make what is
needed 60-80% of genes constitutively expressed: “housekeeping
genes”
-genes not involved in normal or continuous processes have
expression regulated feedback inhibition regulates enzymes that
have already been synthesized
Genetic Control Mechanism
1.Induction
2.Repression

54. Bacterial Operons:
Gene Regulation Model
Genes grouped into operons
- Promoter to help initiate transcription
- Operator: DNA sequence acts as on-off switch
- Genes encode metabolic enzymes
Operon regulated by repressors and/or activators
in response to environment.

55. DNA lacl
mRNA
5
3
Lac operon
Lactose present, repressor inactive, operon on
lacZ lacY lacA
RNA
polymerase
mRNA 5
Protein
Allolactose
(inducer)
Inactive
repressor
-Galactosidase Permease Transacetylase
Enzymes facilitate lactose import and breakdown
for cellular energy
Lac operon:
group of genes involved in catabolism of lactose

56. DNA lacl
Regulatory
gene
mRNA
5
3
RNA
polymerase
Protein
Active
repressor
No
RNA
made
lacZ
Promoter
Operator
Lactose absent, repressor active, operon off

57. • Inducible gene products
– usually function in catabolic pathways (lactose metabolism)
• Repressible gene products
-products usually function in anabolic pathways
(trp synthesis)
Trp and lac operons (similarities):
- Negatively controlled
- Blocked by a repressor

58. Positive Gene Regulation
• Activator protein turns on Lac operon
– catabolite activator protein (CAP)
Glucose high cAMP low
Glucose low cAMP high
CAP-cAMP binds Lac promoter and induces transcription
When would this occur, when glucose is high or low?
Low

59. DNA
cAMP
lacl
CAP-binding site
Promoter
Active
CAP
Inactive
CAP
RNA
polymerase
can bind
and transcribe
Operator
lacZ
Inactive lac
repressor
Lactose present, glucose scarce (cAMP level high): abundant lac
mRNA synthesized

60. DNA lacl
CAP-binding site
Promoter
RNA
polymerase
can’t bind
Operator
lacZ
Inactive lac
repressor
Inactive
CAP
Lactose present, glucose present (cAMP level low): little lac
mRNA synthesized

61. Mutation
Is a change of the nucleotide sequence of the genome of an
organism, virus, or extra chromosomal genetic element.
 Types of Mutation:-
 Substitution
 Insertion
 Deletion
 Frameshift

62. Substitution: A substitution is a mutation that exchanges one base
for another (i.e., a change in a single "chemical letter" such as
switching an A to a G).

63. Insertion: Insertions are mutations in which extra base pairs are
inserted into a new place in the DNA.

64. Inversion: a DNA sequence of nucleotides is reversed. Inversions
can occur among a few bases within a gene or among longer
DNA sequences that contain several genes.

65. Deletion: Deletions are mutations in which a section of DNA is
lost, or deleted.

66. Frameshift: Since protein-coding DNA is divided into codons
three bases long, insertions and deletions can alter a gene so
that its message is no longer correctly parsed.

68. Natural Cause
DNA fails to copy accurately
when a cell divides, it makes a copy of its DNA and sometimes the
copy is not quite perfect.
External Influences/Mutagens
In genetics, a mutagen is a physical or chemical agent that changes
the genetic material, usually DNA, of an organism and thus increases
the frequency of mutations above the natural background level.

69. Effects of mutagens
• can affect the transcription and replication of the DNA,
• can lead to cell death.
• produces mutations that can result in aberrant, impaired or loss
of function for a particular gene,
• accumulation of mutations may lead to cancer.
• (chromosomal instability)chromosomal breakages and
rearrangement of the chromosomes such as translocation,
deletion, and inversion. (clastogens)
• Some mutagens can cause aneuploidy and change the number
of chromosomes in the cell.

71. Physical Mutagens
• Ionizing radiations such as X-rays,
gamma rays and alpha particles
–may cause DNA breakage and other
damages. The most common sources
include cobalt-60 and cesium-137.
• Radioactive decay

72. • Ultraviolet radiations with
wavelength above 260 nm
–are absorbed strongly by bases, producing
pyrimidine dimers, which can cause error in
replication if left uncorrected.
• DNA reactive chemicals
–A large number of chemicals may interact
directly with DNA. However, many are not
necessarily mutagenic by themselves, but
through metabolic processes in cells they
produce mutagenic compounds.

73. • Reactive oxygen species (ROS)
– These ROS may result in the production of many
base adducts, as well as DNA strand breaks and
crosslinks.
• Deaminating agents
– for example nitrous acid which can cause
transition mutations by converting cytosine to
uracil.
• Aromatic amines and amides
– have been associated with carcinogenesis, may
cause cancer of the bladder, liver, ear, intestine,
thyroid and breast

74. Biological Agents
• Virus
–Virus DNA may be inserted into the genome and
disrupts genetic function. Infectious agents have
been suggested to cause cancer.
• Bacteria
–some bacteria such as Helicobacter pylori cause
inflammation during which oxidative species are
produced, causing DNA damage and reducing
efficiency of DNA repair systems, thereby
increasing mutation.

75. Protection Against
Mutagens
• Fruits and vegetables that are
rich in antioxidants
–Antioxidants are an important group of
anticarcinogenic compounds that may help
remove ROS or potentially harmful
chemicals.

76. • An effective precautionary measure
an individual can undertake to
protect themselves is by limiting
exposure to mutagens such as UV
radiations and tobacco smoke.

77. Genetic Transfer and Recombination
genetic recombination = exchange of genes between two DNA
molecules to form new combinations of genes on chromosome
involves crossing over

78. • Discovered by F. Griffith 1928 who studied streptococcus pneumoniae
• Virulent strain had capsule
• Non-virulent stain did not
• In mouse, dead virulent strain could pass “virulence factor” to live
non virulent strain
• Competent cells can pick up DNA from dead cells and incorporate it
into genome by recombination (e.G. Antibiotic resistance)
• Transformed cell than passes genetic recombination to
progeny competent = permeable to DNA: alterations
in cell wall that allow large molecule like DNA to get
through (in lab we use chemical agents to poke holes)

80. 2. Conjugation
• Genes transferred between two live cells via sex pilus (gram -
) or surface adhesion molecules (gram +)
• Transfer mediated by a plasmid: small circle of DNA separate
from genome that is self
• Replicating but contains no essential genes
• Plasmid has genes for its own transfer
• Gram negative plasmids have genes for pilus
• Gram positive plasmids have genes for surface adhesion
molecules
• Conjugation requires cell to cell contact between two cells of
opposite mating type, usually the same species, must be same
genus

81. • During conjugation plasmid is replicated and single stranded copy is
transferred to recipient. Recipient synthesizes complementary strand
to complete plasmid
• Plasmid can remain as separate circle or
• Plasmid can be integrated into host cell genome resulting in
permanent chromosomal changes

82. Transduction mechanism
1. Phage attaches to donor cell and injects phage DNA
2. Phage DNA directs donor cell to synthesize phage proteins and
DNA, phage enzymes digest the bacterial chromosome
3. New phages are assembled: phage DNA is packaged into
capsids Occasionally bacterial DNA is packaged by mistake
4. Capsid containing bacterial DNA “infects” new host recipient
cell by injecting the DNA
5. Donor DNA does not direct viral replication (not viral DNA):
instead integrates into recipient genome

83. DNA entities used for genetic change: (in both prokaryotes and eukaryotes)
1.Plasmids = self-replicating circle of DNA containing “extra” genes
 conjugative plasmids: used in bacterial conjugation, at minimum contain genes
for pili or adhesion molecules
 dissimilation plasmids: carry genes that code for enzymes to trigger catabolism
of unusual carbs or hydrocarbons
 pathogenicity plasmids: carry genes that code for virulence traits capsules,
toxins, adhesion molecules, bacteriocins
 resistance factor plasmids: carry genes for resistance to antibiotic and toxins
 plasmids can be transferred between species:
-allows spread of antibiotic resistance between different pathogens
-wide use of antibiotics has put selective pressure on microbes to “develop”
and “share” resistance genes
2. Transposons = small segments of DNA that can move independently from one
region of DNA to another
 transposons pop out and randomly insert at rate of 10-5 to 10-7 per generation
 integration is random: can disrupt genes
 at minimum transposons carry genetic info to carry out own transposition, may
also carry other genes.

84.  complex transposons have inverted repeats outside other genes
 genes will get carried with transposon when it moves
 transposons can be carried between cells on plasmids or by viruses, even
between species depending on where it inserts and what genes it carries it
can mediate good or bad genetic changes
-