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
2001-03-16
2003-12-02
Romeo, David S. (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069100, C435S325000, C435S252300, C435S252330, C435S320100, C530S350000, C536S023500, C536S023510, C536S024100
Reexamination Certificate
active
06656708
ABSTRACT:
FIELD OF INVENTION
This invention relates to the field of genetic engineering, and, in particular, relates to the nucleotide sequence of a novel human gene. More particularly, this invention relates to the cDNA sequence of a novel human Growth/Differentiation Factor (hGDF3-2), which is a splice variant of hGDF3. The invention also relates to the polypeptides encoded by the nucleotide sequence, the uses of these polynucleotides and polypeptides, and the methods for producing them.
PRIOR ART
Transforming Growth Factor-&bgr; (TGF-&bgr;) was discovered about 15 years ago, using biochemical means. It is a protein with many biological regulatory activities. Shortly after the discovery, it was found that TGF-&bgr; represented a group of growth factors with various functions. In different organisms, these factors exert important regulatory functions on cell growth, differentiation and tissue morphogenesis. (Handbook of Experimental Pharmacology, 1990, Vol.95, p419-475, Springer Verlag, Geidelberg). TGF-&bgr;, together with these related proteins, forms a superfamily named TFG-&bgr; superfamily. Up to now, the TGF-&bgr; superfamily has over 30 different members. There are four major families in the TGF-&bgr; superfamily (Proc Soc Exp Biol Med, 1997, 214(1), 27-40), which are: (1) the Mullerian inhibitory substance (MIS) family—MIS regulates Mullerian duct regression in male embryos; (2) the inhibin/activin family—Inhibins block the follicle stimulating hormone (FSH) release by the pituitary cell, and activins stimulate FSH release; (3) Vg-related family, which includes bone morphogenic protein (BMP), dorsalin-1 (which regulates the differentiation of neural tube), growth/differentiation factor GDF-1, DPP, Vgl in the Xenopus and the murine homolog Vgr-1, etc.; (4) TGF-&bgr; family, which includes five isoforms of TGF-&bgr; (TGF-&bgr; 1-5).
As a representative of this superfamily, TGF-&bgr; has been extensively and intensively studied. The investigation indicates that TGF-&bgr; is a strong endogenous mediators of tissue repair via their stimulatory effects on chemotaxis, angiogenesis, and extracellular matrix (ECM) deposition within the wound: environment. (Clin Immunol Immunopatol, 1997,83(1), 25-30). TGF-&bgr; also regulates the growth and differentiation of various cells (Bioessays,1997, 19(7), 581-591), either positively or negatively. Most of the evidences suggest that TGF-&bgr; exerts its regulatory effects at the G1 phase of cell cycle. Besides, it is reported that TGF-&bgr; can induce cell death of some sensitive cell types, including hepatoma, myeloid, and osteoclast cells. In vitro experiments also show TGF-&bgr; regulates the differentiation of various cell strains, though the mechanism is still unknown. The regulatory activity of TGF-&bgr; on cell growth and differentiation naturally leads to considerations on the potential application in chemotherapy and cancer therapy. There have been considerable amount of reports concerning these topics (Clin Immunol Immunopathol, 1997, 83(1), 25-30; Bioessays, 1997, 19(7),581-591).
Members of TGF-&bgr; family have been found in many species, e.g., Xenopus, fowl, mice, swine, bovine, etc. Human TGF-&bgr; (−1,−2,−3) were cloned in the late 1980's. Among them, the sequencing of TGF-&bgr;
1
, was finished by Derynck R et al. in 1985. (Nature, 1985, 316(6030), 701-705). By analyzing the sequence encoding TGF-&bgr;
1
, they found that functional TGF-&bgr; was produced by splicing a precursor that was much longer than the mature protein. Later, people found this phenomenon was common in TGF-&bgr; superfamily. In 1988, the TGF-&bgr;
2
and TGF-&bgr;
3
nucleotide sequences were obtained by Madisen L et al. and Ten Dijke P et al., respectively. (Proc Natl Acad Sci USA, 1988, 85(13), 4715-4719; DNA, 1988, 7(1), 1-8). Sequence comparison showed the homology between TGF-&bgr;
2
, TGF-&bgr;
3
and TGF-&bgr;
1
was 70%-80%.
Along with the steady improvements of gene cloning and sequencing techniques, more and more members of TGF-&bgr; superfamily have been cloned since 1990. Alexandra. C reported in 1993 that they found a novel member of TGF-&bgr; superfamily—murine Growth/Differentiation Factor 3, GDF-3 (J. Biol. Chem., 1993, 268(5), 3444-3449). The homology between GDF-3 and other members of the TGF-&bgr; superfamily is not very high. But it still contains the unique conservative sequence of the TGF-&bgr; superfamily. In particular, it lacks the fourth cystein of the seven conservative cysteins of the superfamily, indicating that it might have some particular property.
The homologue of GDF-3 in human was cloned in 1998 (Oncogene, 1998, 16, 95-103). This protein is highly homologous to the murine GDF-3, and thus named hGDF-3. Nevertheless, it is noteworthy that hGDF-3 is much shorter than GDF-3, mainly due to the lack of nearly 50 residues in the N-terminal. Moreover, two residues corresponding to residues 128 and 248 in the murine GDF-3 are also deleted in hGDF-3. This change of hGDF-3 is supposed to be the result of alternative splicing variation or genetic evolution.
Prior to this invention, no other forms of hGDF3 has been isolated or disclosed.
SUMMARY OF INVENTION
One purpose of the invention is to provide a new polynucleotide which encodes a splice variant of human growth/differentiation factor hGDF3. The splice variant of hGDF3 of the invention is named hGDF3-2.
Another purpose of the invention is to provide a novel protein, which is named hGDF3-2.
Still another purpose of the invention is to provide a new method for preparing said new hGDF3-2 protein by recombinant techniques.
The invention also relates to the uses of said hGDF3-2 protein and its coding sequence.
In one aspect, the invention provides an isolated DNA molecule, which comprises a nucleotide sequence encoding a polypeptide having human hGDF3-2 protein activity, wherein said nucleotide sequence shares at least 70% homology to the nucleotide sequence of nucleotides 14-1105 in SEQ ID NO: 5, or said nucleotide sequence can hybridize to the nucleotide sequence of nucleotides 14-1105 in SEQ ID NO: 5 under moderate stringency. Preferably, said nucleotide sequence encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 6. More preferably, the sequence comprises the nucleotide sequence of nucleotides 14-1105 in SEQ ID NO: 5.
Further, the invention provides an isolated hGDF3-2 polypeptide, which comprises a polypeptide having the amino acid sequence of SEQ ID NO: 6, its active fragments, and its active derivatives. Preferably, the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 6.
The invention also provides a vector comprising said isolated DNA.
The invention further provides a host cell transformed with said vector.
In another aspect, the invention provides a method for producing a polypeptide with the activity of hGDF3-2 protein, which comprises:
(a) forming a hGDF3-2 protein expression vector comprising the nucleotide sequence encoding the polypeptide having the activity of hGDF3-2 protein, wherein said nucleotide sequence is operably linked with an expression regulatory sequences, and said nucleotide sequence shares at least 70% homology to the nucleotide sequence of positions 14-1105 in SEQ ID NO: 5;
(b) introducing the vector of step (a) into a host cell, thereby forming a recombinant cell of hGDF3-2 protein;
(c) culturing the recombinant cell of step (b) under the conditions suitable for the expression of hGDF3-2 polypeptides;
(d) isolating the polypeptides having the activity of hGDF3-2 protein.
In one embodiment of the present invention, the isolated polynucleotide has a full length of 1141 nucleotides, whose detailed sequence is shown in SEQ ID NO: 5. The open reading frame (ORF) is located at nucleotides 14-1105.
In the present invention, the term “isolated” or “purified” or “substantially pure” DNA refers to a DNA or fragment which has been isolated from the sequences which frank it in a naturally occurring state. The term also applies to DNA or DNA fragment which has been isolated from other components naturall
Dai Fangyan
Fu Qiang
Yu Long
Zhang Honglai
Zhao Shouyuan
Merchant & Gould P.C.
Romeo David S.
Yu Long
LandOfFree
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