WO2003014340A2 - Human histone deacetylase-related gene and protein hdac10 - Google Patents

Abstract

Disclosed is an HDAC related gene and gene product. In particular, the invention relates to a protein that is highly homologous to known HDACs and referred to herein as HDAC10, nucleic acid molecules that encode such a protein, antibodies that recognize the protein, and methods for diagnosing conditions related to abnormal HDAC10 activity or gene expression.

Description

HlSTONE DEACETYLASE - RELATED GENE AND PROTEIN

This invention relates to a histone deacetylase gene and gene product. In particular, the invention relates to a protein that is highly homologous to known mammalian histone deacetylases (HDACs), nucleic acid molecules that encode such a protein, antibodies that recognize the protein, and methods of use which include assays screening for modulators of HD AC activity and for diagnosing conditions related to abnormal HDAC activity, inciting, for example, abnormal cell proliferation, cancer, atherosclerosis, inflammatory bowel disease, host inflammatory or immune response or psoriasis.

BACKGROUND

Histone acetylation is a major regulatory mechanism that modulates gene expression by altering the accessibility of transcription factors to DNA. Acetylation of histones is a reversible modification of the free ε-amino group of lysine that occurs during the assembly of nucleosomes and during DNA synthesis.

HDACs have been shown to play an important role in the regulation of transcription. HDACs function as components of complexes that are involved in transcriptional repression. This is mediated through interactions of HDACs with multi-protein complexes and requires deacetylase activity. Changes in histone acetylation levels also occur during transcriptional activation and silencing. Acetylation of histones is generally associated with transcriptional activity, whereas deacetylation is associated with transcriptional repression.

HDAC complexes may contain the co-repressor mSin3A and mSin3A-associated proteins, silencing mediators NcoR and SMRT, transcriptional repressors, Rb-like proteins pi 07 and pi 30, Rb- associated proteins, nuclear hormone receptors, nucleosome remodeling factors, methyl-binding proteins, DNA repair machinery proteins, and the like. Furthermore, HDACl has been found to bind directly to YY1 and Spl and HDACs 4 and 5 bind to MEF2. In addition, HDACs have been found together in complexes.

Two distinct classes of yeast histone deacetylases have been identified based upon size and sequence. Yeast class I HDACs include Rpd3, Hoslp, and Hos2p. Class II contains yeast HDAlp. Furthermore, members of these two classes were found to form different complexes. Human HDACs have been classified based upon their similarity to yeast sequences. Class I human HDACs include HDACsl-3 and 8. Class ϋ HDACs include HDACs 4-7. The deacetylase core of class I HDACs reside in the first ~390 amino acids. Class II HDAC catalytic domains are located in the C-terminal of these peptides, with the exception of HDAC6 that contains a second catalytic domain in the N- terminus. Here we report the isolation and characterization of a new HDAC, referred to herein as HDAClO.

An important approach that has been used to study the function of chromatin acetylation is the use of specific inhibitors of histone deacetylase. Several classes of compounds have been identified that inhibit HDAC. Histone deacetylase inhibitors have been found to have anti-proliferative effects, including induction of Gl/S and G2 M cell cycle arrest, differentiation and apoptosis of transformed and normal cells and reversal of transformation. These effects, along with the presence of HDAC in complexes with fusions of unliganded retinoic acid receptors PML-RARαand PLZF-RARα indicate a role for HDACs in tumorigenicity. Furthermore, histone deacetylase inhibitors, phenylbutyrate and trichostatin A have shown promise in the treatment of promyelocytic leukemia and several other HDAC inhibitors are being studied as treatments for cancers.

SUMMARY OF THE INVENTION

The present invention relates to a novel histone deacetylase designated HDAClO.

In a first aspect, the invention provides an isolated polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:l. Furthermore, the invention provides an isolated polypeptide consisting of an amino acid sequence as set forth in SEQ ID NO:l. The amino acid sequence as set forth in SEQ ID NO:l shows a considerable degree of homology to that of known members ofthe family of HDACs in the catalytic domain. For convenience, the polypeptide consisting ofthe amino acid sequence as set forth in SEQ ID NO:l will be designated as histone deacetylase 10 or HDAClO. Fragments ofthe isolated polypeptide having an amino acid sequence as set forth in SEQ ID NO:l also form a part of the present invention. Preferably, fragments will encompass the catalytic domain, which is predicted to exist between amino acid number 15 to 323. In accordance with this aspect of the invention there are provided novel polypeptides of human origin as well as biologically, diagnostically or therapeutically useful fragments, variants and derivatives thereof, variants and derivatives of the fragments, and analogs of the foregoing. In a second aspect, the invention provides an isolated DNA comprising a nucleotide sequence that encodes a polypeptide as mentioned above. Li particular, the invention provides (1) an isolated DNA comprising the nucleotide sequence as set forth in SEQ ID NO:2; (2) an isolated DNA comprising the nucleotide sequence set forth in SEQ ID NO:3; (3) an isolated DNA capable of hybridizing under high stringency conditions to the nucleotide sequence set forth in SEQ ID NO:2; and (4) an isolated DNA comprising the nucleotide sequence set forth in SEQ ID NO:4. Also provided are nucleic acid sequences comprising at least about 15 bases, preferably at least about 20 bases, more preferably a nucleic acid sequence comprising about 30 contiguous bases of SEQ ID NO:2 or SEQ ID NO:3. Also within the scope ofthe present invention are nucleic acids that are substantially similar to the nucleic acid with the nucleotide sequence as set forth in SEQ ID NO:2 or SEQ ID NO:3. In a preferred embodiment, the isolated DNA takes the form of a vector molecule comprising at least a fragment of a DNA ofthe present invention, in particular comprising the DNA consisting of a nucleotide sequence as set forth in SEQ ID NO:2 or SEQ ID NO:3.

A third aspect ofthe present invention encompasses a method for the diagnosis of conditions associated with abnormal regulation of gene expression which includes, but is not limited to, conditions associated with abnormal cell proliferation, cancer, atherosclerosis, inflammatory bowel disease, or psoriasis in a human which comprises detecting abnormal transcription of messenger RNA transcribed from the natural endogenous human gene encoding the novel polypeptide consisting ofthe amino acid sequence set forth in SEQ ID NO:l in an appropriate tissue or cell from a human, wherein such abnormal transcription is diagnostic ofthe human's affliction with such a condition. In particular, the said natural endogenous human gene encoding the novel polypeptide consisting ofthe amino acid sequence set forth in SEQ ID NO:l comprises the genomic nucleotide sequence set forth in SEQ ID NO:4. In one embodiment ofthe present invention, the diagnostic method comprises contacting a sample of said appropriate tissue or cell or contacting an isolated RNA or DNA molecule derived from that tissue or cell with an isolated nucleotide sequence of at least about 15 - 20 nucleotides in length that hybridizes under high stringency conditions with the isolated nucleotide sequence encoding the novel polypeptide having an amino acid sequence set forth in SEQ ID NO:l.

Another embodiment of the assay aspect ofthe invention provides a method for the diagnosis of a condition associated with abnormal HDAClO activity in a human, which comprises measuring the level of deacetylase activity in a certain tissue or cell from a human suffering from such a condition, wherein the presence of an abnormal level of deacetylase activity, relative to the level thereof in the respective tissue or cell of a human not suffering from a condition associated with abnormal HDAC activity, is diagnostic ofthe human's suffering from said condition.

In accordance with one embodiment of this aspect ofthe invention there are provided anti- sense polynucleotides that can regulate transcription ofthe gene encoding the novel HDAClO; in another embodiment, double stranded RNA is provided that can regulate the transcription ofthe gene encoding the novel HDAClO.

Another aspect ofthe invention provides a process for producing the aforementioned polypeptides, polypeptide fragments, variants and derivatives, fragments ofthe variants and derivatives, and analogs ofthe foregoing. In a preferred embodiment of this aspect ofthe invention there are provided methods for producing the aforementioned HDAClO comprising culturing host cells having incorporated therein an expression vector containing an exogenously-derived nucleotide sequence encoding such a polynucleotide under conditions sufficient for expression ofthe polypeptide in the host cell, thereby causing expression ofthe polypeptide, and optionally recovering the expressed polypeptide. In a preferred embodiment of this aspect ofthe present invention, there is provided a method for producing polypeptides comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:l, which comprises culturing a host cell having incorporated therein an expression vector containing an exogenously-derived polynucleotide encoding a polypeptide comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:l, under conditions sufficient for expression of such a polypeptide in the host cell, thereby causing the production of an expressed polypeptide, and optionally recovering the expressed polypeptide. Preferably, in any of such methods the exogenously derived polynucleotide comprises or consists ofthe nucleotide sequence set forth in SEQ ID NO:2, the nucleotide sequence set forth in SEQ ID NO:3, or the nucleotide sequence set forth in SEQ ID NO:4. In accordance with another aspect ofthe invention there are provided products, compositions, processes and methods that utilize the aforementioned polypeptides and polynucleotides for, inter alia, research, biological, clinical and therapeutic purposes.

In certain additional preferred embodiments of this aspect ofthe invention there is provided an antibody or a fragment thereof which specifically binds to a polypeptide that comprises the amino acid sequence set forth in SEQ ID NO:l, i.e., HDAClO. In certain particularly preferred embodiments in this regard, the antibodies are highly selective for human HDAClO polypeptides or portions of human HDAClO polypeptides. In a further aspect, an antibody or fragment thereof is provided that binds to a fragment or portion ofthe amino acid sequence set forth in SEQ ID NO:l.

In another aspect, methods of treating a condition in a subject, wherein the condition is associated with abnormal HDAClO gene expression, an increase or decrease in the presence of HDAClO polypeptide in a subject, or an increase or decrease in the activity of HDAClO polypeptide, by the administration of an effective amount of an antibody that binds to a polypeptide with the amino acid sequence set out in SEQ ID NO: 1, or a fragment or portion thereof to the subject are provided. Also provided are methods for the diagnosis of a disease or condition associated with abnormal HDAClO gene expression or an increase or decrease in the presence ofthe HDAClO in a subject, or an increase or decrease in the activity of HDAClO polypeptide.

In yet another aspect, the invention provides host cells which can be propagated in vitro, preferably vertebrate cells, in particular mammalian cells, or bacterial cells, which are capable upon growth in culture of producing a polypeptide that comprises the amino acid sequence set forth in SEQ ID NO: 1 or fragments thereof, where the cells contain transcriptional control DNA sequences, where the transcriptional control sequences control transcription of RNA encoding a polypeptide with the amino acid sequence according to SEQ ID NO:l or fragments thereof. This includes, but is not limited to, the propagation of HDAClO in a plasmid and the production of DNA, RNA or protein in human or insect cells or bacteria using the endogenous HDAClO promoter or any other transcriptional control sequence.

In yet another aspect ofthe present invention there are provided assay methods and kits comprising the components necessary to detect above-normal expression of polynucleotides encoding a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 1, or polypeptides comprising an amino acid sequence set forth in SEQ ID NO:l, or fragments thereof, in body tissue samples derived from a patient, such kits comprising e.g., antibodies that bind to a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1 , or to fragments thereof, or oligonucleotide probes that hybridize with polynucleotides ofthe invention. In a preferred embodiment, such kits also comprise instructions detailing the procedures by which the kit components are to be used. In another aspect, the invention is directed to use of a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:l or fragment thereof, polynucleotide encoding such a polypeptide or a fragment thereof, or antibody that binds to said polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1 or a fragment thereof in the manufacture of a medicament to treat diseases associated with abnormal HDAC activity or gene expression.

Another aspect is directed to pharmaceutical compositions comprising a polypeptide comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 1 or fragment thereof, a polynucleotide encoding such a polypeptide or a fragment thereof, or antibody that binds to such a polypeptide or a fragment thereof, in conjunction with a suitable pharmaceutical carrier, excipient or diluent, for the treatment of diseases associated with abnormal HDAC activity or gene expression.

In another aspect, the invention is directed to methods for the identification of molecules that can bind to a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1 and/or modulate the activity of a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1 or molecules that can bind to nucleic acid sequences that modulate the transcription or translation of a polynucleotide encoding a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:l. Molecules identified by such methods also fall within the scope ofthe present invention.

In a related aspect, the invention is directed to use ofthe novel HDAClO to identify associated proteins in HDAC biologically relevant complexes. At present, the proteins that associate with HDAClO are not known. However, these may be characterized by determining whether HDAClO associates with proteins that have been previously shown to interact with other HDACs (see Introduction). For example, components of HDAClO complexes may be determined using conventional methods, including co-immunoprecipitation.

In yet another aspect, the invention is directed to methods for the introduction of nucleic acids of the invention into one or more tissues of a subject in need of treatment with the result that one or more proteins encoded by the nucleic acids are expressed and or secreted by cells within the tissue.

Other objects, features, advantages and aspects ofthe present invention will become apparent to those of skill from the following description. It should be understood, however, that the following description and the specific examples, while indicating preferred embodiments ofthe invention, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following description and from reading the other parts ofthe present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows amino acid sequence (SEQ ID NO:l) of HDAClO.

Figure 2 shows the full-length cDNA sequence (SEQ ID NO:2) of HDAClO. The full-length cDNA sequence starts at nucleotide position 1 and ends at nucleotide position 1755.

Figure 3 shows the open reading frame of HDAClO cDNA sequence (SEQ ID NO:3). The sequence starts at nucleotide position 25 and ends at nucleotide position 1065 as indicated in SEQ ID NO:2.

Figure 4 shows HDAClO genomic DNA sequence (SEQ ID NO:4).

DETAILED DESCRIPTION OF THE INVENTION

In practicing the present invention, many conventional techniques in molecular biology, microbiology, and recombinant DNA are used. These techniques are well known to one of ordinary skill in the art. The following abbreviations used throughout the disclosure are listed herein below: histone deacetylase (HDAC), histone deacetylase-like protein (HDLP)

In its broadest sense, the term "substantially similar", when used herein with respect to a nucleotide sequence, means a nucleotide sequence corresponding to a reference nucleotide sequence, wherein the corresponding sequence encodes a polypeptide having substantially the same structure and function as the polypeptide encoded by the reference nucleotide sequence, e.g. where only changes in amino acids not affecting the polypeptide function occur. Desirably the substantially similar nucleotide sequence encodes the polypeptide encoded by the reference nucleotide sequence. The percentage of identity between the substantially similar nucleotide sequence and the reference nucleotide sequence desirably is at least 80%, more desirably at least 85%, preferably at least 90%, more preferably at least 95%, still more preferably at least 98 or 99%. Sequence comparisons are carried out using Clustalw (see, for example, Higgins, D.G. et al. Methods Enzymol. 266:383-402 (1996)). Clustalw alignments were performed using default parameters.

A nucleotide sequence "substantially similar" to reference nucleotide sequence hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C with washing in IX SSC, 0.1% SDS at 50°C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C, preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 50°C, more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C with washing in 0.1 X SSC, 0.1% SDS at 65 °C, yet still encodes a functionally equivalent gene product.

"Elevated transcription of mRNA" refers to a greater amount of messenger RNA transcribed from the natural endogenous human gene encoding the novel polypeptide ofthe present invention present in an appropriate tissue or cell of an individual suffering from a condition associated with abnormal HDAClO activity than in a subject not suffering from such a disease or condition; in particular at least about twice, preferably at least about five times, more preferably at least about ten times, most preferably at least about 100 times the amount of mRNA found in corresponding tissues in humans who do not suffer from such a condition. Such elevated level of mRNA may eventually lead to increased levels of protein translated from such mRNA in an individual suffering from a condition associated with abnormal cellular proliferation as compared with a healthy individual. It is also understood that "elevated transcription of mRNA" may refer to a greater amount of messenger RNA transcribed from genes the expression of which is modulated by HDAClO either alone or in combination with other molecules.

A "host cell," as used herein, refers to a prokaryotic or eukaryotic cell that contains heterologous DNA that has been introduced into the cell by any means, e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, and the like.

"Heterologous" as used herein means "of different natural origin" or represent a non-natural state. For example, if a host cell is transformed with a DNA or gene derived from another organism, particularly from another species, that gene is heterologous with respect to that host cell and also with respect to descendants of the host cell which carry that gene. Similarly, heterologous refers to a nucleotide sequence derived from and inserted into the same natural, original cell type, but which is present in a non-natural state, e.g. a different copy number, or under the control of different regulatory elements.

A "vector" molecule is a nucleic acid molecule into which heterologous nucleic acid may be inserted which can then be introduced into an appropriate host cell. Vectors preferably have one or more origin of replication, and one or more site into which the recombinant DNA can be inserted. Vectors often have convenient means by which cells with vectors can be selected from those without, e.g., they encode drug resistance genes. Common vectors include plasmids, viral genomes, and (primarily in yeast and bacteria) "artificial chromosomes."

"Plasmids" generally are designated herein by a lower case p preceded and/or followed by capital letters and/or numbers, in accordance with standard naming conventions that are familiar to those of skill in the art. Starting plasmids disclosed herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids by routine application of well-known, published procedures. Many plasmids and other cloning and expression vectors that can be used in accordance with the present invention are well known and readily available to those of skill in the art. Moreover, those of skill readily may construct any number of other plasmids suitable for use in the invention. The properties, construction and use of such plasmids, as well as other vectors, in the present invention will be readily apparent to those of skill from the present disclosure.

The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all ofthe coexisting materials in the natural system, is isolated, even if subsequently reintroduced into the natural system. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.

As used herein, the term "transcriptional control sequence" refers to DNA sequences, such as initiator sequences, enhancer sequences, and promoter sequences, which induce, repress, or otherwise control the transcription of protein encoding nucleic acid sequences to which they are operably linked. As used herein, "human transcriptional control sequences" are any of those transcriptional control sequences normally found associated with the human gene encoding the novel HDAClO polypeptide ofthe present invention as it is found in the respective human chromosome. It is understood that the term may also refer to transcriptional control sequences normally found associated with human genes the expression of which is modulated by HDAClO either alone or in combination with other molecules.

As used herein, "non-human transcriptional control sequence" is any transcriptional control sequence not found in the human genome.

The term "polypeptide" is used interchangeably herein with the terms "polypeptides" and "protein(s)".

As used herein, a "chemical derivative" of a polypeptide ofthe invention is a polypeptide of the invention that contains additional chemical moieties not normally a part ofthe molecule. Such moieties may improve the molecule's solubility, absorption, biological half life, etc. The moieties may alternatively decrease the toxicity ofthe molecule, eliminate or attenuate any undesirable side effect ofthe molecule, etc. Moieties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Pa. (1980).

As used herein, "HDAClO" refers to the amino acid sequences of substantially purified HDAClO obtained from any species, particularly mammalian, including bovine, ovine, porcine, murine, equine, and preferably human, from any source, whether natural, synthetic, semi-synthetic, or recombinant.

As used herein, "HDAC activity", including "HDAClO activity" refers to the ability of an HDAC polypeptide to deacetylate histone proteins, including ³H-labeled H4 histone peptide. Such activity may be measured according to conventional methods. A biologically "active" protein refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.

The term "agonist", as used herein, refers to a molecule which when bound to HDAClO, causes a change in HDAClO which modulates the activity of HDAClO. Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules that bind to HDAClO. The terms "antagonist" or "inhibitor" as used herein, refer to a molecule which when bound to HDAClO, blocks or modulates the biological activity of HDAClO. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecules, natural or synthetic that bind to HDAClO.

The full-length cDNA for HDAClO is 1755 base pairs in length and it predicts a protein of 347 amino acids. The predicted HDAClO protein possesses a putative catalytic domain which encompasses approximately 317 amino acids (~6 to 323) based upon alignments of HDAClO with the putative catalytic domains of all ofthe other known HDACs. To identify the catalytic domain of HDAClO, Clustalw alignments were performed separately using HDAClO complete peptide and catalytic domain sequences from class I HDACs (1-3 and 8) or class II HDACs (4-7).

Table 2 below shows the catalytic domain amino acids of HDACs 1-10 that align with histone deacetylase-like protein (HDLP), a bacterial protein that shares 35.2% homology with HDACl and possesses deacetylase activity (Finnin, M. S., Doniglan, J. R., Cohen, A., Richon, V. M., Rifkind, R. A., Marks, P. A., Breslow, R., and Pavletich, N. P. (1999) Nature 401, 188-193).

Table 2. HDAC catalytic amino acids

As a member ofthe HDAC family, HDAClO may form biologically relevant complexes with proteins and display functions that have been described for other HDACs. For example, it is likely to be involved in transcription repression as a component of multi-protein complexes that often include transcription co-repressors. Thus, increased activity or expression of HDAClO may be associated with numerous pathological conditions, including but not limited to, abnormal cell proliferation, cancer, atherosclerosis, inflammatory bowel disease, host inflammatory or immune response, or psoriasis.

Thus, the identification of HDAClO is useful for designing agents (e.g. antagonists or inhibitors) useful to ameliorate conditions associated with abnormal HDAC activity. These may include, for example, antiproliferative or antiinflammatory agents either through the use of small molecules or proteins (e.g. antibodies) directed against it or its associated proteins in the HDAC transcription repressor complexes. In addition, protein derived from the HDAClO sequence may also be used as a therapeutic to modify host cell proliferative or inflammatory responses.

To determine the tissue distribution of HDAClO in human, Northern analyses were performed using a blot containing mRNA isolated from various human tissues. The results indicate that overall expression level of HDAClO is low and the highest expression level is restricted to brain, heart, skeletal muscle and kidney. Furthermore, real-time PCR experiments reveal that HDAClO is also highly expressed in testis as well as several human cancereous cell lines. Thus, HDAClO represents a transcribed gene.

In one aspect, the present invention relates to a novel histone deacetylase (HDAC). As outlined above, HDAClO is clearly a member ofthe HDAC family since it is highly similar to other HDAC proteins, especially in the catalytic domain.

The present invention relates to an isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1. For example, such a polypeptide may be a fusion protein including the amino acid sequence ofthe novel HDAClO. In another aspect the present invention relates to an isolated polypeptide consisting ofthe amino acid sequence set forth in SEQ ID NO:l, which is, in particular, the novel HDAClO.

The invention includes nucleic acid or nucleotide molecules, preferably DNA molecules, in particular encoding the novel HDAClO. Preferably, an isolated nucleic acid molecule, preferably a DNA molecule, ofthe present invention encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:l. Likewise preferred is an isolated nucleic acid molecule, preferably a DNA molecule, encoding a polypeptide consisting ofthe amino acid sequence set forth in SEQ ID NO: 1. Such a nucleic acid or nucleotide, in particular such a DNA molecule, preferably comprises a nucleotide sequence selected from the group consisting of (1) the nucleotide sequence as set forth in SEQ ID NO:2, which is the full-length cDNA sequence encoding the polypeptide consisting ofthe amino acid sequence set forth in SEQ ID NO: 1 : (2) the nucleotide sequence set forth in SEQ ID NO:3, which corresponds to the open reading frame ofthe cDNA sequence set forth in SEQ ID NO:2; (3) a nucleotide sequence capable of hybridizing under high stringency conditions to a nucleotide sequence set forth in SEQ ID NO:3; and (4) the nucleotide sequence set forth in SEQ ID NO:4, which corresponds to the endogenous genomic human DNA encoding the polypeptide consisting ofthe amino acid sequence set forth in SEQ ID NO:l. Such hybridization conditions may be highly stringent or less highly stringent, as described above. In instances wherein the nucleic acid molecules are deoxyoligonucleotides ("oligos"), highly stringent conditions may refer, e.g., to washing in 6X SSC/0.05% sodium pyrophosphate at 37 °C (for 14-base oligos), 48 °C (for 17-base oligos), 55 °C (for 20-base oligos), and 60 °C (for 23-base oligos). Suitable ranges of such stringency conditions for nucleic acids of varying compositions are described in Krause and Aaronson (1991), Methods in Enzymology, 200:546-556.

These nucleic acid molecules may act as target gene antisense molecules, useful, for example, in target gene regulation and/or as antisense primers in amplification reactions of target gene nucleic acid sequences. Further, such sequences may be used as part of ribozyme and/or triple helix sequences, also useful for target gene regulation. Still further, such molecules may be used as components of diagnostic methods whereby the presence of an allele causing a disease associated with abnormal HDAClO expression or activity, for example, abnormal cell proliferation, cancer, atherosclerosis, inflammatory bowel disease, host inflammatory or immune response, or psoriasis, may be detected.

The invention also encompasses (a) vectors that contain at least a fragment of any ofthe foregoing nucleotide sequences and/or their complements (i.e., antisense); (b) vector molecules, preferably vector molecules comprising transcriptional control sequences, in particular expression vectors, that contain any ofthe foregoing coding sequences operatively associated with a regulatory element that directs the expression ofthe coding sequences; and (c) genetically engineered host cells that contain a vector molecule as mentioned herein or at least a fragment of any ofthe foregoing nucleotide sequences operatively associated with a regulatory element that directs the expression of the coding sequences in the host cell. As used herein, regulatory elements include, but are not limited to, inducible and non-inducible promoters, enhancers, operators and other elements known to those skilled in the art that drive and regulate expression. Preferably, host cells can be vertebrate host cells, preferably mammalian host cells, such as human cells or rodent cells, such as CHO or BHK cells. Likewise preferred, host cells can be bacterial host cells, in particular E.coli cells.

Particularly preferred is a host cell, in particular ofthe above described type, which can be propagated in vitro and which is capable upon growth in culture of producing an HDAClO polypeptide, in particular a polypeptide comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 1, wherein said cell contains some fragment or complete sequence of HDAClO coding sequence in a construct that is controlled by one or more transcriptional control sequences that is not a transcriptional control sequence ofthe natural endogeneous human gene encoding said polypeptide, wherein said one or more transcriptional control sequences control transcription of a DNA encoding said polypeptide. Possible transcriptional control sequences include, but are not limited to, bacterial or viral promoter sequences.

The invention includes the complete sequence ofthe gene as well as fragments of any ofthe nucleic acid sequences disclosed herein. Fragments ofthe nucleic acid sequences encoding the novel HDAClO polypeptide may be used as a hybridization probe for a cDNA library to isolate other genes which have a high sequence similarity to the HDAClO gene or similar biological activity. Probes of this type preferably have at least about 30 bases and may contain, for example, from about 30 to about 50 bases, about 50 to about 100 bases, about 100 to about 200 bases, or more than 200 bases. The probe may also be used to identify a cDNA clone that correspond to a full-length transcript and a genomic clone or clones that contain the complete HDAClO gene including regulatory and promoter regions, exons, and introns. An example of a screen comprises isolating the coding region ofthe HDAClO gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that ofthe gene ofthe present invention may be used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library to which the probe hybridizes.

In addition to the gene sequences described above, homologs of such sequences, as may, for example, be present in other species, may be identified and may be readily isolated, without undue experimentation, by molecular biological techniques well known in the art. Furthermore, there may exist genes at other genetic loci within the genome that encode proteins that have homology to one or more domains of such gene products. These genes may also be identified via similar techniques. For example, the isolated nucleotide sequence ofthe present invention encoding the novel HDAClO polypeptide may be labeled and used to screen a cDNA library constructed from mRNA obtained from the organism of interest. Hybridization conditions will be of a lower stringency when the cDNA library is derived from an organism different from the type of organism from which the labeled sequence was derived. Alternatively, the labeled fragment may be used to screen a genomic library derived from the organism of interest, again, using appropriately stringent conditions. Such low stringency conditions will be well known to those of skill in the art, and will vary predictably depending on the specific organisms from which the library and the labeled sequences are derived.

Further, a previously unknown differentially expressed gene-type sequence may be isolated by performing PCR using two degenerate oligonucleotide primer pools designed on the basis of amino acid sequences within the gene of interest. The template for the reaction may be cDNA obtained by reverse transcription of mRNA prepared from human or non-human cell lines or tissue known or suspected to express a differentially expressed gene allele. The PCR product may be subcloned and sequenced to ensure that the amplified sequences represent the sequences of a differentially expressed gene-like nucleic acid sequence. The PCR fragment may then be used to isolate a complete cDNA clone by a variety of conventional methods. For example, the amplified fragment may be labeled and used to screen a bacteriophage cDNA library. Altematively, the labeled fragment may be used to screen a genomic library.

PCR technology may also be utilized to isolate full-length cDNA sequences. For example, RNA may be isolated, following standard procedures, from an appropriate cellular or tissue source. A reverse transcription reaction may be performed on the RNA using an oligonucleotide primer specific for the most 5' end ofthe amplified fragment for the priming of first strand synthesis. The resulting RNA/DNA hybrid may then be "tailed" with guanines using a standard terminal transferase reaction, the hybrid may be digested with RNAase H, and second strand synthesis may then be primed with a poly-C primer. Thus, cDNA sequences upstream ofthe amplified fragment may easily be isolated.

In cases where the gene identified is the normal, or the wild type gene, this gene may be used to isolate mutant alleles ofthe gene. Isolation of mutant alleles is preferable in processes and disorders that are known or suspected to have a genetic basis. Mutant alleles may be isolated from individuals either known or suspected to have a genotype which contributes to disease symptoms related to abnormal HDAC activity, including, but not limited to, conditions such as abnormal cell proliferation, cancer, atherosclerosis, inflammatory bowel disease, host inflammatory or immune response, or psoriasis. Mutant alleles and mutant allele products may then be used in the diagnostic assay systems described below.

A cDNA of the mutant gene may be isolated, for example, using PCR, a technique that is well known to those of skill in the art. In this case, the first cDNA strand may be synthesized by hybridizing an oligo-dT oligonucleotide to mRNA isolated from tissue known or suspected to be expressed in an individual putatively carrying the mutant allele, and by extending the new strand with reverse transcriptase. The second strand ofthe cDNA is then synthesized using an oligonucleotide that hybridizes specifically to the 5' end ofthe normal gene. Using these two primers, the product is then amplified via PCR, cloned into a suitable vector, and subjected to DNA sequence analysis through methods well known to those of skill in the art. By comparing the DNA sequence ofthe mutant gene to that ofthe normal gene, the mutation(s) responsible for the loss or alteration of function of the mutant gene product can be ascertained.

Alternatively, a genomic or cDNA library can be constructed and screened using DNA or RNA, respectively, from a tissue known to or suspected of expressing the gene of interest in an individual suspected of or known to carry the mutant allele. The normal gene or any suitable fragment thereof may then be labeled and used as a probe to identify the corresponding mutant allele in the library. The clone containing this gene may then be purified through methods routinely practiced in the art, and subjected to sequence analysis as described above.

Additionally, an expression library can be constructed utilizing DNA isolated from or cDNA synthesized from a tissue known to or suspected of expressing the gene of interest in an individual suspected of or known to carry the mutant allele. In this manner, gene products made by the putatively mutant tissue may be expressed and screened using standard antibody screening techniques in conjunction with antibodies raised against the normal gene product, as described below. In cases where the mutation results in an expressed gene product with altered function (e.g., as a result of a mis-sense mutation), a polyclonal set of antibodies are likely to cross-react with the mutant gene product. Library clones detected via their reaction with such labeled antibodies can be purified and subjected to sequence analysis as described above. The present invention includes those proteins encoded by nucleotide sequences set forth in any of SEQ ID NOs:2, 3 or 4, in particular, a polypeptide that is or includes the amino acid sequence set out in SEQ ID NO: 1 , or fragments thereof.

Furthermore, the present invention includes proteins that represent functionally equivalent gene products. Such an equivalent differentially expressed gene product may contain deletions, additions or substitutions of amino acid residues within the amino acid sequence encoded by the differentially expressed gene sequences described, above, but which result in a silent change, thus producing a functionally equivalent differentially expressed gene product. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature ofthe residues involved.

Nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. "Functionally equivalent," as utilized herein, may refer to a protein or polypeptide capable of exhibiting a substantially similar in vivo or in vitro activity as the endogenous differentially expressed gene products encoded by the differentially expressed gene sequences described above. "Functionally equivalent" may also refer to proteins or polypeptides capable of interacting with other cellular or extracellular molecules in a manner substantially similar to the way in which the corresponding portion ofthe endogenous differentially expressed gene product would. For example, a "functionally equivalent" peptide, the sequence of which was modified from the endogenous peptide to achieve "functional equivalency, would be able, in an immunoassay, to diminish the binding of an antibody to the corresponding peptide within the endogenous protein, or the binding to the endogenous protein itself, against which the antibody was raised. An equimolar concentration ofthe functionally equivalent peptide will diminish the aforesaid binding ofthe corresponding peptide by at least about 5%, preferably between about 5% and 10%, more preferably between about 10% and 25%, even more preferably between about 25% and 50%, and most preferably between about 40% and 50%.

The polypeptides of the present invention may be produced by recombinant DNA technology using techniques well known in the art. Therefore, there is provided a method of producing a polypeptide ofthe present invention, which method comprises culturing a host cell having incorporated therein an expression vector containing an exogenously-derived polynucleotide encoding a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:l under conditions sufficient for expression ofthe polypeptide in the host cell, thereby causing the production ofthe expressed polypeptide. Optionally, said method further comprises recovering the polypeptide produced by said cell. In a preferred embodiment of such a method, said exogenously-derived polynucleotide encodes a polypeptide consisting of an amino acid sequence set forth in SEQ ID NO:l. Preferably, said exogenously-derived polynucleotide comprises the nucleotide sequence as set forth in any of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4. In case of using the nucleotide sequence set forth in SEQ ID NO:3, i.e. the open reading frame, the sequence, when inserted into a vector, may be followed by one or more appropriate translation stop codons, preferably by the natural endogenous stop codon TGA beginning at nucleotide 1066 in the cDNA sequence.

Thus, methods for preparing the polypeptides and peptides ofthe invention by expressing nucleic acid encoding respective nucleotide sequences are described herein. Methods which are well- known to those skilled in the art can be used to construct expression vectors that contain protein coding sequences and appropriate transcriptional/translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. Alternatively, RNA capable of encoding differentially expressed gene protein sequences may be chemically synthesized using, for example, synthesizers.

A variety of host-expression vector systems may be utilized to express the HDAClO gene coding sequences ofthe invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, exhibit the HDAClO gene protein ofthe invention in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing differentially expressed gene protein coding sequences; yeast (e.g. Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the differentially expressed gene protein coding sequences; insect cell systems infected or transfected with recombinant virus expression vectors (e.g., baculovirus) containing the differentially expressed gene protein coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant vectors, including plasmids, (e.g., Ti plasmid) containing protein coding sequences; or mammalian cell systems (e.g. COS, CHO, BHK, 293, 3T3) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothioneine promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter, or the CMV promoter).

Expression ofthe HDAClO ofthe present invention by a cell from an HDAClO encoding gene that is native to the cell can also be performed. Such methods are known in the art. Cells that have been induced to express HDAClO can be implanted into a desired tissue in a living animal in order to increase the local concentration of HDAClO in the tissue.

i bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the protein being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of antibodies or to screen peptide libraries, for example, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. In this respect, fusion proteins comprising hexahistidine tags may be used, such as EpiTag vectos including pCDNA3.1 His (Invitrogen, Carlsbad, CA). Other vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the protein coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors; and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene protein can be released from the GST moiety. Fusion proteins containing Flag tags, such as 3X Flag (Sigma, St. Louis, MO) or myc tags, for example pCDNA3.1/myc-His (Invitrogen, Carlsbad, CA) may be used. These fusions allow coimmunoprecipitation and Western detection of proteins for which antibodies are not yet available.

Promoter regions from any desired gene can be introduced into vectors containing a reporter transcription unit, such as a chloramphenicol acetyl transferase ("CAT"), or the luciferase transcription unit, which also lack a promoter region. Restriction site or sites in the vector can be used for introducing a candidate promoter fragment; i.e., a fragment that may contain a promoter. For example, introduction into the vector of a promoter-containing fragment at the restriction site upstream of the cat gene engenders production of CAT activity, which can be detected by standard CAT assays. Vectors suitable to this end are well known and readily available. Two such vectors are pKK232-8 and pCM7. Thus, promoters for expression of polynucleotides ofthe present invention include not only well-known and readily available promoters, but also promoters that readily may be obtained by the foregoing technique, using a reporter gene.

Among known bacterial promoters suitable for expression of polynucleotides and polypeptides in accordance with the present invention are the E. coli lad and lacZ promoters, the T3 and T7 promoters, the T5 tac promoter, the lambda PR, PL promoters and the trp promoter. Among known eukaryotic promoters suitable in this regard are the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those ofthe Rous sarcoma virus ("RSV"), and metallothionein promoters, such as the mouse metallothionein-I promoter. For example, a plasmid construct could contain a HDAClO transcriptional control sequence fused to a reporter transcription unit that encodes the coding region of jS-Galactosidase, chloramphenicol acetyltransferase, green fluorescent protein or luciferase . This construct could be used to screen for small molecules that modulate HDAClO transcription. Such molecules are potential therapeutics. Furthermore, using fluorescence microscopy or Biophotonic in vivo imaging, a technology that produces visual and quantitative measurements in real time (Xenogen, Palo Alto, CA), expression of a fluorescent HDAClO reporter gene could be examined to determine the effects of an HDAClO therapeutic in mammalian cells or xenografts. Changes in these reporters in normal, diseased or drug-treated tissue or cells would be indicators of changes in HDAClO expression or activity.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is one of several insect systems that can be used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) ofthe virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful insertion ofthe coding sequence will result in inactivation ofthe polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed.

In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the coding sequence of interest may be ligated to an adenovirus transcription translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region ofthe viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the desired protein in infected hosts (e.g., See Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:3655-3659). Specific initiation signals may also be required for efficient translation of inserted gene coding sequences. These signals include the ATG initiation codon and adjacent sequences. Li cases where an entire gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion ofthe gene coding sequence is inserted, exogenous translational control signals, including, perhaps, the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame ofthe desired coding sequence to ensure translation ofthe entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc.. Other common systems are based on SV40, retrovirus or adeno-associated virus. Selection of appropriate vectors and promoters for expression in a host cell is a well known procedure and the requisite techniques for expression vector construction, introduction ofthe vector into the host and expression in the host per se are routine skills in the art. Generally, recombinant expression vectors will include origins of replication, a promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence, and a selectable marker to permit isolation of vector containing cells after exposure to the vector.

In addition, a host cell strain may be chosen which modulates the expression ofthe inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function ofthe protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing ofthe foreign protein expressed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing ofthe primary transcript, glycosylation, and phosphorylation ofthe gene product may be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, etc. and are well known to one of skill in the art.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express a differentially expressed protein product of a gene may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction ofthe foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines that express the differentially expressed gene protein. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the endogenous activity ofthe expressed protein.

A number of selection systems may be used, including but not limited to, the herpes simplex virus thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase genes can be employed in tk^", hgprt^" or aprf cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate, gpt, which confers resistance to mycophenolic acid; neo, which confers resistance to the aminoglycoside G-418; and hygro, which confers resistance to hygromycin genes.

An alternative fusion protein system allows for the ready purification of non-denatured fusion proteins expressed in human cell lines. Li this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the gene's open reading frame is translationally fused to an amino- terminal tag consisting of six histidine residues. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni²⁺ nitriloacetic acid-agarose columns and histidine-tagged proteins are selectively eluted with imidazole-containing buffers.

When used as a component in assay systems such as those described below, a protein ofthe present invention may be labeled, either directly or indirectly, to facilitate detection of a complex formed between the protein and a test substance. Any of a variety of suitable labeling systems may be used including, but not limited to, radioisotopes such as ¹²⁵I; enzyme labeling systems that generate a detectable calorimetric signal or light when exposed to substrate; and fluorescent labels.

Where recombinant DNA technology is used to produce a protein ofthe present invention for such assay systems, it may be advantageous to engineer fusion proteins that can facilitate labeling, immobilization, detection and/or isolation Indirect labeling involves the use of a protein, such as a labeled antibody, which specifically binds to a polypeptide ofthe present invention. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by an Fab expression library.

In another embodiment, nucleic acids comprising a sequence encoding HDAClO protein or functional derivative thereof, may be administered to promote normal biological function, for example, normal transcriptional regulation, by way of gene therapy. Gene therapy refers to therapy performed by the administration of a nucleic acid to a subject. L this embodiment ofthe invention, the nucleic acid produces its encoded protein that mediates a therapeutic effect by promoting normal transcriptional regulation.

Any ofthe methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.

In a preferred aspect, the therapeutic comprises a HDAClO nucleic acid that is part of an expression vector that expresses a HDAClO protein or fragment or chimeric protein thereof in a suitable host. In particular, such a nucleic acid has a promoter operably linked to the HDAClO coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific. Li another particular embodiment, a nucleic acid molecule is used in which the HDAClO coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression ofthe HDAClO nucleic acid.

Delivery ofthe nucleic acid into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vector, or indirect, in which case, cells are first transformed with the nucleic acid in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid is directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, for example, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by infection using a defective or attenuated retroviral or other viral vector, or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering it in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (which can be used to target cell types specifically expressing the receptors), etc. Li another embodiment, a nucleic acid-ligand complex can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. Li yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor. Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination

L a specific embodiment, a viral vector that contains the HDAClO nucleic acid is used. For example, a retroviral vector can be used. These retroviral vectors have been modified to delete retroviral sequences that are not necessary for packaging ofthe viral genome and integration into host cell DNA. The HDAClO nucleic acid to be used in gene therapy is cloned into the vector, which facilitates delivery ofthe gene into a patient.

Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Adeno-associated virus (AAV) has also been proposed for use in gene therapy.

Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions ofthe recipient cells are not disrupted. The technique should provide for the stable transfer ofthe nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by various methods known in the art. In a preferred embodiment, epithelial cells are injected, e.g., subcutaneously. In another embodiment, recombinant skin cells may be applied as a skin graft onto the patient. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.

In a preferred embodiment, the cell used for gene therapy is autologous to the patient.

L an embodiment in which recombinant cells are used in gene therapy, a HDAClO nucleic acid is introduced into the cells such that it is expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. Li a specific embodiment, stem or progenitor cells are used. Any stem-and/or progenitor cells that can be isolated and maintained in vitro can potentially be used in accordance with this embodiment ofthe present invention. Such stem cells include but are not limited to hematopoietic stem cells (HSC), stem cells of epithelial tissues such as the skin and the lining ofthe gut, embryonic heart muscle cells, liver stem cells, and neural stem cells. Epithelial stem cells (ESCs) or keratinocytes can be obtained from tissues such as the skin and the lining ofthe gut by known procedures. Li stratified epithelial tissue such as the skin, renewal occurs by mitosis of stem cells within the germinal layer, the layer closest to the basal lamina. Stem cells within the lining ofthe gut provide for a rapid renewal rate of this tissue. ESCs or keratinocytes obtained from the skin or lining ofthe gut of a patient or donor can be grown in tissue culture. If the ESCs are provided by a donor, a method for suppression of host versus graft reactivity (e.g., irradiation, drug or antibody administration to promote moderate immunosuppression) can also be used.

With respect to hematopoietic stem cells (HSC), any technique which provides for the isolation, propagation, and maintenance in vitro of HSC can be used in this embodiment ofthe invention. Techniques by which this may be accomplished include (a) the isolation and establishment of HSC cultures from bone marrow cells isolated from the future host, or a donor, or (b) the use of previously established long-term HSC cultures, which may be allogeneic or xenogeneic. Non- autologous HSC are used preferably in conjunction with a method of suppressing transplantation immune reactions ofthe future host/patient. In a particular embodiment ofthe present invention, human bone marrow cells can be obtained from the posterior iliac crest by needle aspiration. In a preferred embodiment ofthe present invention, the HSCs can be made highly enriched or in substantially pure form. This enrichment can be accomplished before, during, or after long-term culturing, and can be done by any techniques known in the art. Long-term cultures of bone marrow cells can be established and maintained by using, for example, modified Dexter cell culture techniques or Witlock-Witte culture techniques.

In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression ofthe nucleic acid is controllable by controlling the presence or absence ofthe appropriate inducer of transcription.

A further embodiment ofthe present invention relates to a purified antibody or a fragment thereof which specifically binds to a polypeptide that comprises the amino acid sequence set forth in SEQ ID NO:l or to a fragment of said polypeptide. A preferred embodiment relates to a fragment of such an antibody, which fragment is an Fab or F(ab')₂ fragment. In particular, the antibody can be a polyclonal antibody or a monoclonal antibody. Methods for the production of antibodies capable of specifically recognizing one or more differentially expressed gene epitopes are known to one of ordinary skill in the art. Such antibodies may include, but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab')₂ fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any ofthe above. Such antibodies may be used, for example, in the detection of a fingerprint, target, gene in a biological sample, or, alternatively, as a method for the inhibition of abnormal target gene activity. Thus, such antibodies may be utilized as part of disease treatment methods, and/or may be used as part of diagnostic techniques whereby patients may be tested for abnormal levels ofthe HDAClO polypeptide, or for the presence of abnormal forms ofthe HDAClO polypeptide.

For the production of antibodies to the HDAClO polypeptide, various host animals may be immunized by injection with the HDAClO polypeptide, or a portion thereof. Such host animals may include but are not limited to rabbits, mice, and rats, to name but a few. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette- Guerin) and Corynebacterium parvum.

Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as target gene product, or an antigenic functional derivative thereof. For the production of polyclonal antibodies, host animals such as those described above, may be immunized by injection with the HDAClO polypeptide, or a portion thereof, supplemented with adjuvants as also described above.

Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production. In addition, techniques developed for the production of "chimeric antibodies" by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable or hypervariable region derived from a murine mAb and a human immunoglobulin constant region.

Alternatively, techniques described for the production of single chain antibodies can be adapted to produce differentially expressed gene-single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments ofthe Fv region via an amino acid bridge, resulting in a single chain polypeptide.

Most preferably, techniques useful for the production of "humanized antibodies" can be adapted to produce antibodies to the polypeptides, fragments, derivatives, and functional equivalents disclosed herein.

Antibody fragments that recognize specific epitopes may be generated by known techniques. For example, such fragments include but are not limited to: the F(ab")₂ fragments which can be produced by pepsin digestion ofthe antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges ofthe F(ab')₂ fragments. Alternatively, Fab expression libraries may be constructed (Huse et al., 1989, Science, 246: 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.

An antibody ofthe present invention can be preferably used in a method for the diagnosis of a condition associated with abnormal HDAClO expression or activity, for example, abnormal cell proliferation, cancer, atherosclerosis, inflammatory bowel disease, host inflammatory or immune response, or psoriasis, in a human which comprises: measuring the amount of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:l, or fragments thereof, in an appropriate tissue or cell from a human suffering from a condition associated with abnormal HDAClO activity, wherein the presence of an elevated amount of said polypeptide or fragments thereof, relative to the amount of said polypeptide or fragments thereof in the respective tissue from a human not suffering from a condition associated with abnormal HDAClO activity is diagnostic of said human's suffering from such condition. Such a method forms a further embodiment ofthe present invention. Preferably, said detecting step comprises contacting said appropriate tissue or cell with an antibody which specifically binds to a polypeptide that comprises the amino acid sequence set forth in SEQ ID NO:l or a fragment thereof and detecting specific binding of said antibody with a polypeptide in said appropriate tissue or cell, wherein detection of specific binding to a polypeptide indicates the presence of a polypeptide that comprises the amino acid sequence set forth in SEQ ID NO:l or a fragment thereof.

Particularly preferred, for ease of detection, is the sandwich assay, of which a number of variations exist, all of which are intended to be encompassed by the present invention.

For example, in a typical forward assay, unlabeled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation time sufficient to allow formation of an antibody-antigen binary complex, a second antibody, labeled with a reporter molecule capable of inducing a detectable signal, is then added and incubated, allowing time sufficient for the formation of a ternary complex of antibody- antigen-labeled antibody. Any unreacted material is washed away, and the presence ofthe antigen is determined by observation of a signal, or may be quantitated by comparing with a control sample containing known amounts of antigen. Variations on the forward assay include the simultaneous assay, in which both sample and antibody are added simultaneously to the bound antibody, or a reverse assay in which the labeled antibody and sample to be tested are first combined, incubated and added to the unlabeled surface bound antibody. These techniques are well known to those skilled in the art, and the possibility of minor variations will be readily apparent. As used herein, "sandwich assay" is intended to encompass all variations on the basic two-site technique. For the immunoassays ofthe present invention, the only limiting factor is that the labeled antibody be an antibody that is specific for the HDAClO polypeptide or a fragment thereof.

The most commonly used reporter molecules in this type of assay are either enzymes, fluorophore- or radionuclide-containing molecules. In the case of an enzyme immunoassay an enzyme is conjugated to the second antibody, usually by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different ligation techniques exist, which are well known to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, among others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. For example, p-nitrophenyl phosphate is suitable for use with alkaline phosphatase conjugates; for peroxidase conjugates, 1 ,2-phenylenediamine or toluidine are commonly used. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. A solution containing the appropriate substrate is then added to the tertiary complex. The substrate reacts with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an evaluation ofthe amount of HDAClO which is present in the serum sample.

Alternately, fluorescent compounds, such as fluorescein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labeled antibody absorbs the light energy, inducing a state of excitability in the molecule, followed by emission ofthe light at a characteristic longer wavelength. The emission appears as a characteristic color visually detectable with a light microscope. Immunofluorescence and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotopes, chemiluminescent or bioluminescent molecules may also be employed. It will be readily apparent to the skilled artisan how to vary the procedure to suit the required use.

This invention also relates to the use of polynucleotides ofthe present invention as diagnostic reagents. In particular, the invention relates to a method for the diagnosis of a condition associated with abnormal HDAClO expression or activity, for example, abnormal cell proliferation, cancer, atherosclerosis, inflammatory bowel disease, host inflammatory or immune response, or psoriasis in a human which comprises: detecting elevated transcription of messenger RNA transcribed from the natural endogeneous human gene encoding the polypeptide consisting of an amino acid sequence set forth in SEQ ID NO: 1 in an appropriate tissue or cell from a human, wherein said elevated transcription is diagnostic of said human's suffering from the condition associated with abnormal HDAClO expression or activity. In particular, said natural endogeneous human gene comprises the nucleotide sequence set forth in SEQ ID NO:4. Li a preferred embodiment such a method comprises contacting a sample of said appropriate tissue or cell or contacting an isolated RNA or DNA molecule derived from that tissue or cell with an isolated nucleotide sequence of at least about 20 nucleotides in length that hybridizes under high stringency conditions with the isolated nucleotide sequence encoding a polypeptide consisting of an amino acid sequence set forth in SEQ ID NO:l.

Detection of a mutated form of the gene characterized by the polynucleotide of SEQ ID NO:4 which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over- expression or altered spatial or temporal expression ofthe gene. Individuals carrying mutations in the gene may be detected at the DNA level by a variety of techniques.

Nucleic acids, in particular mRNA, for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques prior to analysis. RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size ofthe amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled nucleotide sequences which encode the HDAClO polypeptide ofthe present invention. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing. Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method. Li another embodiment, an array of oligonucleotides probes comprising nucleotide sequence encoding the HDAClO polypeptide ofthe present invention or fragments of such a nucleotide sequence can be constructed to conduct efficient screening of e.g., genetic mutations. Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability.

The diagnostic assays offer a process for diagnosing or determining a susceptibility to disease through detection of mutation in the HDAClO gene by the methods described. In addition, such diseases may be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of polypeptide or mRNA. Decreased or increased expression can be measured at the RNA level using any ofthe methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods. Assay techniques that can be used to determine levels of a protein, such as a polypeptide ofthe present invention, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.

Thus in another aspect, the present invention relates to a diagnostic kit which comprises: (a) a polynucleotide ofthe present invention, preferably the nucleotide sequence of SEQ ID NO:2, 3 or 4, or a fragment thereof;

(b) a nucleotide sequence complementary to that of (a);

(c) a polypeptide ofthe present invention, preferably the polypeptide of SEQ ID NO:l or a fragment thereof; or

(d) an antibody to a polypeptide ofthe present invention, preferably to the polypeptide of SEQ ID NO: 1.

It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component. Such a kit will be of use in diagnosing a disease or susceptibility to a disease, particularly to a disease or condition associated with abnormal HDAClO expression or activity, for example, abnormal cell proliferation, cancer, atherosclerosis, inflammatory bowel disease, host inflammatory or immune response, or psoriasis.

The nucleotide sequences ofthe present invention are also valuable for chromosome localization. The sequence is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome. The mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene-associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position ofthe sequence on the chromosome can be correlated with genetic map data. The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).

The differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined. If a mutation is observed in some or all ofthe affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent ofthe disease.

An additional embodiment of the invention relates to the administration of a pharmaceutical composition, in conjunction with a pharmaceutically acceptable carrier, excipient or diluent, for any ofthe therapeutic effects discussed above. Such pharmaceutical compositions may consist of HDAClO, antibodies to that polypeptide, mimetics, agonists, antagonists, or inhibitors of HDAClO function. The compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones.

Li addition, any ofthe therapeutic proteins, antagonists, antibodies, agonists, antisense sequences or vectors described above may be administered in combination with other appropriate therapeutic agents. Selection ofthe appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention ofthe various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects. Antagonists and agonists of HDAClO may be made using methods that are generally known in the art.

The pharmaceutical compositions encompassed by the invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-articular, intra- arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.

In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing ofthe active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymefhylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.

Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. L soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration may be formulated m aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity ofthe suspension, such as sodium carboxymefhyl cellulose, sorbitol, or dextran. Additionally, suspensions ofthe active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Non-lipid polycationic amino polymers may also be used for delivery. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility ofthe compounds to allow for the preparation of highly concentrated solutions.

For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

The pharmaceutical compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee- making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation may be a lyophilized powder which may contain any or all ofthe following: 1-50 mM histidine, 0. l%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. For administration ofthe HDAClO, such labeling would include amount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art.

For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of active ingredient, for example HDAClO or fragments thereof, antibodies of HDAClO, agonists, antagonists or inhibitors of HDAClO, which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% ofthe population) and LD50 (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity ofthe patient, and the route of administration. The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity ofthe disease state, general health ofthe subject, age, weight, and gender ofthe subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate ofthe particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. Pharmaceutical formulations suitable for oral administration of proteins are known in the art.

All patent applications, patents and literature references cited herein are hereby incorporated by reference in their entirety.

The following Examples illustrate the present invention, without in any way limiting the scope thereof.

Example 1 : HDAClO protein expression in vivo

An expression vector containing HDAClO's coding sequences plus the Flag-epitope encoding sequences at the C-terminus is transfected into 293 embryonic kidney cells using the GenePORTER2 transfection reagent (Gene Therapy System Inc., San Diego, CA). Forty-eight hr. after transfection, cell lysates are prepared from the transfected cells and 10 μg of total protein is subjected to SDS- PAGE on a 10% Tris-glycine gel. The proteins are then transferred onto a PVDF membrane and probed with an anti-Flag antibody, followed by a secondary antibody that is conjugated with horseredish peroxidase, which allows for detection of signal using enhanced luminescence reagents. The anti-Flag antibody detects the HDACIO-Flag fusion protein as a single band of 39 kDa in size, which agrees with the estimated size of HDAClO protein based on its amino acid composition. Example 2: Distribution of HDAClO mRNA in normal human tissues and cancer cell lines

A multiple human tissue Northern blot is purchased from Clontech (Palo Alto, CA). A ³²P- labeled probe corresponding to HDAClO cDNA (nucleotide no.181 to no.l 122) is prepared using the Rediprime DNA labeling system (Amersham Pharmacia Biotech) according to the manufacturer's instructions. The Northern blot is pre-hybridized and hybridized in the presence ofthe ³²P-labeled probe under stringent conditions according to the manufacturer's protocol. A probe corresponding to human actin cDNA (Clontech) is used as a control for the relative amount of mRNA in each lane. Results of Northern analyses indicate that there are two spliced variant forms of HDAClO mRNA, one is ~1.7kb, which agrees with the size ofthe full-length cDNA (SEQ ID NO:2); the other is ~3.2kb and is expressed at a higher level. The larger transcript agrees with the size of a Macac fascicularis brain cDNA clone (GenBank™ accession #AB052134), which encodes a truncated HDAClO polypeptide (minus the first 29 amino acids) with 3 conservative amino acid substitutions. Northern analyses also show that overall expression level of HDAClO mRNA is low and high expression level is restricted to brain, heart, skeletal muscle and kidney. These findings imply that the HDAClO gene is expressed in normal human tissues and that HDAClO's function may be tissue-specific.

In addition to Northern blotting, the Real-time PCR technique is used to examine HDAClO mRNA distribution in normal human tissues as well as several human cancer cell lines. These experiments confirm findings ofthe Northern analyses; in addition, they reveal high expression level of HDAClO in testis. Furthermore, our data indicate that large amount of HDAClO mRNA is also found in a non-small cell lung carcinoma cell line, a rhabdomyosarcoma muscle tumor line, a urinary bladder cancer cell line and an osteosarcoma cell line. Taken together, these results indicate that HDAClO may function not only in normal human tissues, but also in the development and/or maintenance of human cancers.

Example 3:In vitro HDAC enzyme assay

To determine whether the putative HDAC "10" is an active deacetylase, transfected Flag epitope-tagged recombinant HDAClO is used to measure the ability of HDAClO to deacetylate histone H4 peptide. Enzymatic activity may be determined according to conventional methods, such as the following techniques: Preparation of HDACl 0-Flag expression vector. Using conventional techniques in molecular biology, a Flag-epitope sequence is added to the C-terminus of HDAClO coding sequences (SEQ ID NO:3) by PCR. The PCR primers are:

Forward: 5'-GAGGATCCACCATGCTACACACAACCCAGCTG- 3'

Reverse: 5'-GCGTCTAGACT CTTGTCATCGTCGTCCTTGTAATCAGCCCGGGGC-

ACTGCAGGGGGAAG- 3'.

The BamHI and Xbal restriction enzyme cutting sites are underlined, the ATG translational start site is bolded in the forward primer and the Flag-epitope endocding sequences are bolded in the reverse primer. The Flag-tagged HDAClO PCR fragment is cloned into the pcDNA3.1(+) expression vector between the BamHI and Xbal sites.

Transfection and Immunoprecipitation. Approximately lxlO⁷293 human embryonic kidney cells were grown in a 15-cm² plate (~50% confluent) on the day of transfection. GenePORTER transfection reagent (Gene Therapy Systems, Inc., San Diego, CA) is used to transfect 30 μg of plasmid DNA per plate of cells according to manufacturer's instructions. Forty-eight hr after transfection, cells are washed twice with ice-cold phosphate-buffered saline (PBS) and resuspended in 1 mL ice-cold lysis buffer (50 mM Tris-Cl, pH 7.4, 120 mM NaCl, 0.5 mM EDTA, 05% NP-40) supplemented with EDTA-free protease inhibitor complete (Roche Molecular Biochemicals, Indianapolis, IN). The lysate is incubated at 4°C for 20 min on a rotator, followed by spinning at 12,000 x g for 20 min at 4°C. The soluble supernatant is collected and used for immunoprecipitation with 20 μl anti-FLAG M2 affinity gel (Sigma, Saint Louis, MI) at 4°C overnight. As a negative control, 1 mL lysis buffer is used instead ofthe cell lysate. The immnuoprecipitated complex is pelleted by centrifugation and washed three times with 1 mL ice-cold lysis buffer, four times with lysis buffer containing 1 M NaCl and three times with 1 mL HDAC assay buffer (10 mM Tris-Cl, pH 8.0, 10 mM NaCl, 10% glycerol).

In vitro HDAC enzyme assay. The immunoprecipitated complex is suspended in 30 μl HDAC assay buffer containing 30,000 cpm ofthe acetylated histone H4 peptide. Histone deacetylase activity is determined after incubation at 37°C for 3 hr as described (Emiliani, S., Fischle, W., Van Lint, C, Al-Abed, Y, and Verdin, E. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 2795-2800). Results ofthe in vitro HDAC enzyme assays show that cells expressing the HDACIO-Flag fusion protein contain 2.5-3 fold higher enzyme activity than cells expressing the pcDNA3.1(+) vector alone. Therefore, HDAClO is likely to contain intrinsic histone deacetylase enzyme activity.

Example 4: Identification of HDAClO associated protein

Using conventional methods, proteins in the same complex as HDAClO may be identified by their ability to coimmunoprecipitate with HDACIO-Flag fusion protein. The HDACIO-Flag expression vector or the vector alone is transfected into 293 cells and cell lysates are prepared as described above. The lysates are precleared with Sepharose A/G plus agarose beads, followed by immunoprecipiation using anti-Flag antibody at 4°C overnight on a rotator as described in example 3. The immune complexes are washed twice with ice-cold lysis buffer (see example 3), twice with lysis buffer containing 1 M NaCl and twice with PBS. The final complexes are separated by SDS-PAGE on 10% Tris-glycine gels, transferred onto a PVDF membrane and probed with antibodies against known HDAC-associated proteins or other HDACs. Conversely, the immunoprecipitation could be done using antibodies of choice, and the resulting immune complexes could be probed with anti-Flag antibody.

Claims

What is claimed is:

1. An isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1.

2. An isolated DNA comprising a nucleic acid sequence that encodes the polypeptide of claim 1.

3. A vector molecule comprising at least a fragment ofthe isolated DNA according to claim 2.

4. The vectqr molecule according to claim 3 comprising transcriptional control sequences.

5. A host cell comprising the vector molecule according to claim 4.

6. The isolated DNA according to claim 2, comprising a nucleotide sequence selected from the group consisting of (1) the nucleotide sequence set forth in SEQ ID NO:2; (2) the nucleotide sequence set forth in SEQ ID NO:3; (3) a nucleotide sequence capable of hybridizing under high stringency conditions to a nucleotide sequence set forth in SEQ ID NO:3; and (4) the nucleotide sequence set forth in SEQ ID NO:4.

7. A vector molecule comprising the isolated DNA molecule according to claim 6, or a fragment thereof.

8. The vector molecule according to claim 7 comprising transcriptional control sequences.

9. A host cell comprising the vector molecule according to claim 8.

10. A host cell which can be propagated in vitro and which is capable upon growth in culture of expressing HDAC 10, wherein said cell comprises at least one transcriptional control sequence that is not a transcriptional control sequence ofthe natural endogeneous human gene encoding HDAC 10, wherein said one or more transcriptional control sequences control transcription of a DNA encoding HDAC 10.

11. A method for the diagnosis of a condition associated with abnormal regulation of gene expression which includes, abnormal cell proliferation, cancer, atherosclerosis, inflammatory bowel disease, host inflammatory or immune response, or psoriasis in a human which comprises: detecting abnormal transcription of messenger RNA transcribed from the natural endogeneous human gene encoding HDAC 10 in an appropriate tissue or cell from a human, wherein said abnormal transcription is diagnostic of said condition.

12. The method of claim 11 , wherein said natural endogeneous human gene comprises the nucleotide sequence set forth in SEQ ID NO:4.

13. A method for the diagnosis of a condition associated with abnormal HDAC 10 expression or activity in a human which comprises: measuring the amount of HDAC 10, or fragments thereof, in an appropriate tissue or cell from a human suffering from said condition wherein the presence of an abnormal amount of said polypeptide or fragments thereof, relative to the amount of said polypeptide or fragments thereof in the respective tissue from a human not suffering from said condition associated with abnormal HDAClO expression or activity is diagnostic of said human's suffering from said condition.

14. The method of claim 13, wherein said detecting step comprises contacting said appropriate tissue or cell with an antibody which specifically binds to a polypeptide that comprises the amino acid sequence set forth in SEQ ID NO:l or a fragment thereof and detecting specific binding of said antibody with a polypeptide in said appropriate tissue or cell, wherein detection of specific binding to a polypeptide indicates the presence of a polypeptide that comprises the amino acid sequence set forth in SEQ ID NO: 1 or a fragment thereof.

15. An antibody or a fragment thereof which specifically binds to a polypeptide that comprises the amino acid sequence set forth in SEQ ID NO:l or to a fragment of said polypeptide.

16. An antibody fragment according to claim 15 which is an Fab or F(ab')₂ fragment.

17. An antibody according to claim 15 which is a polyclonal antibody.

18. An antibody according to claim 15 which is a monoclonal antibody.

19. A method for producing an HDAC 10 polypeptide, which method comprises: culturing a host cell having incorporated therein an expression vector comprising an exogenously-derived polynucleotide encoding a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 1 or a nucleotide sequence capable of hybridizing under high stringency conditions to a complement of said polynucleotide, under conditions sufficient for expression ofthe polypeptide in the host cell, thereby causing the production ofthe expressed polypeptide.

20. The method according to claim 19, wherein said exogenously-derived polynucleotide hybridizes under stringent conditions to the nucleotide sequence as set forth in SEQ ID NO:2.

21. The method according to claim 19, wherein said exogenously-derived polynucleotide comprises the nucleotide sequence as set forth in SEQ ID NO:3.

22. A histone deacetylace which comprises the catalytic domain of HDAC 10.

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SEQ ID NO: 1

MLHTTQLYQH VPETPWPIVY SPRYNITFMG LEKLHPFDAG KWGKVINFLK EEKLLSDSML 60

VEAREASEED LLWHTRRYL NELKWSFAVA TITEIPPVIF LPNFLVQRKV LRPLRTQTGG 120

TIMAGKLAVE RGWAINVGGG FHHCSSDRGG GFCAYADITL AIKFLFERVE GISRATIIDL 180

DAHQGNGHER DFMDDKRVYI MDVYNRHIYP GDRFAKQAIR RKVELEWGTE DDEYLDKVER 240

NIKKSLQEHL PDVWYNAGT DILEGDRLGG LSISPAGIVK RDELVFRMVR GRRVPILMVT 300 SGGYQKRTAR IIADSILNLF GLGLIGPESP SVSAQNSDTP LLPPAVP

SEQIDNO:2

1 agctttggga gggccggccc cgggatgcta cacacaaccc agctgtacca gcatgtgcca

61 gagacaccct ggccaatcgt gtactcgccg cgctacaaca tcaccttcat gggcctggag

121 aagctgcatc cctttgatgc cggaaaatgg ggcaaagtga tcaatttcct aaaagaagag

181 aagcttctgt ctgacagcat gctggtggag gcgcgggagg cctcggagga ggacctgctg

241 gtggtgcaca cgaggcgcta tcttaatgag ctcaagtggt cctttgctgt tgctaccatc

301 acagaaatcc cccccgttat cttcctcccc aacttccttg tgcagaggaa ggtgctgagg

361 ccccttcgga cccagacagg aggaaccata atggcgggga agctggctgt ggagcgaggc

421 tgggccatca acgtgggggg tggcttccac cactgctcca gcgaccgtgg cgggggcttc

481 tgtgcctatg cggacatcac gctcgccatc aagtttctgt ttgagcgtgt ggagggcatc

541 tccagggcta ccatcattga tcttgatgcc catcagggca atgggcatga gcgagacttc

601 atggacgaca agcgtgtgta catcatggat gtctacaacc gccacatcta cccaggggac

661 cgctttgcca agcaggccat caggcggaag gtggagctgg agtggggcac agaggatgat

721 gagtacctgg ataaggtgga gaggaacatc aagaaatccc tccaggagca cctgcccgac

781 gtggtggtat acaatgcagg caccgacatc ctcgaggggg accgccttgg ggggctgtcc

841 atcagcccag cgggcatcgt gaagcgggat gagctggtgt tccggatggt ccgtggccgc

901 cgggtgccca tccttatggt gacctcaggc gggtaccaga agcgcacagc ccgcatcatt

961 gctgactcca tacttaatct gtttggcctg gggctcattg ggcctgagtc acccagcgtc

1021 tccgcacaga actcagacac accgctgctt ccccctgcag tgccctgacc cttgctgccc

1081 tgcctgtcac gtggccctgc ctatccgccc cttagtgctt tttgttttct aacctcatgg

1141 ggtggtggag gcagccttca gtgagcatgg aggggcaggg ccatccctgg ctggggcctg

1201 gagctggccc ttcctctact tttccctgct ggaagccaga agggcttgag gcctctatgg

1261 gtgggggcag aaggcagagc ctgtgtccca gggggaccca cacgaagtca ccagcccata

1321 ggtccaggga ggcaggcagt taactgagaa ttggagagga caggctaggt cccaggcaca

1381 gcgagggccc tgggcttggg gtgttctggt tttgagaacg gcagacccag gtcggagtga

1441 ggaagcttcc acctccatcc tgactaggcc tgcatcctaa ctgggcctcc ctccctcccc

1501 ttggtcatgg gatttgctgc cctctttgcc ccagagctga agagctatag gcactggtgt

1561 ggatggccca ggaggtgctg gagctaggtc tccaggtggg cctggttccc aggcagcagg

1621 tgggaaccct gggcctggat gtgaggggcg gtcaggaagg ggtacaggtg ggttccctca

1681 tctggagttc cccctcaata aagcaaggtc tggacctgca aaaaaaaaaa aaaaaaaaaa

1741 aaaaaaaaaa aaaaa

SEQ ID NO: 3

25 atgcta cacacaaccc agctgtacca gcatgtgcca

61 gagacaccct ggccaatcgt gtactcgccg cgctacaaca tcaccttcat gggcctggag

121 aagctgcatc cctttgatgc cggaaaatgg ggcaaagtga tcaatttcct aaaagaagag

181 aagcttctgt ctgacagcat gctggtggag gcgcgggagg cctcggagga ggacctgctg

241 gtggtgcaca cgaggcgcta tcttaatgag ctcaagtggt cctttgctgt tgctaccatc

301 acagaaatcc cccccgttat cttcctcccc aacttccttg tgcagaggaa ggtgctgagg

361 ccccttcgga cccagacagg aggaaccata atggcgggga agctggctgt ggagcgaggc

421 tgggccatca acgtgggggg tggcttccac cactgctcca gcgaccgtgg cgggggcttc

481 tgtgcctatg cggacatcac gctcgccatc aagtttctgt ttgagcgtgt ggagggcatc

541 tccagggcta ccatcattga tcttgatgcc catcagggca atgggcatga gcgagacttc

601 atggacgaca agcgtgtgta catcatggat gtctacaacc gccacatcta cccaggggac

661 cgctttgcca agcaggccat caggcggaag gtggagctgg agtggggcac agaggatgat 45

721 gagtacctgg ataaggtgga gaggaacatc aagaaatccc tccaggagca cctgcccgac 781 gtggtggtat acaatgcagg caccgacatc ctcgaggggg accgccttgg ggggctgtcc 841 atcagcccag cgggcatcgt gaagcgggat gagctggtgt tccggatggt ccgtggccgc 901 cgggtgccca tccttatggt gacctcaggc gggtaccaga agcgcacagc ccgcatcatt 961 gctgactcca tacttaatct gtttggcctg gggctcattg ggcctgagtc acccagcgtc 1021 tccgcacaga actcagacac accgctgctt ccccctgcag tgccctga

SEQ ID NO: 4 ctacacacaa cccagctgta ccagcatgtg ccagagacac gctggccaat cgtgtactcg 17663325 ccgcgctaca acatcacctt catgggcctg gagaagctgc atccctttga tgccggaaaa 17663265 tggggcaaag tgatcaattt cctaaaaggt atggaaggtc ccccttggac tctcatctgc 17663205 ttcctccaac ccacctgtcc tctccgtcct catccccaac ataagcctca ggctctctcc 17663145 catcttcagt ttcagccctc ggatggcctt ccacccatgc ttccgcccaa aatgattttt 17663085 ccaacacaga ctcctaatca cgatatgatg tccctgactc agactctccc tggctcccca 17663025 tcctgtgggc ctaagtcctg cctctgccca agaggcctag tggaaaggta gctgattact 17662965 gatgggcaca gggaaggtga agcttggagg agtccatttc ctaaggttca gagagtcagg 17662905 aggtagagca cctccaccgc acctctcttg attacagatg ggggaaattg tgtcctagaa 17662845 tgattaggaa acatgtgcac ccaattccag tccagtcctc acagcagccc tcggggtagg 17662785 caccacaatc gcagcagagg ctcaggagct cactgtaacc tccgcctttc aggttcaaac 17662725 aatttttctg cctcagcctc ccaagtagct ggaattacag gcgtgagcca ccacacccgg 17662665 ccctgatttc ttaatatggc actcattata agattgtaaa agcccacctg tagaccgaac 17662605 tgggcacact ggctgcctgc ttgtgacctc tttccaggga aggacacagc tcccattagt 17662545 ggctgaagta acacagttac aagaggcgga gttgggtttg gaactcagag ctccaggcgc 17662485 cctaccttta gggctcatcc ccttgagcaa aatgatgctt cgaagagcat atcgttttaa 17662425 ctgtggtttg taatcaaggg gcctgattta ggtgggaaat tcacttaaac ttgttttaaa 17662365 aggaaacatt atgtcatcaa aatgggaaaa ggcagtttca cttgccataa ataggtcatg 17662305 gtaaaaaagt aaatgcaatg aaaacaacag tataattcaa tccaggctgg ttactattgc 17662245 ctgcaggctg tgagactgat tagtggtttg aacggaagat gagcaaagca caggcaggtg 17662185 ttgcgaggcc atgccacact gagcctcctg taatatcatc agaaggtgga gggaggccgg 17662125 gcgcagtggc tcgtgcctgt aatcccagca ctctgggagg ccaaggctag gagaacactt 17662065 gaggccggga gtttgagacc agcttgggca acatagcaag atcctgtctc tacaaaataa 17662005 aaataaaaaa cttagctggg ggtggtggta tgcacctata gtcctagcta cttgaaatgc 17661945 tgaggcagga ggatcacttg agcccagaag ttcgagggtg cagtgagcta tggttgtgcc 17661885 actgcactcc agcctgggct acagagcaag accttgtctt gcatttattt atttgtttat 17661825 ttatttattg agacagggtc tcactcccat cacccaggct agagtgcagt ggcggaatca 17661765 aggctcattg ccacctcaac ctccctggct taagtgatcc tcccacctca tgtttttgat 17661705 attttgtaga gatgcggtct cactatgttg tctaggctgg tcttgaactc ctgggctcaa 17661645 gcaatccacc tgcctcaacc tcccaaagtg ctgggattac aggcgtgaac caccacacct 17661585 ggccaagacc ctgtctcttt aaatgaatta aaaaaaaaaa aagggcgggg ggaaggtgga 17661525 gggggaattc ctaagaagag tttttctcac tctgagggtc aacatccctg acccttgtgc 17661465 cacctgctcc tgaaggttgt ctagcacacc tgagctctcc ttgtgactat cagtggcttg 17661405 ggaaacatgg ggattgctgt gtgtacgatg ttcattgctc cctggccaga gggactggcc 17661345 actgtccaca gtggctgggg aggctacccc ttctcagaag gcccacaagc cagcagtgcc 17661285 tacctacccc tggggcaggg gctgccacag gccaagtctg cagcctgtgg gagggtctgg 17661225 ggctggccct ggccttgagg tcagtgggga agcaggatgc tccctctgtg gtttcagaag 17661165 agaagcttct gtctgacagc atgctggtgg aggcgcggga ggcctcggag gaggacctgc 17661105 tggtggtgca cacgaggcgc tatcttaatg agctcaaggt acaggatgtc gggcctgggg 17661045 ggctgcgggc ctggggcagg gggctgctgg ccaggagtgg ccagaggcag gaggtgactc 17660985 agcctgggga agccaagtct cacagggcac ccattcatgt ccctagtgtt ggaggaacat 17660925 gggagtctgt ggtccccaag agaaggagag aggtcataaa aaggcagacc tcagtttggg 17660865 ccaggccact ctgagggtgg tgtcctcccc ttctccaggg cgtatgaaag ccttcataga 17660805 attttaggct tctacattat gactttcaag ctgtgctctg tcgacacgcc tccgagaccc 17660745 cagcccctgt cctccaacca tacatagctc tttcactttg gtctattttg tttgtttgtt 17660685 tgttcatttt tttgttggtt tttgttcttg aaatggagtc tcactctgtc gcccaggctg 17660625 gagtgcagtg atgtgatctc ggctcattgc aacctccgcc ttccgggttc aagcaattat 17660565 cctgtctcac cctcctgagt gagtagctag gattacaggc gcgtgccacc atgcctggct 17660505 aatttttttt gtattttagt agagatgtgg tttcgccgtg ttggccaagc tggtctcgaa 17660445 ctcctgaact caggtcatct gcccacctcg gcctcccaaa gtgctggggt tacaggcgtg 17660385 46

agccaccgca cccaccctat tttttatatt gggctgaagt ttaagactct ggtctaagta 17660325 cttctgctga agttttgttg aaaattgttg gtctaaaaac taatttgaaa ccctcagggc 17660265 tcagcagaga agagaaacaa gtgggagggc cggtggtaga gtctgaggtg aactcctgcc 17660205 ccttcccaag gggcggctcc tcagctccac tgtgggcccg gcatggccag agcacctggt 17660145 cttcaaagag aagccaggaa tccagattat taagtgacat ttcctgattt tttttttgag 17660085 actgagtctc gctcttgttg agcaggctga agtgcagtgg cacgatctca gctcactgta 17660025 acctccgcct cccgggttca aacaattttt ctgcctcagc ctccgaagta gctggaatta 17659965 taggggtgag ccaccacacc cggccctgat ttcttaatgt ggcactcatt ataagattgt 17659905 aaaagcccac ctgtagacca aactgggcac actggctgcc tgcttgtgac ctctttccag 17659845 agaaggacac agctcctatt agtggctgaa gttctgaggg ctgaggcatt cagttcagtg 17659785 ctctttgtag gaacagaggg gaggttgggg cgggggcttg cattggaatc tggtactgcc 17659725 agcctgcctt ggtgggggtg gggtcaggga tgcctcaggt tatctgcccc aagagtgtgg 17659665 gagccctgac ccccaggctc cctggctgag ctcaccttag actcagagcc acagtggatg 17659605 cctgaggcca gcaggcccct ctgctccaca ggtggaaaag cctaggtcca gaaagaggct 17659545 gtgctcaagg tcacctggga agttggccgg gccttgggga gacccctggc aggtcatcca 17659485 gtccagtctt ctaggttccc agtcagggct gctgcctccc tgctccccaa ccgcagcctg 17659425 aggtgtgaga attctagata gggccacgac agtgtgagca catgaaagat taccaggaag 17659365 aggttgaaac ctggctcctg ggagagagag gggtgtgagg ccttggcagg aagcccagtg 17659305 cttggctgcc ctggtttcct ggggcccagg catgcgtggt cacagtccac agcctagggc 17659245 tgggccagga ggacatgcct gccagagtcc cgagggtgag gggaaggaag ggacaggagg 17659185 cgctcagctg gggcagggag aaaccaaaac agaatggtgt gattgaacca ggctgggggt 17659125 ggggggccta ggttccaggg ccccacccat ttgaggggcc ttcaggggaa ctgtgttggg 17659065 caggctgcat gcctggcctt ggtcccccaa aagcctgaaa gcagcttact atgtgatata 17659005 taataataca aaatagctgg gtgtagtggc atgcacttgt agtcctagct acttgggagc 17658945 ctgaggcagg agaccttgag cccaggagtt tgaagctgta gtgagctatg attgcaccac 17658885 tgcactccag ctggtatgac agagtgagac tgtctcttaa aaaaaataat aaaagtatta 17658825 acaggtagag tcccaagtag aaaactgagg ttgagggtag gaggagaatt caggtatgtc 17658765 cactgaaaaa gttaaccaag atggtgatcc agctgcatat ttggcttgga gctccctggc 17658705 agtcagaaca aaaggagaaa catgatggtt tctacggcac ctattaagat gaagaagtag 17658645 gccgggtgca gtgactcatg cctgtaatcc cagcactttg ggagaacgag gcgggcggat 17658585 cacttgaggt cgggagtttg agatcagcct ggccaacatg gagaaaccct gtctctacta 17658525 aaactacaaa attagccagg catggtagtg catgcctgta atcccagcta cctgggaggc 17658465 tgaggcagga aaatcacttg aacctgggag gtagaggttg cagtgagccg agattgcgcc 17658405 attgcactcc agcttgggca ataagagtga aactccatct caaaaaaaaa aaaaaaaaaa 17658345 aaagaaaaag atgaagaagt agtcagtcat tcaacacatc tgtattgaat gccaactgta 17658285 cagagagaat aagacagcag ggctctctgc caccatggat ttgcatttga gtcgtggaag 17658225 attaaaatta aggaagcaac cacccaagag cattttagag agcaccaagg gctatgaaga 17658165 aagtgaaaaa tagagggtaa ttggatggtc agggagggcc tcacagagga ggtgatgttt 17658105 gagttgagac taaacaaagg agcaggtgat actcatgtag aggtgttttt tttttttttt 17658045 tttttttttg gagaaggaat ctcgctttgt tgcccaggct cgagtacagt ggtgcgatct 17657985 cagctcacag caacctctgc ctcttggttc aagcgattct cctgcctcag cctcccaagt 17657925 agctaggatt acaggcacct gccaccatgc ccggctaatt tttgtatttt tagtagagac 17657865 ggagttttca ccatgttggc caggctggtc tcgaactcct gacctcaagc aatccatctg 17657805 cctcggcctc ccaaagtgct gggattacag gcatgagcca ctgctcctgg ccctcatgta 17657745 tagctttgaa ggaagaatgt ttcagaatcc caggcctgga gggtggaggg gacttgatct 17657685 tccaaagggg agaagaatgc ttgggaggcc ggatggaagg gaataaaaca ttgtggctcg 17657625 tacacggtgc agttagggag gccagagccc caggccacac aaggtcttgc aggccgtggg 17657565 aggagtgtat atgttgttcc agggaccttg gacagtcacg agggggtttt cagcaggagg 17657505 gtgatatggt gtgacatgcc cttgctgccc aggtgggacc caagcccgtt tcagacatca 17657445 tctggcacct aaggctgcag ctcaggaaca tctcccacct ccctgcagat gtctgcaatg 17657385 tttcttttct ccttcctctg ctgtgggcgc ccagagagtg ccctagagag tccttcaggt 17657325 ttctcaggct gcttttccct ggtcattctg tgtgtgctgt gtaacatcca ccgtctcccc 17657265 tgcctcatcc cattctaccc ccaacccctg cctggggctc atgcctgact ctgcactggt 17657205 gtggcctttg atacttaata aacagggcac tgaaggagaa gcaggagctg gacgtttgca 17657145 agatgtcaat tcagggaaac ccatgtttat caagctcctg ctgtgtgcaa ggtccagggt 17657085 tggcccctct gagggtagct gttgagctcc ccagtgcccc agcactgggc tcttgccttt 17657025 ggttgattct cgggtgacag tttgccatgg agtgtcggtt agtgctgggc agcatctgac 17656965 atcctgcccc tgtgcactct gcactggaca gtgctcagaa cacgtggatc cagcaagtgc 17656905 tcagagggca ccactctgtg atctaggtgc tgcagggatg ggatggagca aaagaccaca 17656845 tccctttcct gctggagctg gcatttaggt gggagagtca gacaataaat gtaataatta 17656785 agtaatgaga taatatgtta gatggtgctg agtgtcgtga agaaaggaag ggacagcaga 17656725 aaaggggtgg ggagagctgg tgagaggatg gcagttttaa atcaggagtc aggaaagggc 17656665 - 47 -

ttactacctg tgatcacagg tgacatgtgg gaagggagtg agggagtggg tgatgtggtc 17656605 atctggggaa gggcattcca agcagaagaa acagcaagtg caaagatccc agggcagaac 17656545 tatctgtcat gagttccagt atagtgtgga gagaaggaga cacagaccat agctccatgg 17656485 agcacctgga gggaccctgg agagtctcta ggggagtgag ctcctcttgg tctccaactc 17656425 tctcttctct tccctgaggg gctcctctct cctttaaaaa aaaatttttt ttaattgtgg 17656365 taaaatttac ataacaaaat tcgccattaa ccactttaaa ctgtacagtt cagtggcctt 17656305 tagtccattc acaaagtgct gcaaccatca tctctagttc caaacatttt catcactcca 17656245 aaaggaaacc ctgtgtcctt taaacacttg ctccccattt atccccccaa gtccccttgg 17656185 taatcactca cctgcattct ctctctatgg atttgcctat cctggatatt tcatataaat 17656125 ggaatcatac aatatgtgac cttttgtgtc tggcttatct cactaagcac agcgttttca 17656065 acattcatct gtgttgtgtt gtagcatgta tcagtacttc attccttttc acagcagaat 17656005 gatattccat tgtaaaacac tacatttttt ttatccattc attagtttat aggccttttg 17655945 gctattgtga gtagtgttgc tgtggacatg tgcatacgag tatttattag aatacctgtt 17655885 ttcagttatt tggggtatac acctaggagt agaattactg ggtcacatgg taattctgtt 17655825 taattttctg aagaaccatc aaggtgatct ccacgggggc tgcaccattt ccaccagtaa 17655765 tgtaccaggg tcccaatttc tctacatcct tttcaatgct tgttattttc tggtgttttt 17655705 ttttttcccc cccagtgtgg ccatcttact ggatgtgaag tggtatctca tggttttaat 17655645 ttgcatttac ctaatggcta attaacactg aggatctttt catgtgctga ttggctattt 17655585 gtatatgtca tttggagaaa tgtttattca agtcctttgt ccatttttaa aattggcttg 17655525 tctttttgtt gagttgtagg gttctttata tattctggat attatttaat ttgtaaataa 17655465 ctcctcccat tctgtgggtt gtcttttttt tgatagtgtc ctttgatgca caaaaatttt 17655405 agttttgctg aagtccaatt tatctttttt tccttttctt taggtgtcat atctaagaat 17655345 ccattgccaa acccaaggtc atgaaggttt accgcatgtg ttttcttcta agagttttat 17655285 agttttcact tatatttagg ccttgataaa ttttgagtta atttttgtat atgtgtgagg 17655225 caagtccaac ttcattgttt tgtactcaga tatccagtta tcccagcacc atttgttagg 17655165 ctgtttttcc cctgttgaat ggtcttggta cctttgtaga aaatcaactg gccatagatg 17655105 tatggattta tttctagact ctcaattcta ttcatttttt tggtttgttt gtttaagaaa 17655045 gggttgcatt ctttcgacag cccaggctgg agtacggtgg ctccatcttg gctcactgca 17654985 acctccgtct cctgggttca agcaattctc ccatctcagc ctcccaggta gctgggacta 17654925 caggcgtgtg ctaccatgcc tggctaattt ttgtgtttct tggtagagat ggggtttcac 17654865 catgttggct aggctggtcc tgaattcgtg acctcaagtg atttgctcac ctcggcctct 17654805 caaagtactg ggattacagg catgtgtgag ccactgcgcc cagccaattc tattcatttg 17654745 atctatatgt caataccaca ctattttggt actgttactg tggcttactg tggttattgt 17654685 ggctttggag caaattttga aattccagat tgtgaggcct ccaactttgt tctttttttt 17654625 tttttgagac gcagtctcgc tttgtcgcct atgctggagt gcaatggcgc gatctcggct 17654565 cactgcaacc tccgccttct ggtttcaggt gattctcctg cctcagcctc ccgagtagct 17654505 gggattacag gcgcccggca ccacgcctag ctaatttttc tatttttagt agagatgagg 17654445 tctcaccatg ttggtcaggt tggtctcaaa ctcctgacct catgatctgc ctgcctctgc 17654385 ctcccaaagt gctgggatta caggcatgag ccaccgtgcc cagccaactt tgttcttttt 17654325 taagatcgtt ttggctgttt gaggtccctt gagattccat gtgaattata gcatcaactt 17654265 ccattttttg caaaaaaggc cattgggatt ttgacaggaa ttgcattgag taaattgctt 17654205 tggggagttt tgccatctta acaatattcg gtctttcaat ccatgaacat gggatgtctt 17654145 tccgtttatt tatgtcttta atttctttca gcaatgtttt gtagctttca atggacaaat 17654085 cttgcacctc ttggttaaat ctattcccat gcattttatt cttttcgatg ttattataaa 17654025 tgaaattgtt tgaatttcct tttaagattg ttcattgctg gtatatacaa taatcagttg 17653965 tatagaaata caactgattt ttttgtgttg atcttgtatc ctacaacttt gctgaatttg 17653905 tttcttagca tttttttctt tttttttttt tttttttttt ttttagacag agtctctctc 17653845 tgttaccagg ctggagtgca gtggcatgat ctcggctcac tgcaacctcc gcctcccagg 17653785 ttcaagcgat ttttctgcct cagcctccca agtagctggg actgcaggtg catgccacca 17653725 tgcccagcta atttttgtat ttttagtaga gatggggttt cgccatgttg gccagtgtgg 17653665 tctcgatctc ttgacctcgt gatctgccca cctcggcctc tcaaagtgct ggtattacag 17653605 gcatgagcca ctgcgcctgg cctgtttctt agctttaata gttgtgtgtg tgtgtgtgtg 17653545 tgtgtgtgtg tgtgtgtgtg tgtgtgtatt ctttaggatc ctctatatat aacatcatac 17653485 cgtctgtgaa gagaggtagc ttcctttcca atttggatgg cttttattta tttttcttgc 17653425 ctaattcctc tgattggaac ttccagtact atgttaaata gcagtagtgg agcaggcatc 17653365 tttgtcttgt tcctgatctt agacagaggg ctttcaatat tttaccattg agtataatgt 17653305 cagctgtggg gttaaatttt ttaacgcctt ttatcatgtt gagggagttc ccttctgttc 17653245 ctagtttgtt gagtgatttt atcacaaaag gctattgaat tttgtcaaag gctttttgtg 17653185 catcaactga gagatcgtgt tttccccttc tctgcttttg ctccccttct actggtagaa 17653125 aggacccacc taaagcaagc agtgggcgcc ctagaggggt tacagcctag ctcttccctg 17653065 agagcagttc ttggtttgaa cctgagggca gcgggtccgc ctgaggaaac caggtgtctg 17653005 gaaggtgaag gcttgtggag ctgagtagat ggggcagtag gtcccagaga tatggccagc 17652945 - 48

cccagtcatg tcctgctctc tgtggagtcc cacagaggct gacgaggtat gggggccctg 17652885 atagctggct acatgcaggc catgcccttt ggcgggtggt ggcgtcagtc tggggcagac 17652825 ctcccatgct cacatagtgt gctcattcac ccagcactgc cttaggttgg gctccctaga 17652765 atggtggctc ttaaacccca gcaagtatct gaaacactgg agggcttgtt ccagcagatg 17652705 gctgggcccc tcccagagtt tctgatccat gttgtcttgg gtagagactg ggaatctgca 17652645 tttctaatac attctcaagt gttgtggatg ctgctggtct gagaaccaca tccctagaag 17652585 cagagtctga gatggtgcag gcgatttcag atgaaccctg caagaggcac aggcagtggg 17652525 gagcgggcag agtgagcagc tgagcacaga tgtggatttg gaagtgtggc ctcagcctga 17652465 ttccatggag atctctgggg cgtgaatgtc accacagggt tgccctgccc agaagcatgt 17652405 ggcctggctg ttacaggccc ttgtcagtca tggctctcct gggatgatgc aggtgaggtg 17652345 gcttctgtca ggagaagggc tctggtgcac cagccagaaa aggggatcaa cggcatgcat 17652285 ggccagcacc tactgtgtgc caggcatggc ctcagcactg tctgcacagc agtgagcaga 17652225 cgcgtgctgt cctcctggag ctggcatcct tttgagggag atagatgcta atcgggacag 17652165 tctgtagcct cagggagaga agtgctatct gggaagatga agccaaggtg tgggctccag 17652105 ggggccccag gtgggagtat tttattttat ttttttgaga cagagtttca ctctgtcacc 17652045 caggctggag tgcagtggtg cgatcttggc tcactgcaac ctccacccct tgggttgaag 17651985 agattctcct ccctcgcctc ctgagtagct gggattacag gcacctgcca ccatgcccgg 17651925 ctaatttttg tgtttttaat ggacaccaga tttcaccatg ttggccaggc tggtcgtgaa 17651865 ctctggacct caagtaatcc gcctacctca gcctcccaaa gttctgggat tacagatgta 17651805 agccaccaag cctggctggg tgtggggatt ttagattaga tgaggaggac aggcctctct 17651745 gactggtttc cacctctaag tcctcatcca aagccttgtt ttatagatga gacagaggca 17651685 cagagaagtg aattctaaat tcacatagcc agtggcagaa cccagacttg gaccagtttg 17651625 gggaacttct gagcctgtcc accccagtcc tagcctcacc cacagtgccc ttgcccaggg 17651565 cgagactatc agggagcctg acctgctgga tctgggcagt cccaccgtgg catgctgcat 17651505 gtcccagaga aggtatctgt cagcagtgca gcacccccca cctgccccac ccacagctcc 17651445 ctcgggggct atccctggaa gtgttggtca gaaagtgaat ctccagatgt cacctggtgg 17651385 tgccctgagc tcctcctacc tgccacctcc tctgaccaca tagagcctgc tctagcccag 17651325 gccctcttcc ctctcctccc ctcacccagg gacccgccac tagtccgccc cacccactct 17651265 gtttatttct caccttggcc actgatgggt ggtttctcct agagcggtgc tgccctgtgg 17651205 aaccttctgc aatgatggaa atgctcagac ctgctctgtg cagtccagtc gccactggcc 17651145 gcatgtggct cttgaaatat ggagagtgta actgaggaac caaacttgaa tttttaaaat 17651085 tttgatgaat ttacaatcac tcgtaagtag ccacctgtgg ctggcagcca ctggattgga 17651025 tggtgctggt ctagggtgtt ggcaaccaca tcactgcctt gtgcagaaac cactgctgca 17650965 ccaggagaag gcccaagtgc cagcctcctc ttcactgccc gaagcctgct gctccgctga 17650905 ggggctcgtc tcgccaacgt tggcacagca aacacacata ctttctcctg tgggggctgg 17650845 tcctgctggc caagtcccgt gcatgctcct gggtggctgc acctggcccc tgcaccaggt 17650785 caggtccaat ctgtggagga taccaaggaa cctctttgag gttcccaagt gtgtcccatg 17650725 ccactgcagt tttgcagaag gttagtgtgt gtgacttaaa aggcaaagag ggcaggcaga 17650665 tcttctgaca tctgggggga gcaaagttag aatggaatat ttgctgcaga acttctcaga 17650605 gcctttagca tgctaggatg tgctgcaaat ctccaggagg caggcggcat aagccatgct 17650545 tcccaaacga cttgccggtg gaagcctcct tgaggagtgc tgtgcgagac ccgtggctgt 17650485 ggagcacacg agagaatgcc tttctcgtgg tttgtgtcca tgctgggctc tcggctgcat 17650425 tgtcttccag tctgtgtccc ctgctggctt cccagggagg gagggaggct gtgactccat 17650365 gtgctccttc agcggctcgt ttgtttgctc attcgttcat ggaaaaccat ggttccatgc 17650305 cagccacacg cggggcctct gccgggcagt gggatgagtg tggtgaacaa gaggagctga 17650245 tgacctcagg cagggacctt cctttctctg ggtctgtccc gcaacataca cacacgcaca 17650185 cacgcacacg gacatacctg tgcacacatg tatacacaag acacatacac acacatacat 17650125 acactcatgg gtgtgtcctg cagctgtctg gctgtgctgg tcccagctct tacactccca 17650065 ccccttccca ggccctgtga tgcctccatg ttaccgccag agggcctggg cttgtggaag 17650005 tggtgccccg tgggcacctc tccttcccga ccatgagtgg gaccctgctc actgccttct 17649945 ctaccagagt gagggagtga tgccagcttc ccccgccttc agccgccctt gccggcctgg 17649885 gctggtggcc atgggcattc cccagcagtg tgggcaggct gggtgcctgg cacccccagg 17649825 actatgacag aagcctcccc tggtggccag ggcctaagcc atgaggcccc tgctggggcc 17649765 tgacttaggg tgtgtcctgc cttttgtccg gccctgagtg gcctggctac agcacctctt 17649705 ggccctctga ggttcgtcac ccctctgcca tcacacccat ccctggccac cctctccctg 17649645 cctgctgcct gctgtctgtc attgaacatg ctcgtgtttc tcccatccta aaactcctcc 17649585 tcctggttgg tgaacgcaat ggccacactt cccactttcc tctcatggaa tgtctgcagc 17649525 ttggtgcctc cctccacctg ctccttccag ccaccctctc tccacctggc ctcctgagca 17649465 ctgcacctta ggtctttcca catctcaccc tgtcccaggg aagcccttga tcgtccccag 17649405 gggttctctc tctgggcctt gcccttcagc atgggaagcc tgcagtccca acccagccct 17649345 tcacctccca ctctcccacc cctgttctga gctccagtct cacttaaacc tcagctgtct 17649285 cacctggctg ccccaggggc tgacttggcc catagagagc agaacctagt gccgcctctg 17649225 49 -

taccctgctt caggttcacc tccaagtgcc attaccctca caggccccag acccgacacc 17649165 tgggccctct accccttgtc cctgcatgct gcctgctaat acctgctcct cttaccaccc 17649105 cagacccttc ttatctcatg cttcctctct agggctgcta cttctctatt cctgttcccc 17649045 taattggttc tccttgctgc agctagtgca gcttgggaca gcaccatcta tggttcccta 17648985 ctgccctgac gacaatgtgt gagcctgtgc taggagacca ggccctgtgt gataagctca 17648925 gcctgccctg ttccagctgc acccaccttc tctagatcat ggactcactt ctctgcccac 17648865 agataccttt ttcccttgac ctctgcatct ggataactcc tattcactct tcacctcctg 17648805 caaatgccat cacccccaga aagcctctct aataaccccc acccagttct cctcttcatc 17648745 accacactca tcacactgca aataagtgtc tgcaagtgtc ctggcatgag aatgggccct 17648685 ccagtgccca cctggggcac ctagcaggca cttagtaaat atttacaaag tgagtggctc 17648625 tgcctcgcgt gggtggggag cagggatgcg ttttcagcca ggagatggct tggggtttgg 17648565 gttcagctgg gcagccagtg ccatggatat ttacctggtg cacttggagg tcacagggca 17648505 cactctgtcc tgatcttagt gcagatacct ttcaggtacc gtagaccccc ccagcctcag 17648445 cagctggaga tgagggcagt gcatcccttt tgccaggaag gtccgattcc caatggacaa 17648385 agaggcaatg cagtgcgagg gagccagagg ccagggctcc cgtcccagct ctgtcagtga 17648325 ctcattgtgt ggccttggga agatcctcgc tgcctaggcc tcagtgtccc cttctgtaca 17648265 gtgggtggtc tagactaatt tgttatccca aagcagtcct agacctgcac tgctgacttg 17648205 gagccctctg cacctcctgt tctgggcaca agagggcagc caagggcctc agaacgctga 17648145 ggaaccctgg ccaactagct ttaagaaatg cattgtgtaa actgctcttt actgagccca 17648085 gagcttgcca ggagcctggt agggttgtgg ctctggctct catttctacc aaaggaagtg 17648025 tgcttgacca gggagttcat ccaagggcac ctggaaactg tcctcaaggc atttcccggg 17647965 gaaccaattt ctcacgggtt gcctcagggt ggggaagcgg aggccaacag cccctgtctt 17647905 tttccgcagt ggtcctttgc tgttgctacc atcacagaaa tcccccccgt tatcttcctc 17647845 cccaacttcc ttgtgcagag gaaggtgctg aggccccttc ggacccagac aggaggaacc 17647785 ataatggtag gtggggtggg ggggcatggc tgggctgggg gcccccacac cccagggtcc 17647725 ttctcacctc ctttgccctg gaatgccctc ctcccactta gtagttgaac agaatcctaa 17647665 atattcctca aggctcggca acaatgaccc tttctccaaa agcctttttt ccccatcttg 17647605 ggacatcaga attctcttct catcgttcct tctcctatga cctcctattt gttaccgtaa 17647545 ttgctagtat ataatatacc tctccaccca ccaaagcgga tatcctagca ctatggcttt 17647485 aaggcacacc ccctcaccag ttttttcttt ctttctttct tttttttttt tgagtagagt 17647425 ctcgttctgt cgcccaggct ggagtgcagt ggtgtgatct tggctcactg caacctctgc 17647365 ctcctaggtt caagcgattc tcttgcctca ggctcctgag tagctgggac tacaggtgtt 17647305 cgccaccatg cctggctaat ttttgtattt ttagtagaga cggggtttta ccatgttggc 17647245 caggctggtc ttgaactcct gacctcaaat gatccactca ccttggcctc ccaaagtact 17647185 gggattacag gctttgagcc accatgccca gccctaatgc acccaaaatt aagatggaga 17647125 actgatcctc catgacttca gtgatgaata agcctccacg tctcccccac tgcgggtgtg 17647065 gcaacaaaga atccccacag caaaattagg tttcacattg tgtgtgtggt ttttttaaaa 17647005 aaatgtgcca cacactgcct agttatttgg agatagagga atgtttcaca tgcaaatgta 17646945 tgaggatcta acccagccct ggatcactac ctactgatcc cctacagttc tgttatgttt 17646885 gtaaatttgt actttttcct ttagcttagt agaatattac tgcccatccc caaaactatg 17646825 atttcctgga agatttcagt atttagtcta ctatatttct ttttttgctt tttttttttt 17646765 tttttttgag acagagtctc actctgtcct ccaggctgga gtgcaggggt gtgaccttgg 17646705 ctcactgcaa cctctgcctc ctgggttcaa gtgattctcc tgcctcagcc tcccgagtag 17646645 ctgggattac aggcacacgc cactctgcct ggctaatttt tgtattttta gtagagacgg 17646585 ggtttcacca tgttggcgag gctggtcttg aactcctgac ctcaagtgat ccgcctgcct 17646525 cggcctccca aagtgctggg attacaggcg tgagccactg cgcctggccc agtctactgt 17646465 atttctgtga gcaaaacttt gcctattttc cctttgaaag ccatatcaaa attattgtca 17646405 gctcatatgt gatggatgat aagtactttt attttttcca gtttccttgc acaatttcaa 17646345 aggtgcttat gcactgtaca tctcatatgc cagccaagct ggcacttact tcctggactg 17646285 ttgcttgggg tagggagttc cttctatacc cctgccttgt agctcagctc atccttcccc 17646225 cagagctggc tagaagcagt gtttatggaa tgagtgcatg aatcagtgaa tgaatgactg 17646165 gtggatcggc tgcctgcgcc ccctcaccct ctgcttgtct ccaaaggcgg ggaagctggc 17646105 tgtggagcga ggctgggcca tcaacgtggg tgagtgctgg gaatgtcctc gggaatgtcc 17646045 agcccggctt ggtggaactg gcctgaaagg gggctggggg agggcgggag gatcctggag 17645985 gtggcagctg tgaattcaga agctctggtt ttcccaagtc accctagcct ccttgtggag 17645925 tggcctggag gttgatgtgt agcctcctag gtacctggga gagactgacc agtgcctcca 17645865 tctgacgtgg gatccttgtc taaggaggtc cccgggtggt tccccagccc cctctttgcg 17645805 tacttccggt ggcagggagc ttcctccctt ccagagagcg tgtgccatcc ttgggcagct 17645745 cagcatggtc tgaagcctgc cttgtgtctt ccctgaagga ctccacctgt gtcctggggc 17645685 ccaggacagc ccacagaggc ttggtcatgt tgggttgggt gggcacatcc tgggtcaata 17645625 ccaccacctt ctcaagggtc cagagggccc gtgctcccca gcccccttga atctcccaca 17645565 agattggctc atgggagggc tgcacgggag tctcccttgt ccctgtcatt gtccctcctg 17645505 50 -

gaggcacagc acttgacaat ttacaaagct ctttttcacc aggctctttt tttctttttc 17645445 gagacgtagt ttcactcttg ttgcccaggc tggagtgcaa tggcgcgatc tcggctcacc 17645385 gcaacctccg cctcccaggt tcaaacaatt ctcctgcctc agcctcctga gtagctgaga 17645325 ttacaggcat gcaccaccat gcccggctaa ttttgtattt ttagtagaga cagggtttct 17645265 ccatgttggt caggctgggt cttgaactcc cgacctcagg tgatacgccc acctcgctcg 17645205 gcctcccaaa gtgctaagat tacagacatg agccaccacg cccggccttc acccagactc 17645145 ttatttgagc tgggcataat tgtcaggcct gtctcactga tgaggaaatg gccatggaaa 17645085 gatgcgtact ggatcgtgta gagccctaaa gcagggtccc ccagcctttg gctctgaact 17645025 ctgcagggga gagtccacct tgggccactg cacagttgag gggagcccca ctctgcaggg 17644965 gctgggtctc ttccatcttg gtattaccag gtgcctagca ttcagtctgg catagtaatg 17644905 atgttatggt actctgctgc acaaacccgg gagtgatctg tgccctgcgt gtctacagca 17644845 gggttccgag gagggcctgg atggccctcc ccatggcagg tgttactgcc tggtagaggt 17644785 taagagcctg gatcctgatc caccctgggt ttgatcctgg ttctgccatt acctggctgt 17644725 gtgaccctgg gcaagttgct gacctcctct gtgggtcagt ctcctcatct gtaaaatggg 17644665 gatggtgatg ctaatgcccc tcctcgggct ggagggagtc ttcagcaagc tcagttgctc 17644605 agtcaggtgt tcactgtggc tgtcttctca tcattaggag ccaacagtag cctcctgggg 17644545 ggtgggagag gcaagttcct ggtatccatg gggccagctg cacactgtct gacggagcag 17644485 ttgttgggct caatttcaga gggcctctgc aattcaggcc atcccagggg ctgcagggga 17644425 gggggtatct atgggcccta gggctctgag gctgtgtctc agggttgagg ggtgatggat 17644365 cccgggctct agggccctcc tcgtggctgt aggcagtcat gaccagcaga gggtgccctt 17644305 cctgaccacc cgctttggcc actggcagaa tccgtgtggc ccccatacca ccactccttc 17644245 ctggagtggg gagccacatg gagccaggcc cagcttggtg gggacaagga gcagctttct 17644185 gcttctggaa tgatgagcta tctgttgctt aggggtgtga gtggcactga ggacttgctg 17644125 gggacaccct gaagatgtgg ctgccttctg gcctggggat ggtgacatgc cccagcactc 17644065 agcttagttt gccaacccag agtccgaggc acaggttcct gagagctgag cagggaggat 17644005 gctgggggag gtgaagggat ggaggagctc ctggactgag cctgggagcc tggctctgag 17643945 cagcaccgct ctctgccctt ccgcaggggg tggcttccac cactgctcca gcgaccgtgg 17643885 cgggggcttc tgtgcctatg cggacatcac gctcgccatc aaggtgtgtc tatgagcaag 17643825 tggggtctcg cctccaagag ccctcctgga atcctcccca tagctccaaa ttaactgttc 17643765 tcaccctgaa ttatagacaa ggggcctatg ctggagcagg gagggggctt gtttgggttg 17643705 ctcagccagg ctggaactga atccagatct gacacttgct cctcttccat gttgcttaga 17643645 agggttgcct gtggtggaag ggagttattc cagcctccca cagagccagg ggactagaga 17643585 gggtcaggat ctgctgtata gccacatatt aagttgtagg aagaagggca tggctggcaa 17643525 agggagtagg gagtggaaag aatgatggtg ctgatagcac ctggcagttc tgcatgctcc 17643465 aacccgcgct gtgctccagg acttactccc tgaatcctcg cagacagaca ggggcccaca 17643405 gaggtgaggg catgcaaata gcaggggcag aattggcgct ggcctctggt ctgtggggcc 17643345 ccacaactcc cctgccactc tgtgcctggc cttgtgctgg gcatcaggaa ctgactgacc 17643285 tgttcctatg tgtgcctgct ctcatggggc acatagactg atggggggaa gcaggccatt 17643225 aggagaaggg ggaagcacag gagaccttcc tggggaggag ggaatgaagg cttcctggaa 17643165 gagggggcat ttaggacttg gccttgtagg ataaggcaga ggttggggac tgaagtccca 17643105 gggctgtggg gattctctcc ttaaccccta cacatttcct agggaatctg ggaaaatcca 17643045 gggcctgagt gacccactta cctcctgacc tatgaccctt cagggcacag gacatgcccc 17642985 ctcctccagg gagccttccc tgaccacctc ctgcatgcac acatggagcc ccacagctgg 17642925 agctgcacag ctctccctgg caagtgacat ctttgctggg tggcctgatt acccacaagc 17642865 attaggcccc cctccccgcc cctcgccagc cagctgggag ttgctgtagg gctgggtcct 17642805. ctgtccgccc cagatcctca tgtctaccct ctcctccctg gcagtttctg tttgagcgtg 17642745 tggagggcat ctccagggct accatcattg atcttgatgc ccatcaggtg agtgccctgc 17642685 aggggctgga ctcttagggg acctgccacc cccagttcca gaatcttccc ggggcaggag 17642625 agtctccctc ctcatgtccc cacggctctc acggcttctg tcttctgtct ctcgggctac 17642565 aaatgcaggg tctgtctttg tcactctgtc caggacagcg ggtcctcctc attgctcccg 17642505 agggtcctcc ctccctcctc ctgactgccc ccacatgagg ctcttcctga agcccactct 17642445 gatgggactg ctctcgtgtg cagagctctg ctgtgggtcc ccattgctta tgaataattt 17642385 ggggcactgc cccctgccca gagctgctga gcactggcca cctgcccctc aggcggatgc 17642325 ccacacacat ggcttggctc gggcacctgg ggtcaccatt taagaactcg gcgcctaggg 17642265 agtaaagtgt caaagcagag ggttacctcc tcctcaggac ccctaatgag gccagtgcct 17642205 ctggtcagac agggagggga cccagtgggc tccggaaggc acccccctgc accattactg 17642145 ctgtggcttt gtgctagttg gggccctgcc ttgggttctt gcgaccccga actcctgagc 17642085 caggtcacat gtggacagtc ctttacagtt tgcttttcac atccctgatc ccaaccagtc 17642025 ccaccacaga cttgagaggg tggcagagcg ggatttcttc ctctgatagg gaacctaaga 17641965 gcactgggct tgctcaagcc catgctagaa ggtgtcgggg cctggtttta aggttgaatc 17641905 ccagctctgc cccttaacag tcatgagacc tgctgccccc gagagcaggc cgtgctgccc 17641845 tggcaaatgg ggagtttcct gaggggtggg tgggtggcag agccccagcc ttgcctaggg 17641785 51

cacctacccg agagcggcta ctgtgacctc cccacagggc aatgggcatg agcgagactt 17641725 catggacgac aagcgtgtgt acatcatgga tgtctacaac cgccacatct acccagggga 17641665 ccgctttgcc aagcgtaagc tgctgcccct accctcatct tgggtgtgtc cttgtggatg 17641605 aggctctctc ctgagtgtct cctgtctgct aggccctgca gaagccactg cagtggttca 17641545 tagcatccct gtgaggtgat cctttccatt ttacagatga ggaaaccgag acctggagaa 17641485 gtcactcgac ccacccaaga tcacataacc cttacaataa acatgcattt gtctggcaaa 17641425 aaacaggaaa gaatgaaaga aaaaaaagaa aaataggata aatttgaaaa tacgaaataa 17641365 gaaataaatt cacataggct gggcgcggtg gctcacgcct gtaatcccag cactttggga 17641305 ggctgaagtg ggcggatcac ctgaggtcgg gagtttgaga ccagcctgac caacatggag 17641245 aaaccccatc tctactaaaa atacaaaatt agctggatgt ggtggcgcat gcctgtaatc 17641185 ccagctactc gggaggctga gacaggagaa ttgcttgaac ctgggaggcg gaggtttcgg 17641125 taagccgaga tcgcgccatt gcactccagc ttgggcaaca agagcgaaac tccatctcga 17641065 aagaaagaaa gaaattcatg tataatcgtt aaaatgaaaa tgcattaaac tcatcaatca 17641005 aaaggcagag actctcagat gagatttaaa aacagggctg ccacctttgc aggtagggga 17640945 catttttgca ccagtcacga tgagtctggt gtggataagt cagcagctag tatggcccaa 17640885 ggaaccaatt tctgaacaga acctcacatg tgctgagcct gggcttaagg gcagggcagg 17640825 gtgtccatgt gtgtaggcaa gacccagagg aggcagtgaa atctgacatt gccgacacag 17640765 atctccacac ccccagggca gtgtctcagc ttcagtgccc cttctctcct ttgagtcccc 17640705 ctttttgcag ctcttggtgc tcttttcacc ttagttttgg gtggaatgag gctgagcagt 17640645 gctgaatctg acagaccagt ttccagtctt gcctggtgtc cacagtcttg tcctgagcct 17640585 cagtttccct tctctataaa ttgaggccat ccatgtctct ctcccagagg ccatcaggcg 17640525 gaaggtggag ctggagtggg gcacagagga tgatgagtac ctggataagg tggagaggaa 17640465 catcaagaaa tccctccagg agcacctgcc cgacgtggtg gtatacaatg caggcaccga 17640405 catcctcgag ggggaccgcc ttggggggct gtccatcagc ccagcggtac gtcctgaccc 17640345 ttggggccac gggagggtct gctctatgga ctcagcagca gcaggaaagg tgggcggcct 17640285 catgtcaggg aggagatgga ctgaagcaac agcagtttgg agcagggcta gccctgcagc 17640225 aggacttcct gacaccatgg gggtctggcc tgcctgagtc accctcctct tcccctaaca 17640165 gggcatcgtg aagcgggatg agctggtgtt ccggatggtc cgtggccgcc gggtgcccat 17640105 ccttatggtg acctcaggcg ggtaccagaa gcgcacagcc cgcatcattg ctgactccat 17640045 acttaatctg tttggcctgg ggctcattgg gcctgagtca cccagcgtct ccgcacagaa 17639985 ctcagacaca ccgctgcttc cccctgcagt gccc