Chemical Senses Vol. 30 No. suppl 1 © Oxford University
Press 2005; all rights reserved
cDNA Microarray Screening for Taste-bud-specific Genes
1 National Food Research Institute2 Bio-oriented Technology Research Advancement Institution3 Asahi Breweries Ltd4 Kyushu University
Correspondence to be sent to: Yuko Kusakabe, e-mail: ykusa{at}nfri.affrc.go.jp
Key words: annotation, expression profile, genetic information, in situ hybridization, screening
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Introduction |
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 Top Introduction Results and discussionReferences |
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Information about taste signaling molecules is still insufficient to understand the mechanisms of taste signal transductions, while novel taste receptors have been found recently. There are two strategies to discover novel taste signaling related genes; one strategy is homology based cloning and another strategy is global gene screening. Homology based cloning has been mainly used to find out the taste related genes, but we cannot expect the discovery of unexpected genes with critical roles in this strategy. Global gene screening can lead to the discovery of unexpected expressed genes. At present, new techniques for global gene screening are developed, for example, differential display-PCR, SAGE (serial analysis of gene expression), DNA array technology and so on. We tried here gene screening by DNA array technology to find taste-bud-specific genes, then predicted their function using gene expression pattern and genetic information.
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Results and discussion |
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TopIntroductionResults and discussionReferences |
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Fabrication of cDNA microarray
To obtain the cDNA microarray containing taste-bud-specific genes at high rate, the
mRNA from the epithelium of mouse circumvallate papillae was used for the cDNA library.
As described previously (Bonaldo et al.,
1996
), the cDNA library was normalized and subtracted with cDNA library from
the tongue epithelium without taste buds to increase the rate of taste-bud-specific genes
in the library (Bonaldo et al.,
1996). Using the subtracted library, each of the cDNA clones in the
subtracted library was printed on slide glass by the arrayer. Our cDNA microarray
contained 3500 clones which were from the cDNA library and known taste related genes.
Probe preparation
Two kinds of fluorescent probes were synthesized from a single taste bud of
circumvallate papillae and from the tongue epithelium without taste cells to compare the
expression profile between these two tissues. The amount of mRNA from one taste bud is
too small to make first strand cDNA (fs-cDNA) probes by standard methods, so we carried
out global amplification of fs-cDNA from the single taste bud by the PCR-based method
(Brady and Iscove, 1993). We confirmed
that the amplified cDNA contained taste-related genes, then labeled it Cy3 or Cy5. The
probes from the tongue epithelium were prepared by the same method.
Microarray image analyses
To identify novel taste cell-specific genes, we carried out subtraction-coupled cDNA
microarray analyses using the cDNA microarray and the probes from a single taste bud (TB)
and epithelium without taste cells (EP) (Figure
1). The method for standard cDNA
microarray image analyses was not suitable for ours, because we used amplified cDNA probe
which did not reflect the level of expression so accurately. Therefore, it was determined
by our original selection criteria, described below, whether each clone was
taste-bud-specific or not. The signal intensity of each clone for the TB or EP probe was
plotted as relative intensity (RI) to that of the liver-specific gene, apoA1. We
confirmed that apoA1 was expressed neither in taste buds nor tongue epithelium, so the
signal intensity of apoA1 was treated as background noise (RI = 1). The
plot area was divided into three sub-areas on the basis of RI (Figure
2). RItb > 1
and RIep 
1 was defined as Area 1. (RItb,
RI for TB probe; RIep, RI for EP probe).
RItb > 1, RIep > 1 and
RItb > 2 x RIep was defined as Area 2.
The remaining area was defined as ‘rejection area’. Using a pilot array with
1172 cDNA fragments, we examined that known taste-bud-specific genes are located in Area
1 or Area 2. Almost all tested genes in the array (e.g. endoA, G
gust,
T1r3) were plotted in area 1 and 2, indicating that our selection method is useful to
select taste-bud-specific genes.
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Gene expression pattern analysis
We considered the cDNA clones in Areas 1 and 2 as the candidates of taste-bud-specific
genes. Then, in situ hybridization analyses were carried out using the probes of
the clones to examine their regional expression patterns (Figure
3). Thirty-seven genes were identified
to be expressed selectively in taste bud and divided into two groups depending on their
expression pattern in taste buds. One group was comprised of the genes expressed in
almost all taste cells in taste buds, e.g. specific taste bud gene (STG) (Neira et al., 2001). Another was comprised of the
genes expressed in a subset of taste cells in taste buds, e.g. the T1r family (Hoon et al., 1999;
Kitagawa et al., 2001). By
comparing the co-expression pattern of the genes expressed in a subset of taste cells
with known taste-related genes, we can predict which kind of taste the genes related to.
For example, the gene co-expressed with T1r2 and T1r3 might be related to sweet taste. We
carried out double-labeled in situ hybridization to compare the expression
patterns of the genes expressed in a subset of taste cells with known taste signal
transduction-related genes. Two genes were co-expressed with T1r2 and T1r3, suggesting
that they might be related to sweet taste. One gene was co-expressed with Trpm5,
suggesting that it might be related to sweet, bitter and umami (Zhang et al., 2003). And another gene was
co-expressed with Mash1, a candidate for a marker of immature taste receptor cells
(Kusakabe et al., 2002).
Therefore, this result suggested that it might be related to taste cell
differentiation.
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Genetic information analysis
All the cDNA clones in Areas 1 and 2 were sequenced. Homology search against Unigene revealed that the cDNA clones contained known the taste-related gene, STG, Trpm5, Gß3 and a pseudogene for bitter taste receptor T2r. From these results, it was confirmed again that Areas 1 and 2 contained taste related genes. About 90% of the clones were annotated by the homology search and all of the genes expressed in a subset of taste cells were annotated. Taken together, these annotations and the information about the gene expression by double in situ hybridization, enabled the prediction of the functions of the genes in taste cells in more detail. These predictions suggested the possibility that the product of one clone (No. 120) might interact with IP3R3, which is the taste signal transduction-related gene. In vitro studies of interacting No. 120 and IP3R3 and a role in taste signal transduction are under investigation.
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References |
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TopIntroductionResults and discussionReferences |
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Bonaldo, M.F., Lennon, G. and Soares, M.B. (1996) Normalization and subtraction: two approaches to facilitate gene discovery. Genome Res., 6, 791–806.
Brady, G. and Iscove, N.N. (1993) Construction of cDNA libraries from single cells. Methods Enzymol., 225, 611–623.[Web of Science][Medline]
Hoon, M.A., Adler, E., Lindemeier, J., Battey, J.F., Ryba, N.J. and Zuker, C.S. (1999) Putative mammalian taste receptors: a class of taste-specific GPCRs with distinct topographic selectivity. Cell, 96, 541–551.[CrossRef][Web of Science][Medline]
Kitagawa, M., Kusakabe, Y., Miura, H., Ninomiya, Y. and Hino, A. (2001) Molecular genetic identification of a candidate receptor gene for sweet taste. Biochem. Biophys. Res. Commun., 283, 236–242.[CrossRef][Web of Science][Medline]
Kusakabe, Y., Miura, H., Hashimoto, R., Sugiyama, C., Ninomiya, Y. and Hino, A. (2002) The neural differentiation gene Mash-1 has a distinct pattern of expression from the taste reception-related genes gustducin and T1R2 in the taste buds. Chem. Senses, 27, 445–451.
Neira, M., Danilova, V., Hellekant, G. and Azen, E.A. (2001) A new gene (rmSTG) specific for taste buds is found by laser capture microdissection. Mamm. Genome, 12, 60–66.[CrossRef][Medline]
Zhang, Y., Hoon, M.A., Chandrashekar, J., Mueller, K.L., Cook, B., Wu, D., Zuker, C.S. and Ryba, N.J. (2003) Coding of sweet, bitter, and umami tastes: different receptor cells sharing similar signaling pathways. Cell, 112, 293–301.[CrossRef][Web of Science][Medline]
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