Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-05-26
2004-03-30
Reynolds, Deborah J. (Department: 1632)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S325000, C435S252300, C435S183000, C435S069100, C435S070100, C435S071100, C536S023100
Reexamination Certificate
active
06713618
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel DNA, more particularly, to a novel DNA which encodes trehalase, and uses thereof.
2. Description of the Prior Art
Trehalose, a non-reducing disaccharide which consists of glucose units as constituent saccharide, is widely distributed in the natural world, for example, in bacteria, fungi, algae, insects, Crustacean, etc. In organisms such as insects which have a relatively-large amount of trehalose in their bodies, trehalose would play an important role as energy source and relate to the maintenance of physiological function such as cold resistance. Mammals including humans have long been utilizing trehalose widely from mushrooms, seaweeds, fermented foods, etc, as reported by Oku et al., in “
Journal of The Japanese Society For Food Science And Technology
”, Vol. 45, No. 6, pp. 381-384 (1998). As disclosed in Japanese Patent Kokai Nos. 143,876/95 and 213,283/95 applied for by the present applicant, the establishment of technologies for industrial-scale production of trehalose has more increased the interest of trehalose in the maintenance and regulation of biological functions in mammals, and this results in energetic and continuous researches in various fields.
Trehalase is an enzyme which specifically hydrolyzes the glucosidic bond in trehalose. Because of this substrate specificity, the enzyme may deeply correlate to the trehalose level in vivo in organisms such as insects and relate to the regulation of their biological functions. Even in mammals with no significant amount of trehalose, trehalase is found in animals such as humans, mice and rats. Major physiological role of trehalase in mammals would be the hydrolysis of externally-ingested trehalose when the saccharide is digested and absorbed by the mammals. It was reported that trehalase is commonly found in specific mammalian organs independently of the intake of trehalose, and hence there still remains many unknown biological roles of trehalase per se. As described above, the roles of mammalian trehalase in the maintenance and regulation of their physiological functions have also been focused recently, along with the increasing interest in trehalose.
Methods in a molecular biological manner are very useful for elucidating the physiological roles of specific enzymes or polypeptides in living bodies. Mice are the animals commonly used widely as models for elucidating the biological functions of mammals including humans. Thus, the techniques and analyses for murine trehalase in a molecular biological manner would be particularly useful for elucidating the physiological roles of mammalian trehalase. Any nucleotide sequence of murine trehalase, which is requisite for its molecular biological engineering, has not been elucidated, and any structure of the enzyme per se has not been disclosed. Urgently expected are as follows: The elucidation of a nucleotide sequence of a DNA for murine trehalase, the establishment of a DNA useful for engineering the enzyme in a molecular biological manner, and uses thereof.
SUMMARY OF THE INVENTION
In view of the foregoing, the object of the present invention is to provide a DNA useful for engineering murine trehalase in a molecular biological manner, and uses thereof.
To attain the above object, the present inventors widely screened cDNAs for RNAs, collected from mice, to isolate a cDNA for murine trehalase. As a result, the present inventors obtained from a murine intestine a cDNA which consists of about 2,000 base pairs (hereinafter abbreviated as “bp”) and expresses a polypeptide with trehalase activity. When compared with nucleotide sequences of conventionally known DNAs, the cDNA contained, as a coding sequence of the polypeptide, a nucleotide sequence of SEQ ID NO: 1 which was clearly different from conventionally known DNAs. The present inventors also found the fact that a gene for the cDNA exists on a murine chromosome. Based on these results, they confirmed that the cDNA was for murine trehalase, and then analyzed murine genomic DNAs with reference to the nucleotide sequence of the cDNA, elucidated the structure of a gene for murine trehalase which consists of a total length of about 20,000 bp and comprises the nucleotide sequence of SEQ ID NO: 1 and introns for splitting the nucleotide sequence, and isolated the gene as a genomic DNA. The present inventors also confirmed that a part or the whole of the isolated DNA can be arbitrarily used to engineer and analyze murine trehalase in a molecular biological manner. For example, such a DNA can be advantageously used to prepare transformants suitable for producing polypeptides used as murine trehalase standard specimens and as antigens for preparing anti-murine trehalase antibodies, and to prepare transgenic animals and trehalase gene knockout animals. The present invention is based on these findings.
The present invention solves the above object by providing a DNA which comprises a part or the whole of the nucleotide sequence of SEQ ID NO: 1 that encodes trehalase, a polypeptide obtainable by the expression of the DNA, a process for producing the polypeptide using the DNA, and a transgenic- and knockout-animals obtainable therewith.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a novel DNA which encodes trehalase and uses thereof. The term “trehalase” as referred to in the present invention means an enzyme, i.e., a protein or polypeptide which specially hydrolyzes the glucosidic bond in &agr;,&agr;-trehalose, and the hydrolysis activity is called “trehalase activity” in the present invention. The DNA of the present invention comprises a part or the whole of the nucleotide sequence of SEQ ID NO: 1 which encodes trehalase, and usually it is mouse origin.
In the present invention, the term “a part or the whole of the nucleotide sequence of SEQ ID NO: 1” generally means “a nucleotide sequence which contains at least ten and several contiguous bases in the nucleotide sequence of SEQ ID NO: 1.”
Preferred examples of the DNA of the present invention include DNAs as cDNAs which comprise a part or the whole of the nucleotide sequence of SEQ ID NO: 1, DNAs as genomic DNAs obtainable by fragmenting chromosomes, and other DNAs obtainable by applying replacement, deletion, and/or addition of bases to the above DNAs. These DNAs do not contain telomeres as a characteristic structure in the terminal region of chromosomes of eukaryotic cells such as mammalian cells, and such DNAs are usually provided in the from of isolated liner DNAs which are composed of not more than about 20,000 bp and which are distinguishable from naturally-occurring chromosomal DNAs and RNAs present in mammalian cells. All of these DNAs according to the present invention may be homologous to the nucleotide sequences of cDNAs for human and rat trehalases, registered in “GenBank”, a database of nucleic acids provided by the National Institute of Health of USA, under the accession Nos. AB000824 and AF038043, respectively, but are not completely coincided with the above registered nucleotide sequences.
A cDNA, as an example of the DNA of the present invention, usually comprises a part or the whole of the nucleotide sequence of SEQ ID NO: 1 as a coding sequence or nucleotide sequence which encodes the polypeptide of the present invention, and may contain a nucleotide sequence as a non-coding region at the 5′ and/or 3′ end regions. Usually, such a cDNA can be obtained by preparing in the usual manner a cDNA using a RNA as a template obtainable from organs such as intestines, kidneys, livers, and lungs from mice or their relative rodents, and cell lines established from these organs; and screening the cDNA with an index of the existence of annealing with at least a part of the nucleotide sequence of SEQ ID NO: 1. Any method such as PCR, colony hybridization, or plaque hybridization method commonly used in this art can be used as the screening method. Although the DNAs thus obtained are varied depending on the nature of species, strains, individuals, and
Ariyasu Harumi
Kurimoto Masashi
Ohta Tsunetaka
Yanai Yoshiaki
Browdy and Neimark
Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo
Reynolds Deborah J.
Woitach Joseph
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