Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-10-16
2004-09-14
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S024100
Reexamination Certificate
active
06790617
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a promoter of a gene expressed in renal cells. The promoter of this invention can be applied to the field such as gene therapy.
BACKGROUND ART
Sixty trillion various cells in a living body have essentially identical genomic DNA. For the normal physiological functions, the expression of these genes is strictly controlled through signals received by cell lines and cells. Therefore, elucidation of genes expressed specifically in each cell type is very important.
Mesangium is located in the center of lobula of capillary loop in glomerulus and is a tissue of a core that connects each lobule. Mesangium is covered by glomerular basal membrane and comprises mesangial cells which are separated from capillary cavity by endothelial cells and amorphous material (mesangial matrix) which is continuous with internal hyaline layer in glomerular basal membarane consisting of three layers.
A mesangial cell is known to play a pivotal role in maintaining the structure and function of a glomerulus and is considered to be the major cause of the onset of glomerular diseases such as glomerulonephritis and glomerulosclerosis. A mesangial cell is a target of disorders for each type of nephritis. For example, proliferation of mesangial cells and accumulation of extracellular mesangial matrix are thought to be the first step in which glomerulosclerosis is developed in a patient suffering from various glomerular diseases such as chronic glomerulonephritis and diabetic nephropathy, the two major causes of the end stage of renal failure [D. Schlondorff, Kidney Int., 49, 1583-1585 (1996); R. B. Sterzel et al., Glomerular mesangial cells. Immunologic Renal Diseases, pp595-626 (1997)]. Therefore, identification of genes expressed specifically in mesangial cells and elucidation of mechanism regulating its expression are helpful for understanding biological characteristics of mesangial cells and the causes of diseases relating to mesangial cells, and in turn, treating or diagnosing diseases relating to mesangial cells.
Through the determination of large-scale DNA sequences and through the database analysis, the present inventor isolated a gene named MEGSIN that is strongly expressed specifically in mesangial cells. The inventor also determined the whole nucleotide sequence of the gene and deduced the amino acid sequence of the novel protein (human MEGSIN) comprising 380 amino acids encoded by the whole cDNA clone of MEGSIN. Furthermore, the homology search in amino acid sequences with FASTA program using SwissProt database revealed that human MEGSIN belonged to SERPIN (serine protease inhibitor) superfamily [R. Carrell et al., Trends Biochem. Sci., 10, 20 (1985); R. Carrell et al., Cold Spring Harbor Symp. Quant. Biol., 52, 527 (1987); E. K. O. Kruithof et al., Blood, 86, 4007 (1995); J. Potempa et al., J. Biol. Chem., 269, 15957 (1994); E. Remold-O'Donnell, FEBS Lett., 315, 105 (1993)] [T. Miyata et al., J. Clin. Invest., 120, 828-836 (1998)].
Human MEGSIN is weakly expressed in human fibroblasts, smooth muscle cells, endothelial cells, and keratinocytes, but is strongly expressed in mesangial cells (that means human MEGSIN gene is expressed specifically in mesangial cells). When compared between IgA nephropathy patients or diabetic nephropathy patients and normal healthy people, the expression level of MEGSIN in renal tissue is significantly larger in IgA nephropathy patients or diabetic nephropathy patients [D. Suzuki et al., J. Am. Soc. Nephrol. 10, 2606-2613 (1999)]. Also, increase in expression level was observed in the model of mesangial proliferative glomerulonephritis using rats.
As indicated above, there is a possibility that the expression of MEGSIN gene is deeply involved in renal disease. Therefore, it is desired to reveal the actual condition of the regulatory mechanisms of the expression of MEGSIN gene, to clarify the function of human MEGSIN in vivo, and to provide useful promoter that is available for diagnosis and treatment of, for example, genetic disease caused by the mutation of MEGSIN or that expresses specifically in mesangial cells.
On the other hand, because the members of SERPIN superfamily to which human MEGSIN belongs are highly similar to each other in their primary structures, they are thought to be derived from the evolutionary common ancestral protein. Namely, as a result of the analysis of the phylogenetic tree constructed based on the number of mutated amino acids in the sequences [K. Suzuki et al., Tanpakushitsu Kakusan Koso, 34, 949-962 (1989)] and on the chromosomal gene structure [J. J. Bao et al., Biochem., 26, 7755 (1987)], it has been revealed that SERPIN superfamily has evolved through not less than 5 million years with various higher vertebrates. It is extreme characteristic of MEGSIN gene that it is expressed specifically in mesangial cells in glomerulus.
Recently, it was reported that genes of ion channels and genes involved in transportation are expressed specifically in kidney [S. J. Lolait et al., Nature, 357, 336-339 (1992); Y. Kanai et al., J. Clin. Invest., 93, 397-404 (1994); S. Uchida et al., J. Biol. Chem., 268, 3821-3824 (1993); S. Adachi et al., J. Biol. Chem., 269, 17677-17683 (1994); K. Fushimi et al., Nature, 361, 549-552 (1993); G. Gamba et al., J. Biol. Chem., 269, 17713-17722 (1994)].
However, these genes are located in epithelial cells of renal tubular and are not expressed in mesangial cells of glomeruli. Therefore, identification of the promoter and the transcription factors of MEGSIN gene can bring important information about the mechanism of gene expression depending on specific cell type. This information can also be applied to target cells in molecular genetics and gene transfer.
DISCLOSURE OF THE INVENTION
An objective of this invention is to provide a promoter of MEGSIN gene and its use.
The present inventor has determined nucleotide sequences of genomic DNA which is about 1.5 kb long including the upstream (5′ terminal) sequences of MEGSIN gene to reveal the regulatory mechanism of expression of MEGSIN gene. As a result of S1 nuclease protection assay, it has been revealed that four transcription initiation sites are existed in MEGSIN mRNA. It has also been revealed that the conserved transcription regulatory sequence that can be the transcription regulatory sites including AP1 binding site, cMyb binding site, and Oct-1 exist upstream of the transcription initiation site. Vectors in which various region of this transcription regulatory sequence were deleted and luciferase gene was integrated into the 3′ region have been constructed and the transcription regulatory region has been determined by detecting luciferase activity in cells transfected with the vector. As a result, two regulatory sequences have been identified that control transcription positively. The transcription regulatory activity of one promoter region that is located at 3′ end has been analyzed in detail by introducing nucleotide substitution based on the method of site specific mutagenesis. As a result, the present inventor has succeeded in determining the nucleotide sequence of the DNA that plays an important role in transcriptional regulation.
Therefore, the present invention relates to a promoter of MEGSIN gene and its use, and more specifically relates to the following:
(1) a DNA comprising the nucleotide sequence of SEQ ID NO: 1 or a part thereof, the DNA having a promoter activity;
(2) a vector comprising the DNA of (1);
(3) the vector of (2), wherein a foreign gene is expressibly ligated downstream of the DNA of (1);,
(4) a cell transfected with the vector of (3);
(5) a method for screening a protein that binds to a DNA comprising the nucleotide sequence of SEQ ID NO: 1 or a part thereof, the method comprising the steps of: (a) contacting a test sample with the DNA comprising the nucleotide sequence of SEQ ID NO: 1 or a part thereof, and (b) selecting a protein that has an activity to bind the DNA comprising the nucleotide seque
Bozicevic Karl
Bozicevic Field & Francis LLP
McKelvey Terry
Toshio Miyata
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