JP2002541850A - Genes and expression products from hematopoietic cells - Google Patents

Abstract

  (57) [Summary] Disclosed are human hematopoietic stem / progenitor cell (hHSPC) polypeptides (referred to as C17 polypeptides), as well as DNA (and RNA) encoding such polypeptides. Also disclosed are markers for chromosomal mapping, DNA fingerprinting and possible roles performed by genetic mutations in the disease process and the generation of polyclonal sera or monoclonal antibodies specific for said polypeptide, etc. This is a method utilizing the polynucleotide and the polypeptide disclosed herein. Further disclosed is a method for increasing the growth rate of hMSCs utilizing a polypeptide of the invention.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD OF THE INVENTION

This application is related to US Provisional Patent Application No. 60/129, filed April 15, 1999,
No. 643, the disclosure of which is incorporated in its entirety into this application.

[0002] The present invention relates to a newly identified polynucleotide corresponding to a transcript of a human gene.
Related to the sequence and its related complete gene sequence and its gene expression product.
And uses of the foregoing, especially when they involve hematopoietic and bone marrow microenvironments
With regard to their use. More specifically, the invention disclosed herein relates to CD34⁺
Expressed on hematopoietic stem cells and or hematopoietic progenitor cells (HSPCs), ^- It relates to a novel gene that is not expressed in hematopoietic cells.

It is well established that circulating blood cells are the product of terminal differentiation of a number of determined progenitor cells. During fetal life, hematopoiesis develops throughout the reticuloendothelial system, whereas in adults, progenitor cells (eg, leukocytes, erythrocytes and platelets) develop in the bone marrow, especially the bone marrow of the axial skeleton. Bone marrow photographs and biopsy specimens contribute a great deal of information about the status of hematopoiesis in living organisms, especially humans.

[0004]

[Prior art]

Researchers have defined pluripotent stem cells that can produce any and all of the blood cells. In particular, this pluripotent stem cell found in the bone marrow differentiates along one of two well-defined pathways. The stem cells thus differentiate into bone marrow stem cells and ultimately produce all terminally differentiated blood cells except B and T lymphocytes,
Or differentiate into T lymphocytes (T cells). Current research into the nature of hematologic diseases as well as the formation of differentiated blood cells tends to focus on these progenitors, giving rise to what has been termed the progenitor basis of hematopoiesis.

[0005] If it was not for the discovery of the presence on the surface of such cells of various cell surface antigens, which are proteins specific to a particular type of cell or a particular group of cell types, the bone marrow It would have been very difficult to follow the growth of various types of blood cells within the fraction. As cells differentiate, the pattern of cell surface antigens on their surface changes as various genes within the cell stop or restart in each successive generation.

[0006] Most such markers are classified using the popular "CD" nomenclature used, inter alia, when describing hematopoietic cells. The term "CD" is understood as describing either the "community name" or the "differentiated colony" and identifies a stage of differentiation of a cell and thus includes a class of cells that operate at different stages of the hematopoietic process. Reference is made to a molecule recognized by a "population" of monoclonal antibodies useful for distinguishing one hematopoietic cell from another.

[0007] These CD proteins are used routinely to identify various types of cells and to follow the hematopoietic transition from progenitor stem cells to terminally differentiated progeny. For example, CD34 is a protein approximately 105-120 kilodaltons in size and is present on hematopoietic stem cells.

[0008] Human hematopoietic stem / progenitor cells (HSPC) are enriched in a rare population of bone marrow (BM) mononuclear cells (MNC) that carry the CD34 surface antigen (CD34 ⁺ ). CD in addition to BM
34 ⁺ HSPC is also found in neonatal cord blood (CB) and is enriched in peripheral blood after mobilization with cytokines and chemotherapy (Klaus et al., Blood, 87, 1 (1996))
. CD34 ⁺ cells account for ~ 1-4% of MNCs in BM and ~ 0.5% for CB and recruited peripheral blood (mPB). Purified CD34 ⁺ cells can be transplanted into the bone marrow and produce blood / lymphoid cells for many years after transplantation in patients. In addition, individual C
D34 ⁺ cells can form colonies of hematopoietic cells in culture. Conversely, CD3
Mononuclear cells lacking 4 expression (CD34 ⁻ ) are large, mature hematopoietic cells of various differentiation lineages and have lost the ability to form colonies in culture. (Klaus et al., 19
96).

The present invention discloses herein the diagnostic and therapeutic use of a novel secreted protein and its encoded gene, the latter gene being specifically expressed in CD34 ^- hematopoietic stem / progenitor cells.

[0010]

Summary of the Invention

Human hematopoietic stem / progenitor cells (hHSPCs) are present in blood from bone marrow and umbilical cord (C
A relatively rare population of mononuclear cells that express the CD34 cell surface antigen (of the cells as represented by D34 ⁺ ). However, the CD34 gene is not exclusively expressed on HSPCs (eg, endothelial cells also express high levels of CD34).

In accordance with the present invention, blood cells obtained from the umbilical cord have been discovered to express a novel protein if the cells had the CD34 surface antigen, and CD34
If was not detected in the cells, it was not expressed.

[0012] The cells are used to prepare cDNA clones of the novel gene, and expression products containing the nucleic acids, the segregating sequences, fragments thereof, and transform them into expression products of the sequences containing these nucleic acids and fragments thereof. Use is the object of the present invention.

Furthermore, protein expression products of cDNAs such as mRNAs identified according to the present invention expressed in hematopoietic stem / progenitor cells, as well as other nucleic acids, especially isolated nucleic acids, nucleotide sequences of such nucleic acids, fragments thereof, Providing is another object of the present invention.

[0014] Provided is a polypeptide expression product of the nucleotide sequences disclosed herein, wherein the polypeptide acts as a growth factor for mesenchymal stem cells, which cells can differentiate into most types of connective tissue cells, It is another object of the invention to provide such a stem cell with a polypeptide expression product that is useful, for example, in replacement therapy and the like.

Furthermore, it is possible to use the cDNAs thus produced, and fragments thereof, as well as their expression products as chromosomal markers to determine the location of such genes, including any of their alleles in the genome. It is an object of the present invention.

[0016] Furthermore, there is provided a DNA sequence for use in human "fingerprinting" wherein different individuals can be identified based on the sequence of a gene identified as completely or partially identical to that disclosed herein. Doing so is another object of the present invention.

Furthermore, such a sequence can be compared with that found at a similar chromosomal location in mammals, especially humans, where such mammals suffer from disease, thereby reducing the mutation of said gene. It is another object of the invention to detect the presence and provide a polynucleotide sequence corresponding to the gene encoding the polypeptide disclosed herein, wherein said mutation probably leads to such a disease.

Furthermore, the replication of one or more nucleic acids, or DNAs, or genes, or nucleotide sequences according to the invention, capable of expressing a peptide, polypeptide or protein according to the invention for their rapid cloning, It is an object of the present invention to provide genetically engineered cells and vectors containing the same.

[0019]

DISCLOSURE OF THE INVENTION

One aspect of the present invention relates to nucleic acids that exhibit sequence homology to or can hybridize to the DNA sequence identified in FIG. 1 (SEQ ID NO: 1), and isolated DNA.
Sequences, as well as molecules, and fragments thereof (further matching isolated RNA sequences, and fragments thereof). The present invention further provides at least 15 bases, preferably at least 3 bases.
Directed to fragments or portions of such sequences containing 0 bases, more preferably at least 50 bases and most preferably at least 80 bases, and at least 60%, preferably at least 80% and most preferably at least 95% A DNA (or RNA) sequence that encodes the same polypeptide as the sequence of FIG. 1, including fragments and portions thereof, and further including its alleles when derived from a natural source, is directed to those sequences that are identical. Can be

According to the present invention, when referring to sequences, the terms “percent identical” or “percent identical” refer to the sequence to be compared or compared to the sequence to be compared (“comparison sequence”) (“the quoted sequence”). Sequence ") means that the sequence is compared to the described or claimed sequence after alignment. Percent identity is determined based on the following formula:

Percent identity = 100 [1-C / R] where C is the number of differences between the reference sequence and the reference sequence over the entire length of the alignment between the reference sequence and the reference sequence, where ( i) each base or amino acid in the reference sequence without the aligned bases or amino acids corresponding to the comparison sequence, and (ii) each gap in the reference sequence, and (iii) the aligned bases in the comparison sequence. Alternatively, each aligned base or amino acid in the quoted sequence that differs from the amino acid constitutes a difference. R is also the number of bases and amino acids in the reference sequence over the length of the alignment with the comparison sequence, along with any gaps created in the reference sequence and also counted as bases or amino acids.

If, as previously calculated, an alignment exists between the comparison sequence and the reference sequence that is about the same as or greater than the specified minimum percent identity, the percent identity calculated here The comparison sequence will have the specified minimum percentage relative to the reference sequence, even if there is an alignment that is less than or equal to the specified percent identity.

A further aspect of the invention is a DNA sequence identical to that contained in FIG. 1 (SEQ ID NO: 1) or a DNA sequence comprising that DNA sequence (also the corresponding RNA
Array). The DNA sequence according to the present invention can be hybridized under stringent conditions to the DNA sequence identified in FIG. 1 and set in the sequence listing (SEQ ID NO: 1). As used herein, the term "stringent conditions" means that hybridization occurs only if there is at least 97% identity between the sequences.

Yet another aspect of the invention is a human gene (or a DNA sequence encoding the same polypeptide, such as a coding region, etc.), especially isolated DNA comprising at least the coding region of an expressed human gene (or RNA) sequence or molecule, the human gene of which is the sequence shown in FIG. 1 (SEQ ID NO: 1).
) Or at least 90%, preferably at least 95%, and most preferably at least 98% identical thereto, and also encodes a polypeptide having a similar function to the polypeptide encoded by said coding region. DNA sequences homologous to or contributing to fragments or portions of the coding region to be converted. Thus, the isolated DNA (or RNA) sequence can include only the coding region of the expressed gene (or a fragment or portion thereof described above), or can contain all or a portion of the non-coding DNA (or RNA) of the expressed human gene. It may further include.

Generally, the sequence shown in FIG. 1 (SEQ ID NO: 1), or at least 9
0%, desirably 95%, and most desirably at least 98% are homologous to those which are identical or homologous, and the sequences contributing thereto are from the coding region of the human gene.

The present invention further relates to vectors or plasmids containing such DNA (or RNA) sequences, as well as to the use of DNA (or RNA) sequences.

The sequence shown in FIG. 1 (SEQ ID NO: 1) is capable of hybridizing to actual DNA and RNA sequences derived from different human tissues. The distribution of this sequence in various human tissues was determined from a database of other human sequences.

The polynucleotide of the present invention may be in the form of RNA or DNA,
NA includes cDNA, genomic DNA and synthetic DNA. DNA is double-stranded or single-stranded, and if single-stranded is the coding strand or non-coding strand (antisense strand). The coding sequence encoding the mature polypeptide is shown in FIG.
The coding sequence is identical to or different from the coding sequence shown in SEQ ID NO: 1 and encodes the same mature polypeptide as the DNA of FIG. 1 (SEQ ID NO: 1) as a result of redundancy or degeneracy.

The polynucleotide encoding the polypeptide of FIG. 2 (SEQ ID NO: 3) is not necessarily limited to the coding sequence of the mature polypeptide only; the mature polypeptide, and the leader or secretory sequence, the proprotein sequence and the membrane Additional coding sequences, such as anchors; coding sequences of the mature polypeptide (and optionally additional coding sequences) and non-coding sequences, such as non-coding sequences 5 'and / or 3' of the coding sequence of the intron or mature polypeptide.

Thus, the term “polynucleotide” as used in the present invention encompasses a polynucleotide comprising only the coding sequence of a polypeptide as well as a polynucleotide comprising additional coding and / or non-coding sequences.

The present invention further relates to variants of the polynucleotides described above, which encode fragments, analogs and derivatives of the polypeptide having the amino acid sequence of FIG. 2 (SEQ ID NO: 3). A variant of the polynucleotide is a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.

Thus, a nucleic acid, or polynucleotide, according to the present invention has a coding sequence that is a naturally occurring allelic variant of the coding sequence shown in FIG. As is known in the art, allelic variants have one or more nucleotide substitutions, deletions or additions, and are alternative forms of the polynucleotide sequence that do not substantially alter the function of the encoded polypeptide. It is.

The present invention further includes polynucleotides, wherein the coding sequence of the mature polypeptide facilitates attachment of the polypeptide to cell membranes, eg, a leader sequence that functions as a secretory sequence that controls transport of the polypeptide from the cell. Fused in the same reading frame to a polynucleotide sequence that facilitates expression and secretion of the polypeptide from the host cell, such as a transmembrane anchor. A polypeptide having a leader sequence is a preprotein that causes the leader sequence to be cleaved by the host cell to form a mature polypeptide. The polynucleotide is the mature protein plus 5 additional
'Encodes a proprotein that is an amino acid residue. Mature proteins with prosequences are proproteins and often are inactive forms of the protein. Once the prosequence is cleaved, the active mature protein remains.

Thus, for example, a polynucleotide according to the invention may be a mature protein, a protein with a prosequence, a protein with a transmembrane anchor, or a polypeptide with a prosequence, prosequence (leader sequence) and a transmembrane anchor. Code.

The polynucleotides of the invention have the coding sequence fused in frame to a marker sequence that allows for purification of the polypeptide of the invention. The marker sequence is hexahistidine provided in a pQE-9 vector that provides for purification of the mature polypeptide fused to the marker in the case of a cellular host, or for example, where the marker sequence is a mammalian host, such as a COS-7 cell. Is a hemagglutinin (HA) label. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (I. Wilson, et al., Cell. 37: 767 (1984)).

The fragments of the full-length polynucleotides of the present invention are used as hybridization probes in cDNA libraries to isolate full-length cDNAs and other cDNAs with high sequence similarity or similar biological activity to the gene. Is done. Probes of this type desirably have at least 15 bases, at least 30 bases and 5
It may even have zero or more bases. The probes are further used to identify cDNA clones corresponding to the full length transcript, and genomic clones or clones containing regulatory and promoter regions, exons, and introns. Examples of screens include separating the coding region of a gene by using a known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence that is complementary to the sequence of the gene of the invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine to which member of the library the probe will hybridize. Is done.

A polynucleotide according to the present invention has at least 15 bases, preferably at least 30 bases, and preferably at least 50 bases, which hybridizes to the polynucleotide of the present invention and, as described herein, the polynucleotide. They may have identity to the peptide and may or may not retain activity. Such a polynucleotide is used, for example, as a probe of the polynucleotide of FIG. 1 for recovery of the polynucleotide, or as a diagnostic probe or as a PCR primer.

The polynucleotide according to the present invention may also be generated in the form of a mixture of polynucleotides capable of hybridizing to some extent to the sequence of FIG. 1 (SEQ ID NO: 1), including any and all fragments thereof, and A polynucleotide mixture consists of any number of such polynucleotides or fragments thereof, including mixtures having at least 10, and possibly at least 30, such sequences or fragments thereof.

Because the coding region contains only a small portion of the human genome, the identification and mapping of the transcribed region and the chromosomal coding region is of significant interest. There is a need for corresponding reagents to identify and label chromosomal coding and transcription regions. In addition, such human sequences can be used to map chromosomes, identify humans, identify tissue types and origins, identify in court, and identify genes associated with disease-related genes (ie, mutations, deletions, or incorrect gene expression). It is valuable for chromosomal positioning of genes related to human diseases).

Various aspects of the invention include partial and complete cDNAs, genomic DNA, mRNA
, Antisense strand, PCR primers, coding region, and each individual sequence corresponding to the construct. Expression vectors and polypeptide expression products are within the scope of the invention, as are antibodies to such expression products, especially monoclonal antibodies.

Except as used herein and otherwise specifically described, all terms are defined as follows.

According to the present invention, the term “gene” or “cistron” refers to a segment of DNA involved in producing polypeptides (ie a DNA segment). It consists of regions surrounding the coding region (5 'and 3' untranslated regions or UTRs, or untranslated regions also called leader and trailer sequences, regions or segments) and intervening sequences (introns) between the individual coding segments (exons). This intron region is typically removed during post-transcriptional RNA processing to form the final translatable mRNA product. Of course, by its nature, cDNA does not contain intron sequences.

According to the present invention, the term “DNA segment” means a DNA polymer in the form of different fragments or as a component of a larger DNA construct, which is at least once in a substantially pure form, ie, impure endogenous. The segments were separated in amounts or concentrations that allowed the identification, manipulation and recovery of the segment and its component nucleotide sequences by standard biochemical methods, for example, using cloning vectors, without any aggressive substances. Such segments are typically provided in the form of internal untranslated sequences present in eukaryotic genes, ie, open reading frames that are not disturbed by introns. The sequence of the untranslated DNA is downstream from the open reading frame, where the same does not interfere with the manipulation or expression of the coding region.

The nucleic acid and polypeptide expression products disclosed in accordance with the present invention, as well as expression vectors containing such nucleic acids, are in “enriched form”. As used herein, the term “enriched” is at least about 2, 5, 10, 100, or 1,000 times the concentration (eg) of its natural concentration; It is meant to be at least preferably 0.01% by weight, preferably at least about 0.1% by weight. Enriched preparations of about 0.5%, 1%, 10% and 20% by weight are also contemplated. Sequences, constructs, vectors, clones, and other materials that include the present invention can be advantageously in enriched or isolated form. Removal of the clones corresponding to the ribosomal RNA gene and the "housekeeping" gene and clones without the human cDNA insert, e.g., via the differential display technique described herein, can be used to "enrich" live clones within the desired clone. Causes a rally.

The DNA and RNA sequences and polypeptides disclosed in accordance with the present invention are generally in a separated form. The term "isolated" means that the substance is removed from its original environment (eg, the natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide or DNA present in a living animal is not separated, but the same polynucleotide or DNA separated from some or all of the coexisting materials in the natural system is separated. Such DNA can be part of a vector, and / or such a polynucleotide can be part of a composition, and such that the vector or polynucleotide is not part of its natural environment. Can be separated.

The DNA and RNA sequences or polypeptides disclosed according to the present invention also exist in “purified” form. The term "purified" does not require absolute purity. Rather, it is intended as a relative definition, and these terms may include highly purified preparations or only partially purified preparations as those skilled in the art will appreciate. Individual clones isolated from the cDNA library were purified by conventional means for electrophoretic homogeneity. cDNA clones are obtained via manipulation of partially purified naturally occurring substances (messenger RNA). By converting the mRNA to a cDNA library, purified individual cDNA clones can be separated from the synthetic library by clone selection. Thus, from RNA to cD
Creating a NA library, followed by separating the individual clones from this library yields about ^106-fold purification of the raw message. Particular consideration is given to purification of the starting material or natural material by at least one order of magnitude, preferably two or three orders of magnitude, and more preferably four or five orders of magnitude. Even more desirably, the claimed polynucleotides having a purity of 0.001% by weight, or at least 0.01% or 0.1%, and even more desirably 1% or more by weight.

The term “coding region” refers to that portion of a human gene that naturally or normally encodes the expression product of the gene in its natural genomic environment, ie, the region that encodes the natural expression product of the gene in vivo. . The coding region can be from a normal, mutated, or altered gene, or
It can also be obtained from a fully synthesized DNA sequence or gene using methods known to those skilled in the art of synthesis.

According to the present invention, the term “nucleotide sequence” refers to a heteropolymer of deoxyribonucleotides. Generally, the DNA segment encoding the protein provided by the present invention comprises a cDNA fragment and a short oligonucleotide to provide a synthetic gene that can be expressed in a recombinant transcription unit containing regulatory elements derived from a microorganism or viral operon. Fragments are assembled from short oligonucleotide linkers or from a series of oligonucleotides.

The term “expression product” refers to a polypeptide or protein that is the natural transcript of a gene and that results from the genetic coding degeneracy and is any nucleic acid sequence coding equivalent that encodes the same amino acid.

The term “fragment” when referring to a coding sequence includes those below the fully human coding region whose expression product retains essentially the same biological function or activity as the expression product of the complete coding region. Means a portion of DNA.

The term “primer” refers to a short nucleic acid sequence paired with a single strand of DNA, at which point a DNA polymerase provides a free 3 ′ OH terminus to begin synthesis of a deoxyribonucleotide chain.

The term “promoter” refers to a region of DNA involved in binding RNA polymerase to initiate transcription.

The term “open reading frame (ORF)” refers to a sequence of three letter codes for amino acids without any stop codons and is a (potentially) translatable protein sequence.

The term “exon” refers to any segment of a discontinuous gene represented by the mature RNA product.

As used herein, references to DNA sequences refer to single-stranded DNA and double-stranded DNA
Including both. Thus, unless the context indicates otherwise, the specific sequence is a single-stranded DNA of such a sequence, the duplex of such a sequence with its complement (double-stranded DNA) and the Reference is made to the complement of such a sequence.

In accordance with the present invention, a holistic approach to the identification of cDNAs involved in the mesenchymal differentiation process of hMSCs involved measuring gene expression during osteogenic differentiation of cells growing in culture. Cells were harvested and their total RNA content was recovered.
Using various primer combinations, a reverse transcriptase and polymerase chain reaction procedure was then used to produce and amplify the corresponding cDNAs, which were then screened to find regulated DNA to be purified and cloned. Was. These clones were then sequenced and used to determine a consensus sequence (the consensus sequence is based on the most commonly occurring base at each nucleotide position in the sequence after the contributing sequences have been aligned by residue position) Array).
The resulting sequences were then subjected to computer database searches for novelty and homology to known sequences using, for example, a blast program and the Genbank database.

In accordance with the foregoing, a cDNA library was generated, which comprises the sequence of FIG.
Used to identify SEQ ID NO: 1). Probes based on these cDNAs were used to identify relevant transcripts using previously known Northern blot analysis.

The nucleotide sequences disclosed in accordance with the present invention have been found to be expressed in cord blood as well as in bone marrow CD34-bearing cells, as included in FIG. 1 (SEQ ID NOs: 1 and 2) (Northern blot hybridization). The complete transcript was about 1.1 kilobases (as determined by analysis). The sequence contained an open / reading frame encoding a polypeptide of 136 amino acids (the latter amino acid did not show any significant homology to any of the known proteins in Genbank and was therefore considered novel). Hydropathic (graph of hydrophobicity versus amino acid sequence) analysis of the deduced peptide sequence (shown in FIG. 2, SEQ ID NO: 3) showed a signal peptide of 19 amino acids at the N-terminus of the protein, which was a CD34 ⁺ cell Suggests that it is secreted by This secretion characteristic is cD
NA was labeled with a histidine-6 label (the latter being easily purifiable by nickel affinity chromatography) and confirmed with an epitope of the human myc oncogene at a position corresponding to the C-terminus of the expressed protein. . When the recombinant protein was expressed in human 293 cells, it was discovered that it was secreted into the medium.

Each of the DNA sequences identified herein (and the corresponding complete gene sequence) can be used as a polynucleotide reagent in a number of ways. The sequence can be used as a diagnostic probe in the presence of specific mRNA in certain cell types as well as in gene linkage analysis (polymorphism). In addition, the sequences can be used as probes to locate gene regions associated with genetic diseases.

The nucleotide and gene sequences of the present invention are further valuable for chromosome identification.
Each sequence specifically labels a particular location on the individual human chromosome and is capable of hybridizing at that location. In addition, there is a current need for sequences to identify specific sites on the chromosome. Mapping of polynucleotides to specific chromosomes according to the present invention is an important first step in correlating these sequences with genes associated with diseases, such as those affecting bone formation or skeletal abnormalities.

In summary, the PCR primers (preferably 15-
(30 base pairs) to prepare a map. Computer analysis of these sequences is used to rapidly select primers so that they do not span more than one exon in the corresponding genomic DNA, which could otherwise complicate the amplification process. These primers are then used for PCR screening of somatic hybrids containing individual human chromosomes. Only hybrid cells containing a human gene corresponding to the sequences or subsequences disclosed herein will produce the amplified fragment.

[0062] PCR mapping of somatic hybrids is an accelerated procedure for assigning specific sequences to specific chromosomes. As is known in the art, a single thermal cycler can be used to assign more than two clones per day. Using the present invention with the same oligonucleotide primers, partial localization can be achieved in a similar manner with a panel of fragments from a particular chromosome or pool of a large genomic clone. Other mapping strategies that can also be used to map a sequence or portion of a sequence to that chromosome are in situ hybridization, pre-screening on labeled flow sorted chromosomes, and constructing chromosome-specific DNA libraries. Pre-sorting by hybridization for

Fluorescence in situ of cDNA clones to metaphase chromosome spread (in situ)
Hybridization (FISH) can be used to provide a precise chromosomal location in one step. This method has a short cD of 500 or 600 bases.
Can be used with NA. However, large clones of 2000 base pairs or more have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. The FISH method requires the use of a clone from which the sequence was derived, the longer the better. For example, 2000 base pairs is better, 4000 base pairs is better, but 4000 or more is probably not necessary to get good results at a reasonable rate of time. For reporting this technique, Vama other, human chromosomes: See between the basic technology documentation, Pergamon Press, New York (1988).

Reagents for chromosome mapping can be either individually (to label a single chromosome or a single site on that chromosome) or separately (to label multiple sites and / or multiple chromosomes). Can be used as a panel. Reagents corresponding to non-coding regions of the gene are desirable for mapping purposes in nature. The coding sequence is certainly more retained within the gene family, thus increasing the chance of cross-hybridization during chromosome mapping.

[0065] Once a sequence has been mapped to a precise chromosomal location, it can be correlated with the physical location of the sequence on the chromosome with genetic map data. (Such data is, for example, V
. McCuesick, found in Mendelian genetics in humans (available online through Johns Hopkins University, Welch Medical Library)). The relationship between the disease and the gene mapped to the same chromosomal region is then identified through linkage analysis (simultaneous inheritance of physically closely related genes).

It is then necessary to determine whether there are differences in the cDNA or genomic sequence between affected and unaffected individuals. If the mutation is observed in some or all of the affected individuals but not in its normal individuals, the mutation will be the causative agent of the disease.

With current solutions in physical and genetic mapping techniques, cDNAs that accurately localize to chromosomal regions associated with the disease can be any of 50 to 500 potential pathogenic disease genes. You can see one (this is a 1 megabase mapping solution, 2
Assume one gene per kilobase).

Comparison of unaffected and unaffected individuals is generally done by first looking for structural alterations in the chromosome, such as deletions or translocations that are visible from chromosomal spreads or detectable using PCR based on cDNA sequences. Accompanied by seeing. Ultimately, complete sequencing of the gene from several individuals is necessary to confirm the presence of the mutation and to distinguish the mutation from a polymorphism.

In addition to the foregoing, the broadly described sequences of the present invention can be used to regulate gene expression through triplex formation or antisense DNA or RNA, both of which methods rely on the binding of the polynucleotide sequence to DNA or RNA. Can be used to Polynucleotides suitable for use in these methods are typically between 20 and 40 bases in length and are designed to be complementary to regions of the gene involved in transcription (triple-Lee et al., Nucleic acids research, 6: 3073 (1979); Cooney et al., Science, 241: 456 (1998); and Durban other, Science, 2 51: 1360 (1991)), or alternatively is complementary to the mRNA itself (Antisense-Okano J. Neurochemistry, 56 :
560 (1991); oligodeoxynucleotides as antisense inhibitors of gene expression, CRC Press, Bocalayton, Florida, (1998)). The triplex format optimally results in termination of RNA transcription from DNA, while the antisense R
NA hybridization blocks the translation of mRNA molecules into polypeptides. Both methods were shown to be effective in the model system. The information contained in the sequences of the present invention is necessary for designing antisense or triplex oligonucleotides.

The present invention is also a tool useful for gene therapy, and requires isolation of a disease-related gene which is a problem as a precondition for inserting a normal gene into a living body in order to correct a gene defect. The high specificity of the cDNA probes according to the invention ensures that they target such gene locations in a very precise manner.

The sequences of the present invention, including their broadly defined subsequences and fragments, are further useful for identifying individuals from microbiological samples. For example, the United States Army is considering using restriction fragment length polymorphisms (RFLPs) to identify its personnel. In this approach, individual genomic DNA is digested with one or more restriction enzymes and probed on a Southern blot to generate unique bands to identify soldiers. This method does not suffer from the current constraints on "recognition tags" that can be lost, switched, or stolen, making confident identification difficult. The sequences of the present invention are useful as additional DNA labels for RFLP.

However, RFLP is a pattern-based technique, which does not require the DNA sequence of the individual to be sequenced. Portions of the sequences of the present invention can be used to provide an alternative approach to determining DNA sequences in virtually base units of selected portions of an individual's genome. In addition, these sequences can be used to prepare PCR primers that amplify and separate such selected DNAs. For example, a part of the sequence of the present invention is removed and two PCs are extracted from the 5 'and 3' ends of the sequence or a fragment of the sequence.
An R primer can be prepared. These are used to amplify the individual DNA corresponding to the sequence. The amplified DNA is sequenced.

The panel of corresponding DNA sequences from the individuals thus produced provides a unique individual identification, as each individual has a unique set of such DNA sequences due to allelic differences. can do. The sequences of the present invention can be used for particular benefit in obtaining such identified sequences from individuals and from tissues. Allelic alterations occur to some extent in the coding regions of these sequences, and to a greater extent in non-coding regions. Allelic alterations among individual humans are 5
Occurs about once every 100 bases. Each of the fragments or complete coding sequences comprising a portion of the invention can be used as a standard to some extent that DNA from an individual can be compared for identification purposes. Because a larger number of polymorphisms occur in non-coding regions, a smaller number of sequences is needed for differentiated individuals.

If a panel of reagents from a sequence according to the invention is used to generate a unique ID database for an individual, these same reagents will later be used to identify tissue from that individual. it can. Unambiguous identification of an individual, whether alive or dead, can be made from a very small recognition sample.

Another use of DNA-based identification techniques is in forensic biology. PCR
The technique can be used to amplify DNA sequences taken from very small biological samples. In one prior art, the gene sequence is amplified at a specific locus known to contain a large number of allelic alterations, such as the DQα class II HLA gene (H. Ehrlich, PCR technology, Freeman & Co.). (1
992)). Once this specific region of the genome has been amplified, DQα class II H
It is digested with one or more restriction enzymes to produce an identified set of bands on a Southern blot probed with DNA corresponding to the LA gene.

The sequences of the present invention can be used to provide polynucleotide reagents that specifically target additional loci in the human genome and increase the confidence in DNA-based legal identification. it can. Those sequences that target non-coding regions are particularly suitable. As described above, the actual nucleotide sequence information can be used for identification as an accurate alternative to the pattern formed by the restriction enzyme-generated fragments. Reagents for obtaining such sequence information are within the scope of the present invention. Such reagents can include the complete sequence, a portion of the gene or the corresponding coding region, or a fragment of at least 15 base pairs, preferably at least 18 base pairs.

There is also a need for reagents that can identify particular tissue sources. For example, such a need arises in court when filing an organization of unknown origin. Suitable reagents can include, for example, DNA probes or primers specific to a particular tissue prepared from the sequences of the present invention. Panels of such reagents can identify tissues by species and / or by organ type. In a similar manner, these reagents can be used to screen tissue cultures for impurities.

Sequences that perfectly match several different genes can be detected by hybridizing to the chromosome. If many chromosomal loci are observed, the sequence (or closed mutant) is within one or more genes. This problem is circumvented by using the 3 'untranslated portion of the cDNA alone as a chromosomal location or full length cDNA or gene probe. The 3 'untranslated region appears to be more uniquely unique within the gene family because there is no evolutionary pressure to preserve the coding function of the region of the mRNA.

The cDNA library disclosed in accordance with the present invention ideally uses directional cloning, so that the 5 ′ end of the cDNA (possibly containing the coding sequence)
Alternatively, either the 3 'end (probably a non-coding sequence) can be selectively obtained.

Using the sequence information provided herein, the polynucleotides of the present invention can be derived from natural sources or synthesized using known methods. Sequences located within the scope of the present invention are not limited to the specific sequences described above, but also include human alleles and species alterations and portions thereof. In addition, the present invention includes all coding sequences associated with specific polynucleotide sequences of the bases listed in the Sequence Listing, as well as portions of all coding sequences. Allelic alteration can be routinely determined by comparing one sequence to the sequence of one individual of the same species. Furthermore, to accommodate codon variability, the present invention includes sequences that encode the same amino acid sequence as the specific sequences disclosed herein. In other words, it is particularly contemplated that one codon in the coding region is replaced by one that encodes the same amino acid (the coding region can be determined through routine sequence analysis).

In a cDNA library, there are many species of mRNA to be displayed. Each c
DNA clones are interesting for their own good reason, but must be isolated from the library before further testing can be performed. To sequence any specific cDNA, it is removed and separated from any other sequence (
I.e., isolated and purified). This can be achieved by a number of methods known to those skilled in the art. These procedures usually involve the cDNA of interest.
With the identification of bacterial colonies containing this and further amplification of the bacteria. Once the cDNA is isolated from the mixed colony library, it can be used as a template for further procedures such as nucleotide sequencing and the like.

The present invention also includes a recombinant construct comprising one or more sequences as described extensively above. This construct comprises a vector, such as a plasmid or viral vector, into which the sequence of the invention has been inserted in a forward or reverse orientation.
In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter operably linked to the sequence. Many suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided as examples. Bacterial: pBs, phasescript,
PsiX174, pBluescript SK, pBs KS, pNH8a,
pNH16a, pNH18a, pNH46a (Stratagene); pTrc9
9A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Prokaryotic system: pWLneo, pSV2cat, pOG44, pXT1,
pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia).

Thus, the invention is not limited to only such constructs or sequences, but includes expression vehicles, including plasmids, viruses, or any expression vectors,
It includes cells and liposomes and contains any of the nucleic acids, nucleotide sequences, DNAs, RNAs, or fragments thereof as disclosed in accordance with the present invention. Furthermore, regardless of whether such sequences are coding or non-coding sequences and whether such coding sequences encode all or a portion of the expression products disclosed herein, such expression products or fragments thereof This is true so long as it shows a utility in harmony with the invention disclosed herein. Thus, while the present invention includes an isolated DNA sequence or nucleic acid that expresses a human protein when in a suitable expression system, such as a cell-free system or an in vitro expression system, such a system may comprise a suitable expression vehicle or Included in the vector, or portions thereof, are cells, plasmids, viruses or other operable expression vectors.

These expression systems, especially those that are part of the expression mediator, generally employ a promoter different from that normally associated with the genes encoding the gene expression products and proteins disclosed in accordance with the present invention in vivo. Requires some promoter region to contain. The promoter region can be selected from any desired gene using a CAT (chloramphenicol transferase) vector or other vector with a selection marker. Two suitable vectors are pKK232-8 and pCM7. Particularly named bacterial promoters are lacl, lacZ, T3
, T7, gpt, lambda P _R and trc. Eukaryotic promoters include the CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-1 promoters. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

[0085] In a further embodiment, the present invention relates to host cells containing the above-described constructs. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into host cells can be accomplished by calcium phosphate transfection, DEAE
, Dextran-mediated transfection, or electroporation (L.
Davis, M .; Jivner, I. Batti, Basic Methods in Molecular Biology (198
6)).

The construct in the host cell can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. As an option, once the sequence is cDN
The encoded polypeptide known from A or known for the isolation of pure products can be produced synthetically by conventional peptide synthesis methods, either manual or automatic.

Thus, according to the present invention, once the coding sequence is known, or once the gene encodes the polypeptide and is cloned, conventional techniques in molecular biology can be used to obtain the polypeptide. . More generally, the invention includes any of the herein disclosed, including fragments of the polypeptides, as well as derivatives and functional analogs thereof, and all polypeptides encoded by individual DNA or RNA sequences.

At the simplest level, amino acid sequences can be synthesized using commercially available peptide synthesizers. This is particularly useful for producing small peptides and fragments of larger polypeptides. (Fragments are useful, for example, to generate antibodies against the native polypeptide).

As an option, the DNA encoding the desired polypeptide can be inserted into the host organism and expressed. The organism is a bacterium, yeast, cell line, or multicellular plant or animal. The literature indicates that there are many suitable host organisms and expression techniques. For example, it allows a polynucleotide (DNA or mRNA) to be injected directly into the muscle tissue of a mammal that expresses it. This method can be used to deliver a polypeptide to an animal or to generate an immune response to an external polypeptide. Wolff et al., Science , 247 : 1465 (1990); Fergner,
Et al., Nature , 349 : 351 (1991). Optionally, the coding sequence can be inserted into a vector with appropriate regulatory regions (ie, constructs),
It is then used to transfect cells. The cell (where the cell is or is not a part of a larger organism) then expresses the polypeptide.

The present invention further relates to polypeptides having the amino acid sequence of FIG. 2 (SEQ ID NO: 3) and fragments, analogs and derivatives of such polypeptides.

“Fragments” and “derivatives” when citing the polypeptide of FIG. 2 (SEQ ID NO: 3)
And the term "analog" means a polypeptide that retains essentially the same biological function or activity as said polypeptide. Thus, analogs include proproteins that can be activated by cleavage of the protein portion to produce an active mature polypeptide. Such fragments, derivatives and analogs must have properties sufficiently similar to the polypeptide of FIG. 2 (SEQ ID NO: 3) to retain the activity of the native polypeptide.

The polypeptide of the present invention is a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.

[0093] As used herein, "recombinant" means that a protein is derived from a recombinant (eg, bacterial or mammalian) expression system. "Microbial" refers to recombinant proteins made in bacterial or fungal (eg, yeast) expression systems. As a product, "recombinant microbial" means a protein that is essentially devoid of natural endogenous material and without associated natural glycosylation. Proteins expressed in most bacterial cultures, such as E. coli, lack glycosylation modifications, and proteins expressed in yeast will have a different glycosylation pattern than those expressed in mammalian cells.

A fragment, derivative or analog of the polypeptide (SEQ ID NO: 3) of FIG.
1.) one or more amino acid residues are replaced with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue), and such a substituted amino acid residue is replaced with the genetic code (
Or (ii) one or more amino acid residues include a substituent, or (iii) a compound in which the mature polypeptide increases the half-life of the polypeptide. Or (iv) additional amino acids are added to a leader or secretory sequence, or a sequence used to purify a mature polypeptide or proprotein sequence. That is fused to a polypeptide. Such fragments, derivatives and analogs are deemed to be within the capabilities of those skilled in the art in light of the teachings herein.

[0095] The polypeptides of the present invention are desirably provided in a separated form, and desirably are purified to homogeneity. When applied to a polypeptide, the term "isolated" has the previously stated meaning.

[0096] The polypeptides of the present invention may comprise the polypeptide of FIG.
Also at least 90% identity to the polypeptide of FIG. 2 (SEQ ID NO: 3), or at least 95% identity to the polypeptide of FIG. 2 (SEQ ID NO: 3), and even more preferably the polypeptide of FIG. It includes polypeptides having at least 98% identity to the peptide (SEQ ID NO: 3) and more generally having at least 30 amino acids or more desirably at least 50 amino acids.

A fragment or portion of a polypeptide of the present invention is used to produce the corresponding full-length polypeptide by peptide synthesis. Thus, this fragment is used as an intermediate to produce a full-length polypeptide. Fragments or portions of a polynucleotide of the present invention are used to synthesize a full-length polynucleotide of the present invention.

According to the present invention, the polypeptide disclosed in FIG. 2 has growth stimulating activity when present in an in vitro growth medium containing human mesenchymal dry cells. Thus, such stem cells are induced to replicate at a faster rate in the presence of the polypeptides disclosed herein (as shown in FIG. 3). The recombinant C17 protein thus expressed in 293 cells was affinity purified and added to human MSC (hMSC) culture. HMSCs maintained in serum-free conditions typically show little basal growth activity. Here, a dos titration of fetal bovine serum (FBS) was used as a positive control. Recombinant C17 protein stimulated hMSC growth about 10-fold at a level comparable to 10% fetal bovine serum compared to serum medium. The C17 polypeptide is present in the medium at a concentration of at least 1 picogram (pg) per ml of medium.

The invention further relates to polynucleotides of the invention, host cells genetically engineered with vectors of the invention, and vectors comprising production of the polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of the present invention, which are, for example, cloning vectors or expression vectors, either of which may be a plasmid, virus particle, phage, etc. In the form of The engineered host cells can be cultured in a conventional nutrient medium suitably modified to activate the promoter of the invention, select transformed cells, or amplify the gene. Temperature, pH and other culture conditions are those previously used in the host cell selected for expression and are well known to those skilled in the art.

The polynucleotides of the present invention are used for recombinantly produced polypeptides. Thus, for example, a polynucleotide is included in any one of various expression vectors that express the polypeptide. Such vectors include chromosomal, non-chromosomal and synthetic DNA sequences, including, for example, derivatives of SV40; bacterial plasmids, phage DNA; baculovirus and yeast plasmids; plasmid and phage DNA, tumor rash, adenovirus, fowlpox virus, and pseudopox. Viral DNA such as rabies
And vectors derived from the combinations of However, other vectors can be used as long as they are replicable and viable in the host.

According to the present invention, a suitable DNA sequence, or segment, is inserted into a vector by various procedures. Generally, DNA is inserted into the appropriate restriction endonuclease site by known procedures. Such procedures and the like are deemed to be well known to those skilled in the art.

[0103] The DNA sequence in the expression vector is operably linked to a suitable expression control sequence (eg, a promoter sequence) to direct mRNA synthesis. Such the following as representative examples of such promoters can be mentioned: LTR or SV40 promoter, the E. coli lac or trp, the phage lambda P _L promoter, known to control expression of genes in prokaryotic or eukaryotic cells or their viruses And other promoters. The expression vector further contains ribosome binding sites for translation initiation and transcription termination factors. The vector also contains appropriate sequences for amplified expression.

In addition, the expression vector desirably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, which is resistant to dihydrofolate reductase or neomycin for eukaryotic cell culture. etc., or a marker gene, such as Kitora cyclin or ampicillin resistance in E. coli.

Vectors containing the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, are used to transform the appropriate host to allow expression of the protein to the host.

Representative examples of suitable hosts include the following:E. coli, Streptomyces ,Salmonella typhimuriumBacterial cells such as yeast; fungal cells such as yeast:Shi Drosophila S2 andSpodoptera Sf9Insect cells such as CHO, COS
Or animal cells such as BOWES melanoma; adenovirus; plant cells, and the like.
The selection of a suitable host is deemed to be within the skill of the art from the teachings herein.

“Recombinant expression vehicle or vector” refers to a plasmid or phage or virus or vector that expresses a polypeptide from a DNA (RNA) sequence. Expression vehicles are (1) genetic elements having a regulatory role in gene expression, such as promoters or enhancers, (2) structural or coding sequences transcribed into mRNA and translated into proteins, (3) appropriate transcription initiation and termination sequences. , A transfer unit consisting of an assembly of Structural units intended for use in yeast or eukaryotic expression systems desirably include a leader sequence that enables extracellular secretion of translated protein by a host cell. Alternatively, if the recombinant protein is expressed without a leader or transport sequence, it will include an N-terminal methionine residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide the final product.

“Recombinant expression system” means a host cell that stably integrates a recombination, transcription unit into chromosomal DNA, or a host cell that transports a recombinant transcription unit extrachromosomally. Cells are either prokaryotic or eukaryotic. A recombinant expression system as defined herein will express a heterologous protein upon the introduction of a regulatory element linked to the expressed DNA segment or synthetic gene.

[0109] The mature protein can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be used to produce such proteins using RNAs derived from the DNA constructs of the present invention.
Suitable cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook et al., Molecular Cloning: Laboratory Manual, Second Edition (Cold
Spring Harbor, New York, 1989), the disclosure of which is incorporated herein by reference.

The transcription of a DNA encoding a polypeptide according to the invention by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. Such enhancers have become known at some time and are usually cis-acting elements of anywhere from 10 to 300 base pairs of DNA that act on the promoter to increase transcription. Common examples are the SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer and the enhancer found in adenovirus.

In general, recombinant expression vectors contain an origin of replication and a selectable marker that allow for the transformation of the ampicillin resistance gene of host cells such as E. coli and the TRP1 gene of S. cerevisiae, and highly expressed genes for direct transcription of downstream structural sequences. And promoters derived from. Such promoters include, for example, 3
-Can be derived from operons encoding glycolytic enzymes such as phosphoglycerate kinase (PGK), alpha factor, acid phosphatase, or heat shock proteins. The heterologous structural sequence is assembled in appropriate phase with a translation initiation and termination sequence, and preferably a leader sequence capable of directing secretion of the translated protein to the periplasmic space or extracellular medium. Alternatively, the heterologous sequence can encode a fusion protein containing an N-terminal identification peptide that confers desired properties, such as, for example, stabilization of the expressed recombinant product or simple purification.

[0112] Useful expression vectors for bacteria include the structural DN encoding the desired protein.
It is constructed by inserting the A sequence into an operable reading phase with a functional promoter along with appropriate translation initiation and termination signals. The vector will include one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desired, provide for amplification in the host. Suitable prokaryotic hosts for transformation include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, and various species in the genera Pseudomonas, Streptomyces, and Staphylococci, although others may be employed as a matter of choice. .

As a representative, but not limiting example, useful expression vectors for bacteria are selection markers and bacterial origins of replication derived from commercially available plasmids containing the genetic elements of the well-known cloning vector pBR322 (ATCC37017). Can be included. Such commercial vectors include, for example, pKK22
3-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GE
M1 (Promega Biotech, Madison, Wisconsin, USA). These pBR322 "backbone" portions are combined with an appropriate promoter and the structural sequence to be expressed.

Following transformation of the appropriate host strain and propagation of the host strain to an appropriate cell density, the selected promoter is activated by appropriate means (eg, temperature transfer or chemical induction) and the cells are treated for additional Cultured for a period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the crude extract to be purified is retained for further purification.

[0115] Various mammalian cell culture systems can also be used to express the recombinant protein. Examples of mammalian expression systems include Grazman, cells , 23 :
175 (1981), and the COS7 line of monkey kidney fibroblasts, and other cell lines capable of expressing compatible vectors, such as C127, 3T3, CHO, HeLa
And BHK cell lines. Mammalian expression vectors will contain an origin of replication, a suitable promoter and enhancer, as well as any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and 5 'flanking non-transcribed sequences. DNA sequences derived from the SV40 viral genome, such as the SV40 origin, early promoter, enhancer, splice, and polyadenylation sites are used to provide the required non-transcribed genetic elements.

The recombinant protein produced in the bacterial culture is conveniently separated from the cell pellet by an initial extraction, followed by one or more steps of salting out, aqueous ion exchange or size exclusion chromatography. A protein refolding step can be used to complete the configuration of the mature protein if necessary. Finally, high performance liquid chromatography (HPLC) can be used in the final purification step. Microbial cells used for protein expression can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

The proteins, fragments or other derivatives thereof, or analogs thereof, or cells expressing them can be used as immunogens to produce antibodies thereto. These antibodies can be, for example, polyclonal, monoclonal, chimeric, single chain, Fab fragments, or the products of a Fab expression library. Various procedures known in the art are used for the production of polyclonal antibodies.

Antibodies purified against a polypeptide corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptide into an animal, or by administering the polypeptide to an animal, preferably a non-human. it can. The antibody thus obtained then binds to the polypeptide itself. In this way, sequences encoding only fragments of the polypeptide can still be used to purify antibodies that still bind to the entire native polypeptide. Such antibodies can then be used to separate the polypeptide from tissues expressing the polypeptide. Panels of such antibodies specific for larger numbers of polypeptides can be used to identify and differentiate such tissues.

For preparation of monoclonal antibodies, any method which provides antibodies produced in continuous cell line cultures can be used. An example is the hybridoma method (Koehler and Milstein, 1975, Nature, 256 : 495-497).
, The trioma technique, the human B cell hybridoma technique (Kozubo other, 1983, today Immunology 4: 72), and human monoclonal antibodies EBV-hybridoma technique to produce (call, other, 1985, Monoclonal Antibodies and Cancer therapy, Alan R. Riss, Inc., pages 77-96).

The method described for the production of single-chain antibodies (US Pat. No. 4,946,778) can be applied to produce single-chain antibodies against the immune polypeptide products of the invention.

The antibodies can be used as a method for the localization and activity of the protein sequences of the present invention, for example, by imaging these proteins and measuring their levels in appropriate physiological samples and the like.

Of course, knowing the sequence of the C17 protein of FIG. 2 would allow one of ordinary skill in the art to readily locate appropriate receptors on the surface of mesenchymal stem cells, as well as other cell types, and thereby to identify such cells. Will confer the ability to regulate growth.

In addition, the present invention further provides for the inclusion of sequences homologous to the disclosed nucleotide and polypeptide sequences, thereby providing C17 without structural and itself proteinization.
It would be possible to derive structurally similar structural analogs containing similar functional regions, including small molecules capable of mimicking the function of proteins. Thus, small organic molecules can easily be mimicked by regions of the C17 protein disclosed herein by molecular modeling using computer programs and algorithms.
Or it may be developed by a combination method. Such mimicry structures are further considered to be encompassed by the present disclosure. Such compounds can be readily synthesized and added to the cell growth medium, thereby stimulating the relevant receptor and enhancing the rate of cell growth. Such methods of enhancing cell growth are considered to be within the scope of the invention disclosed herein.

Such proliferative effects can be used to position cell growth stimulating receptors on the surface of cells. Here, the cells can be grown in a suitable medium, to which is added an appropriate amount of labeled C17 protein, or a homolog thereof, or a small chemical analog thereof, and then the homolog or analog is added to the cell. To determine if it can stimulate the growth of If stimulable, the analog can be further introduced into a suitable labeled form; typically in a labeled form by conventional means well known to chemists, and such labeling can be accomplished by radiolabeling methods. It involves both non-radioactive labeling methods and then stays in the medium at various times to bind the surface receptor to the cell surface, such as at the surface receptor. Following this, conventional separation techniques can be used which allow the separation, identification and characterization of the receptors, whether they are surface receptors or others. In this way, growth stimulating receptors for various cell types can be determined.

In practicing the procedures of the present invention, the particular buffer, media, reagents, cells, culture conditions, and the like are not limiting and those of ordinary skill in the art will appreciate in the particular context in which the discussion is presented. It should be understood that it should be read to include all relevant substances that it perceives to be valuable. For example, it is often possible to replace one buffer system or culture medium with one and still achieve similar, if not identical, results. Those of skill in the art will have sufficient knowledge of such systems and methods, and will therefore make use of the methods and procedures disclosed herein without undue experimentation to optimally serve this purpose for those skilled in the art. Could be done.

Specific embodiments of the present invention will now be described in more detail by way of the following non-limiting examples, in which additional and different embodiments of the teachings of the present invention will undoubtedly suggest themselves to those skilled in the art, It will be appreciated that the embodiments of the present invention are fully inferred from those disclosed herein.

Methods and Materials Separation of Cord Blood Cells and CD34 ⁺ Cells Frozen cord blood cells from neonatal neonates were purchased from the University of Arizona cord blood bank. Mononuclear cells (MNC) from 3-4 units of CB (umbilical cord blood)
) Are pooled and CD34 progenitor cell isolation kit (Auburn, CA)
Anti-CD34 antibody (Clone QBEND) provided by Milteny Biotech
/ 10). Up to 2 million MNCs were passed through an LS ⁺ column assembled on a VarioMACS system (Miltenyi Biotech). CD34 ⁺ cells labeled with a magnetic bead and retained on the column were separated from the column by eluted cells after migration from the magnet. These FT cells were previously passed through a second column to ensure removal of CD34 ^- cells in the flow (FT) fraction. The depleted FT fraction in FIG. 2 was used as the CD34 ⁻ cell population. CD34 ⁺ cells separated from the first and second columns were pooled. The content of CD34 ⁺ cells in each population was monitored using fluorescence activated cell sorter (FACS) staining (see below).
. Most cells are immediately prepared with TRIzol reagent (Gaithersburg, MD,
Gibco / BRL).

Other human cells CD34 ⁺ cells from bone marrow of a healthy donor were similarly isolated by Pure Cell Company (San Mateo, Calif.) According to federal and state regulations. We also used human CD34 ⁺ cells from mPB from healthy volunteers. Five days after continuous G-CSF treatment, leukocyte exchange cells were obtained. CD34 ⁺ and CD34 ^- cells were purchased from the Isolex 300 system (Nexel / Irvine, CA)
Baxter). Bone marrow-derived mesenchymal stem cells (MSCs) have been isolated and described in the literature (Pittanger, Science, 284: 143 (1999)).
And expanded in culture as described in

FACS analysis of CD34 ⁺ cells CB cells before and after cell separation were R-phycoerythrin (R-PE) complex CD
34 antibody (Becton Dickinson Immune System [BDIS], clone HPCA-2, San Jose, CA). HPCA-2 is QBEN
Recognizes a different CD34 epitope than that recognized by D / 10, which is used to purify cells. Antibody labeled cells were analyzed on a BDIS FACS caliber or Vantage instrument equipped with an ion argon laser tuned to 488 nm. Specific CD34 staining of solid MNC was recorded in FL2 channel (relative to R-PE). Non-specific staining (background) was 0.1%.
The content of CD34 ⁺ cells before cell separation was 0.4 +/− 0.1% (n = 3), which was consistent with the previously published information (Cairo and Wagner, blood, 90: 4665-
4678 (1997)). Typically, the proportion of CD34 ⁺ cells in the CD34 ⁺ preparation was 85.0%, and in the CD34 ⁻ fraction it was about 0.1% (
Background level). Among the 30 CB samples processed, a few cells did not meet this criteria and were discarded.

Complementary DNA (cDNA) synthesis Total RNA was isolated using TRIzol reagent (Gibco / BRL). 2
00 micrograms of total RNA was pooled from some preparations to 4,000
Isolated from 10,000 CD34 ⁺ cells. About 2 micrograms of poly A ⁺ RNA
Purified using RNA isolation midi kit (Qiagen, Valencia, CA). Double-stranded cDNA was prepared using a cDNA synthesis system containing Superscript II reverse transcriptase and random hexamers (Gibco / BRL). RNA samples derived from CD34 ⁺ and CD34 ⁻ cells were constantly processed in parallel.

Generation and Analysis of RDA Gene Fragments Representative difference analysis (RDA) amplicon preparation and subtractive hybridization methods were performed as described in the literature (Licitin et al., Science 259: 946-951 (1).
993); Hubank and Schatz, Nucleic Acids Research, 22: 5640-5648.
(1994)) but with a shorter PCR cycle (95 ° C, 30 seconds, 72 ° C, 2 minutes).
Only acted for the preparation of the amplicon (before subtraction) and the product of the subtraction (after subtraction). After three subtractions, another band became apparent on the agarose gel. The third (and final) subtraction product (DP3) was digested with Dpnll (which removes the adapter and creates a GATC overhang) and then BamHI
It was cloned into the digested pUC18 vector. More than 500 clones were obtained after transforming a strain of E. coli DH5 with small aliquots of continuous DNA. Initially, 55 individual clones were randomly picked. The insert of the individual clone is P
CR amplified and sequenced. Sequences were first searched against the Genbank non-redundant (NR) database using the BLASTN and BLASTX algorithms (
Altschul et al., Nucleic Acids Research, 25: 3389-3402 (1997); http: // w ww.ncbi.nlm.nih.gov/BLAST/). Those without significant associations were considered new. The new sequence is then genbank E using the BLASTN algorithm.
A search was performed against the ST database (dbEST).

The primers (298 base pairs) for amplification of the oligonucleotide CD34 cDNA for RT-PCR are as follows.

CD34-5 ': CTGTGTCTCAACATGGCA-3' (SEQ ID NO: 4) CD34-3 ': GCCTTGATGTCACTTAGG-5' (SEQ ID NO: 5) Primers (286 base pairs) for amplification of C17 cDNA are as follows. is there.

C17-5 ': GATCACCCGCGACTTCAACC (SEQ ID NO: 6) C17-3': TGGCAGGACCGTAGTCACTG (SEQ ID NO: 7) Primers for amplification of beta-2-microglobulin (β2M as control) cDNA (270 base pairs) Is:

Β2M-5 ′: TCTGGCCTTGAGGCTATCCAGCGT (SEQ ID NO: 8) β2M-3 ′: GTGGTTCACACGGCAGGCATACTC (SEQ ID NO: 9) In addition to our RDA clone containing the plasmid C17 cDNA fragment (290 bp) containing the C17 cDNA We are the International Molecular Analysis of Gene
Five plasmids containing human ESTs were purchased from Research Genetics, Inc. (Huntsville, Alabama), a seller of EST clones of the Expression (IMAGE) consortium (Gene Expression International Molecular Analysis Loan) (Table 1). . IMAGE clone 786066 is the longest insert (~
1 kilobase) and was used as the source of the C17 coding sequence for subsequent analysis.

Recombinant Gene Expression in Human Cells The complete coding region of C17 from IMAGE clone 786066 is PC
R and amplified at the EcoRI and BamHI sites with the mammalian expression vector pC
DNA 3.1 / myc-HisB (Invitrogen, Carlsbad, CA) was cloned in a framed state. The resulting plasmid is p
CMV. Named C17 / myc / his. The recombinant C17 protein expressed from this construct was labeled with a human C-myc epitope at the C-terminus and six histidine residues (His6) (in italics in the sequence below).

As a result, 31 amino acids

Was added to the C-terminus of C17.

Human 293-derived BOSC23 cells were transfected by calcium phosphate precipitation (Chen et al., Nature Biotechnology, 14: 606 (1996)).
Chen et al., Gene Therapy, 4: 1013 (1997)). 48 hours after transfection, cells and conditioned media were collected. Cells were scraped from culture dishes in the presence of a protein inhibitor cocktail (Complete ^™ , Roche Biochemicals). Cells were eluted in a buffer containing 150 mM sodium chloride, 20 mM Tris-HCl (pH 7.4), glycerol 10%, NP40, 1%, EDTA 10 mM, NaVO ₃ , 2 mM, sodium fluoride 100 mM, and Complete ^™ . The eluate was clarified by centrifugation at 14,000 rpm for 30 minutes at 4 ° C. Cell extracts and conditioned culture media were denatured with sample buffer under reducing conditions and electrophoresed on a 4-20% polyacrylamide gel with SDS-Tris / glycine buffer.
rC17 production was monitored by Western blot with antibodies to the c-myc or His6 epitope (Invitrogen).

Chromosome mapping A Stanford G3 human hamster radiohybrid cell (RH) panel was purchased from Research Genetics. Two pairs of PCR primers are C17cD
Designed based on the 5 'untranslated region of NA.

TTTGATTTTCATCACCTTTC (SEQ ID NO: 11) and CTGGTTTAATGGAGTAATGG (SEQ ID NO: 12) GTTAGATACACAGCATGTTGA (SEQ ID NO: 13) and GACAGTGAAGAAAGTCTGTG (SEQ ID NO: 14) Each pair is ２００200 bases in genomic DNA template from human but not hamster The pair fragment was specifically amplified. The PCR reaction was performed under the following conditions: 94 ° C., 20 seconds;
This was performed using 25 ng (nanogram) of DNA template under 30 cycles of 5 ° C., 20 seconds; 72 ° C., 30 seconds. Both sets of primers showed identical results. The results of the PCR reactions are tabulated (1 as positive and 0 as negative) and are
Submitted to the Human Genome Center RH server (http: //www.shgc.
stanford.edu/). LOD scores of 6 and above are considered important.

[0142]

[Table 1]

The C17 gene fragment (290 base pairs) was generated using the BLASTN algorithm.
Searched against bEST. See http://www.ncbi.nlm.nih.gov/blast/ for more details on the cDNA library used, scores and E values.
ND: Not determined by the depositor who partially sequenced the insert from either the 5 'or 3' end. At the time of the search (July 1998), a total of 2,07
A 2,964 EST (human and non-human) entry has been deposited.

[0144]

【Example】

Example 1 Desired gene expression of C17 on CD34 ⁺ hematopoietic cells was established as follows. RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction) is CD34 ⁺ or CD
Performed using total RNA from 34 ^- cells and two C17-specific primers (based on the sequence of the 290 base pair C17RDA fragment). C17 gene expression was readily detected in CB CD34 ⁺ cells but not in the CD34 ^- cell population. Similar RT-PCR results were obtained with cells from mPB, also with cells from bone marrow. Thus, C17 gene expression is known to involve HSPC3.
Are restricted to a population of CD34 ⁻ cells separated from a single source, CB, BM and mPB.

The C17 gene expression of untreated cultured CD34 ⁺ cells was analyzed by Northern blot. BM CD34 ⁺ cells were cultured under different culture conditions with different cytokines. Under the first condition, BM CD34 ⁺ cells were treated with TPO, SCF and a combination of Flt3 / Flk2 ligand (FL) known to support stem cell maintenance and progenitor cell expansion (Luens et al., Blood , 9
1: 1206-1215 (1998); Kaushansky, Blood, 92: 1-3.
(1998)). Under the second condition, the cells had 5 hematopoietic colony stimulating factors (IL
-3, IL-6, G-CSF, GM-CSF and EPO). They are known to expand the committed cells and stimulate cell differentiation, leading to the differentiation of HSPCs into the myeloid / erythroid lineage and CD34 ⁺ cell loss (Ruens et al., 1998). C17 in cultured and untreated CD34 ⁺ cells
Gene expression was analyzed by Northern blot using the C17 RDA fragment (290 base pairs) as a probe. Single ~ 1.0 kb base band untreated B
It was observed in M CD34 ⁺ cells as well as in cultured cells, which expressed the C17 gene at various levels. After 7 to 15 days of culture, C17 mRNA levels increased under condition # 1, while it decreased slightly under condition # 2.

In normal tissues, C17 expression was detected in human bone marrow and very weakly in lymph nodes using Clontech multiple tissue blots containing purified poly A + RNA, whereas in spleen, thymus, fetal liver and PBL, Northern No hybridization was detected. With the same hybridization, C17 is used in several human cancer cell lines: He
la S3 (cervical carcinoma), A549 (lung carcinoma), G-361 (melanoma), SW4
80 (colorectal adenocarcinoma), HL-60 (promyelocytic leukemia), K-562 (chronic myelogenous leukemia), Molt-4 (lymphoblastic leukemia) and Raji (Burkit's lymphoma), these cancer cells One blot containing poly A + RNA from the system was not detectable. In addition, Northern blot and RT-PCR analysis showed that C17
This indicates that gene transcripts were absent from mesenchymal stem cells, and these were
It is believed to be very close to the PC (Bittanger et al., 1999)
). Thus, C17 expression is regulated by hematopoietic cytokines.

Example 2 The full-length DNA sequence of C17 cDNA was obtained by purchasing five plasmid clones containing a C17-related EST sequence (Table 1). The inserts of these plasmids were partially sequenced from either the 5 'or 3' end by IMAGE consortium members. The size of the insert in these plasmids was determined from either end and sequenced. The insert in IMAGE clone 786066 is the longest (〜1 kilobase), and the other four plasmids and C17 RDA
Includes all sequences from the fragment. The insert sequence of IMAGE clone 786066 was used in subsequent analyses. AAT, a putative mRNA polyadenylation signal
AAA is found near the 3 'end of the C17 cDNA (e.g., residues 979-9
(See SEQ ID NO: 84). Based on the size of the transcript (〜1 kilobase) indicated by the sequence from the Northern blot and the multiple ESTs, IMAGE clone 786066 contains an almost full length cDNA of the C17 gene. C17 cD
NA contains an open reading frame of 408 nucleotides that encodes a protein of 136 amino acids (SEQ ID NO: 3). The presence of the Kozak sequence immediately around the first ATG suggests that it is the desired translation initiation (Kozak, Journal of Cell Biology, 115: 887-903 (1991)).
. Hydrophobic analysis of the putative peptide sequence shows a putative signal peptide at the N-terminus. Furthermore, the elaborated signal peptide revealed that the secretory peptide cleavage site was between its 19th and 20th amino acids (Nielsen et al., Protein Engineering, 10; 1-6 (1997)). There are no other hydrophobic transmembrane or GPI anchoring signal domains in the rest of the sequence, indicating that C17 is a secreted protein. Secondary structure analysis predicts that the C17 peptide contains a 4α helix that is characteristic of hematopoietic cytokines and interleukins (Bazan, Immunology Today, 11 (10): 350-354).
(199); Well and De Boss, Biochemistry Manual Review, 65; 609.
-634 (1996)). C17 diene bank, access number AF1937
66.

Example 3 The C17 protein was pCMV. To make C17 / myc / his C17
It was characterized by cloning the cDNA coding region into a mammalian expression vector. Recombinant C17 protein is labeled at the C-terminus with both the 9E10 c-myc epitope and six histidine residues (His6), which results in rC17
Detection and purification was facilitated. Human 293T cells were transfected with the vector to allow expression of the labeled C17 gene. Forty-eight hours post-transfection, both cell detection from transfection and conditioned media (supernatant) were analyzed by Western blot using antibodies against either of the two labels. The anti-myc antibody recognized a cell extract unique to C17 transfected cells and a specific protein in the supernatant. In the cell extract, the major 19 kilodalton protein band is specifically recognized, which is the untreated labeled C17 protein (167 amino acids in total including the signal peptide).
Of the expected 19 kilodalton. A single protein band was detected in the supernatant collected from C17 transfected cells, indicating that the C17 protein was indeed secreted. The visible molecular weight (approximately 26 kilodaltons) is larger than the expected 17.2 kilodalton size (processed C17 protein without the 19 amino acid signal peptide), which indicates that the secreted protein is modified during or after secretion. It indicates that it was done. Based on the amino acid sequence, there are no potential N-glycosylation sites within C17 or the epitope tag. Digestion with a panel of glycosidases also failed to shift protein migration on SDS-PAGE.

Example 4 rC17 was produced in large quantities by cloning C17 into the prokaryotic expression vector pBAD / gIII (of Invitron). In this expression vector, the putative C17 signal sequence was removed and the remainder of the coding sequence was ligated into the framework of the bacterial leader sequence. This allows the recombinant protein to be secreted into the pericyte space. The C17 protein expressed in this vector is also labeled with c-myc and His6 epitope. Upon introduction with arabinose (as preheated with full length rC17), the 19 kilodalton protein was 0.00
It was induced to express at high levels at arabinose concentrations of 2% or more.

Example 5 Radiation fibrous cell (RH) technology was used to map the location of the C17 gene in the human genome. A G3 human-hamster hybrid chromosomal DNA sample was used as a template for PCR amplification with primers specific for human C17. If the human genome (
Primer (if not the hamster genome) is present as a template.
It can only amplify a 0 base pair fragment. P in certain G3 RH DNA templates
The CR reaction produced the expected DNA fragment, while others failed. The generated data was used to determine its chromosomal localization based on the Stanford Human Genome Center database and algorithms. (http: // www.
shgc.stanford.edu/). A unique pattern mapped the C17 gene to a single locus on human chromosome 4P between D4S412 and D4S1601 (http: //
www.hcbi.nlm.nih.gov/genemap98/map.cgi?MAP=G3&BIN=130&MARK=SHGC.33462).
This result shows that related ESTs performed by others (Hs in the Unigene database)
.13872). This region is the human chromosome 4p15-1
6 is co-localized by cell genetic mapping. Several other genes associated with hematopoiesis are located in this region as well. These include CD38 (between D4S412 and 74S1601 as C17 / Hs.13872) and AC133 (disposed around D4S1601 through D4S1608). The latter is a recently discovered cell surface protein that is preferentially expressed on CD34 ^- HSPC (In et al., Blood, 90: 5002-5012 (1997); Miraria et al., Blood, 90
: 5013-5021 (1997)).

[Sequence list]

[Brief description of the drawings]

FIG. 1A shows the nucleotide sequence of the novel gene disclosed according to the present invention (
FIG. 1 shows SEQ ID NO: 1), which is an open reading frame (sequence identification) consistent with the putative protein of FIG. 2 and two relevant dpnII restriction sites that clone the gene and provide the fragments utilized. No. 2).

FIG. 1B shows a continuation of FIG. 1A.

FIG. 2 shows the deduced amino acid sequence (SEQ ID NO: 3) for the open reading frame of the sequence disclosed in FIG.

FIG. 3 shows the effect of C17 protein on the proliferation of mesenchymal stem cells in serum-free culture. The C17 protein allows a growth rate equal to that supported by the serum-containing medium.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/21 C12Q 1/02 5/06 G01N 33/53 M 5/10 33/566 C12Q 1/02 C12P 21/08 G01N 33/53 C12N 15/00 ZNAA 33/566 5/00 A // C12P 21/08 E (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR , GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD) , TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM) , AL AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH , GM, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA , ZWF term (reference) 4B024 AA01 AA11 BA41 BA80 CA04 CA11 EA04 GA11 4B063 QA01 QQ79 QQ91 QR48 QR69 QS03 QS12 QS24 4B064 AG27 CC24 DA13 4B065 AA26X AA46X AA50X AA72X AAAAAAAAAC ACAA ABC19 BA10 CA40 DA75 DA76 EA20 EA50 FA72 FA73 FA74

Claims (29)

[Claims] 1. An isolated nucleic acid comprising a polynucleotide that is at least 90% identical to a polynucleotide encoding a polypeptide comprising the amino acid sequence of FIG. 2. 2. An isolated nucleic acid comprising a polynucleotide that is at least 95% identical to a polynucleotide encoding a polypeptide comprising the amino acid sequence of FIG. 2. 3. An isolated nucleic acid comprising a polynucleotide that is at least 98% identical to a polynucleotide encoding a polypeptide comprising the amino acid sequence of FIG. 2. 4. The DN according to claim 1, which is one isolated nucleic acid,
An isolated nucleic acid comprising RNA complementary to either the A sequence or a fragment. 5. An isolated nucleic acid which is identical to the DNA sequence of FIG.
An isolated nucleic acid comprising an NA sequence. 6. An isolated nucleic acid, comprising an RNA that is complementary to the DNA sequence according to claim 5, which is an isolated nucleic acid. 7. An isolated nucleic acid comprising at least the polypeptide coding region of a human gene, wherein said human gene is a DNA according to claim 1.
An isolated nucleic acid comprising a sequence. 8. An isolated nucleic acid comprising at least a polypeptide coding region of a human gene containing the DNA sequence according to claim 5, which is an isolated nucleic acid. 9. The isolated nucleic acid according to claim 8, which expresses a human protein when it is in an appropriate expression system. 10. An expression vehicle comprising the DNA sequence according to claim 3. 11. An expression carrier comprising the DNA sequence according to claim 5. 12. An expression carrier comprising the DNA sequence according to claim 7. 13. An expression carrier comprising the DNA sequence according to claim 9. 14. A polypeptide characterized by being encoded by the DNA sequence according to claim 7 and its active fragments, derivatives and functional analogues. 15. A polypeptide characterized by being encoded by the DNA sequence according to claim 8 and active fragments, derivatives and functional analogs thereof. 16. A polypeptide comprising the amino acid sequence shown in FIG. 2 (SEQ ID NO: 3). 17. One genetically engineered cell, which is inserted into the genome of the DNA according to claim 7. 18. A process comprising using the engineered cell of claim 17 to produce a cell for expressing the polypeptide. 19. An isolated DNA sequence comprising a fragment of the DNA of FIG. 1 (SEQ ID NO: 1), wherein said fragment comprises at least 15 contiguous bases of said sequence. DNA sequence. 20. An isolated DNA sequence comprising a fragment of the DNA of FIG. 1 (SEQ ID NO: 1), wherein said fragment comprises at least 30 contiguous bases of said sequence. DNA sequence. 21. An isolated DNA sequence comprising a fragment of the DNA of FIG. 1 (SEQ ID NO: 1), wherein said fragment comprises at least 50 contiguous bases of said sequence. DNA sequence. 22. An isolated DNA sequence comprising a fragment of the DNA of FIG. 1 (SEQ ID NO: 1), wherein said fragment comprises at least 80 contiguous bases of said sequence. DNA sequence. 23. An antiserum prepared by immunizing a mammal with the polypeptide according to claim 14 or 15. 24. A monoclonal antibody against the polypeptide according to claim 14. 25. One monoclonal antibody against the polypeptide according to claim 15. 26. A method for increasing the proliferation rate of human mesenchymal stem cells in vitro, comprising adding an effective amount of the polypeptide of claim 16 to an extracellular growth medium in which such cells are suspended. A method comprising: 27. The method of claim 26, wherein the polypeptide is present in said growth medium at a concentration of at least 1 pg / ml. 28. A chemical compound having a structure similar to the domain of the C17 polypeptide of FIG. 29. A method for determining the presence of a growth-stimulating receptor on the cell surface, comprising using a chemically labeled C17 polypeptide of FIG. 2 or an analog thereof in a suitable medium. Incubating such analogs with the cells, determining if there is a stimulation of growth in the presence of such analogs, detecting analogs that bind to the cell surface, and binding to such analogs Detecting the presence of such a receptor on the surface of such cells by isolating the receptor.