The malvolio (mvl) gene of Drosophila melanogaster encodes a protein with a high degree of homology to natural resistance-associated macrophage proteins (Nramps). This family of integral membrane proteins, many of which appear to function as cation transporters, is remarkably conserved in several phylogenetically distinct species. In Drosophila melanogaster, the protein Mvl is expressed in macrophages and in differentiated neurons; loss-of-function mutations lead to defects in gustatory behaviour. The human Nramp-1 protein was expressed in Drosophila melanogaster using the hsp70 promoter. Overexpression in normal animals does not lead to any alterations in their behaviour or physiology. In mutants, however, ubiquitous expression of human Nramp-1 can totally rescue the taste defect. This finding that Nramp-1 can complement the taste defect in mvl mutants provides a potent means of exploiting behavioural genetics to dissect the function of Nramp-1 and to identify other molecules involved with this transport system.

The response of a fly to chemical stimuli involves the correct functioning of sensory as well as motor elements within gustatory pathways. Hence, taste behaviour can serve as a sensitive index of brain function, allowing subtle defects within functioning circuits to be detected. The feeding preference test measures the response of flies to sugars and salts and has been used to isolate mutations in more than a dozen genes affecting taste perception (Tanimura et al., 1982; Arora et al., 1987; Siddiqi et al., 1989; Rodrigues et al., 1991; VijayRaghavan et al., 1992; Inamdar et al., 1993). Most of the genes that have been analysed encode molecules that play rather general roles in the development or function of the nervous system; their apparently specific effect on gustatory behaviour results from hypomorphic alleles that allow adult viability (Murugasu-Oei et al., 1996).

Adults of the malvolio (mvl) of Drosophila melanogaster strain show a reduced acceptance of sucrose, trehalose and fructose and an increased acceptance of low concentrations of sodium chloride (Rodrigues et al., 1995). Electrophysiological recordings from the labellar taste neurons demonstrated that the mutation does not affect the peripheral level of stimulus detection. It was proposed that Mvl plays a role in the integrative synapses between sensory neurons and postsynaptic partners in the suboesophageal ganglion. This was supported by the demonstration that mvl is expressed in differentiated neurons of both the peripheral and central nervous systems. mvl encodes a molecule that belongs to a family of integral membrane proteins defined by natural resistance-associated macrophage proteins (Nramps) (Cellier et al., 1995). Members of this family, which have been identified in yeast, mycobacteria, plants, nematodes, insects and mammals, all possess very similar hydropathy profiles and are typified by the presence of 10 transmembrane domains (Cellier et al., 1995, 1996).

The first mammalian Nramp (Nramp-1) was identified through analysis of inbred strains of mice (BcgR and BcgS) with altered sensitivity to infection by mycobacterial species (Gros et al., 1981; Vidal et al., 1993). Nramp-1 is expressed in the late endocytic compartment in macrophages and has been shown to be recruited to phagosomal membranes (Gruenheid et al., 1997). While Nramp-1 is macrophage-specific, the highly homologous protein Nramp-2 (78 % identity) is more generally expressed (Gruenheid et al., 1995). A large body of evidence favours the hypothesis that Nramp-2 is a transporter for divalent cations (Vulpe and Gitschier, 1997; Fleming et al., 1997, 1998; Supek et al., 1997; Orgad et al., 1998). Nramp-2 can functionally complement the defect of the plasma-membrane-associated transporter (SMF1) in yeast Sacchromyces cerevisiae mutants (Supek et al., 1996; Pinner et al., 1997). Recent genetic evidence has shown that mutations in Nramp-2 were causative of the iron-deficiency syndromes seen in the mk mouse and the Belgrade rat. Both rodent models show defects in Fe2+ uptake in the intestine and into reticulocytes (Vulpe and Gitschier, 1997; Fleming et al., 1997, 1998).

It is not yet clear whether Nramp-1, like Nramp-2, transports metal ions, and the mechanism by which the killing of parasites is mediated is still controversial. However, the linkage between susceptibility to tuberculosis and leprosy in human populations and polymorphisms in the Nramp-1 gene make it crucial to study the mechanism of action of this putative transporter (Bellamy et al., 1998; Abel et al., 1998). The Drosophila Mvl protein shows a very high degree of similarity with the Nramps and, like Nramp-1, is also associated with macrophages (Rodrigues et al., 1995; Cellier et al., 1995). The fly can therefore serve as a very important experimental system in which to study the mechanisms of Nramp-1 action and the systems with which it participates. The effect of hypomorphic mutations in the mvl gene on macrophage function has not been investigated in Drosophila. In addition to macrophages, however, Mvl expression has been demonstrated in differentiated neurons of the central and peripheral nervous systems, and loss-of-function allelic mutations lead to defects in taste behaviour. Interestingly, these aberrant behaviour patterns could be completely suppressed when flies were grown on media supplemented with Fe2+ or Mn2+ for a minimum of 2 h before testing (Orgad et al., 1998). This striking result suggests that Mvl also participates in the transport of certain divalent cations that are essential for the proper functioning of the taste circuits.

This finding allows us to test whether Nramp-1 can also function as a metal transporter. We expressed human Nramp-1 cDNA in mvl97f under the control of the hsp70 promoter. Constitutive expression of Nramp-1 could functionally complement the taste defect in mutant flies. This finding therefore provides an in vivo assay system for human Nramp-1 function, allowing an analysis of the properties of this molecule which has direct relevance to human infectious disease.

All standard molecular biology procedures were performed according to the method described by Sambrook et al. (1989). Protocols for Drosophila melanogaster were as described by Ashburner (1989).

Drosophila stocks

The Canton-S strain of Drosophila melanogaster was used as the wild type, except where specifically mentioned. The mvl97f strain carries a P(w+ lacZ) insertion in the malvolio (mvl) gene and results in a partially dominant taste defect (Rodrigues et al., 1995). The strain Cy/Sp; (Δ2-3 ry+)ry Sb/TM6-Ubx contained a constitutively expressing transposase insertion at 99B (Robertson et al., 1988). The attached-X stock C(1)DX y w f allows the segregation of the X chromosome of males to their sons. Details of strains and markers are available in the review by Lindsley and Zimm (1992).

Generation of hs-Nramp-1 trangenic flies

The full-length cDNA of human Nramp-1 was cloned into pBluescript SK (M13-) (P. Gros, unpublished results). The complete coding sequence was released by digestion with KpnI and EcoRI and subcloned into pCaSpeR-hs in frame with the hsp70 promoter. The recombinant vector P(w+ hs-Nramp-1) was microinjected with Pπwc into yw1118 embryos following standard protocols (Ashburner, 1989). Germline transformants were selected by monitoring the w+ marker in subsequent generations. One transgenic line (hs-Nramp-1) was obtained in which the mutation mapped on the third chromosome and allowed homozygous viability.

Mobilisation of the P(w+ hs-Nramp-1) transposon

To mobilise the insertion onto the X chromosome, y w; hs-Nramp-1/hs-Nramp-1 females were crossed to CyO/Sp; (Δ2-3 ry+) Sb/TM2-Ubx males. Jumpstarter males [y w; P(hs-Nramp-1)/(Δ2-3 ry+) Sb] from among the progeny were crossed to C(1)DX y w f; CyO/+ virgin females. Progeny were screened for males with w+ eye colour; single males were mated with C(1)DX; y w f virgin females and set up as lines. Insertions on the X chromosome were recognised by the segregation of the w+ marker in the progeny of these individual lines; in such cases, only the males would be w+. Two of these lines (designated hs-Nramp-1 line 1 and hs-Nramp-1 line 4) were analysed further.

Feeding preference test

The feeding preference test described by Tanimura et al. (1982) was carried out with some modifications (Balakrishnan and Rodrigues, 1991). Alternate wells of a 6×10 μl plate were filled with 1 % agar containing the stimulus. The remainder of the wells contained 0.2 % Carmoisine Red in agar. Control experiments established that the food dye did not interfere with the test. Flies were starved in humid conditions for 18 h prior to testing. Approximately 100 flies were introduced into each test plate and left for 1 h in the dark. Following the test, flies were immobilised by cooling and scored by visual inspection for colour in their abdomen. The acceptance response was calculated from the percentage of flies in the population with an uncoloured abdomen. Means and standard deviations of each data point were obtained from a minimum of 10 independent tests.

Excision of the P element

To generate revertants, y w; P(hs-NRAMP-1) males were mated with C(1)DX y w f; CyO/+; (Δ2-3 ry+)Sb/+ virgin females. Jumpstarter males of genotype y w P(hs-NRAMP-1); (Δ2-3 ry+)Sb/+ were crossed to C(1)DX y w f virgin females. Progeny males were scored for the eye colour marker. White-eyed males were crossed to C(1)DX y w f; mvl/mvl virgin females and bred as lines. In subsequent generations, y w (Pex); mvl/mvl flies were selected and tested in feeding preference assays. Eleven such lines were analysed in detail.

Southern blot analysis

Genomic DNA was prepared from adult flies, and Southern hybridisation was carried out as described by Sambrook et al. (1989). DNA was transferred to nylon membranes and hybridised with labelled probes. An 800 base pair (bp) internal fragment of Nramp-1 cDNA was used as the probe. Probes were labelled with [32P]dATP by random priming reactions.

In situ hybridization to RNA in whole-mount embryos

Localisation of mRNA to embryos was carried out as described by Ashburner (1989). Nramp cDNA was labelled with digoxygenin-dUTP using random-priming reactions. Embryos of different ages were collected on yeast agar plates, fixed with 4 % paraformaldehyde and washed in phosphate-buffered saline containing 0.1 % Triton X-100 (PBT). They were treated with proteinase K for 3 min and hybridized for 16 h with digoxygenin-labelled probes. The embryos were washed extensively in PBT, and hybridised probe was detected using anti-digoxygenin antibody conjugated to alkaline phosphatase (Boehringer Mannheim). The enzymatic reaction was visualised using Nitroblue Tetrazolium and X-phosphate. Stained embryos were dehydrated through increasing concentrations of ethanol and mounted in DPX.

The high degree of homology between Mvl and Nramp-1 suggested the attractive possibility that Drosophila genetics could be used to analyse the structure and function of the human Nramp-1 protein. As a first step towards this goal, we created transgenic flies carrying the complete human Nramp-1 cDNA cloned downstream from an hsp70 promoter. Adult flies bearing viable hypomorphic alleles of mvl demonstrate a lowered sensitivity to sucrose and trehalose as measured in feeding preference assays (Rodrigues et al., 1995). Using this assay, we tested whether human Nramp-1 could functionally complement for Mvl in the neural circuits underlying taste behaviour.

Transgenic flies in which human Nramp-1 was ubiquitously expressed showed normal development and gustatory behaviour

The complete human Nramp-1 coding sequence was subcloned downstream from and under the control of the hsp70 promoter and microinjected into Drosophila embryos of the y w1118 strain. We obtained a single transformant from this experiment, and the insertion P(w+ hs-Nramp-1) (hereafter called hs-Nramp-1) was located on the third chromosome. Since insertions of ectopic genes often result in positional effects, we carried out a remobilisation experiment to generate strains bearing insertions at different positions on the genome. To facilitate testing for rescue of the behavioural defect in mvl, we biased our selections for insertions on the X chromosome.

We selected X chromosome insertions using the attached-X [C(1)DX y w f] strain (Lindsley and Zimm, 1992). This strain allows the segregation of paternal X chromosomes to male progeny; hence, only males will carry the hs-Nramp-1 transgene. Female progeny will be C(1)DX, y w f and will not carry the transgene. We selected such two independent lines (lines 1 and 4) for further analysis. Southern analysis of genomic DNA from transformant flies probed with an 800 bp internal fragment of human Nramp-1 verified the presence of the hs-Nramp-1 transgene in male but not in female genomes (Fig. 1A).

Transcription of the Nramp-1 gene in the transformants was tested by RNA in situ hybridization. Embryos were collected from hs-Nramp-1 mvl97f and Canton-S parents at 16 °C and 29 °C; the transcription of the transgene was monitored by hybrization with digoxygenin-labelled Nramp-1 probes (Fig. 1B). There was no native expression of Nramp-1, nor any cross-hybridizing gene, in wild-type embryos at any temperature of growth. The hs-Nramp-1 strain showed ubiquitous expression of the transgene when reared at 29 °C but not when reared at 16 °C. These data show that the human Nramp-1 gene could be transcribed in Drosophila under regulation of the hsp70 promoter.

We examined transgenic flies reared at 25 °C and 27 °C for any apparent morphological changes as well as their behavioural responses to 1 mmol l−1 sucrose. The ratio of males to females [hs-Nramp1 males:C(1)DX, y w f females] of the transgenic strain was comparable with that in normal flies [+ males:C(1)DX, ywf females], indicating that ubiquitous expression of hs-Nramp-1 did not result in lethality. The hsp70 promoter is leaky when flies are reared at 25 °C but, to induce higher protein levels, we heat-pulsed embryos, larvae and pupae at 37 °C for 1 h at different times during development and examined the flies under the light microscope. No defects were observed. Flies reared at 25 °C and 27 °C were tested in the feeding preference assay, and these showed responses comparable with those of the wild-type strains (see below).

Human Nramp-1 can substitute for Mvl in the taste pathway of Drosophila

To test for functional complementation of the taste phenotype, we generated hs-Nramp-1; mvl97f/mvl97f males and maintained them with C(1)DX; y w f; mvl97f/mvl97f females. Male progeny in such a strain are homozygous for the mvl97f mutation as well as carrying hs-Nramp-1. The females serve as controls and are genotypically mvl97f/mvl97f.

Flies were reared at different temperatures and tested in the feeding preference assay for their responses to sucrose. When adults are given a choice between agar containing 1 mmol l−1 sucrose and control agar, more than 90 % of normal flies preferentially eat from the sucrose-containing wells. In mvl97f/mvl97f mutants, however, only approximately 30 % of flies preferentially choose sucrose, irrespective of their temperature of rearing. Induction of transgene expression by rearing Nramp-1; mvl97f/mvl97f flies at either 25 °C or 27 °C resulted in a higher acceptance of sucrose in the feeding preference assay (P<0.001; Fig. 2). The hsp70 promoter is not active at 16 °C, and flies reared at this temperature did not show any alteration in behavioural response compared with mvl97f/mvl97f mutants. At 25 °C, a low level of hsp70 activity is observed which is increased at 27 °C (data not shown). Transgenic mvl97f flies showed a slightly greater acceptance of sucrose when reared at 27 °C than at 25 °C (P=0.1). We were unable to test the effects of increasing Nramp-1 levels further since rearing flies at 29 °C or subjecting them to pulses of 37 °C affects the behavioural responses of wild-type controls themselves (V. Rodrigues, unpublished results).

In mvl97f females, hs-Nramp1 showed a dose-dependent rescue of the taste defect (Fig. 3). Mutant flies carrying one copy of hs-Nramp-1 (Nramp-1/+; mvl/mvl) showed a 50 % acceptance response to 1 mmol l−1 sucrose (compared with 40 % in the absence of the transgene), while those with two copies showed a 65 % response (Fig. 3).

An independent hs-Nramp-1 line (line 4) showed results comparable with those of line 1. These results demonstrate that ubiquitous expression of human Nramp-1 can rescue the taste defect of mvl mutants. The observation that two independent insertions can rescue the behavioural defect argues against the possibility that insertion itself led to mutation of genes leading to second-site modification of the mvl phenotype. However, this possibility could be ruled out more directly by generating lines in which the transposon was excised by transposase activity.

Excision of P(w+ hs-Nramp-1) reverted the rescue of the taste defect in mvl mutants

To generate strains in which P(w+ hs-Nramp-1) was excised, we crossed line 4 to a strain carrying transposase activity. The presence of the transposon could be monitored by following the w+ eye marker. Excision of P elements has been shown to occur by a ‘cut-and-paste’ mechanism leaving sequences of DNA of varying length within the gene (Engels, 1989). Hence, if the locus into which the P element was located itself acted as second-site suppressor, some of the excision lines would continue to show this effect. We tested 11 excision lines for their ability to rescue the mvl mutant phenotype when reared at 27 °C, and no such lines were found. A representative line is shown in Fig. 4.

These data together provide compelling evidence that the ubiquitous expression of human Nramp-1 can complement for Mvl in the taste pathway of Drosophila melanogaster.

We have shown that expression of human Nramp-1 under control of the heat-shock promoter can rescue the taste defect in mvl97f mutant flies. Previous molecular analysis has demonstrated that the insertion of P(w+) in mvl97f strain is located 313 bp 5′ to the translation initiation site in the transcribed but untranslated region of the transcription unit (Rodrigues et al., 1995). This observation, together with northern analysis and genetic data, suggests that P element insertion results in a partial loss of mvl function. We favour the hypothesis that the Mvl protein is translated, but is present in smaller amounts than in the wild type. The mvl locus encodes a protein with a high degree of homology to a family of integral membrane proteins, many of which have been demonstrated to transport metal ions. It is therefore interesting that the behavioural defect in mvl mutants can be completely suppressed by supplementing growth media with 10 mmol l−1 MnCl2 or FeCl2 but not with CaCl2 or MgCl2 (Orgad et al., 1998). This implies that the transport function in mvl mutants is less efficient than in the wild type but can be compensated for by increasing the amount of Mn2+ or Fe2+ available in the external milieu.

Mvl is expressed in differentiated neurons, and loss of function leads to a reduction in the sensitivity of the gustatory circuits to stimuli. The experiments of Orgad et al. (1998) strongly suggest that the absence of specific divalent cations in cells is causative of the behavioural defects and that Mvl is involved in the transport of these ions (Supek et al., 1997; Orgad et al., 1998). The role of metal ions in regulating the sensitivity of gustatory circuits is not clear. One possibility is that Mn2+ and Fe2+ directly or indirectly modulate the effects of neurotransmitter receptors found at synapses between sensory neurons and their postsynaptic partners in taste circuits (Shuto et al., 1997). Alternatively, the effect of metal ions on neural function could result from an indirect effect of their role as cofactors for various metalloproteins. In mammalian macrophages, the role of Nramps is linked to the killing of parasites, which is presumably mediated through reactive oxygen or nitrogen species. It is conceivable that increased levels of these radicals induced by metalloproteins are also crucial for proper function of Nramps within neural circuits.

Mvl is also expressed in macrophages, although the effect of loss of function on these cells has not yet been unassayed. It is amazing that species separated by 540 million years of evolutionary history (flies and humans) show not only a conservation of molecular homologies but a similarity in the cell types in which these molecules are found. It is tempting to speculate that human and Drosophila phagocytic cell types have a common evolutionary history. Increasingly it is being shown that mechanisms detected in one system are not far removed from those occurring in another. The observation that human Nramp-1 can fully complement the defect in mvl mutants provides a unique opportunity for exploiting Drosophila genetics to study a transport mechanism in humans. This is of particular medical value since Nramp-1 polymorphisms have been shown to be involved in susceptibility to mycobacterial infection in natural populations (Abel et al., 1998; Bellamy et al., 1998). It therefore becomes relevant to understand the function of the transporter and to identify other molecules with which Nramp-1 collaborates to bring about its physiological effects. The fact that human Nramp-1 can function in the taste pathway of Drosophila provides a means of using the easily assayable taste behaviour of the fly as an in vivo model system for Nramp-1. Site-directed mutagenesis of the Nramp-1 coding region and subsequent transformation into mvl flies is an effective means of dissecting the structure–function relationship of this molecule. The isolation of second-site modifiers is a potent means of identifying other molecules in the transport mechanism in which human Nramp-1 functions.

We are grateful to all members of the Chia laboratory for their discussion and interest in this project. V.R. is grateful to the Institute of Molecular and Cell Biology for a Visiting Fellowship where part of this work was carried out.

Abel
,
L.
,
Sanchez
,
F. O.
,
Oberti
,
T. N. V
,
Hoa
L. V.
,
Lap
,
V. D.
,
Skamene
,
E.
,
Lagrange
,
P. H.
and
Schurr
,
E.
(
1998
).
Susceptibility to leprosy is linked to the human NRAMP1 gene
.
J. Infect. Dis.
177
,
133
145
.
Arora
,
K.
,
Rodrigues
,
V.
,
Shanbhag
,
S.
and
Siddiqi
,
O.
(
1987
).
A gene affecting the specificity of the chemosensory neurons of Drosophila
.
Nature
330
,
62
63
.
Ashburner
,
M.
(
1989
).
Drosophila: A Laboratory Manual. Cold Spring Harbor
,
New York
:
Cold Spring Harbor Laboratory Press
.
Balakrishnan
,
R.
and
Rodrigues
,
V.
(
1991
).
The shaker and shaking-B genes specify elements in the processing of gustatory information in Drosophila melanogaster
.
J. Exp. Biol.
157
,
161
181
.
Bellamy
,
R.
,
Ruwende
,
C.
,
Corrah
,
T.
,
McAdam
,
K. P.
,
Whittle
,
H. C.
and
Hill
,
A. V.
(
1998
).
Variations in the NRAMP1 gene and susceptibility to tuberculosis in West Africans
.
N. Engl. J. Med.
338
,
640
644
.
Cellier
,
M.
,
Belouchi
,
A.
and
Gros
,
P.
(
1996
).
Resistance to intracellular infections: comparative genomic analysis of Nramp
.
Trends Genet
.
12
,
201
204
.
Cellier
,
M.
,
Prive
,
G.
,
Belouchi
,
A.
,
Kwan
,
T.
,
Rodrigues
,
V.
,
Chia
,
W.
and
Gros
,
P.
(
1995
).
Nramp defines a family of membrane proteins
.
Proc. Natl. Acad. Sci. USA
92
,
10089
10093
.
Engels
,
W. R.
(
1989
).
P elements in Drosophila melanogaster
. In
Mobile DNA
(ed.
D. E.
Berg
and
M.
Howe
), pp.
437
484
.
Washington, DC
:
American Society for Microbiology
.
Fleming
,
M. D.
,
Romano
,
M. A.
,
Su
,
M. A.
,
Garrick
,
L. M.
,
Garrick
,
M. D.
and
Andrews
,
N. C.
(
1998
).
Nramp2 is mutated in the anemic Belgrade (b) rat: evidence of a role for Nramp2 in endosomal iron transport
.
Proc. Natl. Acad. Sci. USA
95
,
1148
1153
.
Fleming
,
M. D.
,
Trenor
,
C. C.
,
Su
,
M. A.
,
Foernzler
,
D.
,
Beier
,
D. R.
,
Dietrich
,
W.
and
Andrews
,
N. C.
(
1997
).
Microcytic anaemia mice have a mutation in Nramp2, a candidate iron transporter gene
.
Nature Genetics
16
,
383
386
.
Gros
,
P.
,
Skamene
,
E.
and
Forget
,
A.
(
1981
).
Genetic control of natural resistance to Mycobacterium bovis (BCG) in mice
.
J. Immunol.
127
,
2417
2421
.
Gruenheid
,
S.
,
Cellier
,
M.
,
Vidal
,
S.
and
Gros
,
P.
(
1995
).
Identification and characterisation of a second mouse Nramp gene
.
Genomics
25
,
514
525
.
Gruenheid
,
S.
,
Pinner
,
E.
,
Desjardins
,
M.
and
Gros
,
P.
(
1997
).
Natural resistance to infection with intracellular pathogens: the Nramp1 protein is recruited to the membrane of the phagosome
.
J. Exp. Med.
185
,
717
730
.
Inamdar
,
M.
,
VijayRaghavan
,
K.
and
Rodrigues
,
V.
(
1993
).
The Drosophila homolog of the human transcription factor TEF-1, scalloped, is essential for normal taste behavior
.
J. Neurogenet.
9
,
123
139
.
Lindsley
,
D. L.
and
Zimm
,
G. G.
(
1992
).
The Genome of Drosophila melanogaster. San Diego, CA: Academic Press
.
Murugasu-Oei
,
B.
,
Balakrishnan
,
R.
,
Yang
,
X.
,
Chia
,
W.
and
Rodrigues
,
V.
(
1996
).
Mutations in masquerade, a novel serineprotease-like molecule, affect axonal guidance and taste behavior in Drosophila
.
Mech. Dev.
57
,
91
101
.
Orgad
,
S.
,
Nelson
,
H.
,
Segal
,
D.
and
Nelson
,
N.
(
1998
).
Metal ions suppress the abnormal taste behavior of the Drosophila mutant malvolio
.
J. Exp. Biol.
201
,
115
120
.
Pinner
,
E.
,
Gruenheid
,
S.
,
Raymond
,
M.
and
Gros
,
P.
(
1997
).
Functional complementation of the yeast divalent cation transporter family SMF by NRAMP2, a member of the mammalian natural resistance-associated macrophage protein family
.
J. Biol. Chem.
272
,
28933
28938
.
Robertson
,
H. M.
,
Preston
,
C. R.
,
Phillis
,
R. W.
,
Johnson-Schlitz
,
D. M.
,
Benz
,
W. K.
and
Engels
,
W. R.
(
1988
).
A stable genomic source of P element transposase in Drosophila melanogaster
.
Genetics
118
,
461
470
.
Rodrigues
,
V.
,
Cheah
,
P. Y. K.
and
Chia
,
W.
(
1995
).
malvolio, the Drosophila homologue of mouse NRAMP-1(Bcg), is expressed in macrophages and in the nervous system and is required for normal taste behavior
.
EMBO J.
14
,
3007
3020
.
Rodrigues
,
V.
,
Sathe
,
S.
,
Pinto
,
L.
,
Balakrishnan
,
R.
and
Siddiqi
,
O.
(
1991
).
Closely linked leisons in a region of the chromosome affect central and peripheral steps in gustatory processing in Drosophila
.
Mol. Gen. Genet
.
226
,
265
276
.
Sambrook
,
J.
,
Frisch
,
E. F.
and
Maniatis
,
T.
(
1989
).
Molecular Cloning: A Laboratory Manual.
New York
:
Cold Spring Harbor Press
.
Shuto
,
M.
,
Ogita
,
K.
,
Minami
,
T.
,
Maeda
,
H.
and
Yoneda
,
Y.
(
1997
).
Inhibition of [3H]MK-801 binding by ferrous (II) but not ferric (III) ions in a manner different from that by sodium nitroprusside (II) in rat brain synaptic membranes
.
J. Neurochem.
69
,
744
752
.
Siddiqi
,
O.
,
Joshi
,
S.
,
Arora
,
K.
and
Rodrigues
,
V.
(
1989
).
Genetic investigation of salt reception in Drosophila
.
Genome
31
,
646
651
.
Supek
,
F.
,
Supekova
,
L.
,
Nelson
,
H.
and
Nelson
,
N.
(
1996
).
A yeast manganese transporter related to the macrophage protein involved in conferring resistance to Mycobacterium
.
Proc. Natl. Acad. Sci. USA
93
,
5105
5110
.
Supek
,
F.
,
Supekova
,
L.
,
Nelson
,
H.
and
Nelson
,
N.
(
1997
).
Function of metal-ion homeostasis in the cell division cycle, mitochondrial protein processing, sensitivity to mycobacterial infection and brain function
.
J. Exp. Biol.
200
,
321
330
.
Tanimura
,
T.
,
Isono
,
K.
,
Takamura
,
T.
and
Shimada
,
I.
(
1982
).
Genetic dimorphism in taste sensitivity to trehalose in Drosophila melanogaster
.
J. Comp. Physiol. A
147
,
265
269
.
Vidal
,
S. M.
,
Malo
,
D.
,
Vogan
,
K.
,
Skamene
,
E.
and
Gros
,
P.
(
1993
).
Natural resistance to infection with intracellular parasites: Isolation of a candidate for Bcg
.
Cell
73
,
469
485
.
VijayRaghavan
,
K.
,
Kaur
,
J.
and
Rodrigues
,
V.
(
1992
).
The east gene of Drosophila is expressed in the developing nervous system and is required for normal olfactory and gustatory responses of the adult
.
Dev. Biol
.
154
,
23
36
.
Vulpe
,
C.
and
Gitschier
,
J.
(
1997
).
Ironing out anaemia
.
Nature Genetics
16
,
319
320
.