Kit for detecting galactose, cell, and transformant

Abstract

To provide a technology capable of detecting galactose in vivo.SOLUTION: A galactose detection kit comprising a nucleic acid encoding a fusion protein composed of a Gal80(G310D) mutant and a reporter protein, and a nucleic acid encoding a Gal3 protein having a nuclear localization signal added.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a galactose detection kit, a cell, and a transformant. [Background technology]

[0002] Glucose metabolism plays an important role in maintaining life. Its dynamics within living organisms are poorly understood compared to its intracellular metabolism. Galactose, a stereoisomer of glucose, is a monosaccharide that is transported via the same transporter as glucose. Galactose is supplied to glycolysis and regulates protein glycosylation. Galactose is known to be important in prokaryotic ecology, mammalian development, and Drosophila physiology (see, for example, Non-Patent Documents 1 to 3). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Conte F., et al., Galactose in human metabolism, glycosylation and congenital metabolic diseases: Time for a closer look, BBA - General Subjects 1865, 129898, 2021. [Non-patent document 2] Daenzer JMI and Fridovich-Keil JL, Drosophila melanogaster models of galactosemia, Curr Top Dev Biol., 121, 377-395, 2017. [Non-patent document 3] Holden HM, et al., Structure and Function of Enzymes of the Leloir Pathway for Galactose Metabolism, J. Biol. Chem., 278, 45, 43885-43888, 2003. Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide a technique for detecting galactose in vivo. [Means for solving the problem]

[0005] The present invention includes the following aspects. [1] A kit for detecting galactose, comprising a nucleic acid encoding a fusion protein of a Gal80(G310D) mutant and a reporter protein, and a nucleic acid encoding a Gal3 protein to which a nuclear localization signal has been added. [2] A galactose detection kit described in [1], in which the fusion protein of the Gal80(G310D) mutant and a reporter protein and the Gal3 protein to which the nuclear localization signal has been added are configured to be expressed from a single mRNA. [3] A cell having a nucleic acid encoding a fusion protein of the Gal80(G310D) mutant and a reporter protein, and a nucleic acid encoding a Gal3 protein to which a nuclear localization signal has been added. [4] A transformant having a nucleic acid encoding a fusion protein of the Gal80(G310D) mutant and a reporter protein, and a nucleic acid encoding a Gal3 protein to which a nuclear localization signal has been added. [5] The transformant according to [4], which is a Drosophila. [6] A kit for detecting galactose, comprising a nucleic acid encoding a Gal4 protein, a nucleic acid encoding a Gal80 protein, a nucleic acid encoding a Gal3 protein, and a nucleic acid in which a nucleic acid encoding a reporter protein is positioned downstream of an Upstream Activation Sequence (UAS). [7] The galactose detection kit according to [6], wherein the Gal4 protein, the Gal80 protein, and the Gal3 protein are expressed from a single mRNA. [8] A cell comprising a nucleic acid encoding a Gal4 protein, a nucleic acid encoding a Gal80 protein, a nucleic acid encoding a Gal3 protein, and a nucleic acid in which a nucleic acid encoding a reporter protein is arranged downstream of UAS. [9] A transformant having a nucleic acid encoding a Gal4 protein, a nucleic acid encoding a Gal80 protein, a nucleic acid encoding a Gal3 protein, and a nucleic acid in which a nucleic acid encoding a reporter protein is arranged downstream of UAS.

[10] The transformant according to [9], which is a Drosophila. [Effects of the Invention]

[0006] According to the present invention, a technique for detecting galactose in vivo can be provided. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 is a schematic diagram illustrating a galactose sensor according to one embodiment. [Figure 2] FIG. 2 is an image showing the results of observing the fluorescence of green fluorescent protein (GFP) in Experimental Example 2. [Figure 3] FIG. 3 is a fluorescence microscope image of the midgut of a fly in Experimental Example 2. [Figure 4] FIG. 4 shows images showing the results of observing GFP fluorescence from second instar fly larvae (L2) and third instar fly larvae (L3) in Experimental Example 3. [Figure 5]FIG. 5 shows fluorescent microscope images of detailed observations of each tissue of a fly larva in Experimental Example 3. [Figure 6] FIG. 6 is a schematic diagram illustrating a galactose sensor according to one embodiment. [Figure 7] The left side of Figure 7 shows fluorescence microscope images showing the results of detailed observation of each fly tissue in Experimental Example 4. The right side of Figure 7 is a graph showing the results of quantifying the fluorescence intensity (relative value) of GFP in the nucleus based on the image on the left side of Figure 7. [Figure 8] The left side of Figure 8 is a fluorescence microscope image of cells in Experimental Example 5. The right side of Figure 8 is a graph showing the results of calculating the ratio of the fluorescence intensity of GFP in the nucleus to the fluorescence intensity of GFP in the cytoplasm based on the image on the left side of Figure 8. DETAILED DESCRIPTION OF THE INVENTION

[0008] [Galactose detection kit] (First embodiment) In one embodiment, the present invention provides a kit for detecting galactose, comprising a nucleic acid encoding a Gal4 protein, a nucleic acid encoding a Gal80 protein, a nucleic acid encoding a Gal3 protein, and a nucleic acid in which a nucleic acid encoding a reporter protein is positioned downstream of an upstream activation sequence (UAS).

[0009] As described later in the Examples, cells transfected with a nucleic acid encoding a Gal4 protein, a nucleic acid encoding a Gal80 protein, a nucleic acid encoding a Gal3 protein, and a nucleic acid encoding a reporter protein downstream of a UAS express the reporter protein in the presence of galactose. Thus, these cells can be referred to as a galactose sensor or galactose detection system, and can be used to detect galactose.

[0010] Therefore, the kit of this embodiment can be called a kit for preparing a galactose sensor, a kit for preparing a galactose detection system, etc. The kit of this embodiment corresponds to GALDAR1 (G1) and GALDAR2 (G2) described below.

[0011] In the kit of this embodiment, the nucleic acid may be DNA or RNA.

[0012] FIG. 1 is a schematic diagram illustrating a galactose sensor. The galactose sensor shown in FIG. 1 is a modified version of a yeast galactose sensor. As described later in the Examples, the inventors have demonstrated that the yeast galactose sensor functions when introduced into species other than yeast. Furthermore, because galactose and glucose enter cells via the same transporter, the galactose sensor shown in FIG. 1 can simultaneously confirm the presence of a transporter that controls the influx of glucose into cells.

[0013] As shown in Figure 1, in the absence of galactose (left), Gal80 binds to Gal4 and inhibits its transcriptional activation ability, resulting in the non-expression of genes transcribed downstream of UAS. In actual yeast cells, the genes transcribed downstream of UAS are genes involved in galactose metabolism, but in Figure 1, a GFP gene is placed as a reporter. In the presence of galactose (right), galactose enters the cell through the transporter and binds to Gal3, relieving the inhibition of Gal4 by Gal80 and inducing the expression of genes downstream of UAS.

[0014] In the kit of this embodiment, the Gal4 protein, Gal80 protein, Gal3 protein, and UAS that can be used include, for example, those that constitute the yeast galactose sensor.

[0015] The amino acid sequence of yeast Gal4 protein is shown in SEQ ID NO: 1. The amino acid sequence of yeast Gal80 protein is shown in SEQ ID NO: 2. The amino acid sequence of yeast Gal3 protein is shown in SEQ ID NO: 3. The nucleotide sequence of yeast UAS is shown in SEQ ID NO: 4.

[0016] In the kit of this embodiment, the Gal4 protein, Gal80 protein, Gal3 protein, and UAS may have a mutation, as long as they function as a galactose sensor.

[0017] For example, the mutant Gal4 protein has one or more amino acid mutations relative to the amino acid sequence of SEQ ID NO: 1, and the amino acid mutations may be substitutions, additions, insertions, or deletions of amino acid residues. The number of amino acid mutations may be 1 to 100, 1 to 80, 1 to 50, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1.

[0018] Alternatively, the mutant Gal4 protein may be a protein consisting of an amino acid sequence having 80% or more but less than 100% sequence identity to the amino acid sequence of SEQ ID NO: 1. The sequence identity of the mutant Gal4 protein to the amino acid sequence of SEQ ID NO: 1 may be 85% or more, 90% or more, 95% or more, 97% or more, or 99% or more.

[0019] The sequence identity of amino acid sequences can be calculated by methods well known to those skilled in the art, for example, using Blastp (protein-protein BLAST), a search program of BLAST (Basic Local Alignment Search Tool).

[0020] Similarly, the mutant Gal80 protein has one or more amino acid mutations with respect to the amino acid sequence of SEQ ID NO: 2, and the amino acid mutations may be substitutions, additions, insertions, or deletions of amino acid residues. The number of amino acid mutations may be 1 to 50, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1.

[0021] Alternatively, the mutant Gal80 protein may be a protein consisting of an amino acid sequence having 80% or more but less than 100% sequence identity to the amino acid sequence of SEQ ID NO: 2. The sequence identity of the mutant Gal80 protein to the amino acid sequence of SEQ ID NO: 2 may be 85% or more, 90% or more, 95% or more, 97% or more, or 99% or more.

[0022] Similarly, the mutant Gal3 protein has one or more amino acid mutations with respect to the amino acid sequence of SEQ ID NO: 3. The amino acid mutations may be substitutions, additions, insertions, or deletions of amino acid residues. The number of amino acid mutations may be 1 to 60, 1 to 50, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1.

[0023] Alternatively, the mutant Gal3 protein may be a protein consisting of an amino acid sequence having 80% or more but less than 100% sequence identity to the amino acid sequence of SEQ ID NO: 3. The sequence identity of the mutant Gal80 protein to the amino acid sequence of SEQ ID NO: 3 may be 85% or more, 90% or more, 95% or more, 97% or more, or 99% or more.

[0024] Similarly, the nucleotide sequence of the mutant UAS may be a nucleotide sequence that has 80% or more but less than 100% sequence identity to the nucleotide sequence of SEQ ID NO: 4. The sequence identity of the mutant UAS to the nucleotide sequence of SEQ ID NO: 4 may be 85% or more, 90% or more, 95% or more, 97% or more, or 99% or more.

[0025] The sequence identity of nucleotide sequences can be calculated by methods well known to those skilled in the art, for example, using Blastn (nucleotide BLAST), a search program of BLAST (Basic Local Alignment Search Tool).

[0026] In the kit of this embodiment, the reporter protein is not particularly limited, and a fluorescent protein, an enzyme, or the like can be used.

[0027] The fluorescent protein is not particularly limited, and examples thereof include green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), mCherry, and the like.

[0028] The enzyme is not particularly limited, and examples thereof include alkaline phosphatase and peroxidase.

[0029] In the kit of this embodiment, the nucleic acid encoding the Gal4 protein, the nucleic acid encoding the Gal80 protein, the nucleic acid encoding the Gal3 protein, and the nucleic acid in which the nucleic acid encoding the reporter protein is located downstream of the UAS may be incorporated into a vector. The vector can be appropriately selected depending on the target to which the kit of this embodiment is to be introduced, and examples of the vector include a plasmid, a phage, a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, and a Sendai viral vector.

[0030] The kit of this embodiment may be configured so that the Gal4 protein, Gal80 protein, and Gal3 protein are expressed from a single mRNA, thereby allowing the Gal4 protein, Gal80 protein, and Gal3 protein to be expressed in equal amounts.

[0031] When a single mRNA expresses these multiple proteins, the proteins may be linked by a 2A sequence or an IRES (Internal Ribosome Entry Site) sequence having a ribosome binding site. Examples of 2A sequences include the P2A sequence (ATNFSLLKQAGDVEENPGP, SEQ ID NO: 5) derived from Porcine teschovirus, the T2A sequence (EGRGSLLTCGDVEENPGP, SEQ ID NO: 6) derived from Thosea asigne, the F2A sequence (VKQTLNFDLLKLAGDVESNPGP, SEQ ID NO: 7) derived from Foot-and-Mouth Disease Virus, and the E2A sequence (QCTNYALLKLAGDVESNPGP, SEQ ID NO: 8) derived from Equine Rhinitis A Virus. The 2A sequence is also referred to as a self-cleaving peptide sequence. The IRES sequence may be derived from a virus such as Encephalomyocarditis virus or Foot-and-Mouth Disease Virus.

[0032] [cell] (First embodiment) In one embodiment, the present invention provides a cell comprising a nucleic acid encoding a Gal4 protein, a nucleic acid encoding a Gal80 protein, a nucleic acid encoding a Gal3 protein, and a nucleic acid encoding a reporter protein arranged downstream of the UAS.

[0033] The cells of this embodiment may be cells into which the above-mentioned galactose detection kit (GALDAR1 or GALDAR2) has been introduced, or may be cells collected from the transformant of the first embodiment described below.

[0034] The nucleic acid encoding the Gal4 protein, the nucleic acid encoding the Gal80 protein, the nucleic acid encoding the Gal3 protein, and the nucleic acid in which the nucleic acid encoding the reporter protein is located downstream of the UAS can be introduced into cells by a conventional method, such as lipofection or electroporation.

[0035] The cells of this embodiment express a reporter protein in the presence of galactose, and therefore can be used to detect galactose, and can be referred to as a galactose sensor, a galactose detection system, or the like.

[0036] In the cells of this embodiment, the Gal4 protein, Gal80 protein, Gal3 protein, UAS, reporter protein, nucleic acid, etc. are the same as those described above.

[0037] Examples of cells include plant cells, insect cells, and animal cells. Examples of plants include Arabidopsis thaliana. Examples of insects include fruit flies and mosquitoes. Examples of animals include mammals and fish. Mammals are not particularly limited, and include mice, rats, pigs, cows, monkeys, and humans.

[0038] [Transformants] (First embodiment) In one embodiment, the present invention provides a transformant having a nucleic acid encoding a Gal4 protein, a nucleic acid encoding a Gal80 protein, a nucleic acid encoding a Gal3 protein, and a nucleic acid encoding a reporter protein arranged downstream of the UAS.

[0039] The transformant of this embodiment may be a transformant into which the above-mentioned galactose detection kit (GALDAR1 or GALDAR2) has been introduced.

[0040] The transformant may be, for example, a microorganism, a plant, an insect, or an animal. Examples of the microorganism include yeast and Escherichia coli. The same applies to plants, insects, and animals as described above.

[0041] Transformants can be obtained by conventional methods, for example, by introducing a nucleic acid encoding a Gal4 protein, a nucleic acid encoding a Gal80 protein, a nucleic acid encoding a Gal3 protein, and a nucleic acid in which a nucleic acid encoding a reporter protein is located downstream of a UAS into stem cells, followed by growing the cells into living organisms.

[0042] Alternatively, the transformant of this embodiment may be obtained by mating transformants having any of a nucleic acid encoding a Gal4 protein, a nucleic acid encoding a Gal80 protein, a nucleic acid encoding a Gal3 protein, or a nucleic acid in which a nucleic acid encoding a reporter protein is positioned downstream of a UAS.

[0043] [Galactose detection kit] (Second embodiment) In one embodiment, the present invention provides a kit for detecting galactose, comprising a nucleic acid encoding a fusion protein of the Gal80(G310D) mutant and a reporter protein, and a nucleic acid encoding a Gal3 protein to which a nuclear localization signal has been added.

[0044] As described later in the Examples, in cells transfected with a nucleic acid encoding a fusion protein of the Gal80(G310D) mutant and a reporter protein and a nucleic acid encoding a Gal3 protein with a nuclear localization signal added, the reporter protein localizes to the nucleus in the presence of galactose. Therefore, these cells can be referred to as a galactose sensor or galactose detection system, and can be used to detect galactose.

[0045] Therefore, the kit of this embodiment can be called a kit for preparing a galactose sensor, a kit for preparing a galactose detection system, etc. The kit of this embodiment corresponds to GALDAR3 (G3) described below.

[0046] Figure 6 is a schematic diagram illustrating the galactose sensor. The galactose sensor shown in Figure 6 is a modified version of the galactose sensor shown in Figure 1. Specifically, Gal4 is removed from the galactose sensor shown in Figure 1, and a Gal80 mutant (G310D) is used. The Gal80 mutant (G310D) has the glycine (G) at position 310 in the amino acid sequence of SEQ ID NO: 2 mutated to aspartic acid (D). This mutant interacts normally with Gal3 but does not bind to Gal4. These changes render G3 independent of the Gal4 / UAS system. In Figure 6, a GFP protein is fused to the mutant Gal80 as a reporter. A nuclear localization signal is also added to Gal3 to localize it to the nucleus. Hereinafter, Gal3 with a nuclear localization signal added will sometimes be referred to as nlsGal3.

[0047] As shown in Figure 6, in the absence of galactose (left), Gal80 * Gal80-GFP remains diffuse within the cell and constantly moves in and out of the nucleus, producing a diffuse fluorescent signal. * -GFP binds to nlsGal3 in the nucleus and is trapped there, resulting in an observable and quantifiable signal.

[0048] In G1 and G2, the time required for transcription, translation, and maturation of the fluorescent protein is relatively long, so the temporal sensitivity for detecting galactose is relatively low, but this point is improved in G3.

[0049] In the kit of this embodiment, the reporter protein, nucleic acid, etc. are the same as those described above.

[0050] In the kit of this embodiment, the Gal80(G310D) mutant protein and the Gal3 protein may have a mutation as long as they function as a galactose sensor. The Gal80(G310D) mutant protein may be a mutant Gal80 protein having a mutation corresponding to the G310D mutation. The mutant Gal80 protein is the same as that described above.

[0051] Here, "the amino acid residue corresponding to the 310th amino acid residue in the amino acid sequence set forth in SEQ ID NO: 2" refers to the amino acid residue determined by homology analysis or other techniques to correspond to the 310th amino acid residue counting from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 2.

[0052] Examples of homology analysis methods include pairwise sequence alignment methods such as the Needleman-Wunsch method and the Smith-Waterman method, and multiple sequence alignment methods such as the ClustalW method. Using the amino acid sequence of SEQ ID NO: 2 as a reference sequence, a person skilled in the art can identify the "amino acid residue corresponding to the 310th amino acid residue in the amino acid sequence of SEQ ID NO: 2" in the amino acid sequence of a mutant Gal80 protein based on these methods.

[0053] A nuclear localization signal is added to the Gal3 protein. The nuclear localization signal is not particularly limited as long as it can localize the Gal3 protein to the nucleus and can bind to the Gal80(G310D) mutant protein in the presence of galactose, thereby allowing the Gal3 protein to localize to the nucleus.

[0054] Examples of nuclear localization signals that can be used include YKSRHRRHRQRSRSRNRSRSRSSERKRRQRSRSRSSERRRYLYSGRPRL (SEQ ID NO: 9), PKKKRKV (SEQ ID NO: 10), and KRPAATKKAGQAKKKK (SEQ ID NO: 11). The nuclear localization signal may be located on the N-terminus or C-terminus of the Gal3 protein.

[0055] The Gal3 protein may be a mutant Gal3 protein having a nuclear localization signal, as described above.

[0056] In the kit of this embodiment, the nucleic acid encoding the fusion protein of the Gal80(G310D) mutant and the reporter protein and the nucleic acid encoding the Gal3 protein to which a nuclear localization signal has been added may be incorporated into a vector, which is the same as that described above.

[0057] In the kit of this embodiment, the fusion protein of the Gal80(G310D) mutant and the reporter protein and the Gal3 protein to which a nuclear localization signal has been added may be configured to be expressed from a single mRNA, thereby allowing the fusion protein of the Gal80(G310D) mutant and the reporter protein and the Gal3 protein to which a nuclear localization signal has been added to be expressed in equal amounts.

[0058] When a single mRNA expresses these multiple proteins, the proteins may be linked by a 2A sequence, an IRES (Internal Ribosome Entry Site) sequence having a ribosome binding site, etc. The 2A sequence, IRES sequence, etc. are the same as those described above.

[0059] [cell] (Second embodiment) In one embodiment, the present invention provides a cell comprising a nucleic acid encoding a fusion protein of a Gal80(G310D) mutant and a reporter protein, and a nucleic acid encoding a Gal3 protein to which a nuclear localization signal has been added.

[0060] The cells of this embodiment may be cells into which the above-mentioned galactose detection kit (GALDAR3) has been introduced, or may be cells collected from the transformant of the second embodiment described below.

[0061] The nucleic acid encoding the fusion protein of the Gal80(G310D) mutant and a reporter protein, and the nucleic acid encoding the Gal3 protein with a nuclear localization signal added thereto can be introduced into cells by conventional methods, such as lipofection and electroporation.

[0062] In the cells of this embodiment, the reporter protein is localized in the nucleus in the presence of galactose, and therefore the cells of this embodiment can be used to detect galactose, and can be referred to as a galactose sensor, a galactose detection system, or the like.

[0063] In the cells of this embodiment, the Gal80(G310D) mutant protein, reporter protein, nuclear localization signal, Gal3 protein, nucleic acid, cells, etc. are the same as those described above.

[0064] [Transformants] (Second embodiment) In one embodiment, the present invention provides a transformant having a nucleic acid encoding a fusion protein of the Gal80(G310D) mutant and a reporter protein, and a nucleic acid encoding a Gal3 protein to which a nuclear localization signal has been added.

[0065] The transformant of this embodiment may be a transformant into which the above-mentioned galactose detection kit (GALDAR3) has been introduced.

[0066] The transformant may be, for example, a microorganism, a plant, an insect, or an animal, as described above for the microorganism, plant, insect, and animal.

[0067] Transformants can be obtained by conventional methods, for example, by introducing a nucleic acid encoding a fusion protein of the Gal80(G310D) mutant and a reporter protein and a nucleic acid encoding a Gal3 protein with a nuclear localization signal added into stem cells, followed by growing them into living organisms.

[0068] Alternatively, the transformant of this embodiment may be obtained by mating transformants having either a nucleic acid encoding a fusion protein of the Gal80(G310D) mutant and a reporter protein, or a nucleic acid encoding a Gal3 protein to which a nuclear localization signal has been added. [Example]

[0069] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0070] [Experimental Example 1] (Preparation of galactose sensor) We heterologously integrated a yeast galactose sensor into Drosophila, carrying the Gal3, Gal4, and Gal80 genes, as well as a fluorescent protein (GFP) driven by a UAS sequence. We named this galactose sensor GALDAR (galactose radar).

[0071] As shown in Figure 1, the first version of GALDAR, GALDAR1 (G1), ubiquitously expressed Gal3 and Gal80 proteins, separated by a self-cleaving P2A sequence, utilizes a tubulin enhancer. G1 can be used as a tissue-specific sensor with an appropriate Gal4 driver.

[0072] During the development of G1, we considered the possibility that an imbalance in the amount of Gal4 and Gal80 proteins in the cells might affect the results, so to address this issue, we created a second version of the sensor, GALDAR2 (G2).

[0073] As shown in Figure 1, in G2, Gal4, Gal3, and Gal80 proteins are expressed from a single transcript separated by a self-cleaving P2A sequence, ensuring equal expression of these proteins.

[0074] The principle of galactose detection by G1 and G2 is the same; in the absence of galactose, no signal is observed from a reporter protein such as GFP, but in the presence of galactose, a signal from the reporter protein is observed.

[0075] [Experimental Example 2] (Detection of galactose in adult flies) Five-day-old Drosophila melanogaster transfected with GALDAR were fed a galactose-supplemented diet (1.5% agar, 10% yeast, 5% sucrose, 5% galactose, 0.3% butyl parahydroxybenzoate, 0.3% propionic acid) or a control diet (1.5% agar, 10% yeast, 5% sucrose, 0.3% butyl parahydroxybenzoate, 0.3% propionic acid) for three days.

[0076] Next, the flies were observed for GFP fluorescence (GALDAR signal). Figure 2 shows images of the GFP fluorescence. The outlines of the flies are indicated by lines. In Figure 2, "galactose" indicates flies fed with galactose-supplemented food, and "sucrose" indicates flies fed with the control food.

[0077] As a result, flies fed the galactose-supplemented diet showed GFP fluorescence, but flies fed the control diet showed no visible GFP fluorescence, demonstrating that GALDAR can detect galactose in vivo in Drosophila.

[0078] Next, we analyzed the fly's midgut, which is the first tissue responsible for the absorption and metabolism of ingested food. Figure 3 shows a fluorescence microscopy image of the midgut. As a result, GFP fluorescence was detected in the anterior midgut (R2 region) of flies fed with galactose-supplemented food. This result further supports the idea that GALDAR can detect galactose in vivo in Drosophila.

[0079] [Experimental Example 3] (Detection of galactose in fly larvae) Galactose metabolism in fly larvae was investigated. It is known that sugar metabolism changes significantly during larval development.

[0080] Larvae into which GALDAR had been introduced were cultured on diets containing 0% and 5% galactose. The larvae were then observed for GFP fluorescence. Figure 4 shows images of GFP fluorescence observed from second-instar larvae (L2) and third-instar larvae (L3). In Figure 4, "galactose" indicates larvae fed diets containing galactose, and "sucrose" indicates larvae fed the control diet.

[0081] As a result, larvae fed the galactose-supplemented diet showed a visible decrease in GFP fluorescence (GALDAR signal) as they progressed from L2 to L3.

[0082] Figure 5 shows detailed fluorescence microscopy images of each larval tissue. In Figure 5, "midgut" refers to the midgut, "fat body" refers to the fat body, and "epidermis" refers to the epidermis. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI).

[0083] The results showed that the midgut and epidermis maintained similar GALDAR signal levels at L2 and L3 stages, whereas the fat body showed a decrease in GALDAR signal during the L2-to-L3 transition, suggesting that the decrease in GALDAR signal in the fat body contributes to the overall decrease in GALDAR signal at L3.

[0084] [Experimental Example 4] (Production of GALDAR3) We created a new version of GALDAR, named GALDAR3 (G3). Figure 6 shows a schematic diagram of G3. In G3, Gal4 was deleted, and a mutant Gal80 (G310D) was used. This mutant interacts normally with Gal3 but does not bind to Gal4. These changes render G3 independent of the Gal4 / UAS system. Furthermore, GFP was fused to the mutant Gal80. A nuclear localization signal was added to Gal3 to localize it to the nucleus.

[0085] A fusion protein of the G310D mutant of Gal80 and GFP (Gal80 * Gal3 carrying a nuclear localization signal (nlsGal3) and a tubulin enhancer is expressed from a single transcript separated by a self-cleaving P2A sequence.

[0086] In the absence of galactose in cells, Gal80 * Gal80-GFP remains diffuse within the cell and continually moves in and out of the nucleus, producing a diffuse fluorescent signal. * -GFP binds to nlsGal3 in the nucleus and is trapped there, resulting in an observable and quantifiable GALDAR signal.

[0087] In G1 and G2, the time required for transcription, translation, and maturation of the fluorescent protein is relatively long, so the temporal sensitivity for detecting galactose is relatively low, but this point is improved in G3.

[0088] Five-day-old Drosophila melanogaster transfected with G3 were fed a galactose-supplemented diet (1.5% agar, 10% yeast, 5% sucrose, 5% galactose, 0.3% butyl parahydroxybenzoate, and 0.3% propionic acid) or a control diet (1.5% agar, 10% yeast, 5% sucrose, 0.3% butyl parahydroxybenzoate, and 0.3% propionic acid) for two days.

[0089] The left side of Figure 7 shows fluorescent microscope images showing the results of detailed observation of each fly tissue. Nuclei were stained with DAPI. The right side of Figure 7 shows a graph showing the results of quantifying the GFP fluorescence intensity (relative value) in the nucleus based on the image on the left side of Figure 7. In Figure 7, "midgut" refers to the midgut, and "fat body" refers to the fat body. "Galactose" refers to flies fed a diet containing galactose, and "sucrose" refers to flies fed a control diet.

[0090] As a result, it was confirmed that galactose was detected in the midgut and fat body by G3.

[0091] [Experimental Example 5] (GALDAR3 in mammalian cells) GALDAR3 nlsGal3-P2A-Gal80(G310D)-GFP was cloned into the mammalian pCMV vector and then transfected into HEK293T cells, a human embryonic kidney-derived cell line, by lipofection.

[0092] Subsequently, 24 hours later, galactose was added to the medium at concentrations of 0%, 0.01%, 0.1%, 0.25%, and 0.5%, and the GFP signal in the nucleus was observed using a confocal fluorescence microscope and the signal intensity was measured.

[0093] The left image of Figure 8 is a fluorescence microscope image of cells. Nuclei were stained with DAPI. The right image of Figure 8 is a graph showing the results of calculating the ratio of GFP fluorescence intensity in the nucleus to that in the cytoplasm (GFP fluorescence intensity in the nucleus / GFP fluorescence intensity in the cytoplasm) based on the image on the left.

[0094] As a result, a galactose concentration-dependent increase in the percentage of GFP localized in the nucleus was observed. This result indicates that G3 functions in mammalian cells. Furthermore, these results suggest that G1 and G2 function in mammalian cells as well as G3. [Industrial Applicability]

[0095] According to the present invention, a technique for detecting galactose in vivo can be provided.

Claims

1. A kit for detecting galactose, comprising a nucleic acid encoding a fusion protein of a Gal80 (G310D) mutant and a reporter protein, and a nucleic acid encoding a Gal3 protein to which a nuclear localization signal has been added.

2. The galactose detection kit of claim 1, wherein the fusion protein of the Gal80 (G310D) mutant and a reporter protein and the Gal3 protein to which the nuclear localization signal has been added are configured to be expressed from a single mRNA.

3. A cell comprising a nucleic acid encoding a fusion protein of a Gal80(G310D) mutant and a reporter protein, and a nucleic acid encoding a Gal3 protein to which a nuclear localization signal has been added.

4. A transformant having a nucleic acid encoding a fusion protein of a Gal80 (G310D) mutant and a reporter protein, and a nucleic acid encoding a Gal3 protein to which a nuclear localization signal has been added.

5. The transformant according to claim 4, which is a Drosophila.

6. A kit for detecting galactose, comprising a nucleic acid encoding a Gal4 protein, a nucleic acid encoding a Gal80 protein, a nucleic acid encoding a Gal3 protein, and a nucleic acid in which a nucleic acid encoding a reporter protein is located downstream of an Upstream Activation Sequence (UAS).

7. The galactose detection kit according to claim 6 , wherein the Gal4 protein, the Gal80 protein, and the Gal3 protein are expressed from a single mRNA.

8. A cell comprising a nucleic acid encoding a Gal4 protein, a nucleic acid encoding a Gal80 protein, a nucleic acid encoding a Gal3 protein, and a nucleic acid in which a nucleic acid encoding a reporter protein is positioned downstream of the UAS.

9. A transformant having a nucleic acid encoding a Gal4 protein, a nucleic acid encoding a Gal80 protein, a nucleic acid encoding a Gal3 protein, and a nucleic acid in which a nucleic acid encoding a reporter protein is arranged downstream of the UAS.

10. The transformant according to claim 9, which is a Drosophila.

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