Raunvísindadeild; Líffræðiskor
Erfðafræði
Verkleg Erfðafræði
Elicit of Mutations in E. coli
Námsbraut: Raunvísindadeild; Líffræðiskor
Námskeið: 09.51.35 Erfðafræði
Nafn kennara: Ólafur S. Andrésson, Zophonías O. Jónsson, Sigríður H. Þorbjarnardóttir og
Bryndís K. Gísladóttir.
Vikudagur, hópur: Fimmtudagur, síðari kennslustund, Hópur 2 og 4.
Tilraun framkvæmd: 25. september, 02., 09., og 16. október, 2003
Skýrsluskil til kennara: 05. nóvember, 2003
Skýrsluskil til stúdenta:
Einkunn:
Nöfn stúdenta: Bjarki Steinn Traustason, Egill Guðmundsson, J. Gabriel-Rios Kristjánsson,
Marcella Manerba og Nicoletta Palmegiani.
________________________________
Bjarki Steinn Traustason
________________________________
J. Gabriel-Rios Kristjánsson
________________________________
Egill Guðmundsson
________________________________
Marcella Manerba
________________________________
Nicoletta Palmegiani
Háskóli Íslands
Raunvísindadeild; Líffræðiskor
5
Erfðafræði
Elicit of Mutations in E. coli
Introduction:
Mutation are herritable variations in the sequences DNA bases. Knowing that specific
sequences have an important biological meaning for protein translation, even a single
base pair change can bring a modification in the nucleotide reading.
Point mutations involve base pair substitution with another, eg, GC to AT and
vice versa. This alternation can change the protein structure and makes it disfunctional. This type of mutation can be categorise into transversion or transition
muations. If a mutation changes a codon, that codes for an amino acid without
changing the amino acid sequence, it is called silent mutation.
On the other hand, if the mutation changes bases in a codon, it can produce a
difference meaning of the wild sequence, and therefore a different amino acid. This is
called missense mutation.
A base substitution can also create a nonsense mutation within the formation
of a stop codon (UAG;UGA;UAA), that results in a premature termination of translation.
This type of mutation could bring a reverse mutation, that restores the wild type DNA
sequences, using a different mutation in another site in the genome. This second
mutation is called suppression mutation.
In our experiment we studied the effect of nonsense intergenic suppression,
and, in particular, the mutation in the tRNA codon which allows the tRNA to read the
stop codon, and insert an amino acid, allowing translation to continue.
All this mutation could be spontaneous or induced. The latter can occur from
external causes, like: UV radiation, chemical coumponds, heat or ionizing radiation.
The aim of our laboratory exercise was to check the effects of a chemicalcoumpond, N-methyl-N´-nitrosoguanidine (NTG) on the frequency of certain
mutations in E. coli.
FIGURE 01, NTG; N-METHYL-N´-NITROSOGUANIDINE
This mutagen replaces one or only few nucleotide bases in the DNA molecule
(eg, GC-AT or AT-GC point mutation). Specifically, NTG induced an alkylation of
guanine O6 and thymine O4, causing mispairing.
The strain of E. coli that we used was GE502 which has the mutations
trpA9605, his-85, pro-48, and ilvD145.
Therefore the bacteria became auxotroph (trp-, his-, pro-, and ilv-), that is, it
requires extra nutrient in it’s medium to survive.
Aims/hypothesis:
The aim of this experiment was to determine the frequency of spontaneous reverse
mutations in E. coli, and to check if mutagenic compound has inducible affects on the
frequency of reverse mutation.
Háskóli Íslands
Raunvísindadeild; Líffræðiskor
Erfðafræði
Design and Methods:
Reference to work sheets in manual booklet, for present exercise (p.21-24). Exception
from the introduction and procedure part, concerning EMS (ethylmethane sulfonate),
where the EMS evaporated during incubation, so NTG was used instead.
Results:
TABLE 01, GIVEN VALUES:
2.2 · 109
7.0 · 108
1.2 · 109
GE502, untreated:
GE502, EMS-treated:
GE502, NTG-treated:
cells/
mL
cells/
mL
cells/
mL
TABLE 02, VIABLE COUNT FOR CELLS, NOT TREATED WITH EMS NOR NTG:
A257 His+
Group:
H1
H2
H3
H4
H5
A578 Ilv+
colonies
viable count
15
10
23
18
18
1.5·102
1.0·102
2.3·102
1.8·102
1.8·102
/plate
colonies
/plate
0
0
0
3
0
viable count
0
0
0
3.0·101
0
16.8
1.7·102
0.6
*Calculations for Viable count are described in Formula 01.
0.6 · 101
mean
FORMULA 01, VIABLE COUNT:
General formula:
Vc = (n ∙ i) / d
Vc: Viable count [cells / mL]
n : Number of cells per plate,
given that single cell entails single colony, [cells / plate]
i : ratio for solution of incubation (Vsolution / Vused for incubation) [mL / mL]
d : dilution, here the dilution used is 10−1
Eg,:
Viable count = (nA257,His+ ∙ (1 mL / 0.1 mL)) ∙ 101 = 1.7 cells / mL
TABLE 03, VIABLE COUNT FOR CELLS, TREATED WITH EMS:
A257 His+
Group:
H1
H2
H3
H4
H5
colonies
/plate
0
0
2
0
0
A578 Ilv+
viable count*
colonies
0
0
2.0·101
0
0
0
3
15
0
0
/plate
0.33
3.3·100
3.6
*Calculations for Viable count are described in Formula 01.
mean
viable count*
0
3.0·101
1.5·102
0
0
3.6 · 101
TABLE 04, VIABLE COUNT FOR HIS+-CELLS, TREATED WITH NTG:
A257 His+
Group:
H1
§
H2
H3
H4
H5
colonies
viable count*
213
0
190
203
211
2.1·104
0
1.9·104
2.0·104
2.1·104
/plate
204
2.0·104
*Calculations for Viable count are described in Formula 01.
mean
§
A mistake in incubation, led up to no colonies growing on the agar, thereby not included in subsequent calculations.
Háskóli Íslands
Raunvísindadeild; Líffræðiskor
Erfðafræði
TABLE 05, MEANS OF VIABLE COUNT:
1.7 · 102 cells
2.0 · 104 cells
0.6 · 101 cells
3.8 · 104 cells
*This was amogst given data, obtain by teachers.
A257, untreated:
A257, NTG-treated:
A578, untreated:
A578, NTG-treated:
/
/
/
/
mL
mL
mL
mL *
TABLE 06, CALCULATED FREQUENCY OF REVERSES: *
7.6 ∙ 10−8
1.7 ∙ 10−6
2.7 ∙ 10−9
3.2 ∙ 10−6
*Formula used for the calculations, is described in Formula 02.
A257, untreated:
A257, NTG-treated:
A578, untreated:
A578, NTG-treated:
FORMULA 02, FREQUENCY OF MUTAION:
General Formula:
frequency = (n  Vc) / i
Eg,:
n : Number of cells per plate,
given that single cell entails single colony [cells/ plate]
Vc : Viable count, given [cells / mL]
i : ratio for solution of incubation (Vsolution / Vused for incubation) [mL / mL]
frequency = (nA257,His+  VcGE502,untr.) / (1 mL / 0.1 mL) = 7.6 ∙ 10−8
TABLE 07, REPLICA PLATING OF A257, NTG-TREATED:
Group:
Type:
His+ Trp+
His+ Trp−
H1
H2
H3
H4
H5
Σ
83
5
93
2
70
1
67
5
82
6
395
19
Σ
88
95
71
72
88
414
True reverse mutation (sannur viðsnúningur): 414 – 395 = 19
TABLE 08, REPLICA PLATING OF A578, NTG-TREATED:
Group:
Type:
Ilv+ His+
Ilv+ His−
H6
Σ
50
0
50
No true reverses.
Conclusion/Discussion:
Based on the viable count in the untreated strains, the frequency of mutations, and
thereby reverse mutations, are very low. On the other hand, when the bacteria strain
was treated with strong mutagenic compound, NTG, the frequency of reverse, in
mutated bacteria, increased greatly. It came clear that reverse mutations in the Hisgene are more prevalent, than in the Ilv-gene. Likewise, some of the mutated bacteria
that could grow in the His–-media could also gropw in the Trp–-media. The point
mutations, that had occurred in the His- and Trp-gene before treatment with NTG, did
not reverse, but mutations in other genes had ‘healed’ the original point mutation.
Such mutations are called suppressor mutations.
Somewhere in the middle of the genes, a point mutation has altered the base
sequence, and formed a stop codon, which terminates the gene translation. A mutation
in tRNA-genes, which alters the tRNA’s anti-codon, can allow them to read one of the
Háskóli Íslands
Raunvísindadeild; Líffræðiskor
Erfðafræði
stop codons, and continue to add an amino acids to the growing polypeptide chain.
This, in turn, allows the gene translation to conclude.
A reversion of both trpA9605 and his85 mutations suggests, that the same stop
codon inhibited the translation of the genes, meaning, the same mutated anti-codon,
can infact read the stop codons. However, the mutated bacteria strains, that could
grow in the Ilv–-media, could not grow in the His–-media after replica plating. In other
words, it was not possible to reverse both IlvD145 and His85 at the same time. Either
the point mutation in the Ilv-gene, has reversed (true reverse), or that the mutations
IlvD145 and His85, contains either two different stop codons, and treatment with
NTG has only mutated an anti-codon in one tRNA molecule, which means that it
could only read the stop codon in the Ilv-gene bot in the His-gene. Indeed, it is not a
possibility that IlvD145-mutation is a deletion, making the mutation impossible to be
reversed.
■
Háskóli Íslands