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
2000-04-25
2003-12-30
Romeo, David (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...
C435S357000, C435S325000, C435S252300, C435S320100, C530S350000, C536S023500
Reexamination Certificate
active
06670151
ABSTRACT:
BACKGROUND OF THE INVENTION
Field of the Invention
This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production and isolation of such polynucleotides and polypeptides. More particularly, the polynucleotides and polypeptides of the present invention have been identified as being variant porcine stem cell factors, and in particular membrane-bound stem cell factors, and still more particularly as being involved in stem cell, useful for supporting proliferation and growth of porcine bone marrow cells.
SUMMARY OF THE INVENTION
The present invention provides in one aspect a novel polypeptide which has been characterized as an active form of porcine stem cell factor cDNA gene sequence(PSCF) that omits exon 6. Preferably, the PSCF is a splice variant wherein exon 6 is omitted. More preferably, exon 6 is removed from the native full-length porcine stem cell factor and is omitted entirely or is replaced with a polynucleotide that encodes one or more amino acids.
In a preferred aspect the invention provides a PSCF encoded by a polynucleotide sequence corresponding to the full-length native porcine stem cell factor cDNA, but in which: (1) the first 70 nucleotides are removed, (2) exon 6 is excised (nucleotides 591 to 654) from the full polynucleotide sequence, (3) the excised exon 6 segment is replaced by a three-nucleotide segment encoding the amino acid “Gly”, and (4) the fifteen-nucleotide C-terminal tail (nucleotides 938-952) is removed and replaced by the six-nucleotide segment 5′-TCTAGA-3′ (SEQ ID NO: 11).
In accordance with another aspect of the present invention, there are provided novel PSCF polypeptides, as well as active fragments, analogs and derivatives thereof. In a preferred aspect the present invention provides novel membrane-bound PSCF polypeptides, wherein the polypeptide segment encoded by exon 6 is omitted or replaced by an inactive polypeptide segment. In another preferred aspect the present invention provides such novel PSCF polypeptides which are soluble PSCF polypeptides.
In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding the polypeptides of the present invention including mRNAs, cDNAs, genomic DNAs as well as active analogs and fragments of such polypeptides.
In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptides by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing a nucleic acid sequence of the present invention, under conditions promoting expression of said polypeptides.
In accordance with a further aspect of the invention, the polypeptide of the invention may be anchored to a cell's surface, and the modified cell, as a feeder cell for culturing porcine cells.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides for analyzing potential agonists to the polypeptides, Another process utilizes the polynucleotides to assay for compounds which bind said polynucleotides and would thus block expression of any products from said polynucleotides.
In accordance with yet a further aspect of the present invention, there are also provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to a nucleic acid sequence of the present invention.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides, for purposes related to scientific research for example, to generate probes for identifying similar sequences which might encode similar polypeptides from other organisms by using certain regions, i.e., conserved sequence regions, of the nucleotide sequence.
In accordance with an aspect of the present invention, there are provided isolated nucleic acids (polynucleotides) which encode PSCF polypeptides, in which polynucleotides exon 6 has been removed, deactivated or replaced by an inactive portion (SEQ ID NO:8) as shown in
FIG. 4
, as well as said encoded mature PSCF polypeptide as shown in
FIG. 4
(SEQ ID NO:9).
These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.
Definitions
In order to facilitate understanding of the following description and examples which follow certain frequently occurring methods and/or terms will be described.
The term “gene” means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening non-coding sequences (introns) between individual coding segments (exons).
A coding sequence is “operably linked to” another coding sequence when RNA polymerase will transcribe the two coding sequences into a single mRNA, which is then translated into a single polypeptide having amino acids derived from both coding sequences. The coding sequences need not be contiguous to one another so long as the expressed sequences ultimately process to produce the desired polypeptide.
“Recombinant” polypeptides refer to polypeptides produced by recombinant DNA techniques; i. e., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide. “Synthetic” polypeptides are those prepared by chemical synthesis.
A DNA “coding sequence of” or a“nucleotide sequence encoding” a particular polypeptide, is a DNA sequence which is transcribed and translated into a polypeptide when placed under the control of appropriate regulatory sequences.
“Plasmids” are designated by a lower case “p” preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
“Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 &mgr;g of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 &mgr;l of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 &mgr;g of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37° C. are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel or an agarose gel to isolate the desired fragment.
“Oligonucleotides” refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5′ phosphate and thus will not ligate to another oligonucleotide without adding a phosphate group with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
“Ligation” refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (
J. Sambrook
et al., 1989, in Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory, NY). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase (“ligase”) per 0.5 &mgr;g of approximately equimolar amounts of the DNA fragments to be ligated.
Unl
BioTransplant, Inc.
Grant Alan J.
OIstein Elliot M.
Romeo David
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