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Although sex-ratio distortion – the abnormally skewed distribution of male and female progeny – has been studied in many Drosophila species,
the genes that control this process have only recently been cloned in the fruit fly D. simulans. This project seeks to elucidate these genes in another
fruit fly species, D. pseudoobscura, through a mutagenesis screen. Males carrying the SR X chromosome normally produce only female progeny.
An induced mutation may knock out the X-linked distorter function to produce a normal sex-ratio (1:1). We need to determine the fertility of SR females
over a range of γ-radiation doses used for mutagenesis. In the mutagenesis protocol, radiated SR females were mated with marked males, and male progeny
were singly mated with marked females to check for later sex-ratio mutations. We saw an expected decrease in fertility as the γ-dose increases, as well as a
larger percentage of males in the higher γ-doses.
In the fruit fly D. pseudoobscura, an unusual X chromosome – the sex-ratio X chromosome (SR) – was first noted by Alfred Sturtevant and Theodosius Dobzhansky
in 1936 [1]. Males carrying the SR chromosome sire broods consisting of almost 100% females, as well as rare males that lack a Y chromosome. The Y
chromosome fails to segregate into a new sperm cell during the second phase of meiosis [2]. This phenomenon is called sex-ratio distortion.
The geographic distribution of D. pseudoobscura includes Mexico and the western region of North America, and the SR phenotype is most prevalent in the
southern areas of this distribution [3]. The fruit flies used in this experiment are the progeny of approximately five SR males (out of 150 total males) gathered
in Tucson, Arizona in summer 2006. Many generations of SR flies have been established using a cousin-cousin mating scheme. In this scheme, homozygous
SR females are crossed with SR males, which are provided from a cross between SR females and standard (ST) males.
The architecture of the SR X chromosome offers some insight into the distortion mechanism (Figure 1). In D. pseudoobscura, three inversions (red in Figure 1)
on the right arm of the SR X chromosome (XR) distinguish it from the ST X chromosome [4,5]. A short un-inverted region lies between the two proximal inversions,
and a much longer un-inverted region occurs between the two distal inversions. These inversions are apparently important for sex-ratio distortion because
suppressed cross-over in XR between SR and ST chromosomes helps to retain the SR loci. Unfortunately, the nature of these inversions also prevents conventional
genetic mapping of the SR genes. This is the main reason for our ignorance about the SR genes after more than 70 years of research.
Figure 1. Location of Inversions on the SR X Chromosome
The primary goal of this project is to produce mutants with a loss of function of the sex-ratio distorter, thus allowing for mapping and cloning of the SR genes.
This strategy depends heavily on the efficient generation of loss-of-function mutations in the distorter genes through γ-ray mutagenesis. For that purpose, we
need to determine the optimal dose of the γ-ray radiation for mutagenesis. We expect that the treated females will have de-creased fertility, and their progeny
will display a higher frequency of mutations if the γ-ray dose increases. At the optimal dose, the total number of progeny carrying mutations will be maximized.
The experimental scheme for mutagenesis is shown in Figure 2. A range of 0 to 12 KR of γ-radiation was applied. The low dose treatment used doses of 0
(control), 200, 500, 700, 1000 and 2000 R while the high dose treatment used doses of 3, 4, 6, 8, 10, and 12 KR. SR females (20 in each vial) were treated in
the γ-cell and then were mated to 10 marked males (se sp). In one batch of the high dose experiment (3-12 KR), only one male was provided accidentally. The
flies were transferred to new vials every two days for a ten day period. These transfers can provide information about stage-specific responses to γ-ray radiation.
When offspring hatched from these vials, they were sexed and counted. Up to 20 male offspring from each transfer of each dose were singly mated with three se
sp virgins. F2 progeny of the tested males were scored for loss of the sex-ratio phenotype.
A large-scale mutagenesis screen using the protocol in Figure 2 has also been started. The SR females were treated with either 3 KR or 4 KR of γ-ray radiation.
I crossed as many of the F1 progeny males singly to se sp females as possible. So far, a total of 965 vials have been set up for screening, and the data collection
from this screen is currently in progress.
Figure 2. Mutagenesis Scheme
Figure 3. Total offspring produced by radiated SR females in the two high γ-dose range experiments. The graph is scaled to show the
offspring produced by 100 SR females.
Figure 4. Total male and female offspring from the two high γ-dose range experiments. Offspring totals based on 100 SR females
Figure 5. Percentage of males and females in the high dose-range experiments. The percentages are scaled for the offspring of 100 SR females
Figure 6. Percentage of males and females in the high dose-range experiments, by γ-dose. The percentages are scaled for the offspring of 100 SR females.
Figure 3 illustrates the detrimental effects of γ-radiation on treated females. This result was expected based on previous studies in D. melanogaster [6]. The data
suggest that excessively high doses of γ-rays are lethal to developing gametes, so the SR females could only produce a very small number of offspring.
Radiated SR females produced almost twice as many males as females (Figure 5). One explanation for this observation involves the poor recovery of radiated X
chromosomes as compared to the Y chromosome. In D. pseudoobscura, the X chromosome is much larger than the Y. Damage caused by γ-radiation, therefore,
should be more significant on the X. If the cell cycle check-point detects any damages and stops the development of fertilized eggs, then we expect to see more
XY embryos survive to adulthood than XX embryos.
The preliminary data from the mutagenesis screen suggest a lower mutation rate of X-linked male fertility genes than expected. In D. melanogaster, the frequency
of recessive lethal mutations on the X chromosome is about 4% [6]. If a male sterile mutation is about 1/5 as frequent as a lethal one, and the X of D.
pseudoobscura is twice as large as the X of D. melanogaster, the expected frequency of male sterile should be 1.6%. The observed rate is 0.7% (7/965).
We should increase the γ-ray dose, therefore, so that a higher (e.g., 1%) rate of X-linked male sterile mutations can be produced. The higher frequency of male
sterility should increase the probability of obtaining a mutation of the SR genes.
I would like to thank Hailian Xiao for assistance in operating the γ-cell. This research was supported by the Howard Hughes Medical Institute under
Grant #52005873 and by a Student Inquiry Research Experience (SIRE) award from the Office of Undergraduate Studies at Emory College.
1. Sturtevant, A.H. and T.H. Dobzhansky, 1936. Geographical distribution and cytology of “sex ratio” in Drosophila pseudoobscura and related species. Genetics 21: 473-490.
2. Novitski, E., W.J. Peacock, and J. Engel, 1965. Cytological basis of “sex ratio” in Drosophila pseudoobscura. Science 148: 516-517.
3. Krimbas, Costas B. and Jeffrey R. Powell, ed. Drosophila Inversion Polymorphism. CRC Press: Boca Raton, 1992.
4. Cobbs, Gary, 1987. Modifier genes of the sex ratio trait in Drosophila pseudoobscura. Genetics 116: 275-283.
5. Cobbs, Gary, L. Jewell, and L. Gordon, 1991. Male-sex-ratio trait in Drosophila pseudoobscura: frequency of autosomal aneuploid sperm. Genetics 127: 381-390.
6. Ashburner, Michael, Kent Golic, and R. Scott Hawley. Drosophila: A Laboratory Handbook. 2nd edition. Cold Spring Harbor Press: Cold Spring Harbor, NY, 2005.
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