Transcript Mutagenesis of the tRNAse Z Gene in Drosophila

Mutagenesis of the tRNAse Z Gene in Drosophila
Bill Maughan, Dr. Edward Dubrovsky
Department of Biological Sciences, Fordham University
MATERIALS AND METHODS
INTRODUCTION
Certain defects in the ELAC2 tRNAse Z enzyme in humans have been shown to
increase prostate cancer susceptibility. The gene for this enzyme is well conserved
through all three kingdoms of life, indicating its importance. The goal of this
project was to create mutations in the Drosophila melanogaster form of the
tRNAse Z enzyme and study its effects on cell growth and proliferation.
tRNAse Z can be found in two general forms. The short form, also known as ELAC1, is
sufficient for tRNA 3’ processing. The long form of the enzyme, ELAC2, which is not
found in bacteria, contains additional domains. The function of these domains is not yet
fully understood.
ELAC2 is found in both humans and D. melanogaster. The domains unique to ELAC2
can be mutated in D. melanogaster and studied in order to better understand their
function in both species.
It has been shown that knocking out ELAC2 in D. melanogaster causes a build up of
immature tRNA molecules with the 3’ trailers still attached, yet the amount of mature
tRNA does not change. Though they had normal levels of mature tRNA, larvae died
once the supply of ELAC2, suggesting that it has an essential function other than tRNA
processing.
A different tRNAse Z mutant, dRNAseZ83 was also shown to be lethal, but it was late
larvae lethal. Instead of taking the normal 4 days to mature to third instar larvae, it took
them 9. Almost all larvae also developed tumors around day 15. None reached
adulthood.
A
C
Ethyl methane sulfonate, or EMS, was used to produce essentially random point
mutations in the flies’ genomes. The following cross was then used to identify which
flies had mutations in the tRNAse Z gene.
Offspring of the final cross were scored for presence of Cy+ flies. Absence of this class
of flies indicates a recessive lethal mutation within the Df12 chromosomal deficiency.
Those flies testing positive for a mutation within the Df12 deficiency were then crossed
to each other and to the strain ED24, a strain of flies containing a deficiency of the
tRNAse Z gene, in order to determine complementation.
RESULTS
Of the nearly 3,000 mutagenized males, roughly 1,000 of them produced enough viable
offspring to be scored. Of the nearly 1,000 mutants screened, 9 had recessive lethal
mutations within the Df12 deficiency. One of those 9 mutations, BM128, is
noncomplementary to the ED24 null allele.
Males
ED24
BM23
BM47
BM105
BM106
BM119
BM127
BM128
ED24
x
112:61
117:74
99:63
94:62
87:50
101:44
137:0
BM23
129:68
x
141:61
138:59
88:44
184:102
124:59
134:71
BM47
126:83
123:64
x
148:89
184:0
104:59
117:54
115:63
BM105
94:43
121:68
120:68
x
128:61
128:0
112:0
122:54
BM106
140:75
112:51
149:0
145:65
x
121:70
122:60
124:70
BM119
98:63
114:47
92:57
74:0
129:69
x
120:0
141:67
BM127
110:56
114:65
101:46
111:0
118:74
111:0
x
105:58
BM128
113:0
110:65
149:67
101:66
136:65
83:57
125:52
x
Key: Cy:Cy+
3’
5’
Females
B
D
CONCLUSIONS AND NEXT STEP
Those crosses which only produce offspring bearing the curly phenotype exhibit
noncomplementation in their second chromosomes. Therefore the two parents strains
have recessive lethal mutations in the same gene. Four complementation groups have
been identified thus far.
The BM128 strain is noncomplementary to the ED24 strain. This indicates that it
contains a mutation that effectively knocks out the tRNAse Z gene.
A)The Df12 deficiency omits a large part of chromosome 2, including the tRNAse Z
gene.
B) D. melanogaster larvae homozygous for tRNAse Z deletion do not develop properly.
C) tRNAse Z performs the final cleavage at the 3’ end of an immature tRNA molecule.
D) tRNAse Z mutants are unable to cleave the 3’ trailer sequence of an immature tRNA
molecule.
14 more strains that do not complement to the Df12 deficiency have been identified.
They will also undergo a complementation test with each other and with the mutant
strains already identified.
All strains that bear mutations in the tRNAse Z gene will be sequenced in order to
determine the location of the mutation. The effects of the mutation on cell growth and
proliferation will then be studied.
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