KR20070114968A - Genetic mutant of Drosophila C. 5161 with black spots at the larval stage and its preparation method - Google Patents

Abstract

The present invention relates to the dark spot phenomena occurring in the mutants of the Drosophila CG5116 gene, jum ⁹ and jum ^73. Specifically, Δ2-3 having an insertional mutation strain (UY3132) and an active transferase of the P transfer factor in the vicinity of the CG5116 gene It relates to a CG5116 cleavage mutant produced by an inaccurate cleavage method and a method for producing the same. The CG5116 cleavage mutant of the present invention can be used as an animal model that is very useful for the study of gene function in humans, the discovery of new genes, and the study of diseases as well as the function of the Drosophila gene by mutation.

Description

Drosophila CG5116 gene mutant generating black maculation in a period of larva and preparation method

1 shows the protein domain of the Drosophila CG5116 gene.

Figure 2 shows the comparison of the similarity between the protein of the Drosophila CG5116 gene and a human protein (GTP binding protein).

3 shows the insertion position of the P transition factor in the UY3132 mutant strain.

4 shows the genetic process of constructing a CG5116 cleavage mutant using an imprecise excision method.

Figure 5 shows the position of the primers used to confirm the deletion position of the cleavage mutant in the UY3132 mutant strain.

6 is a diagram showing the product of the PCR reaction by electrophoresis on 1% agarose gel.

FIG. 7 shows the deletion sites in the two incorrect cleavage mutants jum ⁹ and jum ⁷³ in the UY3132 mutant strain.

8 is a photograph comparing the phenotype between the normal larvae and inaccurate truncated mutant larvae with dark spots.

Figure 9 is a photograph comparing the blood cells of the normal larva and CG5116 mutant jum ⁹ larvae.

The present invention relates to the dark spot phenomena in jum ⁹ and jum ^{73 which} are mutants of the Drosophila CG5116 gene. More specifically, the mutant 2 of the P transition factor in the periphery of the CG5116 gene (UY3132) and Δ2 having an active transferase. It relates to a CG5116 cleavage mutant produced by inaccurate cleavage using -3 and a preparation method thereof.

There are several factors that contribute to the prosperity of insects on the planet. For example, fruit flies have a long reproductive capacity in a short period of 10 to 14 days, and silkworms in about 50 days. It has dormant, metamorphic, and effective biological defense mechanisms to protect itself from external enemies. These are all adaptations to the environment acquired during the evolution of a long period of time. Insects of infinite variety due to these various adaptations are also studied in terms of biological resources.

In addition, most of the various biomaterials that maintain the specific function of insects are unknown and are unexplored resources as biological resources. Therefore, interpreting the functions of various insects that produce such biological resources not only contributes to life science research, but also contributes to industrial development through applied research using biological resources.

Also important is the study of insects as applied research in the medical and industrial fields. Research on insects, which are the host of disease, such as malaria mosquitoes, a mosquito mosquito, and the somatic fly, which mediates the insects trypanosoma, which is the cause of the sleeping disease, is also an important area of research worldwide. In particular, in genus mosquitoes, the entire genome was decoded after insect flies for the second time. The study of insect hormones is expected to help the use of these hosts if it is possible to artificially control the development of insects.

In addition, since insects find various bioactive substances and useful biopolymers, such insects may be attracting attention as searchers for new compounds useful for industrial development. For example, useful substances produced by the symbiotic microorganisms of insects are expected as a search source for medicines such as humans and livestock. Therefore, to develop the interpretation of specific functions of insects, such as research on the development of insects, research on hormones, research on biodefense function of insects, and research on symbiotic microorganisms of insects, etc. As will be very important.

Thus, in the present invention, the function of a specific gene was investigated through mutation studies in the fruit flies in the functional study of the insects. In particular, fruit flies are the most researched insects in the field of molecular biology, and these fruit flies attracted attention as experimental materials for biology when they were used as materials for genetic research by Dr. Morgan (University of Columbia) in the United States in the 1910s. From. Since then, it has been increasing its superiority as a biological test material such as mutagenesis, gene mapping using other chromosomes, and the use of a balancer chromosome to hold mutations that have occurred once.

Furthermore, in the research of fruit flies, transfection technology was established in the 1980s by the discovery and use of a transposon called a P transposable element, thereby allowing the gene transduction technology to be freely used. In 2000, the first genome was deciphered and established as a representative molecular biology research model for insects.In addition to gene function analysis using genomic bioinformatics based on genome decoding, gene expression analysis using microarray, RNAi (RNA interference) Research on gene function using methods such as inhibition of gene expression and gene targeting by RNA interference) have been conducted. In particular, functional research using fruit flies has the following advantages.

First, mutation detection and genetic engineering methods are highly developed. In particular, mutagenesis is essential for functional studies of genes. In this case, malfunctions using ethyl methane sulphonate (EMS), jumping-out of P transfer factors, RNAi, and defective stocks in fruit flies Gene function can be studied through the effects of loss-of-function mutations and gain-of-function mutations using the upstream activation sequence (UAS) / GAL4 system.

Second, by using enhancer trap mutagenesis, gene expression in specific tissues or cells can be systematically obtained by confirming its expression before cloning a specific gene.

Third, by overexpressing unknown genes in specific tissues or cells using the P transfer factor jumping system and the upstream activation sequence (UAS) / GAL4 system, mutant phenotypes can be observed and induced. Specific genes can be studied.

Fourth, mutation can be induced by overexpressing an apoptosis gene using an upstream activation sequence (UAS) -hid.

Fifth, since the fruit fly genomic sequence is completed, genetic information can be compared with each other smoothly, and the necessary mutation strain can be easily searched and obtained through the search, and thus the fruit fly function research can be performed very efficiently. In particular, the UY3132 mutant strain in which one P transition factor was inserted at the 5 'upper portion of the Drosophila CG5116 gene of the present invention was searched from the flybase database (http://flybase.bio.indiana.edu) using this method. Once found, the mutant was available at the Bloomington Drosophila stock center (USA).

In addition, one of the most important tasks in the study of organisms after the gene sequence is revealed by the Drosophila genome project is the functional study of genes. To this end, several mutations have been made in model animals, and the ideal method is to induce mutations in situ by homologous recombination. However, since there are no organisms that can be used in this way except for yeast, attempts were made in Drosophila to produce as many mutations at once as possible by making mutation aggregates by inserting transition factors into arbitrary positions. This is called the P transfer factor mutation project. Currently, 3,200 30% of the mutations among the 14,000 genes of the Drosophila gene have been established on chromosomes 2 and 3 of the 4 chromosomes of Drosophila (Spradling, AC, et. al ., Genetics , 153, 135-177, 1999). The mutation is characterized by a single transfer factor for each gene and the sequence surrounding the transfer factor is analyzed and stored in the database. Once a mutation is selected, the most similar gene is compared to the expressed sequence tag (EST) sequence. Is found in species. In addition, recently, a complete cDNA sequence corresponding to 40% of the Drosophila gene has been determined, and a project to directly obtain a cDNA similar to the insertion sequence from a transmutant mutation is underway, and 80% of the cDNA will be secured in the future. Rubin, GM, et al ., Science , 287, 2204-2215, 2000).

On the other hand, since there are about 3,200 mutations showing expression traits among 14,000 genes, a method for confirming the function of the remaining genes is needed. Therefore, in Drosophila, the electronic factor GAL4 and its target gene higher activation factor in Drosophila A method has been devised to study the function of genes by overexpressing them at the original expression site or at a completely different site using the upstream activation element (UAE) system (Rorth, P., et. al ., Development , 125, 1049-1057, 1998). That is, a transgenic Drosophila group expressing a GAL4 transcription factor regulated by enhancers of various types of genes by inserting the P transfer factor promoter and the GAL4 gene and inserting it into an arbitrary position of the Drosophila chromosome using the P transfer factor vector. In order to establish and detect their expression, upstream activation sequence (UAS) with five consecutive GAL4 binding sites can be combined with lacZ gene to analyze the expression effect of the modified gene. In addition, the gene can be cross-linked with a transgenic Drosophila in which the gene is linked to a higher activating sequence (UAS) site as needed to analyze the expression effect of the modified gene. In particular, in this method, various Drosophila groups that can be expressed in a variety of specific cells or tissues can be easily prepared without isolation and purification of specific genes, and the gene UAS-X including the transcription factor GAL4 and its binding site can be easily prepared. Since maintaining the transformant fruit fly separately, there is an advantage that the effect of genes expressing harmful products can be easily investigated. On the other hand, the disadvantage is that the expression of GAL4 can be detected only after the early development stage of fruit flies (stage 6), and it is not expressed in germ cells of females. Due to its excellent accessibility, it has recently been used to study the mechanism of fate determination and differentiation of various cells such as neurons, muscle cells and epidermal cells, and the study of the development of structures and organs.

Recently, the Berkeley Drosophila Genome Project (BDGP, USA, http://www.fruitfly.org) has revealed the sequence of the Drosophila CG5116 gene. First, a gene map was prepared to determine the location of the Drosophila CG5116 gene. Specifically, the Drosophila genomic gene sequence was amplified by PCR using a nucleotide sequence of 200 bp to 500 bp, and then the position of a gene or a specific indicator was identified. physical map to guide its original position to the position of the amplified gene in the (physical map) hybridize (in The location of specific genes on the Drosophila genome was determined by situ hybridization. In particular, DNA sequencing must be performed to generate such a genetic map. In the above project, chromosomes are randomly cut using DNase or sonicator using whole genome shotgunning, and the DNA is then randomized. The fragments were cloned into a suitable vector by PCR and amplified, and then the DNA was stretched out in a capillary manner on a gel. The sequence was analyzed using an automated sequencing system. Comparing the Drosophila gene DNA database with nematodes ( C. elegans ) or yeast ( S. cerevisiae ), etc., they were completed by rearranging them at the correct position of the gene map.

In addition, the CG5116 gene actually predicted by synthesizing cDNA of mRNA expressed in the Drosophila larva stage by the EST (expressed sequence taq) project, which shows the nucleotide sequence that is actually expressed on the genome, which was simultaneously performed together with the above project. It was confirmed that it is expressed in.

Therefore, the present inventors produced a UY3132 mutation, in which a P transition factor was inserted at the 5 'upper portion of the Drosophila CG5116 gene by an inaccurate cleavage method, to study a function using a phenotypic difference of the mutant, namely, a function of a human gene, a new one. The present invention has been completed by confirming that the model animal is very useful for the discovery of functional genes and the study of diseases.

It is an object of the present invention to provide a mutant of the Drosophila CG5116 gene. In addition, another object of the present invention is to build a model animal useful for studying the function of the human gene corresponding to the CG5116 gene, the discovery of a new functional gene and the study of disease through gene function research using the mutant.

In order to achieve the above object, the present invention provides a Drosophila mutant lacking the CG5116 gene.

The present invention also provides a method for preparing the mutant.

In addition, the present invention provides these phenotypes.

The present invention provides a Drosophila mutant lacking the CG5116 gene.

The Drosophila CG5116 gene has a DNA sequence of 2459 bp as shown in SEQ ID NO: 1, and has been identified to encode a protein having a 526 amino acid sequence as shown in SEQ ID NO: 2 , and since the CG5116 protein has an Hf1X protein region, it is a biosignal carrier. It is expected to perform the function of GTPase to decompose phosphorus guTP (guanosine triphosphate) to guanosine diphosphate (GDP) ( see FIG. 1 ). In addition, homology assays were performed using NCBI (http://www.ncbi.nlm.nih.gov), and at the amino acid level, CG5116 protein was expressed in human GTP binding protein, mouse GTP binding protein, and frog ( Xenopus). laevis ) and MGC83645 protein and the C. elegans F46B6.4 protein, etc. were found to have high homology. Especially, CG5116 protein has about 64% similar amino acid sequence to human GTP binding protein. It was confirmed ( see FIG. 2 ).

Accordingly, the present inventors have found that flybase (http: //flbase.bio.indiana) of the insertion mutation of the P transition factor, a Drosophila DNA transposable element produced by the Berkeley Drosophila Genome Project (BDGP, USA). .edu) UY3132 (stock number, BL7355; http://flybase.bio.indiana.edu/.bin/fbidq.html?FBal0155675) with one P transfer factor inserted at the top 5 'of the CG5116 gene. After screening for mutants, the mutants were obtained from the Bloomington Drosophila Stock Center (USA). Figure 3 shows the position where the P transfer factor is inserted in the UY3132 mutant strain, specifically, it can be seen that the P transfer factor is inserted 219 bp ahead of the transcription start site of the CG5116 gene in the UY3132 mutant strain.

In addition, the present inventors used an imprecise excison method using a P transfer factor to produce a truncated mutant of the CG5116 gene including the UY3132 mutant strain (Robertson, HM, et al ., Genetics , 118, 461-470, 1988; And Adams, MD & Sekelsky, JJ, Nat . Rev. Genet ., 3, 189-198, 2002). Specifically, the P transfer factor causes metastasis by transposase, which is largely composed of an excision and an insertion process. In particular, during the cleavage process, DNA fragments surrounding the P transition factor, ie, a portion of the Drosophila genomic DNA, are inaccurately cleaved, resulting in a deletion mutant. The inventors use this property to generate a P mutant around the CG5116 gene. CG5116 mutant was constructed using Δ2-3 mutant strain (Laski, FA, et al ., Cell , 44, 7-19, 1986) containing UY3132 mutant and transposase transfectase . ( See FIG. 4 ).

At this time, the truncated Drosophila genomic DNA preferably contains 150 to 1500th base from the CG5116 gene transcription start site, more preferably includes 159 to 1219th base from the CG5116 gene transcription start site, and more More preferably, the P transfer factor cleavage site is preferably 754 bp before the transcription initiation site and 1219 bp after the initiation of transcription, or the P transition factor cleavage site is 754 bp before the transcription initiation site and 159 bp after the transcription initiation, but is not particularly limited thereto.

The present invention also provides a method for preparing the mutant.

In the present invention, the method for preparing a CG5116 mutation comprises the following 1 to 5 steps:

1) a method of crossing uncrossed female Drosophila with UY3132 mutant strain and male Drosophila Δ2-3 strain;

2) a method for screening fruit flies having a P transition factor and a Δ2-3 mutant among the progeny of step 1;

3) a method of mating with the selected Drosophila of step 2 with an uncoated female Drosophila having TM6B and a phenotype of Tb (Tubby);

4) a method of selecting Drosophila, which is Tb and Pb of the descendants of step 3, and not Sb (Stubble); And

5) Incorrect cleavage mutations by PCR method The method comprising the step of constructing and constructing.

Subsequently, the present inventors performed PCR on 200 cleaved mutant strains in the same manner as in FIG. 5 to find a defective region in the cleavage mutant of the CG5116 gene produced using the above-described imprecise excison method . In this case, the primers used were SEQ ID NO: 3 (F4) and SEQ ID NO: 4 (3'-1). As a result, as shown in FIG. 6 , Δ9 (jum ⁹ ) and Δ72 (jum ⁷³ ) having defective sites were amplified by fragments of 1.3 kb and 2.3 kb, respectively, compared to the control (3.2 kb). The inventors were able to confirm that the defective site occurred at the site of the UY3132 inserted with the CG5116 gene and the P transfer factor around it ( see FIG. 7 ).

The present invention also provides their phenotypes.

The inventors found the defective regions of the CG5116 gene by the above PCR method and named them jum ⁹ and jum73 (jum, abbreviation for spotting), and compared their genetic phenotypes with normal larvae. As a result, as shown in Fig. 8 , the normal larvae were not observed, while black spots were observed in jum ⁹ and jum ⁷³ , which are cleavage mutants of CG5116. In particular, the two truncated mutants showed a different phenotype difference, and the more defects on the CG5116 gene, the larger the size of the black spots, it was confirmed that the larger number ( see Fig. 8 ).

Drosophila blood cells are also composed of three cells that are functionally similar to myeloid lineage blood cells in vertebrates: plasmaatocytes, crystal cells, and lamellocytes. (Meister, M., Curr. Opin . Immunol ., 16, 10-15, 2004). In particular, when the hematopoiesis between Plasmasite and Lamelosite in Drosophila blood cells is abnormal, a case similar to human disease has been found. It has been reported that the rapid increase in the number of plasmatosite results in an increase in lamelosite, resulting in melanoma (Evans, CJ, et. al , Dev . Cell , 5, 673-690, 2003).

As mentioned above, despite the fact that Drosophila blood cell formation process and its molecular mechanism are very similar to that of vertebrates, and that Drosophila cell hyperplasia and melanoma are formed, which is a symptom similar to human disease, factors related to the present And the mechanism of action is not elucidated. The present inventors confirmed that the Drosophila mutant of the present invention exhibits a hyperdivision of blood cells, a symptom similar to that of human disease (see FIG. 9 ), which indicates that a new GTP binding protein mutant encoded by the CG5116 gene of the present invention. It is suggested that the use may suggest a new method for elucidating the mechanism of proliferation and differentiation of blood cells.

Accordingly, the present invention provides genetic characteristics that may be caused by the difference in genetic phenotype, namely, the study of gene function using mutations of Drosophila and related human gene function studies, the discovery of new functional genes, and the study of diseases. It is suggested that a useful model animal can be constructed.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

< Example  1> Drosophila by Inaccurate Cutting Method CG5116  Construction of Gene Mutations

)초파리를 Δ2-3 변이주 수컷(

)초파리와 교배한 후, 교배를 통해 나온 자손 중 유전학적 마커인 Tb(Tubby, 몸이 짧고 어깨 털이 정상에 비해 많은 특성을 가짐, http://flybase.bio.indiana.edu/.bin/fbidq.html?FBgn0003668)와 Sb(Stubble, 털이 정상에 비해 짧은 특성을 가짐, http://flybase.bio.indiana.edu/.bin/fbidq.html?FBgn0003319) 등을 유전학적 마커로 사용하여 P 전이인자와 Δ2-3 염색체를 동시에 가진 초파리를 해부현미경하에서 관찰하여 선별하였으며, 이때 이 초파리의 생식세포(정원세포, 후에 정자가 되는 세포임)에서 P 전이인자는 절단(excision) 또는 도약(jumping)된다.In the present invention, in order to produce a CG5116 cleavage mutant by an imprecise excision method through the genetic design method as shown in Figure 4 , first uncrossed females of UY3132 mutant strain (

) Drosophila Δ2-3 mutant male (

After mating with Drosophila, the genetic marker Tb (Tubby, shorter body and more hairy shoulders than normal, among the offspring from breeding, http://flybase.bio.indiana.edu/.bin/fbidq .html? FBgn0003668) and Sb (Stubble, hair shorter than normal, http://flybase.bio.indiana.edu/.bin/fbidq.html?FBgn0003319) as genetic markers for P transfer Drosophila with both factor and Δ2-3 chromosome were observed under the dissecting microscope, and the P transition factor in the germ cells of the fruit flies (spontaneous cells, later sperm cells) were cut or jumped. do.

)초파리(Bloomington stock center, http://flybase.bio.indiana.edu, number, BL3703)와 교배한 다음 이 교배를 통해 나온 자손 중 P 전이인자의 유전학적 마커인 w(white, 초파리 눈 색깔이 흰색, http://flybase.bio.indiana.edu/.bin/fbidq.html?FBgn0003996) 음성이고, Tb이며 Sb가 아닌 초파리를 표현형을 기반으로 선별하였다.The fruit flies were then isolated from uncoated females with the balancer chromosomes TM6B and Tb .

After crossing the fruit fly (Bloomington stock center, http://flybase.bio.indiana.edu, number, BL3703), the genetic marker of the P transfer factor, w (white, fruit fly eye color) White, http://flybase.bio.indiana.edu/.bin/fbidq.html?FBgn0003996) negative, Tb and not Sb Drosophila were selected based on phenotype.

At this time, the fruit flies were negative in both the markers ( w and white) and the transposase markers ( Sb and Stubby) of the P transfer factor, respectively. In addition, many of the fruit flies that had cleavage had a precise excision or because the cleavage site was too wide, resulting in cleavage of genes other than the CG5116 gene, resulting in only 200 CG5116 truncated mutations. P transfer factor cleavage mutants were constructed.

Subsequently, in order to select the CG5116 cleavage mutation among the P transfer factor cleavage mutants obtained from the above, as shown in FIG. 5 , the F4 primer (5'-AAAACTGTCGCTTGAACTG-3 ', SEQ ID NO: 3 ) and the 3'-1 primer (5) PCR was performed using '-TTGCGACTTTTGCGAGCCG-3', SEQ ID NO: 4 ), and when the excise of the excision mutations occurred among the cutting mutations of the UY3132 mutant, the 3.2 kb DNA fragment was amplified, whereas the P transfer factor site. If cleavage occurred at, it was predicted that smaller DNA fragments would be amplified.

< Example  2> Identify the missing part of the produced mutant

<2-1> PCR Defective part check by

In order to perform the PCR reaction of <Example 1>, the present inventors extracted genomic DNA from a control mutant (Bloominton stock center number, BL19889) without a P transfer factor and a truncation mutation having a P transfer factor. In this case, the extraction method used was a method devised by the Berkeley Drosophila Genome Project (BDGP) (www.fruitfly.org). Specifically, black spotted larvae (5 mice) were selected and placed in a 1.5 mL tube, frozen on ice, and then 100 μl of buffer A (100 mM Tris-HCl, pH 7.5; 100 mM EDTA; 100 mM NaCl). 0.5% SDS), followed by pulverization, and additionally 100 µl of buffer solution A was added and suspended at 65 ° C for 30 minutes. Then, 400 μl of LiCl / KAc (5 M KAc concentrate and 6 M LiCl concentrate were mixed at a ratio of 1: 2.5, respectively) was added thereto, suspended in ice for 10 minutes, and centrifuged at 12,000 rpm for 15 minutes at room temperature. The supernatant was transferred to a new 1.5 mL microtube, and then mixed with 600 μl of isopropanol, followed by centrifugation at 12,000 rpm for 15 minutes at room temperature. After removing the supernatant, the remaining precipitate was added to 600 μl of 70% alcohol and centrifuged at 12,000 rpm for 10 minutes at room temperature. After removing the supernatant, the remaining precipitate was dried and 50 μl of distilled water was added thereto. Eluted.

Subsequently, the product obtained by the above method was subjected to PCR in the following reaction composition and reaction conditions, specifically 50 μl [1 μl of each Drosophila genomic DNA (1 μg / μl); 1 μl F4 primer (10 pmol / μl); 1 μl 3'-1 primer (10 pmol / μl); 0.25 μl of Taq ™ polymerase (5 unit / μl); 5 μl of Taq ™ buffer; 4 μl of dNTP mixture (2.5 mM each); 37.75 μl of distilled water] was then reacted for 2 minutes at 98 ° C. using Applide Biosystem 2700 (Applied Biosystems, USA), then 10 times at 98 ° C., 30 seconds at 60 ° C., and 35 times at 72 ° C. for 4 minutes. After repeating, the reaction was performed at 72 ° C. for 5 minutes.

As a result, PCR reactions were performed on 200 cleaved mutant strains as described above. As shown in FIG. 6, in the Δ9 (1.3 kb, mutant strain 3) and Δ73 (2.3 kb, mutant strain 4) control group (3.3 kb, mutant strain 2) Smaller fragments could be identified.

<2-2> jum ⁹  And jum ⁷³ Defects in

Subsequently, the inventors gel-eluted the bands of Δ9 and Δ73 identified in the agarose-gel electrophoresis of FIG. 6 to isolate and purify the genes of the respective fragments (Gene All Gel SV kit, General biosystem). The sequence was analyzed.

As a result, the cleavage site in Δ9 was cleaved from 754bp before the start of transcription of CG5116 gene to 1219bp after the start of transcription (jum9 site in Fig. 7 ), and the cleavage site in Δ73 was started from 754bp before transcription initiation. After 159bp site was confirmed that the cleavage (jum ⁷³ site of Figure 7 ). In particular, these mutants have a phenotype showing black spots, so they are named "jumbagi" and abbreviated "jum". In the present invention, the two mutants are named "jum ⁹ " and "jum ⁷³ ", respectively. ( FIG. 7 ).

< Example  3> CG5116  Mutants ( jum ⁹  And jum ⁷³ Phenotype analysis

<3-1> Phenotype Comparison of Normal and Mutant Larvae

In the present invention, the normal larvae and black spots appearing at the larvae of jum ⁹ and jum ⁷³ mutants were compared. As a result, the normal larvae showed black spots and a smooth phenotype, whereas the mutants (jum ⁹ and jum ⁷³ ) mainly showed black spots that were randomly located or sized behind the body ( Fig. 8 ).

<3-2> jum ⁹  and jum ⁷³  Comparison of black spot pattern and hematopoietic traits of mutants

Subsequently, the inventors examined the appearance of black spots in the mutants, ie jum ⁹ and jum ⁷³ . As a result, it was observed that in the CG5116 mutant Δ9 (jum ⁹ ), the size of the black spots is not only larger and larger in number than the size of the black spots of Δ73 (jum ⁷³ ) but also shows various phenotypes from small to large. On the other hand, at Δ73 (jum ⁷³ ), the size of the black spots was small and no more than one could be found. This result means that Δ9 (jum ⁹ ) can generate larvae with a greater number of black spots than Δ73 (jum ⁷³ ).

In addition, when comparing the cells of the mutant Drosophila with those of normal larvae under an optical microscope, it was observed that the amount of blood cells increased about three times, and the size of the blood cells was also increased compared to that of the normal larvae ( FIG. 9 ). These results indicate that CG5116 is a substance that inhibits hematopoietic cell proliferation and, as mentioned above, may be useful for the study of human leukemia, which exhibits traits similar to the proliferation of Drosophila blood cells and the formation of melanotic masses. Suggests that you can.

As described above, the difference in the phenotype of the mutant of the Drosophila CG5116 gene, ie, jum ⁹ and jum ⁷³ , in which the dark spots generated by the inaccurate cleavage method of the present invention are generated is Drosophila using the deletion mutation of Drosophila This suggests that it can be used as a very useful animal model not only for gene function studies but also for functional studies by human genetic mutations, discovery of new functional genes and disease studies.

<110> Konkuk University <120> Drosophila CG5116 gene mutant generating black maculation in a          period of larva and preparation method therof <130> 6P-04-14 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 2549 <212> DNA <213> cDNA sequence of Drosophila CG5116 gene <400> 1 aattgcataa taattgctca aactacaggt ttttccggca cctatttcgc ccagaagata 60 gtacaatcaa aaattcgcta cccaatcaag tacaacatac ttatctcctc actggtggcc 120 accggcgtgt cctaccaaat cacatcgaca cggactaaag cctgccaagc ggcgtggatg 180 gcctttgagg acaagcattc ggtgctcaaa gagaaaacct tttaacacct tcaaaattcc 240 atttagcttt agtcatgtct gctctacgaa tattggcccg atttgtcagc actaggactc 300 tgcgaattcg tccgattctg gagattcccg cacgctgcaa gtacacccaa catcaaggag 360 ttaagggcat tcggaacagg aagagcttct tcgaagccca aattcaggca ggtcaacaag 420 gtgaggagga tgcggactta gagcggacag ccaatagcga tctggcggat atgcggttct 480 tggatgatcg gtaggcttaa aaatcaaaga aaaaaaaaga gatattacta accttttatt 540 taagagccta cgacgaggtg gctggaggag ccatgagaat cacacgtgac atagccagct 600 ctcagcaggt gctcatcctc caaccgtatg taaaatgggc agccaaacga cagaacgcac 660 ccgtcgatgt tcggcccgag gatcaactcg cggaggccac agcgctgatc cactccctgc 720 ccaattggca ggtggccagg gcattaaagg ttcccctcga aagcctcgaa aagaagacgc 780 ttttcggcag cggcaagctg gtggaactca aggaactggt cgccgaattg cggcaggaac 840 ggcatctaac ttgcctgttt gtcagcaagg ggacactatc attcgcccaa aaacgtttcc 900 tggaggcgga gtttcgcctg ccagtaatgg atcgctattc cgttgttata cagatactaa 960 gactgcatgc caccagcgcc gaagcaaaac tgcaggttac tatactcatt gaacaagcac 1020 tataacgaat tagatactga tcatgttatt taggtagcca tggctgaact tccttacatt 1080 tgggcgcagg ctaaggatgc cagtgttacg cagacgcgtc gtcagggcta cgccttgacc 1140 gatctgcaga aggagattct gcgaaccagg gagcgaaagt tgcgagcgga attagatcgt 1200 gtccggcgac agaggcagtt gcttcgccag aaaagaaaac agcaaaatta tcccatcgtt 1260 gcagtggtgg gttacacaaa tgctggcaag acttccttga tcaaggcact cacagtcgag 1320 gatgcactgc agcccaggaa tcagttgttc gcaacgcttg atgtgactgc tcatgctggt 1380 tgtttgccat gcaatctgga ggtgatctat atggacacgg tgggctttat gtccgatctg 1440 ccaacgggac tgtttgaatg ctttgtggcc actttggagg atgccatgct ggcggtaagt 1500 aaaatcttct gccattaaaa agttctatac ataaaaacct ttttccttcg caggatgtaa 1560 tagtccacgt ccaggatctt tcccatccgt gtcacgctgc tcaacgcagc catgtggagg 1620 ccacgcttcg ttctttggca ttcaacgtgg ctggcggaga ttccacggcc agccagttgc 1680 cgccgatcat taatgtgtac aacaaatgtg atcttgtttc ccaagaggct cagtcgtctg 1740 cagacccagt gcatcatata tcagccagag cacagactgg gctggaaccc ctgctagatg 1800 acatcgagca acagatattg accgccaccg ggcgaagaaa acttcagatg cgagtgccca 1860 gcggcggtcc agaaatggct tggctgtaca aaaacgcagc tgtggtggaa acgacagcgg 1920 atgcggagaa tcccgaacgc ctcatgatgc acgtggttat cagccaacgc acactggatc 1980 agttcaagcg acagttttgc cgatgaattt aaagtgccat ggataaatat ctgaggcgca 2040 actggcaccg actaaatgtt tattattgct ctcgtttttt ttgttggttt attagcgggt 2100 gtagttagga ctggttttct tttcttgttt taaattctat acaaatataa aagtatctgc 2160 cgaaaactaa aacgaaacaa tctctaaaaa tattttttaa cataatattt gtattgtatt 2220 gcacttctct gacatttttg tgctgtggtt ttctctagga ataatcgaaa ctaaaccctt 2280 gtgggatcag ttttcgcatc aaccattttt gtttatataa ctagggcaag ttttttttat 2340 ttatttttct ttgtgtggtg ctgacttctc tattggcaca atggaatatt taaaaataat 2400 gcagggttga tgagcataaa caacttaaaa tacagaaact gaaacaaact taacaccagc 2460 ttaaacctaa agtaacttaa aattaaacaa aaatgcgttc aacactctag tgtacaaatt 2520 aaaaacccat ttatgcgtgt gaaaattga 2549 <210> 2 <211> 526 <212> PRT <213> Amino acid sequence of Drosophila CG5116 gene <400> 2 Met Ser Ala Leu Arg Ile Leu Ala Arg Phe Val Ser Thr Arg Thr Leu   1 5 10 15 Arg Ile Arg Pro Ile Leu Glu Ile Pro Ala Arg Cys Lys Tyr Thr Gln              20 25 30 His Gln Gly Val Lys Gly Ile Arg Asn Arg Lys Ser Phe Phe Glu Ala          35 40 45 Gln Ile Gln Ala Gly Gln Gln Gly Glu Glu Asp Ala Asp Leu Glu Arg      50 55 60 Thr Ala Asn Ser Asp Leu Ala Asp Met Arg Phe Leu Asp Asp Arg Ala  65 70 75 80 Tyr Asp Glu Val Ala Gly Gly Ala Met Arg Ile Thr Arg Asp Ile Ala                  85 90 95 Ser Ser Gln Gln Val Leu Ile Leu Gln Pro Tyr Val Lys Trp Ala Ala             100 105 110 Lys Arg Gln Asn Ala Pro Val Asp Val Arg Pro Glu Asp Gln Leu Ala         115 120 125 Glu Ala Thr Ala Leu Ile His Ser Leu Pro Asn Trp Gln Val Ala Arg     130 135 140 Ala Leu Lys Val Pro Leu Glu Ser Leu Glu Lys Lys Thr Leu Phe Gly 145 150 155 160 Ser Gly Lys Leu Val Glu Leu Lys Glu Leu Val Ala Glu Leu Arg Gln                 165 170 175 Glu Arg His Leu Thr Cys Leu Phe Val Ser Lys Gly Thr Leu Ser Phe             180 185 190 Ala Gln Lys Arg Phe Leu Glu Ala Glu Phe Arg Leu Pro Val Met Asp         195 200 205 Arg Tyr Ser Val Val Ile Gln Ile Leu Arg Leu His Ala Thr Ser Ala     210 215 220 Glu Ala Lys Leu Gln Val Ala Met Ala Glu Leu Pro Tyr Ile Trp Ala 225 230 235 240 Gln Ala Lys Asp Ala Ser Val Thr Gln Thr Arg Arg Gln Gly Tyr Ala                 245 250 255 Leu Thr Asp Leu Gln Lys Glu Ile Leu Arg Thr Arg Glu Arg Lys Leu             260 265 270 Arg Ala Glu Leu Asp Arg Val Arg Arg Gln Arg Gln Leu Leu Arg Gln         275 280 285 Lys Arg Lys Gln Gln Asn Tyr Pro Ile Val Ala Val Val Gly Tyr Thr     290 295 300 Asn Ala Gly Lys Thr Ser Leu Ile Lys Ala Leu Thr Val Glu Asp Ala 305 310 315 320 Leu Gln Pro Arg Asn Gln Leu Phe Ala Thr Leu Asp Val Thr Ala His                 325 330 335 Ala Gly Cys Leu Pro Cys Asn Leu Glu Val Ile Tyr Met Asp Thr Val             340 345 350 Gly Phe Met Ser Asp Leu Pro Thr Gly Leu Phe Glu Cys Phe Val Ala         355 360 365 Thr Leu Glu Asp Ala Met Leu Ala Asp Val Ile Val His Val Gln Asp     370 375 380 Leu Ser His Pro Cys His Ala Ala Gln Arg Ser His Val Glu Ala Thr 385 390 395 400 Leu Arg Ser Leu Ala Phe Asn Val Ala Gly Gly Asp Ser Thr Ala Ser                 405 410 415 Gln Leu Pro Pro Ile Ile Asn Val Tyr Asn Lys Cys Asp Leu Val Ser             420 425 430 Gln Glu Ala Gln Ser Ser Ala Asp Pro Val His His Ile Ser Ala Arg         435 440 445 Ala Gln Thr Gly Leu Glu Pro Leu Leu Asp Asp Ile Glu Gln Gln Ile     450 455 460 Leu Thr Ala Thr Gly Arg Arg Lys Leu Gln Met Arg Val Pro Ser Gly 465 470 475 480 Gly Pro Glu Met Ala Trp Leu Tyr Lys Asn Ala Ala Val Val Glu Thr                 485 490 495 Thr Ala Asp Ala Glu Asn Pro Glu Arg Leu Met Met His Val Val Ile             500 505 510 Ser Gln Arg Thr Leu Asp Gln Phe Lys Arg Gln Phe Cys Arg         515 520 525 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> F4 primer sequence for PCR <400> 3 aaaactgtcg cttgaactg 19 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> 3'-1 primer sequence for PCR <400> 4 ttgcgacttt tgcgagccg 19

Claims (15)

 Drosophila mutant lacking the CG5116 gene. The Drosophila mutant of claim 1, which is prepared from UY3132 mutant strain. The Drosophila mutant of claim 2, wherein the UY3132 mutant strain is inserted with a P-transfer factor at the top of the CG5116 gene. 4. The Drosophila mutant of claim 3, wherein the P transfer factor is inaccurately truncated.    2. The Drosophila mutant according to claim 1, wherein the DNA fragment comprising the 150th to 1500th base is cut from the CG5116 gene transcription start site. The Drosophila mutant according to claim 5, wherein the DNA fragment comprises 159th to 1219th bases from the transcription start site. The Drosophila mutant according to claim 5, wherein the P transfer factor cleavage site is from 754 bp before the transcription start site to 1219 bp after the transcription start. The Drosophila mutant of claim 1, wherein the CG5116 gene encodes a sequence of the amino acid set forth in SEQ ID NO: 2. The Drosophila mutant of claim 1, wherein the CG5116 gene has a nucleotide sequence set forth in SEQ ID NO: 1. 8. The Drosophila mutant of claim 7, wherein the mutant is jum ⁹ . The Drosophila mutant according to claim 5, wherein the P transfer factor cleavage site is from 754bp before the transcription start site to 159bp after the transcription start. 12. The Drosophila mutant according to claim 11, wherein the mutant is jum ⁷³ . The Drosophila mutant of claim 1, wherein the fruit fly mutant has a phenotype representing black spots. 1) crossing the uncrossed female fruit flies with UY3132 mutant strain and the male Δ2-3 mutant strain Drosophila; 2) selecting a Drosophila having a P transfer factor and a Δ2-3 mutant among the progeny of Step 1; 3) mating with the selected Drosophila of step 2 with an uncoated female Drosophila having a TM6B and a phenotype of Tb (Tubby); 4) and having a third phase of the transition of the children of P factor Tb, the method comprising sorting fruit flies than the Sb (Stubble); And 5) Drosophila mutant manufacturing method according to claim 1, comprising the step of selecting and constructing the inaccurate cleavage mutations by the PCR method in the fruit fly of step 4. 15. The method according to claim 14, wherein the UY3132 mutant strain of step 1 is a mutant in which one P transition factor is inserted at the top 5 'of the CG5116 gene.

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