Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-08-14
2002-11-26
LeGuyader, John L. (Department: 1635)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S325000, C435S387000, C536S023100, C536S023500, C536S024500, C530S350000
Reexamination Certificate
active
06485936
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a heat shock transcription factor binding protein. More particularly, the present invention relates to a DNA encoding a protein possessing a heat shock transcription factor 2 binding activity; a protein encoded by the DNA; an expression vector carrying the DNA; a transformant harboring the expression vector; a process for preparing the protein; an antibody against the protein; an antisense DNA or antisense RNA complementary to the DNA; and an oligonucleotide probe or primer capable of specifically hybridizing to the DNA.
BACKGROUND ART
Heat shock transcription factors (heat shock factor: HSF) bind to the heat shock element (HSE) located upstream of the promoter for heat shock protein (HSP) genes in response to various stresses, thereby regulating the transcription from the promoter.
In HSF of higher eukaryotes, there have been so far known four kinds, namely HSF1, HSF2, HSF3 and HSF4. Among them, HSF1 and HSF3 are activated by stresses including heat, heavy metals and amino acid analogues. HSF2 does not respond to these stresses but is thought to play an important role during differentiation and development. In addition, HSF4 does not have any transcriptional activation domain, and is believed to negatively regulate DNA binding of other HSFs.
The DNA binding activity of HSF2 has been observed in hemin-treated human K562 erythroleukemia cells, in spermatids of mouse testis, in mouse F9 embryonal carcinoma cells, and during early embryogenesis in mice. While the mechanism of the transcriptional regulation of HSP genes by HSF1 has been studied extensively and well understood, very little is known about the transcriptional regulation by HSF2. Concretely, there have been reported that in hemin-treated K562 cells, HSF2 binds to HSE on the HSP70 gene (Sistonen, L. et al., (1992)
Mol. Cell. Biol
. 12, 4104-4144), and that in mouse spermatogenic cells, HSF2 binds constitutively to HSE on the testis-specific HSP70.2 gene (Sarge, K. D. et al., (1994)
Biol. Reprod
. 50, 1334-1343). In each case, HSF2 is deduced to be involved in expression of HSP70 and HSP70.2. On the other hand, in F9 cells, the binding of HSF2 to HSE located upstream of the HSP70 promoter has not been found Murphy, S. P. et al., (1994)
Mol. Cell. Biol
. 14, 5309-5317), and moreover in mouse embryos, no correlation in the expression patterns of the main HSP and HSF2 has been found (Rallu, M. et al., (1997)
Proc. Natl. Acad. Sci. USA
94, 2392-2397).
Since HSF2 can actually bind to HSE located upstream of the HSP70 promoter in K562 cells or in spermatogenic cells, it is believed that there is a possibility that there exists a factor which regulates activation of HSF2 in these cells. However, the regulatory factor has not yet been known at present.
The term “spermatogenesis” refers to a process in which spermatogonia, immature masculine reproductive cells, proliferate by mitosis to form spermatocytes; meiosis of the spermatocytes takes place to form spermatoblasts; and further complicated morphological changes result in maturing to sperm. During the spermatogenesis process, expression of HSP is strictly regulated. In general, when exposed to deleterious environmental conditions for cells (under stress), such as high temperature, HSP is rapidly induced in order to prevent irreversible denaturation of intracellular proteins and to protect the cells from disorder caused by the stress. Besides the HSP induced by the stress, there are HSPs (i.e. molecular chaperone) having a role of aiding in folding, association and intracellular translocation of nascent proteins even under usual conditions, and these proteins are constitutively expressed in cells. In the process of spermatogenesis, the gene expression of reproductive cells greatly changes, and as a result, intracellular proteins dramatically change. Therefore, HSPs required for folding and localization of these proteins have been regulated so as to be correctly expressed at the necessary timing (Sarge, K. D. and Cullen, K. E., (1997)
Cell. Mol. Life Sci
. 53, 191-197).
Many of HSPs have been identified also in testis as in other tissues and cells. The HSP which is expressed at the highest level in mouse testis is HSP70.2, belonging to the HSP70 family, and its expression is testis-specific and constitutive, and is not induced by heat shock. There have been reported that HSP70.2 is expressed at a high level in meiosis phase of the spermatogenetic process, namely from spermatocytes to spermatoblasts (Allen et al., (1988)
Mol. Cell. Biol
. 8, 828-831; Zakeri, Z. F. et al., (1988)
Mol. Cell. Biol
. 8, 2925-2932), and that meiosis does not take place in mice of which HSP70.2 gene is disrupted, and consequently the spermatoblast is not formed, resulting in infertility (Dix, D. J. et al., (1996)
Proc. Natl. Acad. Sci. USA
93, 3264-3268). On the other hand, HSF2 is expressed from spermatocytes to spermatoblasts in the spermatogenetic process, and can bind to the HSP70.2 promoter in vitro. Therefore, it is suggested that HSF2 is involved in the transcriptional regulation of HSP70.2 which has a role essential in the spermatogenesis as a molecular chaperone (Sarge, K. D. et al., (1994)
Biol. Reprod
. 50, 1334-1343).
DISCLOSURE OF INVENTION
The present invention has been accomplished in view of the above prior arts, and an object of the present invention is to provide an HSF2 binding factor as an activation regulatory factor of HSF2, which is thought to be involved in the transcriptional regulation of HSP70.2, which has a role essential for spermatogenesis by testis-specific expression. Another object of the present invention is to provide a DNA encoding the HSF2 binding factor. A still another object of the present invention is to provide an expression vector carrying the above DNA or a part thereof, and a transformant harboring the expression vector. A still another object of the present invention is to provide a process for preparing a recombinant protein, comprising culturing the above transformant. A still another object of the present invention is to provide an antibody or a fragment thereof capable of specifically binding to the regulatory factor. A still another object of the present invention is to provide an antisense DNA or antisense RNA of which sequence is complementary to the above DNA. A still another object of the present invention is to provide an oligonucleotide probe or primer capable of specifically hybridizing to the above DNA.
Concretely, the gist of the present invention relates to:
[1] a DNA encoding a protein having a heat shock transcription factor (HSF) 2 binding activity, which is selected from the group consisting of:
(a) a DNA encoding a peptide comprising the amino acid sequence of SEQ ID NO: 1;
(b) a DNA encoding a peptide comprising an amino acid sequence resulting from deletion, substitution, insertion or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 1;
(c) a DNA comprising the nucleotide sequence of SEQ ID NO: 2;
(d) a DNA comprising a nucleotide sequence resulting from deletion, substitution, insertion or addition of one or more bases in the nucleotide sequence of SEQ ID NO: 2;
(e) a DNA capable of hybridizing with a DNA of any one of (a) to (d), under stringent conditions;
[2] a protein encoded by the DNA of item [1] above;
[3] an expression vector carrying all or a part of the DNA of item [1] above;
[4] a transformant harboring the expression vector of item [3] above;
[5] a process for preparing a recombinant HSF2 binding protein, comprising the step of culturing the transformant of item [4] above under conditions capable of expressing a protein from the expression vector of item [3] above;
[6] an antibody or a fragment thereof capable of specifically binding to the protein of item [2] above;
[7] an antisense DNA or antisense RNA comprising 8 or more bases, of which sequence is complementary to the DNA of item [1] above; an
Yanagi Hideki
Yoshima Tadahiko
Yura Takashi
Birch Stewart Kolasch & Birch, LLP.
HSP Research Institute, Inc.
LeGuyader John L.
Schmidt M
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