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Isogenic autosomes to be applied in optimal screening for novel mutants with

viable phenotypes in Drosophila melanogaster

Article in Journal of Neurogenetics · July 2009

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ISOGENIC AUTOSOMES TO BE APPLIED IN OPTIMAL SCREENING FOR NOVEL MUTANTS WITH VIABLE PHENOTYPES IN DROSOPHILA MELANOGASTER 11/1/07 16:10

Journal Neurogenetics, 19:57-85, 2005

Taylor & Francis Inc.
ISSN: 0167-7063
DOI: 10.1080/01677060591007155

ISOGENIC AUTOSOMES TO BE APPLIED IN OPTIMAL SCREENING FOR NOVEL MUTANTS WITH VIABLE
PHENOTYPES IN DROSOPHILA MELANOGASTER
Punita Sharmaa, 1, Zoltan Asztalos a* 1, Champakali Ayyub 2, Marien De Bruyne †3, Anthony J. Dornan4, Araceli Gomez-Hernandez 5, John Keane 1, James Killeen 6, Susanne Kramer 7, Mayur Madhavan 2, Helen
Roe8, Pradeep Dagadu Sherkhane2, Khalid Siddiqi2, Elizabeth Silva 1, John R. Carlson3, Stephen F. Goodwin4, Martin Heisenberg 7, Kits Krishnan2, Charalambos P. Kyriacou 8, Linda Partridge6, Juan Riesgo-
escovar5, Veronica Rodrigues2, Tim Tully9 and Cahir J. O’Kane*2
1 Department of Genetics, University of Cambridge, Cambridge, UK.

2 Tata Institute of Fundamental Research, Department of Biological Sciences, Colaba, Mumbai, India.

3 Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT, USA.

4 Institute of Biomedical Life Sciences – Division of Molecular Genetics, University of Glasgow, UK.

5 Neurobiology Institute, UNAM, Campus UNAM Juriquilla, Querétaro, Mexico, USA.

6 The Galton Laboratory, Department of Biology, University College London, UK.

7 Universität Würzburg, Biozentrum, Würzburg, Germany.

8 Department of Genetics, University of Leicester, Leicester, UK.

9 Beckman Neuroscience, CenterCold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.

Received: January 19, 2005; Accepted: April 24, 2005

*Correspondence: Cahir J. O’Kane, Department of Genetics, University of Cambridge, Cambridge, CB2 3EH, UK; E-mail: [email protected]

ABSTRACT
Most insertional mutagenesis screens of Drosophila performed to date have not used target chromosomes that have been checked for their suitability for phenotypic screens for viable phenotypes. To address this, we
have generated a selection of stocks carrying either isogenized second chromosomes or isogenized third chromosomes, in a genetic background derived from a Canton-S wild-type strain. We have tested these stocks for a
range of behavioral and other viable phenotypes. As expected, most lines are statistically indistinguishable from Canton-S in most phenotypes tested. The lines generated are now being used as target chromosomes in
mutagenesis screens, and the characterization reported here will facilitate their use in screens of these lines for behavioral and other viable phenotypes.

aThese authors contributed equally to this work.

† Current address: Freie Universität Berlin, Neurobiologie, Königin Luise Str. 28-30, D-14195 Berlin, Germany.

Keywords: Mutant screens; Behavior; Learning; Canton-S wild type.

INTRODUCTION
The isolation of novel mutants is a standard approach to dissecting the molecular and cellular mechanisms of biological processes, including behavior. Typically this strategy involves generation of many mutations induced
by various kinds of mutagenic agents at arbitrary chromosomal sites throughout a given genome, followed by screening of these for mutant phenotypes, and subsequent identification and cloning of the gene. However,
this strategy can be confounded if the line being mutagenized already exhibits significant genetic variation affecting the phenotype being screened for.
Such variation has been observed in both laboratory and wild populations, affecting such characteristics as foraging behavior (Osborne et al., 1997), properties of the clock protein Period (Rosato et al., 1994), and the
severity of the anatomical defects of structural brain mutants (de Belle & Heisenberg, 1996). Such variation has a number of consequences in mutant screens. First, many apparently novel mutants will in fact carry pre-
existing mutations, thus multiplying the work required to identify and characterize each truly novel mutant of interest. Second, the presence of additional variation that is superimposed on a novel mutation makes it more
difficult to identify a novel mutant phenotype and to determine whether that mutation is in fact the cause of the mutant phenotype. Third, mutant phenotypes may fade over time as the result of selection for polymorphic
modifiers, or be enhanced or suppressed as genetic background changes inadvertently in laboratory crosses. Effects of strain background have been documented for learning mutants in the laboratory mouse (Gerlai,
1996), for mutations affecting brain anatomy in Drosophila (de Belle & Heisenberg, 1996), and for a gain-of-function allele of sevenless (Polaczyk et al., 1998).
One way to circumvent these problems is to generate mutations in a defined genetic background, and in particular to ensure that all copies of a given chromosome are identical in the population to be mutagenized (i.e., are
isogenic). Increasing numbers of homozygous viable insertions are becoming available from large-scale mutagenesis projects (e.g., Bellen et al., 2004; Ryder et al., 2004; Thibault et al., 2004), and if these are to be useful
for mutant screens, it is preferable if they are available on isogenic chromosomes. However, a potential disadvantage of the isogenic approach is that individuals homozygous for a single chromosome sampled from the
population may not function in a way that is representative of the starting population, both because of inbreeding and sampling. Even if a non-isogenized stock is mutagenized, chromosomes subjected to mutagenic agents
are almost invariably used to make stocks in which all individuals carry the same mutagenized chromosome, and so the problem of chromosome sampling is almost impossible to avoid when a genetic approach is used to
dissect a phenotype. It is therefore important that isogenized chromosomes are representative of those found in the starting stock.
In recent years, most novel mutant collections made available to the wider Drosophila community have been generated by insertional mutagenesis, principally using the P or piggyBac transposable elements. At least for P
elements, an additional problem during mutagenesis is the occurrence of second-site mutations that are not caused by the marked P element used in the experiment, even when the target chromosomes have been isogenized
(Deak et al., 1997). Reasons for this could include either unmarked P elements or deletions that are derived from remobilization of a new insertion, or inadvertent mobilization of other elements that can also cause hybrid
dysgenesis, such as hobo and I (Bucheton et al., 1976; Periquet et al., 1994).
In this work, we have therefore aimed to generate isogenic lines that can be used for P mutagenesis, free of inadvertent mobilization of hobo, I , or P elements other than the marked P element being mobilized. We have
also tested them for a range of behaviors and viable phenotypes, and identified lines that are similar to the parental Canton-S wild-type stock in most of the phenotypes assayed. Together, this makes them suitable for
screens for behavioral and other viable mutations, with respect to the 80% of the Drosophila genome carried on the major autosomes.

MATERIALS AND METHODS

Southern Analysis and PCR
P element probes for Southern blots were made by PCR using DIG-High-Prime-labeled dNTPs (B Boehringer Mannheim) and 1.25 pg of Carnegie 2 plasmid template (Rubin & Spradling, 1983). For the 130-bp p3' probe,
the primers were 2750-5'-CCC CAC GGA CAT GCT AAG GG-3'-2769 and 2856-5'-GCA ATT CAC CTA CAG AGA ACG GC-3'-2879. A second 562-bp probe, p5', was made using primers 24-5'-TCC CGT CGA TAG
CCG AAG-3'-42 and 568-5'-CGC AGC AAA TCT CGT CGT CGT-3'-586. Co-ordinates for all P probes and primers refer to positions within the 2907-bp P element (O’Hare & Rubin, 1983).
730 bp of the 5'-end of the hobo element was PCR-amplified using primers 71-5'- TCG CCA CGC ACA TCG CGG G-3'-89 and 779-5'-CGG CAA AAG ACG TCA TTG GCC GCC GTT TTC TGC AGT AAC CGG-3'-800.
Co-ordinates for primers refer to positions within the 2959-bp hobo element, (GenBank ID: AF516512).
Similarly, 3111 bp of the 5'-end of the I element was PCR-amplified using primers 117-5'-CCG AAG AGA TAA GTC GTG CC-3'-136 and 3210-5'-CGC TTT TCT GAG ACC ACC GC-3'-3228. Co-ordinates refer to
positions within the 6231-bp I element (GenBank ID: AF182948).

Fly Strains and Isogenic Stock Generation

A laboratory stock of Canton-S (de Belle & Heisenberg, 1994; 1996; Dura et al., 1993) was used as wild-type. Stocks, isogenic for either second or third chromosomes from this stock, and retaining the same background
as the Canton-S stock on the non-isogenized chromosomes, were made as shown in Figure 1.

Figure 1 Crossing schemes to generate isogenic second or third chromosome lines.

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ISOGENIC AUTOSOMES TO BE APPLIED IN OPTIMAL SCREENING FOR NOVEL MUTANTS WITH VIABLE PHENOTYPES IN DROSOPHILA MELANOGASTER 11/1/07 16:10

Chemosensory Responses
Odor-induced Jump Response to Benzaldehyde (O’Kane Laboratory)
All strains were raised in glass bottles in large populations on standard cornmeal medium. Stocks were maintained at 25°C, 55% relative humidity on a 12/12 h light/dark cycle. For behavioral experiments 10 pairs of flies
were set up in small food vials, transferred to new vials every 3–4 days, and maintained as above. Flies eclosing were collected and separated by gender under light CO2 anesthesia on the same day. Males were stored as
above in vials in groups of about 10, and tested 2 days later. Experiments were run in an environmentally controlled room at 25°C and at 65–75% relative humidity.
Jump responses of single males to pulses of benzaldehyde vapor were measured using a modified version of the olfactory jump test (McKenna et al., 1989), in which a pulse of benzaldehyde was admitted to an airflow of
1000 ml/min, using a computer-controlled valve. Highest purity benzaldehyde (FLUKA) dissolved in heavy mineral oil (Fisher Scientific) was used. A positive response was defined as a jump during the 4-second odor
presentation or during the following 3 sec. Data were collected on three different days and an equal number of flies (13–42) were tested per day for each isogenic line and Canton-S. Jump frequencies were calculated for
each day, and student’s t-test was used to estimate the difference between frequencies of isogenic and control lines.

Adult Olfactory Choice Test (Rodrigues Laboratory)

Two- to six- day old flies were placed in bottles with moistened filter paper for an hour prior to testing. We tested for repulsion of flies by benzaldehyde (10-4 dilution in liquid paraffin) in an olfactometer, applied via the
“fast mode” in which the maze is held vertically under a light for two minutes. Flies move upwards under the influence of negative geotaxis and positive phototaxis. Approximately 95% of them typically move out of the
start tube. Numbers of flies in the odorant (S) and control (C) arms were counted, and a response index (RI) was calculated as (S - C)/(S + C). An RI of +1 indicates total attraction, and -1 indicates total repulsion.

Larval Olfactory Assay (Riesgo Escovar Laboratory)

This was performed as described (Lilly & Carlson, 1989). Groups of 50–100 third instar larvae were placed in the middle of a Petri plate between two filter discs, one soaked in odorant and one in water, and the Petri plate
was covered. After 5 minutes, the number of larvae on the stimulus half (S) and control half (C) were counted. A Response Index (RI) is then calculated as (S - C)/(S + C).

NaCl Aversion and Sucrose Preference Tests (Rodrigues Laboratory)

Taste preference tests were performed as described (Rodrigues et al., 1991). Briefly, 100 two- to three-day old flies were kept in microtiter plates for 1 h in the dark and allowed to feed on agar wells. For measurement of
NaCl tolerance, wells with no additives alternated with wells containing 2% carmoisine red and 0.5 M NaCl; for sucrose preference testing, alternate wells had 2% carmoisine red and 1 mM sucrose. The percentage of flies
having a white abdomen is taken as % repulsion in the case of NaCl tolerance and as percentage acceptance in the case of sucrose tolerance. The data from five such plates were used to calculate the average of the
percentage of flies responding.

Proboscis Extension Assay (Riesgo Escovar Laboratory)

Proboscis extension in response to glucose applied to the tarsi was measured as described in Connolly & Tully (1998). Flies were 2–10 days old and were kept without food in vials with moistened stoppers for 12 h prior to
testing. Before each test and between tests, flies were offered a deionized water-saturated cotton swab and allowed to drink ad libitum , putatively to clean their tarsi. For tests, flies were offered cotton swabs to their tarsi
soaked in glucose solutions in deionized water. When a fly extended its proboscis completely for the duration of the test (three sec) and tried to make contact with the cotton swab, this behavior was scored as 2. If a fly
extended partially, or extended and retracted the proboscis during the test period, this was scored as 1. Finally, if a fly did not extend the proboscis during the test period, a “0” was scored. All flies were tested in, 0.001,
0.01, 0.1 and 1 M glucose, starting with the lowest concentration; and flies from the same line were tested on different days, to control for day-to-day differences in responses. For each line, between 8 and 15 female flies
were used. For some lines, males were also used, and the results were similar (data not shown).

Electroantennograms (Carlson Laboratory)

Electroantennograms were recorded from the proximomedial region of antennae of one- to seven- day old males as described in Ayer & Carlson (1992). Odorants were chosen to evoke responses from several olfactory
receptor neuron classes with different response spectra (cf. de Bruyne et al., 2001). All odorants were dissolved (10-2) in paraffin oil. Each set of flies (n = 6) from a particular isogenized line was compared to a set of
Canton-S control flies tested intermittently that same day.

Learning
Olfactory Jump Reflex Habituation (O’Kane Laboratory)
Each trial consisted of a benzaldehyde jump test as described above, with a one-minute inter trial interval (ITI). A habituation score was expressed as trials to criterion (TTC), the number of trials before there were four
consecutive failures to jump. Upon reaching criterion, the chamber containing the fly was vortexed for 75 sec and then returned to the habituation machine; the fly was scored for jump response once again, 2 min after the
last training trial (dishabituation test). To test for spontaneous recovery (SR), separate flies were left undisturbed during this 2 min period, presented with odor, and tested for jump response. Six flies of each line were tested
per day for 5–7 days, and daily means were used as raw data in statistical comparisons.

Operant Conditioning Paradigm (Heisenberg Laboratory)

Flies were maintained at 25°C on standard cornmeal/molasses medium in a 16-hr light/8-hr dark cycle at 60% humidity. One- to four-day old flies homozygous for a given isogenized chromosome were selected under
cold-anesthesia at least 12 h prior to the experiment, and tested for operant conditioning essentially as described in Wustmann & Heisenberg (1997) and Zars et al. (2000). Briefly, an individual fly was placed in a chamber.
During a four-min training phase, when the fly entered a predetermined “punished” half of the chamber, the entire chamber was heated to 39 ± 2°C. When the fly returned to the “unpunished” half, the temperature
returned to 19 ± 2°C. The training phase is preceded by a preference test of 30 seconds and followed by a memory test of 3 minutes. During the preference and memory tests the temperature was fixed at 19 ± 2°C. The
memory test measures the fly’s memory of training as evidenced by a continued avoidance of the formerly “punished” side. A Performance Index (PI) is calculated as the time spent on the unpunished side of the chamber
minus the time spent on the punished side, divided by the total time; so that a PI of 0 indicates no preference, and a PI of 1 indicates full avoidance. During the preference test, the PI measures a fly’s spontaneous
preference for either side; during training it indicates heat avoidance; and during the memory test it is a memory score. As no significant difference was observed between Canton-S males and females (Wustmann &
Heisenberg, 1997), equal numbers of males and females were measured, and the data were pooled. 15 flies were tested in parallel in most experiments; each line was measured twice on each of three consecutive days and
the data were pooled. For statistical evaluation the last three minutes of training, and the first three minutes of the memory test were used.

Locomotor Behaviors
Paralysis (Krishnan Laboratory)
Flies were tested for paralytic behavior at 2–3 days old (Krishnan et al., 1996), in a Sushi cooker in the temperature range of 38°C to 43°C. 10 flies were tested at a time. 8 trials were done for each line (4 males and 4
females).

Sensitivity/Resistance to Volatile Anesthetics (Krishnan Laboratory)

Flies were tested at 3 days old for sensitivity/resistance to volatile anesthetics (Krishnan & Nash, 1990). Males and females were tested separately. Flies were loaded into an “inebriometer,” which was equilibrated with 0.5%
halothane. Fractions of anesthetized flies were collected every minute for up to 40 min. The Mean Elution Time (MET) was calculated as:

where nt is the number of flies anaesthetized in time t, and Σ n is the total number of flies. Each isogenic line was tested four times and Canton-S six times.

Circadian Locomotor Behavior (Kyriacou Laboratory)

Cultures were maintained at 18°C in 12:12 hour light : dark (LD) conditions. Locomotor activity analysis of two- to seven- day-old male flies at 18°C was performed as described in Peixoto et al. (1998) and Sawyer et al.
(1977). Activity recording was initiated at ZT 18:00 (6 hours after lights off) in a LD12:12 cycle, and continued for 4 days. Subsequently the lights were turned off permanently so that each fly was “free running” in DD
conditions for a further 4.5 days. The activity events occurring during 30-minute time bins were summed and recorded. At the end of the experiment, flies that died during the observation period were removed from the
analysis. The LD data from all flies that were active for 4 days were used, but only those flies that remained alive for at least 7 days were included in the analysis of DD behavior.
For each individual, activity for each 30-min time bin was averaged over 3 days. A genotype profile was obtained by averaging each individual fly’s scores. The free-running circadian period of each fly was determined
by autocorrelation (Peixoto et al., 1998; Sawyer et al., 1977). For each individual, the DD data were “wrapped around” the free-running period, to the nearest half hour, determined by the autocorrelogram. To pool data
from several individuals within a genotype, each of which may have different periods, each fly’s profile was divided into 96 equidistant virtual bins, by predicting via linear regression from the actual adjacent bins what
the locomotor score of the virtual bin would be.
Experiments were performed in one of two environmental conditions, one a quiet basement room, far away from any human interference; separate tests were carried out in a room adjacent to a busy corridor, very close to
human activity. These conditions were forced upon us due to extensive building works. Canton-S controls were analyzed in both sets of conditions, and isogenic performances were compared only with the relevant control
scores.

Courtship Behavior (Goodwin Laboratory)

Flies were maintained and assays were performed as described in Villella et al. (1997). However the dimensions of the mating chambers were 2.0-cm diameter, 0.4-cm depth with a central removable divider. All courtship
observations were made in the morning within 4 hours of lights on, and transfer of flies to courtship chambers was made by aspiration, without the use of anesthesia. Latency was scored as the first observable wing
extension the male directed toward the target female. The Courtship Index is the proportion of the observation period that the male engages in any of the observable courtship modalities: orientation, following, wing
extension, attempted copulation and full copulation with the target female (reviewed by Hall, 1994). Recording was halted once full copulation was initiated or after a maximum time limit of 10 minutes. Tests on isogenic
males and wild-type control males were performed under the same conditions on separate days.

Wing Measurement (Partridge Laboratory)

Females were allowed to oviposit on grape juice/agar medium. Progeny were picked as first instar larvae, which were then transferred to new vials of unyeasted standard medium at a constant density of 40 larvae per vial,
conditions under which competition is minimal and body size maximized. Five replicate vials were established from each line. Flies from the isogenized lines were reared simultaneously and on the same batch of food as
the controls. After 14 days, all flies that had emerged in each vial were frozen for later measurement. Wing area was measured as described in Gilchrist & Partridge (2001), using a polygon generated from wing landmarks,
except that we used a constant density of 50 (rather than 30) larvae per vial and established 6 replicate vials for each line.

RESULTS

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Generating Isogenic Lines for P–Element Transposon Mobilization

Our first step was to generate isogenic lines that we could be confident would not lead to inadvertent mobilization of unmarked P or other transposable elements when crossed. We therefore subjected all stocks that were
involved in generation of the isogenic chromosomes (see crosses in Figure 1) to Southern and/or PCR analysis to test them for the presence of any such mobile elements.
First, to detect any elements that could be mobilized, we used short probes in Southern blots to detect the 5' and 3' regions of the P element that are present in most P vectors and include the sequences that are
necessary in cis for transposition (Mullins et al., 1989). By excluding vector-specific and most internal P sequences from the probes, they were made more specific for short P elements that still retained their terminal
sequences. No such P elements were found in any of the strains used to generate the isogenic lines (Figure 2a).

Figure 2 Southern blot and PCR analysis to check for presence of transposable elements. A,
Southern blots of Canton-S(lane 1), Sp/ CyO in a Canton-S background (lane 2), Sb/ TM3, Ser in a
Canton-S background (lane 3) and a P{GT1}-containing stock (lane 4) were hybridized with probes specific for the P 5' and 3'-ends. B, PCR amplification of 5'-ends of hobo and I elements.
Genotypes 1–3 are as in A.

Second, we wished to avoid inadvertently mobilizing hobo, by crossing a hobo-containing ( H) strain to a hobo-free ( E) strain, or mobilizing the I element, by crossing an I strain to a R strain (Bucheton et al., 1976;
Periquet et al., 1994). We therefore performed PCR amplifications of the 5' ends of hobo and I from all strains used and detected both elements in all stocks tested (Figure 2b). Since this meant that we would not be
crossing strains carrying any of these elements to strains lacking them, it was unlikely that these elements would be mobilized at a significant frequency.
Using these “mobile-element-safe” fly stocks, we generated lines isogenic for either second or third chromosomes (Figure 1), derived from a Canton-S stock used previously in behavioral experiments (de Belle &
Heisenberg, 1994; de Belle & Heisenberg, 1996; Dura et al., 1993). All 25 isogenic second-chromosome lines generated, and 31 out of 39 third chromosome lines generated, were homozygous viable. Each of these lines
was maintained with a mixture of w+ and w1118 X chromosomes (both X chromosomes carrying a Canton-S genetic background), to allow them to be crossed subsequently to either w+ or w stocks; however, w+ flies were
used for all behavioral characterizations. Also, we maintained these isogenic lines in a large population (at least 100 individuals) to preserve the genetic variability typical for the non-isogenized chromosomes in the
parental strain.

Chemosensory Responses
As an initial step to identify homozygous lines most suited for behavioral work, we measured their jump responses to two concentrations of benzaldehyde. All of the 25 Iso-2 lines, and 28 of the 29 Iso-3 lines tested were
not significantly different from Canton-S (p > 0.002; corrected using Bonferroni method for an experimentwise p > 0.05) in their jump response to 0.75% and 1% benzaldehyde (diluted in mineral oil) (Table I). To bias
our choice of lines to those most representative of the Canton-S control, we ranked them in order of similarity to Canton-S performance at each benzaldehyde concentration, and then averaged the two ranks for each line to
arrive at a final rank. The ten top-ranking isogenic second chromosome and ten top-ranking isogenic third chromosome lines (Table I) were then tested in other assays.
Table I Chemosensory behavioral responses
Adult olfactory jump assay Adult olfactory Y-maze Larval olfactory plate assay Adult taste response Adult proboscis extension
assay
0.75% BA 1.00% BA

Line Isogenic Canton-S Isogenic RI 1% EA 10% BA NaCl repulsion Sucrose attraction 0.001 M 0.01 M 0.1 M 1 M

2A 0.38 ± 0.07 0.40 ± 0.10 0.89 ± 0.06 -0.33 ± 0.01 0.78 ± 0.04 0.03 ± 0.06 96.92 ± 1.10 83.62 ± 1.66
2B 0.16 ± 0.00 0.21 ± 0.03 0.89 ± 0.01 -0.37 ± 0.01 0.78 ± 0.03 -0.25 ± 0.09 97.60 ± 1.50 84.60 ± 3.20
2C 0.25 ± 0.10 0.30 ± 0.05 0.85 ± 0.05 -0.38 ± 0.01 0.73 ± 0.03 0.21 ± 0.04 92.20 ± 4.32 82.80 ± 2.33 0.55 0.81 1.09 2.00

2D 0.26 ± 0.02 0.41 ± 0.09 0.90 ± 0.02 -0.38 ± 0.01 0.86 ± 0.03 -0.18 ± 0.10 98.82 ± 1.05 86.20 ± 1.39
2E 0.27 ± 0.02 0.43 ± 0.09 0.89 ± 0.02 -0.36 ± 0.01 0.91 ± 0.01 -0.32 ± 0.09 95.92 ± 0.47 84.40 ± 2.18
2F 0.20 ± 0.06 0.30 ± 0.05 0.85 ± 0.04 -0.38 ± 0.01 0.82 ± 0.03 -0.16 ± 0.08 93.22 ± 3.54 80.20 ± 1.85
2G 0.27 ± 0.09 0.41 ± 0.09 0.90 ± 0.03 -0.37 ± 0.01 0.76 ± 0.03 0.19 ± 0.07 95.00 ± 2.58 82.00 ± 1.41
2H 0.22 ± 0.07 0.44 ± 0.11 0.86 ± 0.05 -0.34 ± 0.02 0.77 ± 0.03 0.19 ± 0.05 92.44 ± 4.31 84.00 ± 1.44 0.15 0.00 0.31 1.54

2I 0.28 ± 0.04 0.21 ± 0.04 0.92 ± 0.04 -0.46 ± 0.11 0.87 ± 0.03 -0.19 ± 0.10 97.08 ± 0.49 83.00 ± 0.83
2J 0.14 ± 0.02 0.25 ± 0.04 0.90 ± 0.04 -0.37 ± 0.01 0.66 ± 0.06 -0.02 ± 0.10 97.00 ± 1.01 80.40 ± 0.87 0.00 0.46 0.77 1.54
3A 0.43 ± 0.10 0.41 ± 0.03 0.90 ± 0.02 -0.38 ± 0.00 0.78 ± 0.05 0.06 ± 0.07 93.64 ± 0.40 83.00 ± 2.30 0.29 0.64 0.71 1.93
3B 0.50 ± 0.09 0.41 ± 0.03 0.90 ± 0.07 -0.40 ± 0.01 0.75 ± 0.04 0.17 ± 0.07 94.56 ± 1.05 83.60 ± 0.92 0.53 0.87 1.13 2.00
3C 0.40 ± 0.07 0.41 ± 0.03 0.85 ± 0.03 -0.38 ± 0.02 0.77 ± 0.05 0.22 ± 0.06 98.60 ± 0.60 83.66 ± 1.14

3D 0.48 ± 0.13 0.41 ± 0.03 0.87 ± 0.02 -0.42 ± 0.02 0.83 ± 0.03 0.09 ± 0.07 96.31 ± 1.79 84.60 ± 0.74 1.14 0.86 0.71 1.00
3E 0.32 ± 0.01 0.41 ± 0.03 0.88 ± 0.02 -0.36 ± 0.02 0.81 ± 0.02 -0.16 ± 0.07 96.52 ± 0.51 80.00 ± 2.07 0.27 0.55 1.09 1.90
3F 0.38 ± 0.01 0.40 ± 0.05 0.79 ± 0.01 -0.43 ± 0.07 0.77 ± 0.04 0.03 ± 0.09 95.42 ± 0.81 84.00 ± 2.00 0.00 0.00 0.44 1.30

3G 0.51 ± 0.15 0.41 ± 0.03 0.92 ± 0.06 -0.38 ± 0.00 0.64 ± 0.05 0.16 ± 0.05 97.40 ± 0.50 83.60 ± 1.88

3H 0.44 ± 0.05 0.40 ± 0.05 0.75 ± 0.04 -0.38 ± 0.01 0.83 ± 0.05 -0.07 ± 0.11 96.36 ± 0.44 81.40 ± 1.32
3I 0.38 ± 0.06 0.28 ± 0.05 0.85 ± 0.06 -0.38 ± 0.01 0.86 ± 0.03 0.01 ± 0.08 96.76 ± 0.37 82.60 ± 1.20 0.56 0.67 0.44 1.56
3J 0.26 ± 0.05 0.41 ± 0.03 0.87 ± 0.04 -0.56 ± 0.09 0.89 ± 0.03 0.32 ± 0.06 98.44 ± 0.41 86.00 ± 1.92 1.10 1.10 1.40 1.80

Canton-S n.a. n.a. 0.87 ± 0.05 -0.35 ± 0.01 0.68 ± 0.05 0.03 ± 0.06 92.04 ± 3.48 85.40 ± 1.40 0.00 0.13 0.88 1.38

Where appropriate, scores are expressed as mean ± standard error; non-parametric distributions are expressed as mean. Where n comparisons to Canton-S were carried out, to obtain an experimentwise α = 0.05, we applied
the Bonferroni correction to obtain α· = 0.05/n for individual comparisons.

Jump response to benzaldehyde (BA) is expressed as daily jump frequencies (N = 3; 13–42 flies per day). Experiments were carried out at different times and so different values were used for the control Canton-S line.
ANOVA showed no significant difference (p > 0.002).

Adult olfactory response to 10-4 benzaldehyde in Y-maze is expressed as response index (RI); n = 4. No p -value was less than 0.01 (Student’st-test).

Larval olfactory response to 1% ethyl acetate (EA) and 10% benzaldehyde is expressed as response index (RI); n = 10–12 assays. An ANOVA test showed some significant differences between genotypes for ethyl acetate;
however, a post-hoc Scheffé test showed no significant differences between individual isogenic lines and Canton-S.

NaCl aversion and sucrose preference of adult flies are expressed as percentages of groups of 100 flies; n = 5–8. No p -value was less than 0.03 (Student’s t-test).

Proboscis extension in response to glucose solutions is shown as average responses, where 2 stands for a full response, 1 for an incomplete response, and 0 for no response. Flies from the same line were tested on different
days, to control for day-to-day differences in responses; n = 8–15. A couple of results were significantly different from Canton-S at α = 0.01 (Mann Whitney U test; shown in bold); however, these differences were not
significant when the Bonferroni correction (0.05/10) was applied.

None of the 20 lines showed a significant difference from Canton-S in attraction or repulsion of adults to benzaldehyde (10-4 in water) in a Y-maze (Table I), or in taste preferences for and against sucrose and NaCl,
respectively (Table I). Larvae were tested for their attraction or repulsion to filter discs of 1% ethyl acetate and to 10% benzaldehyde; flies from no individual isogenic lines behaved in a significantly different manner
compared to Drosophila from the Canton-S strain (Table I). Females of some of the lines were tested for their ability to extend their proboscis in response to increasing concentrations of glucose applied to their tarsi (Table
I). No lines showed a significant difference from Canton-S.
Consistent with the results of the tests for adult olfactory behavior, none of the isogenized lines tested showed electroantennogram amplitudes that deviated significantly from the controls for any odorant (Table II).
Table II Electroantennogram amplitudes in Response to odors
Ethyl acetate Ethyl butyrate Pentyl acetate Benzaldehyde 1-0cten-3-ol Heptanone Geranyl acetate Paraffin Oil

Line Canton-S Iso Canton-S Iso Canton-S Iso Canton-S Iso Canton-S Iso Canton-S Iso Canton-S Iso Canton-S Iso

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ISOGENIC AUTOSOMES TO BE APPLIED IN OPTIMAL SCREENING FOR NOVEL MUTANTS WITH VIABLE PHENOTYPES IN DROSOPHILA MELANOGASTER 11/1/07 16:10

2A 16.9 ± 1.4 17.5 ± 1.4 17.4 ± 1.3 19.7 ± 1.5 17.0 ± 1.0 16.7 ± 1.1 10.0 ± 0.6 10.3 ± 0.7 13.9 ± 1.1 12.8 ± 1.1 13.6 ± 1.2 13.3 ± 1.1 7.4 ± 0.6 6.3 ± 0.7 1.9 ± 0.3 1.5 ± 0.4
2B 18.7 ± 1.3 18.2 ± 0.8 21.1 ± 1.1 20.2 ± 0.7 19.1 ± 1.2 18.5 ± 1.1 10.7 ± 0.6 9.5 ± 0.6 15.6 ± 0.7 14.5 ± 0.8 16.6 ± 1.0 15.5 ± 1.3 6.3 ± 0.7 6.3 ± 0.5 1.3 ± 0.4 1.3 ± 0.2
2C 16.6 ± 1.1 16.8 ± 2.0 19.3 ± 2.1 19.5 ± 2.2 18.4 ± 2.1 17.2 ± 1.5 9.8 ± 0.6 10.2 ± 0.7 14.4 ± 2.2 14.7 ± 1.1 16.0 ± 2.3 14.2 ± 1.7 5.5 ± 0.7 6.0 ± 0.5 1.1 ± 0.3 1.3 ± 0.3

2D 18.7 ± 0.7 18.1 ± 0.7 19.7 ± 0.9 20.4 ± 0.9 18.4 ± 0.5 19.8 ± 0.9 11.3 ± 1.8 10.0 ± 0.8 16.2 ± 0.6 16.5 ± 1.1 16.2 ± 0.6 16.5 ± 1.3 7.9 ± 0.4 7.5 ± 0.7 2.8 ± 0.3 2.3 ± 0.3

2E 18.7 ± 1.3 18.5 ± 0.6 21.1 ± 1.1 20.3 ± 0.8 19.1 ± 1.2 19.5 ± 0.6 10.7 ± 0.6 9.7 ± 0.4 15.6 ± 0.7 15.0 ± 0.7 16.6 ± 1.0 15.3 ± 0.3 6.3 ± 0.7 7.0 ± 0.6 1.3 ± 0.4 1.7 ± 0.2

2F 16.9 ± 1.4 17.2 ± 0.7 17.4 ± 1.3 18.8 ± 0.7 17.0 ± 1.0 19.5 ± 0.8 10.0 ± 0.6 10.3 ± 0.3 13.9 ± 1.1 15.7 ± 0.6 13.6 ± 1.2 14.2 ± 0.5 7.4 ± 0.6 8.2 ± 0.2 1.9 ± 0.3 1.8 ± 0.2

2G 18.0 ± 0.6 18.2 ± 0.9 20.6 ± 0.5 21.5 ± 0.8 17.5 ± 0.5 18.8 ± 0.5 8.6 ± 0.4 9.2 ± 0.5 15.9 ± 0.4 15.8 ± 0.3 15.8 ± 0.4 16.0 ± 0.4 6.0 ± 0.4 5.8 ± 0.5 1.1 ± 0.2 1.3 ± 0.2
2H 18.0 ± 0.6 17.0 ± 1.7 20.6 ± 0.5 19.0 ± 2.0 17.5 ± 0.5 17.2 ± 1.1 8.6 ± 0.4 10.2 ± 0.3 15.9 ± 0.4 14.3 ± 0.5 15.8 ± 0.4 14.0 ± 1.0 6.0 ± 0.4 5.7 ± 0.5 1.1 ± 0.2 1.1 ± 0.3
2I 18.7 ± 0.7 18.8 ± 0.9 19.7 ± 0.9 20.5 ± 0.7 18.4 ± 0.5 18.5 ± 1.2 11.3 ± 1.8 10.3 ± 1.1 16.2 ± 0.6 14.7 ± 1.4 16.2 ± 0.6 13.3 ± 1.1 7.9 ± 0.4 6.8 ± 0.3 2.8 ± 0.3 2.1 ± 0.3

2J 16.9 ± 1.4 15.7 ± 1.4 17.4 ± 1.3 17.7 ± 1.5 17.0 ± 1.0 16.0 ± 1.3 10.0 ± 0.6 9.3 ± 0.7 13.9 ± 1.1 13.2 ± 1.1 13.6 ± 1.2 14.0 ± 1.1 7.4 ± 0.6 5.7 ± 0.3 1.9 ± 0.3 1.0 ± 0.4
3A 16.6 ± 1.1 19.7 ± 1.6 19.3 ± 2.1 22.8 ± 0.7 18.4 ± 2.1 20.0 ± 0.6 9.8 ± 0.6 9.5 ± 0.8 14.4 ± 2.2 14.7 ± 0.8 16.0 ± 2.3 17.2 ± 0.7 5.5 ± 0.7 6.7 ± 0.5 1.1 ± 0.3 1.2 ± 0.2

3B 18.7 ± 1.3 19.7 ± 0.6 21.1 ± 1.1 22.0 ± 0.6 19.1 ± 1.2 19.8 ± 0.5 10.7 ± 0.6 10.5 ± 0.9 15.6 ± 0.7 15.8 ± 0.7 16.6 ± 1.0 16.8 ± 1.0 6.3 ± 0.7 6.8 ± 0.5 1.3 ± 0.4 1.3 ± 0.3
3C 18.0 ± 0.6 17.7 ± 0.9 20.6 ± 0.5 20.3 ± 0.8 17.5 ± 0.5 17.3 ± 0.7 8.6 ± 0.4 8.7 ± 0.7 15.9 ± 0.4 13.7 ± 0.9 15.8 ± 0.4 13.7 ± 0.8 6.0 ± 0.4 5.2 ± 0.3 1.1 ± 0.2 1.3 ± 0.2

3D 18.0 ± 0.6 18.0 ± 0.5 20.6 ± 0.5 20.7 ± 0.6 17.5 ± 0.5 18.3 ± 0.4 8.6 ± 0.4 8.9 ± 0.4 15.9 ± 0.4 15.5 ± 0.3 15.8 ± 0.4 16.2 ± 0.3 6.0 ± 0.4 6.5 ± 0.5 1.1 ± 0.2 1.3 ± 0.2
3E 18.7 ± 1.3 17.7 ± 1.0 21.1 ± 1.1 20.2 ± 0.9 19.1 ± 1.2 19.5 ± 0.8 10.7 ± 0.6 9.3 ± 0.7 15.6 ± 0.7 15.8 ± 0.6 16.6 ± 1.0 16.8 ± 0.6 6.3 ± 0.7 6.7 ± 0.5 1.3 ± 0.4 1.2 ± 0.3

3F 18.7 ± 0.7 19.8 ± 1.2 19.7 ± 0.9 22.2 ± 1.1 18.4 ± 0.5 18.3 ± 0.8 11.3 ± 1.8 9.5 ± 0.4 16.2 ± 0.6 16.7 ± 0.5 16.2 ± 0.6 15.2 ± 0.6 7.9 ± 0.4 7.7 ± 0.3 2.8 ± 0.3 3.4 ± 0.2
3G 18.0 ± 0.6 18.3 ± 1.0 20.6 ± 0.5 20.0 ± 1.0 17.5 ± 0.5 18.5 ± 0.8 8.6 ± 0.4 9.3 ± 0.7 15.9 ± 0.4 15.5 ± 0.7 15.8 ± 0.4 16.2 ± 0.9 6.0 ± 0.4 6.5 ± 0.8 1.1 ± 0.2 1.8 ± 0.4
3H 18.7 ± 1.3 19.3 ± 0.9 21.1 ± 1.1 21.2 ± 1.1 19.1 ± 1.2 19.2 ± 0.7 10.7 ± 0.6 9.7 ± 0.4 15.6 ± 0.7 13.8 ± 0.4 16.6 ± 1.0 16.0 ± 0.5 6.3 ± 0.7 5.7 ± 0.3 1.3 ± 0.4 1.3 ± 0.2

3I 16.6 ± 1.1 17.0 ± 1.0 19.3 ± 2.1 19.8 ± 0.8 18.4 ± 2.1 17.5 ± 0.5 9.8 ± 0.6 10.0 ± 0.7 14.4 ± 2.2 13.2 ± 0.9 16.0 ± 2.3 15.5 ± 0.3 5.5 ± 0.7 6.7 ± 0.4 1.1 ± 0.3 1.3 ± 0.3

3J 18.7 ± 0.7 16.9 ± 1.0 19.7 ± 0.9 20.8 ± 1.3 18.4 ± 0.5 17.8 ± 0.7 11.3 ± 1.8 9.8 ± 0.4 16.2 ± 0.6 16.6 ± 0.9 15.2 ± 0.7 14.3 ± 0.7 7.9 ± 0.4 6.8 ± 0.5 2.8 ± 0.3 3.3 ± 0.4

Values are expressed in mV as mean ± standard error (n = 6). No significant differences within odors were found using ANOVA (p > 0.0025; corrected using Bonferroni method for an experimentwise p > 0.05).

Learning
On repeated presentation of a pulse of benzaldehyde, the initial jump response gradually decreases (Boynton & Tully, 1992), an example in Drosophila of habituation. This phenomenon helps to prevent inappropriate
responses and can be viewed as simple form of learning (Rankin, 2002). We measured the following habituation parameters: trials to criterion (TTC, the number of trials before four consecutive failures to jump),
spontaneous recovery from habituation (SR, jumping frequency two minutes after habituation has occurred), and dishabituation (DIS, jumping frequency after vortexing, which followed habituation) for all lines (Table
III). All isogenic lines behaved similarly to Canton-S in all of these parameters. Three lines were also generated with different combinations of second and third isogenic chromosomes (2A + 3A, 2C + 3I, 2C + 3J), and
tested for TTC. None was significantly different from Canton-S (Table III), showing that different isogenic chromosome pairs could be recombined and still generate strains with a wild-type behavior.
Table III Learning scores
Operant conditioning Operant conditioning

Jump reflex habituation Activity Performance Index

Habituation Score SR DIS Training Memory Training Memory

Iso line Iso Canton-S Iso Canton-S Iso Canton-S 3 min 4 min 5 min 1 min 2 min 3 min 3 min 4 min 5 min 1 min 2 min 3 min

2A 6.80 ± 0.57 8.14 ± 0.65 0.08 ± 0.050.03 ± 0.050.80 ± 0.080.80 ± 0.080.45 ± 0.020.39 ± 0.020.36 ± 0.020.35 ± 0.020.36 ± 0.020.36 ± 0.020.51 ± 0.050.59 ± 0.050.67 ± 0.050.52 ± 0.060.43 ± 0.060.40 ± 0.06
2B 6.30 ± 0.58 8.20 ± 0.89 0.20 ± 0.080.20 ± 0.080.87 ± 0.060.67 ± 0.060.43 ± 0.020.38 ± 0.020.35 ± 0.020.35 ± 0.020.37 ± 0.020.38 ± 0.020.54 ± 0.050.57 ± 0.050.63 ± 0.050.39 ± 0.060.28 ± 0.060.20 ± 0.06

2C 6.20 ± 0.48 8.03 ± 0.65 0.07 ± 0.050.05 ± 0.050.88 ± 0.080.80 ± 0.080.44 ± 0.020.41 ± 0.020.38 ± 0.020.37 ± 0.020.38 ± 0.020.34 ± 0.020.48 ± 0.060.61 ± 0.060.67 ± 0.060.48 ± 0.060.46 ± 0.060.47 ± 0.07
2D 4.17 ± 0.59 8.20 ± 0.89 0.07 ± 0.070.20 ± 0.070.80 ± 0.090.67 ± 0.090.42 ± 0.020.38 ± 0.020.35 ± 0.020.36 ± 0.020.39 ± 0.020.37 ± 0.020.43 ± 0.050.53 ± 0.050.59 ± 0.050.30 ± 0.060.22 ± 0.060.26 ± 0.06
2E 7.18 ± 0.59 8.03 ± 0.65 0.17 ± 0.060.03 ± 0.060.87 ± 0.080.80 ± 0.080.44 ± 0.020.44 ± 0.020.42 ± 0.020.43 ± 0.020.44 ± 0.020.48 ± 0.020.51 ± 0.050.57 ± 0.050.57 ± 0.050.24 ± 0.060.17 ± 0.060.08 ± 0.06
2F 6.63 ± 0.66 9.50 ± 0.90 0.25 ± 0.080.25 ± 0.080.75 ± 0.110.67 ± 0.110.46 ± 0.020.42 ± 0.020.39 ± 0.020.38 ± 0.020.40 ± 0.020.41 ± 0.020.38 ± 0.060.49 ± 0.060.60 ± 0.050.44 ± 0.060.33 ± 0.070.27 ± 0.07
2G 6.67 ± 0.63 8.20 ± 0.89 0.20 ± 0.080.20 ± 0.080.87 ± 0.060.67 ± 0.060.44 ± 0.020.38 ± 0.020.36 ± 0.020.39 ± 0.020.39 ± 0.020.39 ± 0.020.54 ± 0.060.69 ± 0.060.75 ± 0.050.43 ± 0.070.38 ± 0.080.37 ± 0.07

2H 6.87 ± 0.74 8.20 ± 0.89 0.00 ± 0.060.20 ± 0.060.67 ± 0.070.67 ± 0.070.45 ± 0.020.43 ± 0.020.41 ± 0.020.4 ± 0.02 0.42 ± 0.020.42 ± 0.020.42 ± 0.060.55 ± 0.060.60 ± 0.060.40 ± 0.060.33 ± 0.070.26 ± 0.06
2I 5.80 ± 0.49 8.00 ± 0.66 0.03 ± 0.030.03 ± 0.030.83 ± 0.080.82 ± 0.080.45 ± 0.020.45 ± 0.010.40 ± 0.010.39 ± 0.020.45 ± 0.020.44 ± 0.020.43 ± 0.040.53 ± 0.040.57 ± 0.040.34 ± 0.050.24 ± 0.050.29 ± 0.05

2J 7.14 ± 0.62 8.09 ± 0.68 0.07 ± 0.050.05 ± 0.050.88 ± 0.080.80 ± 0.080.47 ± 0.020.41 ± 0.020.38 ± 0.020.39 ± 0.020.42 ± 0.020.42 ± 0.020.41 ± 0.060.50 ± 0.060.58 ± 0.060.28 ± 0.070.23 ± 0.070.21 ± 0.07

Canton-S 0.42 ± 0.020.36 ± 0.020.34 ± 0.020.36 ± 0.020.37 ± 0.020.37 ± 0.020.48 ± 0.070.64 ± 0.060.67 ± 0.060.42 ± 0.070.27 ± 0.070.34 ± 0.08
3A 8.36 ± 0.68 9.50 ± 0.67 0.07 ± 0.070.13 ± 0.070.87 ± 0.060.90 ± 0.060.43 ± 0.020.39 ± 0.020.37 ± 0.020.36 ± 0.020.36 ± 0.020.36 ± 0.020.43 ± 0.060.55 ± 0.050.59 ± 0.050.34 ± 0.070.36 ± 0.060.32 ± 0.06

3B 8.71 ± 0.67 9.64 ± 0.78 0.17 ± 0.060.00 ± 0.060.88 ± 0.060.92 ± 0.060.45 ± 0.020.42 ± 0.020.41 ± 0.020.36 ± 0.020.39 ± 0.020.38 ± 0.020.47 ± 0.060.52 ± 0.060.56 ± 0.060.29 ± 0.070.26 ± 0.080.30 ± 0.08
3C 5.58 ± 0.92 8.95 ± 1.17 0.07 ± 0.050.00 ± 0.050.87 ± 0.091.00 ± 0.090.54 ± 0.020.49 ± 0.020.46 ± 0.020.46 ± 0.020.47 ± 0.020.49 ± 0.020.35 ± 0.060.43 ± 0.060.56 ± 0.060.28 ± 0.060.24 ± 0.070.21 ± 0.07

3D 7.57 ± 0.73 9.38 ± 0.66 0.03 ± 0.060.15 ± 0.060.80 ± 0.050.90 ± 0.050.50 ± 0.020.47 ± 0.020.46 ± 0.020.43 ± 0.020.46 ± 0.020.45 ± 0.020.52 ± 0.060.56 ± 0.060.61 ± 0.050.32 ± 0.060.28 ± 0.060.22 ± 0.06

3E 6.60 ± 0.86 10.23 ± 0.960.13 ± 0.060.00 ± 0.060.87 ± 0.070.93 ± 0.070.45 ± 0.020.42 ± 0.020.37 ± 0.020.34 ± 0.020.38 ± 0.020.39 ± 0.020.35 ± 0.050.38 ± 0.060.48 ± 0.060.34 ± 0.070.29 ± 0.070.26 ± 0.07
3F 8.22 ± 1.17 8.93 ± 0.95 0.07 ± 0.050.00 ± 0.051.00 ± 0.060.92 ± 0.060.49 ± 0.020.42 ± 0.020.40 ± 0.020.38 ± 0.020.40 ± 0.020.40 ± 0.020.44 ± 0.060.64 ± 0.060.66 ± 0.050.48 ± 0.070.40 ± 0.070.42 ± 0.07

3G 14.11 ± 1.1510.50 ± 0.990.07 ± 0.050.00 ± 0.051.00 ± 0.050.93 ± 0.050.46 ± 0.020.42 ± 0.020.39 ± 0.020.35 ± 0.020.36 ± 0.020.38 ± 0.020.40 ± 0.060.51 ± 0.060.60 ± 0.060.34 ± 0.070.32 ± 0.070.30 ± 0.07
3H 8.90 ± 1.06 10.23 ± 0.960.13 ± 0.060.00 ± 0.060.73 ± 0.100.93 ± 0.100.44 ± 0.020.38 ± 0.020.37 ± 0.020.34 ± 0.020.34 ± 0.020.34 ± 0.020.42 ± 0.060.54 ± 0.060.58 ± 0.060.39 ± 0.070.36 ± 0.070.34 ± 0.07

3I 9.30 ± 0.74 9.63 ± 0.68 0.05 ± 0.060.13 ± 0.060.97 ± 0.040.90 ± 0.040.47 ± 0.020.44 ± 0.020.41 ± 0.020.37 ± 0.020.39 ± 0.020.39 ± 0.020.43 ± 0.060.51 ± 0.060.61 ± 0.060.37 ± 0.070.31 ± 0.070.26 ± 0.07

3J 8.58 ± 0.80 9.63 ± 0.68 0.12 ± 0.070.13 ± 0.070.87 ± 0.050.90 ± 0.050.50 ± 0.020.45 ± 0.020.43 ± 0.020.42 ± 0.020.45 ± 0.020.46 ± 0.020.39 ± 0.050.51 ± 0.050.55 ± 0.050.35 ± 0.060.31 ± 0.060.28 ± 0.06
Canton-S 0.39 ± 0.020.33 ± 0.020.32 ± 0.020.33 ± 0.020.34 ± 0.020.33 ± 0.020.61 ± 0.070.78 ± 0.060.84 ± 0.060.61 ± 0.080.46 ± 0.080.54 ± 0.07

2C + 3J 8.29 ± 0.56
(10)

2C + 3I 10.53 ± 0.82
(38)

2A + 3A 11.08 ± 1.37
(19)
Canton-S9.04 ± 0.49
(41)

Scores are expressed as mean ± standard error. To obtain an experimentwise α = 0.05, we applied the Bonferroni correction to obtain α· = 0.05/20 = 0.0025 for individual comparisons. Lines different from their

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ISOGENIC AUTOSOMES TO BE APPLIED IN OPTIMAL SCREENING FOR NOVEL MUTANTS WITH VIABLE PHENOTYPES IN DROSOPHILA MELANOGASTER 11/1/07 16:10

appropriate control at α = 0.0025 are in bold.

Habituation of jump response to repetitive benzaldehyde pulses: habituation scores are expressed as trials to criterion (n = 7–9 days, except where indicated). Isogenic lines were compared to Canton-S scores obtained on
the same days. No p-value was less than 0.009 (Wilcoxon/Kruskal-Wallis test). Habituation indices from a separate set of experiments over the space of a few months, of 3 lines isogenic for both main autosome pairs, and
of Canton-S are also shown as mean ± standard error (n); ANOVA for these scores and Canton-S scores gave a p-value of 0.18 (not significant). For spontaneous recovery and dishabituation, daily jump frequencies were
calculated (n = 7–9 days); no p-value was less than 0.04 (Student’s t-test).

Activity and Performance Index in the Operant Conditioning Paradigm: The first six columns show the activity of flies (measured in relative units) for the last three minutes of a training period and the first three minutes of
the memory test. The last six columns show the performance index (PI) for the same six time intervals. The PI of the preference test was subtracted to obtain the PIs shown. As the data do not show a normal distribution,
they were tested for significant differences (p > 0.0025) from Canton-S using a Mann Whitney U-test.

The lines were also tested for performance in an operant conditioning paradigm (Wustmann & Heisenberg, 1997; Zars et al., 2000). Performance Indices (PIs) were calculated as the time spent on the unpunished side of
the chamber minus the time spent on the punished side, divided by the total time (see Materials and Methods). During the preference test, the PI measures a fly’s spontaneous preference for either side, during training it
indicates heat avoidance, and during the memory test it is a memory score.
The ten second-chromosome lines showed no significant difference from the parental Canton-S strain in their operant conditioning PIs. Only two lines (2E and 2I) gave a significant increase in their activity in one of the
training or the memory test phases (when compared to Canton-S), but this was not repeated at other time points.
Overall, the lines isogenized for the third chromosome performed worse than the lines isogenized for the second chromosome. Only four of the former showed no significant difference to Canton-S both in their PI and in
their activity (at p > 0.0025; corrected using Bonferroni method for an experiment-wise p > 0.05). Three of them (3B, 3C, 3D) had a lower PI in the memory test at least one time point. Three further lines (3E, 3I, 3J)
showed significantly lower heat avoidance and memory scores that were lower than average, but not significantly so. All six lines that showed lower PIs in either training or memory tests also showed increased activity,
whereas only two of the four lines with normal PIs did so, and usually at fewer time points, suggesting a correlation between the increased activity and the low PI.

Locomotor Behavior
Paralysis and Response to an Anesthetic
We measured the time taken for a shift to 42°C to induce paralysis in each line. The lines showed no difference from Canton-S in the temperature required for 100% paralysis (42°C, data not shown) or in the time required
for 100% paralysis at that temperature (Table IV). No statistically significant differences in halothane sensitivity were found (Table IV).
Table IV Paralysis and sensitivity to anesthetics
Mean time to paralysis Mean elution time on halothane inebriometer

Line Males Females Males Females All

2A 135 ± 17 150 ± 0 8.96 ± 0.08 11.24 ± 0.30 10.10 ± 0.45

2B 105 ± 30 128 ± 29 9.03 ± 0.43 9.47 ± 0.26 9.25 ± 0.25

2C 83 ± 15 90 ± 24 9.77 ± 0.10 11.75 ± 0.24 10.76 ± 0.39
2D 113 ± 15 143 ± 15 7.36 ± 0.11 10.34 ± 0.66 8.85 ± 0.64
2E 135 ± 17 120 ± 0 9.37 ± 0.43 12.66 ± 0.44 11.01 ± 0.68
2F 90 ± 24 120 ± 35 10.79 ± 0.36 13.71 ± 0.56 12.25 ± 0.63

2G 105 ± 17 120 ± 0 7.66 ± 0.57 13.29 ± 1.07 10.47 ± 1.20

2H 120 ± 0 135 ± 17 9.19 ± 0.76 13.01 ± 0.62 11.10 ± 0.85
2I 120 ± 24 128 ± 15 9.76 ± 0.36 11.93 ± 0.41 10.84 ± 0.48
2J 127 ± 15 113 ± 15 8.57 ± 1.05 11.82 ± 1.03 10.19 ± 0.98
3A 135 ± 17 150 ± 0 9.82 ± 0.44 12.31 ± 0.55 11.06 ± 0.57

3B 90 ± 24 83 ± 15 8.82 ± 0.72 11.86 ± 0.89 10.34 ± 0.78

3C 120 ± 0 143 ± 15 8.59 ± 0.61 9.20 ± 0.49 8.90 ± 0.38
3D 128 ± 15 143 ± 15 10.35 ± 0.70 12.06 ± 0.39 11.20 ± 0.49
3E 150 ± 0 150 ± 24 8.78 ± 0.86 10.21 ± 0.53 9.49 ± 0.54
3F 90 ± 24 128 ± 15 8.65 ± 0.60 11.08 ± 0.46 9.86 ± 0.57

3G 135 ± 17 143 ± 15 9.42 ± 0.78 15.41 ± 0.81 12.41 ± 1.24

3H 90 ± 24 105 ± 17 10.59 ± 0.59 12.12 ± 0.98 11.36 ± 0.60
3I 128 ± 15 135 ± 17 8.16 ± 0.71 10.58 ± 1.13 9.37 ± 0.77
3J 68 ± 15 105 ± 17 7.61 ± 1.33 9.58 ± 1.12 8.60 ± 0.88
Canton-S 105 ± 17 120 ± 0.00 7.63 ± 0.42 9.35 ± 0.45 8.49 ± 0.39

Time to paralysis at 42° in seconds is expressed as mean ± SEM (N = 8 trials, with 10 flies per trial. ANOVA showed no significant difference (p > 0.05) of the isogenic lines from Canton-S.

Sensitivity/resistance to halothane: Data points are mean elution time (MET, in minutes) through an inebriometer equilibrated with 0.5% halothane. Each isogenic line was tested four times and Canton-S six times. No
significant differences between genotypes were seen with ANOVA (p > 0.05).

Circadian Locomotor Behavior

Three parameters of circadian locomotor behavior were studied: strength of circadian rhythmicity, length of circadian period, and pattern of locomotor activity (measured as activity time interaction) under light:dark
(LD) and constant-dark (DD) conditions.
All isogenic lines showed circadian rhythmicity and periods similar to the relevant Canton-S control in both LD and DD (Table V). However, analysis of the ANOVA activity time interaction identified some significant
differences in the circadian patterns of locomotor activity (Table V). The patterns of all lines in LD showed typical circadian behavior (Collins et al., 2004) of an increase in activity in anticipation of lights-on, a morning
peak of activity soon thereafter, followed by a lunch-time siesta, and a second peak late in the day, anticipating lights-off for most of the lines. However, lines 2F and 3D, behaved with a peak of activity in the night phase,
30–60 min after the lights had gone off (data not shown).
Table V Circadian locomotor behavior
N Circadian locomotor behavior Pattern of locomotor activity (A T)

Line LD DD Strength of circadian rhythmicity % (DD) Length of circadian period Mean (h) LD DD

2A 12 12 75% 23.83 ns ns

2B 19 19 100% 23.61 ns ns
2C 13 13 100% 23.31 *** ns

2D 14 14 79% 23.95 *** ns

2E 12 10 90% 23.22 ns **

2G 10 8 100% 23.62 ns ns

2H 7 5 100% 23.50 ns *
2J 12 12 100% 23.21 *** ns

3B 23 22 96% 23.66 ns ns
3D 16 16 100% 23.81 ** **

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3E 19 18 100% 23.61 ns ***

3F 14 15 93% 23.50 ns **

3G 9 9 70% 24.14 ns **
3H 15 15 100% 23.60 ns ns

3I 12 10 60% 23.75 ns ns
3J 13 13 92% 23.62 ns ns

Canton-S 40 40 98% 23.55 – –

2F 17b 17b 82%b 22.82b *** ns

2I 28b 24b 79%b 23.00b ns ns

3A 21b 21b 76%b 23.16b ns ns
3C 23b 20b 95%b 23.45b ns ns

Canton-S 21b 18b 89%b 22.91b – –

Circadian locomotor activity, and results of ANOVA using each isogenic strain compared with the Canton-S strain tested in the same environment. Strains analyzed in a busy location are marked with a “b”. Strength refers
to the proportion of statistically rhythmic individuals. Only the activity x time (A T) interaction terms are shown. ns non-significant, * p < 0.05, ** p < 0.01, *** p < < 0.01 (df, 1, 47). LD – Light-Dark cycle; DD –
dark-dark cycle.

In DD conditions, most lines showed an activity peak early in the subjective day, and in some cases, evidence of a second peak later in the afternoon/evening, consistent with previous findings performed at the same
relatively low temperature of 18°C (Majercak et al., 1999). Lines 2E, 2H, 3E, 3F, and 3G showed slight variations in activity (e.g., the size of their afternoon peaks) compared to Canton-S. Line 3D shows a more
qualitative difference in that its primary peak falls late in the subjective day (data not shown). This line also shows the most dramatic night-time peak in activity after lights-off in LD, suggesting some phenotypic
correlation between the two characters.

Courtship Behavior
Three components of courtship were analyzed: the Courtship Index (CI; see Materials and Methods), courtship-initiation latency, and the time taken to achieve copulation. All isogenized lines are able to copulate and
produce viable progeny, and all were similar to Canton-S in their CI values (Table VI). Compared with Canton-S, only one line (3C) showed a significantly different (shorter) courtship latency, whereas 4 of the 20 lines
(2A, 2H, 3A and 3E) showed a significantly different (also shorter) time to full copulation (Table VI).
Table VI Courtship behavior
Line Courtship index median (N) Courtship latency median (N) Time to full copulation median (N)

2A 64.30 (10) 27.5 (10) 55.5 (8)

2B 89.30 (10) 28.0 (10) 297.0 (9)
2C 71.20 (10) 36.0 (10) 86.0 (10)

2D 78.75 (10) 29.5 (10) 72.0 (9)

2E 79.85 (10) 16.0 (10) 79.0 (10)
2F 80.70 (10) 22.5 (12) 65.0 (9)
2G 79.30 (11) 17.0 (11) 73.0 (11)
2H 73.20 (10) 17.0 (10) 76.5 (10)

2I 77.65 (10) 28.0 (10) 144.5 (10)

2J 80.15 (10) 15.5 (10) 93.0 (10)
3A 69.60 (13) 30.0 (13) 86.0 (11)
3B 61.20 (11) 21.0 (11) 104.0 (11)

3C 74.10 (11) 18.0 (11) 63.0 (9)

3D 70.90 (11) 20.0 (11) 86.0 (11)
3E 66.70 (11) 22.0 (11) 108.0 (11)
3F 83.20 (11) 26.0 (11) 149.0 (9)
3G 69.95 (12) 24.0 (12) 100.0 (10)

3H 78.70 (10) 21.0 (10) 61.0 (8)

3I 70.90 (11) 17.5 (10) 79.0 (11)

3J 71.50 (11) 28.0 (11) 93.5 (10)

Canton-S 87.60 (21) 30.0 (21) 154.63 (21)

Courtship index, time to full copulation and courtship latency are expressed as medians. The isogenic lines were compared to Canton-S scores using Mann-Whitney Confidence and Interval Test. For courtship index, no p-
value was less than 0.03, for time to full copulation no p-value was less than 0.0002 and for courtship latency no p-value was less than 0.001. Using Bonferroni correction to obtain α· = 0.05/20 = 0.0025 for individual
comparisons, results that are significantly different from Canton-S are in bold.

Wing Measurement
Wing area is a reflection of factors that include cell determination and differentiation, and cell and organismal size, which can reflect the nutritional state of the animal (Robertson 1962; Sokoloff 1966; Cavicchi et al.
1981). Out of 10 lines for each chromosome, flies from four isogenic second and five isogenic third chromosome lines were found to have a wing area not significantly different from that of Canton-S. The remaining lines
(six isogenic second and five isogenic third chromosome lines) had a wing area significantly larger than Canton-S (Table VII).
Table VII Wing area measurements
Line Male wing area mean ± SE Female wing area mean ± SE P-value relative to control (t test) Significance after correction

Canton-S 1.086 ± 0.011 1.397 ± 0.020 – –

2A 1.157 ± 0.011 1.479 ± 0.015 <0.0001 <0.0001

2B 1.112 ± 0.012 1.384 ± 0.013 Not significant Not significant

2C 1.120 ± 0.006 1.409 ± 0.011 Not significant Not significant

2D 1.141 ± 0.006 1.434 ± 0.009 <0.0001 <0.0001

2E 1.135 ± 0.005 1.437 ± 0.011 <0.0001 <0.0001

2F 1.167 ± 0.009 1.453 ± 0.016 <0.0001 <0.0001

2G 1.110 ± 0.013 1.423 ± 0.011 0.0297 Not significant

2H 1.150 ± 0.006 1.446 ± 0.018 <0.0001 <0.0001

2I 1.103 ± 0.007 1.406 ± 0.014 Not significant Not significant

2J 1.166 ± 0.009 1.483 ± 0.008 <0.0001 <0.0001

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3A 1.111 ± 0.009 1.417 ± 0.011 0.0478 Not significant

3B 1.131 ± 0.009 1.411 ± 0.024 0.0097 Not significant

3C 1.105 ± 0.008 1.399 ± 0.010 Not significant Not significant

3D 1.134 ± 0.007 1.449 ± 0.009 < 0.0001 < 0.0001

3E 1.136 ± 0.008 1.454 ± 0.008 <0.0001 <0.0001

3F 1.137 ± 0.010 1.438 ± 0.014 <0.0001 <0.0001

3G 1.113 ± 0.015 1.440 ± 0.022 0.0023 <0.05

3H 1.101 ± 0.003 1.400 ± 0.007 Not significant Not significant

3I 1.131 ± 0.012 1.462 ± 0.011 <0.0001 <0.0001

3J 1.129 ± 0.007 1.415 ± 0.009 0.0067 Not significant

Mean area of polygons (defined by wing landmarks) are shown (+ SE, n = 4). The mean wing polygon area of each isogenized line was compared by t-test to that of Canton-S control. For an experiment-wise p-value of
0.05, the p-value used for significance of individual comparisons, using the Bonferroni correction, is 0.0025. Lines significantly different from Canton-S are shown in bold.

DISCUSSION
In this work we have generated a collection of stocks with isogenized autosomes, which behave similarly to the commonly used Canton-S stock in a range of behavioral phenotypes. Given that Canton-S has been
maintained in laboratory stocks that are relatively small and isolated compared with wild populations, one would expect most of the original genetic variation to have disappeared. However, maintenance of Drosophila in
laboratory stocks still imposes selective pressures (that differ from those in wild populations), and it is still possible that these maintain some of the original variation. Contributing factors could include heterozygote
advantage; new spontaneous mutations may also contribute to variation.
Most of the isogenic second chromosome lines behave similarly to Canton-S in most behavioral phenotypes tested. All are normal in most behavioral phenotypes tested, and only two of the 10 lines performed differently
from Canton-S in more than one test. 2E has a slightly higher attraction to ethyl acetate, a slightly abnormal circadian pattern in DD conditions, and an increased activity in the operant conditioning test, compared with
Canton-S; 2H has a slightly abnormal circadian pattern in DD conditions and faster time to initiate copulation compared with Canton-S.
There may be more variation in the third chromosomes, given the homozygous lethality observed in some lines and the greater divergence from Canton-S in some behaviors, principally those associated with aspects of
performance in the operant conditioning assay. While most lines tested were indistinguishable from Canton-S in most behavioral phenotypes, it is notable that most of the lines that diverged from Canton-S did so in more
than one behavior: 3C, in operant conditioning and courtship; 3D in operant conditioning and in LD plus DD patterns of daily locomotion; 3E in operant conditioning, DD circadian locomotor pattern, and courtship; 3F in
activity during operant conditioning, and LD and DD circadian locomotor pattern; 3J in larval chemosensory responses, adult proboscis extension, and operant conditioning. In these lines the phenotypes may be different
manifestations of the same underlying genotype.
Many lines diverged from Canton-S in wing area, for which most isogenic lines had a value higher than that of Canton-S. The reasons for this are unknown, but could include some change in wing shape in the isogenized
lines, or a sampling error that gave an uncharacteristically low reading for Canton-S.
While Canton-S may or may not be representative of wild populations, it has been widely used for behavioral studies in the laboratory (e.g. Boynton & Tully, 1993; Dura et al., 1993; Broughton et al., 2004). The isogenic
lines derived from it here are therefore potentially useful stocks for mutagenesis experiments that are accompanied by screens for behavioral phenotypes. They have now been used as target chromosomes in at least two
mutageneses. First, The Cambridge FlySeq Project (http://131.111.146.35/ pseq/) has mobilized a gene-trap element (Lukacsovich et al., 2001) onto three isogenized autosome combinations: 2C + 3J, 2C + 3I and
2A + 3A. Second, the Berkeley Drosophila Genome Project has mobilized the same GT element onto six combinations (2A + 3A to 2F + 3F) to generate several hundred insertions referred to as BG lines (Bellen et al.,
2004). While we have not exhaustively tested the phenotypes of such combinations of isogenic chromosomes, and it is in principle possible that they might behave differently from the lines with single isogenic
chromosomes, the three combinations used by the FlySeq project do not differ significantly from Canton-S in their olfactory jump habituation indices (Table III). If particular combinations of isogenized chromosome pairs
do have any phenotype different from that of each chromosome pair alone in a Canton-S background, then it is often easy to solve the problem by exchanging one isogenic chromosome pair for an outbred pair.
Finally, the availability of mutagenic insertions in a uniform genetic background has already made these lines useful in studies of quantitative traits, including bristle number (Norga et al. 2003) and starvation resistance
(Harbison et al. 2004). Therefore, the phenotypic characterization of the lines presented here will enable their use in a wide range of screens for phenotypic defects of anomalies involving viable Drosophila.

ACKNOWLEDGEMENT
AJD and SFG are supported by grants from the Welcome Trust and the Royal Society and gratefully acknowledge the assistance of Dr. Niall Anderson in the statistical analysis. We thank Pierre Far for help with
programming. This work was supported by a grant and fellowship from the BBSRC to C.J.O’K.

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