1509630104Microbial+GeneVVVVVVtics(3).ppt

RFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFRFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFGRFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFGRFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFGRFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFGGRFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFRFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFGRFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFGRFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFGRFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFGGRFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFRFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFGRFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFGRFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFGRFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFGGRFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFRFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFGRFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFGRFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFGRFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFGGRFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFRFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFGRFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFGRFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFGRFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFGGRFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFRFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFGRFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFGRFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFGRFHSGDGWGDGKAWGKKAGFKDAJMAMFGKGFKGFKGFKQFUKGQEFKGFUKGDSGAMSAGSAGAGGFDAGFJAGFAGHJSAGGFGFGFGFGFGFGFGFGG

1. Microbial Genetics

2. Definitions
Genetics
the study of heredity, genes and the
mechanisms that they carry this information
Replication
Expression
Genome
Complete genetic information of the cell

3. Definitions
Chromosome
The structures that are composed of DNA that carry
the hereditary information
Gene
Segments of the chromosome that code for a specific
product (usually a protein)
Genomics
Sequencing and molecular characterization of
genomes

4. Definitions
DNA (deoxyribose nucleic acid)
Nucleotides
3 components

Phosphate

Deoxyribose sugar
Nitrogenous base
Adenine, thiamine, cytosine or guanine
Double helix (complementary strands)

Base pairs
A-T
C-G
A-U (RNA)

Hydrogen bonds

5. DNA
Base sequence codes for protein
4 letter alphabet (A, T, G and C)
Genetic code
Determines how nucleotide sequence is
converted into amino acid sequences
Complementary strand allow precise
duplication

6. DNA to proteins
Gene on DNA
Converted to mRNA
mRNA on ribosome
tRNA brings amino acids to ribosome for
protein synthesis

7. Definitions
Genotype
Genetic information of the organism
Information that codes for characteristics of
the organism
Phenotype
The expressed or physical characteristics of
the organism
The expression of the genotype

8. Bacterial Chromosome (DNA)
Bacterial
chromosome
Single
Circular
Attached one or many
sites to plasma
membrane

9. Bacteria chromosome
Escherichia coli
4.6 million base pairs
4300 genes
1mm long
1,000 X length of cell
Supercoiled
Topoisomerase II
DNA gyrase

10. Bacterial chromosome
Genetic map
Mapped in minutes
Based on time for
chromosome
exchanged between
two cells

11. DNA replication
Parental strand
Two new “daughter
strands”
Each strand acts as
template for new
strands
Semiconservative
replication

12. DNA Replication
Carbons in nucleotide numbered 1`-5`
Complementary sugars are upside down to one
another
Strands run 5`3` on each side

13. DNA Replication
Steps in replication
DNA unwinds
DNA polymerase
Adds nucleotides to 3`
end
Replication fork forms
Leading strand forms
towards the fork
5`3`

14. DNA Replication
DNA replication
Lagging strand

Needs RNA primer
Removed by DNA
polymerase

Synthesized
discontinuously

Moves away from fork

Okazaki fragments
1000 nucleotides
DNA ligase fuses
segments

15. Bacterial DNA Replication
Bacterial DNA
replication
E. coli
Occurs bidirectionally
Two replication forks
Continues until forks
meet

16. RNA Synthesis
Transcription
Process of taking DNA code and converting to
RNA code
Translation
Converting RNA (mRNA) with tRNA to form
amino acid sequences and proteins
Occurs at ribosome

17. Protein Synthesis
Three types of RNA
mRNA - messenger
tRNA - transfer
rRNA – ribosomal
DNA unzips at gene

18. Transcription
RNA polymerase
binds to DNA at
promoter
Only coding strand of
DNA is template
5`3` direction
RNA polymerase
assembles RNA
nucleotides

19. Transcription
RNA chain grows
RNA stops growing at
terminator site
mRNA strand released
from DNA
DNA zips up
mRNA intermediate
between DNA and
translation

20. Translation
Bacterial translation
Protein synthesis
Decoding mRNA to amino
acids and proteins
Codons

Groups of 3 nucleotides

Sequence of codons
determines amino acid
sequence

Several codons for a
single amino acid
Degeneracy
Allows for
mutations

21. Translation
64 codons
(4
3
)
Sense codons

Code for amino acids

61 codons
Nonsense codons

Stop codons

UAG, UAA, UGA

Signal end of protein
synthesis
AUG
Start codon

Formylmethionine

Usually removed from protein

22. Translation
tRNA
Transfer RNA
Anticodon
Complementary to
codon
Amino acid attached
Brings amino acid to
ribosome

23. Translation
1 – components
needed come
together
Ribosome
tRNA
mRNA
2 – tRNA carries first
amino acid ( ?) to
ribosome and mRNA

24. Translation
3 – second amino
acid brought to
ribosome
P – site
Site of first amino acid
A – site
Site of second amino
acid
Peptide bond forms

25. Translation
4 – after peptide bond
first tRNA is released
to find amino acid

26. Translation
5 – ribosome moves
along mRNA until
tRNA is in P site
Process continues
down mRNA

27. Translation
6 – ribosome
continues down
mRNA
Peptide chain
elongates

28. Translation
7- polypeptide
(protein) released
Ribosome moves
down mRNA until stop
codon
UAG, UAA, UGA
Polypeptide released

29. Translation
8 – tRNA is released
and ribosome
disassembles
tRNA, mRNA, and
ribosome can be used
again

30. Review

31. Other points
Ribosome moves
5`3` direction
Additional ribosome
may attach and begin
synthesizing protein
Prokaryotes can start
translation before
transcription is
complete

32. Eukaryotic differences
Transcription takes place
in nucleus
mRNA completed prior to
entry in cytoplasm
Exons – Expressed DNA,
code for protein
Introns – intervening DNA,
do not code for protein
Removed by ribozymes

33. Regulation of Bacterial Gene
Expression
All metabolic reactions are catalyzed by
enzymes (proteins)
Feedback inhibition stops a cell from
performing unneeded chemical reactions
Stops enzymes that are already synthesized
What prevents synthesis of enzymes that
are not needed?

34. Regulation of Bacterial Gene
Expression
Protein synthesis requires tremendous
energy
Cell does not waste energy
Regulating protein synthesis economizes cells
energy

35. Regulation of Bacterial Gene
Expression
Genes
60-80% are constitutive

Not regulated
Products produced at fixed rate

Genes turned on all the time
Code for enzymes essential to major life processes
Enzymes needed for glycolysis

36. Regulation of Bacterial Gene
Expression
Genes
Inducible genes

Production of enzymes is regulated
Inducible enzymes
Present only when needed
Trypanosoma
Surface glycoproteins
Produces one glycoprotein at a time
Eludes immune system

37. Regulation of Bacterial Gene
Expression
Regulation of
transcription
Repression
Decreases gene
expression
Decrease enzyme
synthesis
Response to
overabundance of an end
product
Regulatory proteins called
repressors
Block RNA polymerase

38. Regulation of Bacterial Gene
Expression
Regulation of transcription
Induction

Turns on genes
Substance that turns on gene
Inducer
Inducible enzymes

39. Regulation of Bacterial Gene
Expression
Induction enzymes
 β-galactosidase (E. coli)
Cleaves lactose
Medium without lactose = little to no β-galactosidase
Lactose added to medium large amounts of β-
galactosidase produced
Lactose is converted to allolactose
Allolactose is the inducer
Enzyme reduction

40. Operon Model
Three genes for lactose
utilization
Located next to each other
on bacterial chromosome
Regulated together
Called structural genes
lac structural enzymes are
transcribed and translated

lac for lactose

41. Operon Model
Operon model
lac operon
Promoter region
Region of DNA where
RNA polymerase
initiates transcription
Operator region
Go or stop signal for
transcription of the
structural genes
Structural genes
Genes for metabolism of
lactose

42. Operon Model
Inducible operon
Near lac operon is
regulatory gene
I gene
Codes for repressor
protein

43. Operon Model
Lactose is absent
Repressor binds to
operator site
RNA polymerase is
inhibited
No transcription of
structural genes
No mRNA
No enzymes are
synthesized

44. Operon Model
Lactose is present
Converted to allolactose
Inducer
Inducer binds to receptor
protein
Receptor protein altered
Does not fit into operator site
RNA polymerase is not
inhibited
Structural genes are
transcribed to mRNA then
translated into enzymes
An inducible operon

45. Operon Model
Repressible operon
Tryptophan synthesis
EDCBA structural
genes
Also has promoter and
operator region

46. Operon Model
Repressible operon
Structural genes
transcribed and
translated
Tryptophan is
synthesized

47. Operon Model
Repressible operon
Excessive tryptophan
accumulates
Tryptophan acts as
corepressor
Corepressor binds to
repressor protein
Repressor protein binds
operator and structural
genes no longer
transcribed

48. Lactose regulation
Lactose operon
Depends on level of glucose in medium
Enzymes for glucose metabolism are constitutive
When glucose is absent cAMP (cyclic AMP) accumulates in cell
cAMP binds to cAMP receptor protein (CRP)

This binds to lac promoter

Initiates transcription by allowing mRNA polymerase to bind to the promoter
Transcription of lac operon requires

Presence of lactose

Absence of glucose
cAMP is an alarmone

Chemical alarm signal the cell uses to respond to environmental or
nutritional stress

49. lac operon

50. Lac operon
Catabolite repression
Inhibition of the metabolism of other carbon
sources by glucose
Glucose effect

51. Mutation
Mutation
Change in the base sequence of DNA

may cause change in the product coded by the
gene
Beneficial
Lethal
Neutral
Occur commonly
Degeneracy

52. Mutations
Types of mutations
Base substitution (point mutation)

AT substituted for CG
mRNA carries incorrect base

Translation
Insertion of incorrect amino acid into protein
Missense mutation, nonsense mutation, frame
shift mutation, and spontaneous mutations

53. Base substitution

54. Mutations
Normal
No mutations
DNA strand properly
transcribed by mRNA
Correct sequence of
amino acids for protein

55. Mutations
Mis sense mutation
Base substitution
results in an amino
acid substitution in
protein
Sickle cell anemia
A to T
Glutamic acid to valine
Hb shape changed
during low oxygen

56. Mutations
Non sense mutation
Base substitution
creates a nonsense or
stop codon
Protein is not
produced
Only a fragment of
protein is produced

57. Mutations
Frame shift mutation
One or a few nucleotide
pairs are deleted or
inserted in the DNA
Shifts the translation
reading frame
Almost always result in a
long stretch of altered
amino acids
Inactive protein

58. Mutations
Insertion of extra bases into a gene
Huntington's disease
Spontaneous mutations
Occur occasionally in DNA replication
Mutagens
Chemically of physically alters DNA and
effects a change is called a mutagen
Radiation, ultraviolet light

59. Mutagens
Chemical Mutagens
Nitrous acid
Converts adenine (A) to a form that doesn’t bind with thymine
(T), but instead binds with cytosine (C)
Alters base pair on DNA, works on random locations

60. Mutagens
Chemical mutagens
(cont)
Nucleoside analogs
Structurally similar to
normal nitrogenous
bases
2 - aminopurine
Adenine
5 – bromouracil
Thymine analog
Will bind with guanine

61. Mutagens
Chemical mutagens
(cont)
During replication
analogs cause base
pairing mistakes
Antiviral and antitumor
drugs

AZT (azidothymidine)

62. Mutagens
Chemical mutagens (cont)
Other chemicals cause deletions, frameshifts,
or insertions
Benzyprene – present in smoke and soot
Frameshift
Aflatoxin – Aspergillus flavus
Frameshift

63. Mutagens
Radiation mutagens
X – rays
Gamma rays
Ultraviolet
Forms covalent bond
between certain bases
Thymine dimers
Death of damage to cell
Light repair enzymes
Photolyases
Use visible light
energy to separate
dimer

64. Mutagens
Ultraviolet damage
Nucleotide excision
repair
Enzymes cut out
distorted thymines
Creates gap
Gap is filled with newly
synthesized DNA
DNA ligase joins strand
to surrounding
backbone

65. Mutation frequency
Mutation rate
Probability that a gene will mutate when a cell divides
Expressed in power of 10
10
-4
mutation rate (1 in 10,000 chance of mutation)
10
-6
( 1 in 1,000,000)
Mutagens

Increase spontaneous mutation by 10 – 10,000 times
10
-6
becomes 10
-3
to 10
-5

66. Identifying Mutants
Positive (direct) selection
Detection of mutant cells by rejection of
unmutated parent cells
Penicillin in agar
Unmutated parental cell will not grow
Only mutated cells grow

67. Identifying Mutants
Negative (indirect)
selection
Replica plating
technique

68. Replica Plating

69. Replica plating
Auxotroph
A mutant microorganism having a nutritional
requirement that is absent in the parent.

70. Identifying Chemical Carcinogens
Carcinogen
A substance found to cause cancer in animals
Often mutagens are carcinogens as well
Previously used animal testing
Time consuming
Expensive

71. Ames test
Ames test utilizes
bacteria to act as
carcinogen indicator
Based on observation
that exposure to
mutant bacteria to
mutagenic substance
may reverse effect of
the original mutation

72. Ames test
These are called reversions
Back mutations
Measures the reversion of Salmonella
Auxotrophs
Have lost there ability to synthesize histidine (his
-
)
(his
+
) bacteria have ability to synthesize histidine
90% of substances that cause reversion
have been shown to be carcinogens

73. Ames Test

74. Genetic Transfer and
Recombination
Genetic recombination
Exchange of genes
between two DNA
molecules to form new
combinations of genes on
a chromosome
Crossing over

Two chromosomes break
and rejoin
Adds to genetic diversity

75. Genetic transfer and recombination
Eukaryotes
Meiosis
Prophase I
Prokaryotes
Numerous different
ways

76. Genetic Transfer and
Recombination
Vertical gene transfer
Genetic information passed from an organism
to its offspring
Plants and animals
Horizontal gene transfer
Bacteria transfer genetic information form one
organism to another in the same generation
Genetic information passed laterally

77. Horizontal Gene Transfer
Horizontal gene transfer
Donor cell
Organism gives up its entire DNA
Part goes to recipient cell
Part is degraded by cellular enzymes
Recipient cell
Receives portion of donor cells DNA
Incorporates donor DNA into its own DNA
Recombinant DNA
Less than 1 % of population

78. Transformation
Genes transferred from one bacterium to
another in solution
Naked DNA
Discovered by Griffith
Used Streptococcus pneumoniae
Two strains
Virulent (pathologic) strain
Had a polysaccharide capsule resists phagocytosis
Avirulent (non- pathogenic) strain
Lacked a capsule

79. Griffith’s Experiment

80. Transformation
Bacteria after cell death and lysis could release
DNA into environment
Recipient cell can take up DNA fragments and
incorporate into their own DNA
Resulting in a hybrid (recombinant cell)
Recombinant cell must be competent
Able to alter cell wall to allow DNA (large molecule) to enter
Bacillus, Haemophilus, Neisseria, Acinetobacter, and some
Staph and Strep

81. Genetic Transformation

82. Conjugation
Conjugation
Involves plasmid
Circular piece of DNA
Replicates independent of
chromosome
Non essential for growth
genes
Requires cell to cell contact
Opposite mating type
Donor cell carries plasmid
Recipient cell lacks
plasmid

83. Conjugation
Gram positive
Sticky surfaces cause
bacteria to come in
contact with one
another
Gram negative
Utilize sex pili

84. Conjugation
E coli model
F factor plasmid
Fertility factor
Donors (F
+
)
Recipients (F
-
)
Converted to (F
+
)
F
+
factor integrated
into chromosome
Becomes Hfr (high
frequency of
recombination) cell

85. Bacterial Conjugation
Hfr conjugates with F
-
cell
Chromosomal strand
replicates and transferred
to recipient
Incomplete transfer of
donor DNA
Recipient integrates new
DNA
Acquires new versions of
chromosome
Remains F
-
cell

86. Conjugation in E. coli

87. Conjugation
Minutes and
conjugation
Identify locations of
various genes
Hfr
His, pro, thr, leu, and
F (+)
F(
-
)
His, pro, thr, leu, and
F(
-
)

88. Transduction in Bacteria
Transfer of bacterial
DNA transferred via
bacteriophage
Bacteriophage
Virus that infects
bacteria

89. Transduction
Steps of transduction
1- bacteriophage infects donor bacterial cell
2- Phage DNA and proteins, and bacterial
chromosome is broken into pieces

90. Transduction
Steps of transduction
3- during phage reassembly, bacterial DNA incorporated
in capsid of bacteriophage
4 – donor cell lysis releasing new bacteriophage particles

91. Transduction
Steps in transduction
5- phage carrying donor DNA infects new recipient cell
6- recombination can occur
Producing bacteria with genotype different than donor and recipient

92. Transduction
Generalized transduction
Previously explained
Specialized transduction
Only certain genes are transferred
i.e. phage codes for toxins to be produced
Cornybacterium diphtheriae – diphtheria toxin
Streptococcus pyogenes – erythrogenic toxin
Escherichia coli – Shiga toxin (hemorrhagic diarrhea)

93. Plasmids
Plasmids
Self replicating rings of DNA
1-5% size of chromosomal
DNA
Non – essential genes
Conjugative plasmid
F factor
Dissimilation plasmids
Code for enzymes to
breakdown unusual sugars
and hydrocarbons
Help in survival of unusual
environments

94. Plasmids
Other plasmids
Toxins (Anthrax, tetanus, Staph)
Bacterial attachment
Bacteriocins
Toxic proteins that kill other bacteria
Resistance factors (R factors)
Resistance to antibiotics, heavy metals, cellular
toxins

95. Plasmids
Resistance factors
Two groups
RTF – resistance transfer factor
Includes genes for plasmid replication and conjugation
r-determinant
Resistance genes
Codes for production of enzymes that inactivate drugs or toxic
substances
Bacteria can conjugate and transfer plasmids between
species
Neisseria
Penicillinase resists penicillin

96. R factor Plasmids

97. Transpoons
Transpoons
Small segments of DNA that move from one region to
another
700-40,000 base pairs
Occur in all organisms
Can insert within genes
Disrupt transcription of gene
Occurs rarely (similar to spontaneous mutation rate)

98. Transpoons
Transpoons
Contain gene for transposition
Insertion sequence (IS)
Codes for transposase
Cuts and seals DNA for transpoons

99. Review