Gene search vector and gene search method

Details Gene search vector and gene search method Gene search vector and gene search method

2000-01-14

2003-05-13

Guzo, David (Department: 1636)

Multicellular living organisms and unmodified parts thereof and

Method of making a transgenic nonhuman animal

Involving breeding to produce a double transgenic nonhuman...

C800S025000, C800S008000, C800S013000, C800S003000, C435S320100, C435S455000, C536S023100

Reexamination Certificate

active

06563018

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel gene search vector for efficiently detecting an unknown gene of a fly, Drosophila, and a gene search method using the vector.
2. Description of the Related Art
Rapid progress has been made in the determination of the full-length genome sequences of various species. So as to effectively utilize the information about these genome sequences, a current investigative issue is the identification of a specific sequence domain practically responsible for a biological function to sequentially determine such domains in the form of “gene” corresponding to biological function.
The essential mechanisms of biological functions are preserved in any species, and genes allowing the mechanisms to function are also analogous in any species. Based on such grounds, the functional elucidation of a gene of a model lower organism is applicable to other numerous organisms including humans. The gene is for example life-span regulatory gene. The evidence up to date indicates that life span characteristically has a close relation with stress resistance, anti-oxidative action and biological protective potency. Because these functions are regarded to belong to the principal mechanisms of any species, the elucidation of the mechanisms in a model organism and the findings about the factors and genes involved in the mechanisms are of significant importance in various industries. For example, it is expected that life-span prolonging factor and stress-resistance factor not only provide information useful for the development of medicinal products but also are directly involved in the development of foodstuffs helping and supporting the control and promotion of human health (foodstuffs enriched with such factors and foodstuffs containing ingredients facilitating the generation of these factors in living organisms). It is also expected that industrially useful animals and plants, given with these ingredients in the forms of feeds or fertilizers or integrated with genes encoding these ingredients, can yield an improved productivity. Furthermore, factors capable of regulating the appearance and growth of insects or the propagating actions thereof provide new strategies for the control of the reproduction of useful insects or harmful insects with deep relation to agriculture and forestry. Otherwise, factors triggering cell growth or differentiation and abnormal actions provide valuable information for the molecular understanding of the fundamental pathogenesis of human genetic diseases or the functions of organisms; and these factors additionally serve as potent materials for the therapies of cancer and nerve diseases and for the development of novel drugs for these diseases.
Drosophila has traditionally been used frequently as a material for genetic research works and is endowed with powerful analytical modalities in the fields of genetics and molecular biology. Thus, Drosophila has been acclaimed as an excellent model organism for searching novel gene. In a practical sense, many novel genes including “homeobox gene” have been isolated from Drosophila.
The mutagenesis process comprising inserting the transposon P-element into the genome has been known as the means for searching novel genes in Drosophila (Science 239: 1121-1128, 1988; Proc. Natl. Acad. Sci. USA 92:10824-10830, 1995).
However, the frequency of the occurrence of a homozygote phenotype recovered through such mutagenesis using P-element is as low as about 15%, which is not sufficient for the detection of a gene in the genome (Science 239:1121-1128, 1988). It is considered that this is due to the facts that the sites for P-element insertion are mostly in the upstream of the coding sequence (Proc. Natl. Acad. Sci. USA 92:10824-10830, 1995) and that the genome per se is functionally redundant (Cell 86:521-529, 1996). Additionally, it is also indicated that the resulting phenotype sometimes has no relation with P-element insertion (Genetics 147:1697-1722, 1997).
As means for the expression of a gene of Drosophila in an enforced fashion further, a method has been known, comprising inserting in the Drosophila genome a vector carrying UAS (upstream activator sequence) enhancer as the target sequence of a yeast-derived GAL4 transcription activator, mating the transformed Drosophila with a Drosophila capable of expressing GAL4 (GAL4 expression line) to create a progeny individual, and allowing the progeny individual to express a gene downstream of the UAS enhancer (Development 118:401-415, 1993).
Another method using a combination of the GAL4-UAS enhancer compulsory expression system and the mutation induction with P-element insertion has been known (Proc. Natl. Acad. Sci. USA 93:12418-12422. 1996. Dros. Inf. Serv. 80;90-92, 1997). According to the method, a pair of the UAS enhancer/promoter is integrated in the multicloning site of a P-element transformation vector pCa SpeR (Gene 74:445-456, 1988) on the side of 3′ P-element; Drosophila with the vector inserted therein is mated with the GAL4 expression line, to enforce the expression of genes downstream of the vector insertion site in a progeny individual to identify phenotypes and the genes corresponding thereto.
The method using a combination of the GAL4-UAS enhancer forced expression system and the mutagenesis with P-element insertion is excellent in that genes can be detected on the basis of gain-of-function phenotype and loss-of-function phenotype. However, the occurrence of the mutant phenotype caused by P-element insertion is so low by the method. Hence, means or methods capable of efficiently allowing a mutant phenotype to emerge have been desired so as to detect a great number of useful novel genes.
In such circumstances, the invention has been accomplished for the purpose of providing a novel gene search vector capable of efficiently identifying a novel Drosophila gene in a manner so as to specify the function corresponding to the novel Drosophila gene; and a gene search method.
SUMMARY OF THE INVENTION
The present invention provides a gene search vector, which carries P-element sequences and two sets of an expression regulatory sequence comprising a UAS sequence for GAL4 transcription activator and a promoter sequence, wherein the expression regulatory sequences are integrated in the P-element sequence in such a manner that their downstreams are in opposite directions.
In one preferable embodiment of the gene search vector, the promoter is the core promoter of heat shock protein hsp70 gene.
In the other preferable embodiment of the gene search vector, the two sets of the expression regulatory sequence are independently integrated in the 5′ P-element sequence and 3′ P-element sequence.
In another preferable embodiment of the gene search vector, a marker gene is present between the two sets of the expression regulatory sequence, while Drosophila white gene is used as the marker gene.
In a still further embodiment of the gene search vector, the full length of the vector DNA is less than 8 kbp.
The present invention also provides a fly carrying the gene search vector set forth above being integrated in the genome thereof.
The present invention further provides a method for searching an unknown gene of Drosophila, comprising the steps of:
(a) inserting the gene search vector of any one of claims 1 to 6 into the genome of Drosophila,
(b) mating the vector-inserted Drosophila with a Drosophila expressing the GAL4 transcription activator to create progenies, and identifying a vector-insertion line with a phenotype different from those of wild-type Drosophila, and
(c) determining the nucleotide sequence of the gene for the phenotype of the mutant individual.
In one preferable embodiment of the gene search method, in step (c), a mRNA fragment including mRNA transcribed from a partial sequence of the gene search vector is amplified by reverse-transcriptase-polymerase chain reaction method to determine the nucleotide sequence of the resulting amplified DNA.


REFERENCES:
G. Toba et al., “The gene sear

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