WO2002031205A2

WO2002031205A2 - Method for rapid subtraction hybridization to isolate a gene which is differentially expressed between two populations of cells

Info

Publication number: WO2002031205A2
Application number: PCT/US2001/031811
Authority: WO
Inventors: Paul B. Fisher; Hongping Jiang
Original assignee: The Trustees Of Columbia University In The City Of New York
Priority date: 2000-10-13
Filing date: 2001-10-11
Publication date: 2002-04-18
Also published as: WO2002031205A3; AU2001296815A1

Abstract

The present invention provides for methods for rapid subtraction hybridization of two pools of double-stranded cDNA to isolate a differentially expressed gene which comprises: isolating RNA from a first and a second population of cells; producing a first and second double-stranded cDNA pool; admixing the first and second pools of cDNA separately with a first restriction enzyme; admixing the digested first pool and second pool of cDNA with an appropriate amount of a double-stranded oligonucleotide; wherein the oligonucleotide sequence comprises (i) an internal recognition site for a second restriction enzyme and (ii) a ligatable cohesive end at one end which is capable of hybridizing with a ligatable cohesive end resulting from digestion of the first pool of cDNA with the first restriction enzyme; performing two separate polymerase chain reactions (PCR) using an aliquot of the ligation mixture (tester cDNA or driver cDNA) admixed with a PCR primer; digesting the PCR products of the tester cDNA with the second restriction enzyme under appropriate digestion conditions; admixing an aliquot of the digestion product (tester cDNA) with an excess of driver cDNA PCR products under hybridization conditions, so as to isolate a differentially expressed gene from among the two pools of double-stranded DNA.

Description

Method for Rapid Subtraction Hybridization to Isolate a Gene Which Is Differentially Expressed Between Two Populations of Cells

The invention disclosed herein was made with Government support under Grant Nos . CA35675 and NS31492 from the U.S. Department of Health and Human Services, National Institutes of Health. Accordingly, the U.S. Government has certain rights in this invention.

Throughout this application, various publications are referenced by author and date within the text. Full citations for these publications may be found listed alphabetically at the end of the specification immediately preceding the claims. All patents, patent applications and publications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

Background of the Invention

Orderly temporal changes in gene expression are major determinants of normal physiological processes and they represent primary mediators of altered cellular properties that define various disease states (1-3) . In these contexts, the ability to elucidate the molecular causes of normal and abnormal cellular changes requires the identification and clarification of function of the spectrum of differentially expressed genes. Attaining this goal continues to represent a major effort in both academic and industrial research laboratories. This task is of high priority and offers promise for discovering target genes regulating specific disease states. Once appropriate genes are recognized, methods such as combinatorial chemistry and high throughput screening can be used to identify and develop new molecules with broad ranges of therapeutic potential.

Several approaches are providing an initial description of potentially important and possibly relevant genes involved in or associated with normal processes and specific disease states, including aging, differentiation, development, cancer, cardiovascular disease and neurodegeneration (1-4) . Specific molecular approaches that have proven especially informative in identifying differentially expressed genes include differential RNA display (4,5), serial analysis of gene expression (6,7), subtraction hybridization (8-11), reciprocal subtraction differential RNA display (12) , representational difference analysis (13), RNA fingerprinting by arbitrarily primed PCR (14), electronic subtraction (15) and combinatorial matrix gene analysis (16) . Subtraction hybridization represents a particularly attractive method for isolating genes that are differentially expressed in diverse target cells, without prior knowledge of their functional or biochemical characteristics. A limitation of this scheme can often be attributed to technical difficulties encountered in performing^' this procedure (11,17). The traditional subtraction hybridization method involves hybridization of first strand cDNAs generated from tester RNAs with mRNAs obtained from drivers . Single stranded unhybridized cDNAs are then selected by hydroxylapatite column chromotography or biotin-avidin extraction and are used as templates for the second strand cDNA synthesis . These approaches can only analyze a fraction of the overall changes in gene expression, require large amounts of mRNA, and are lengthy and labor intensive procedures. To circumvent some of these problems, cDNA libraries in phage plasmid vectors have been used as both testers and drivers leading to successful construction of subtracted cDNA libraries by a number of investigators (8,18,19). However, constructing cDNA libraries and preparation of cDNA fragments for hybridization are laborious, some times difficult, processes. PCR-based cDNA subtraction considerably accelerates the procedures for cDNA library preparation and provides a new direction for subtraction, although it also involves several tedious steps during or after hybridization (13,20) .

Summary of the Invention

The present invention provides for methods for rapid subtraction hybridization of two pools of double-stranded cDNA to isolate a differentially expressed gene which comprises: (a) isolating RNA from a first population of cells (driver cells) and separately from a second population of cells (tester cells) ; (b) producing a first double- stranded cDNA pool (driver cDNA) from mRNA isolated from the first population of cells; (c) producing a second double- stranded cDNA pool (tester cDNA) from mRNA isolated from the second population of cells; (d) admixing the first pool of cDNA (driver cDNA) with a first restriction enzyme which recognizes a restriction site at about every 200 to 500 bases of DNA under appropriate conditions for restriction digestion of the first pool of cDNA; (e) admixing the second pool of cDNA (tester cDNA) with an amount of the first restriction enzyme under appropriate conditions for restriction digestion of the second pool of cDNA; (f) admixing the digested first pool of cDNA (driver) from step

(d) with an appropriate amount of a double-stranded oligonucleotide; wherein the oligonucleotide sequence comprises (i) an internal recognition site for a second restriction enzyme and (ii) a ligatable cohesive end at one end which is capable of hybridizing with a ligatable cohesive end resulting from digestion of the first pool of cDNA with the first restriction enzyme, wherein the second restriction enzyme recognizes a restriction site at about every 4000 bases of DNA, under suitable conditions for ligation of the oligonucleotide to the cDNA; (g) admixing the digested second pool of cDNA (tester) from step (e) with an appropriate amount of a double-stranded nucleotide; wherein the oligonucleotide sequence comprises (i) an internal recognition site for a second restriction enzyme and (ii) a ligatable cohesive end at one end which is capable of hybridizing with a ligatable cohesive end resulting from digestion of the second pool of' cDNA with the first restriction enzyme, wherein the second restriction enzyme recognizes a restriction site at about every 4000 bases of DNA, under suitable conditions for ligation of the oligonucleotide to the cDNA; (h) performing a polymerase chain reaction (PCR) using an aliquot of the ligation mixture resulting from step (f) (tester cDNA) admixed with a PCR primer, wherein the first PCR primer has a sequence which allows it to anneal to the oligonucleotide-derived region of the resulting ligation product from step (f) ; (i) performing a polymerase chain reaction (PCR) using an aliquot of the ligation mixture resulting from step (g) (driver cDNA) mixed with the PCR primer; (j) digesting the PCR products from the PCR reaction performed in step (h) (tester cDNA) with the second restriction enzyme under appropriate digestion conditions; (k) admixing an aliquot of the digestion product (tester cDNA) from step (j ) with an excess of PCR products from step (i) (driver cDNA) under hybridization conditions; and (1) ligating the hybridization mixture from step (k) with a sufficient amount of a vector previously digested with the second restriction enzyme, so as to isolate a differentially expressed gene from among the two pools of double-stranded DNA.

In one embodiment of invention, the driver cells are untreated cells and the tester cells are cells treated with a compound . Brief Description of the Figures

Figure 1: Schematic outline of the RaSH protocol. This scheme involves construction of tester (IFN-β + MEZ) and 5 driver (control) HO-1 libraries followed by digestion of only the tester library with Xho I . Following hybridization, differentially expressed sequences are cloned into Xho I digested vectors resulting in a subtracted cDNA library enriched for mda genes displaying elevated expression. By 0 using the control HO-1 library as the tester and the IFN-β + MEZ library as the driver, RaSH can also be used to produce a subtracted cDNA library enriched for genes downregulated during terminal differentiation.

5 Figure 2 ; Reverse Northern blot analysis of differentially expressed sequence tags identified by RaSH. PCR amplified products from bacterial clones of both RaSH-derived subtracted libraries, Dpn-sLib and EcoR-sLib, were dot-blotted onto nylon membranes and were probed with 0 ³²P-cDNA reverse transcribed from RNA samples of control or IFN-β + MEZ treated HO-1 cells.

Figure 3 : Differential expression of representative types of mda genes identified by RaSH and reverse Northern blotting. Ξ Northern blots of total RNA were prepared from HO-1 cells untreated (control) (lanes 1 and 7) or treated for 2 hr (lane 2) , 4 hr (lane 3) , 8 hr (lane 4) or 24 hr with IFN-β (lanes 5 and 8) , 24 hr with MEZ (lane 9) , 24 hr with IFN-β + MEZ (lanes 5 and 10) or 72 hr with IFN-β + MEZ (lane 6) . C Membranes were probed with radiolabeled (³²P) expressed mda sequence tags, identified by RaSH in the Dpn-sLib and EcoR-sLib. Equal loading of samples was confirmed by hybridization with a ³²P-labeled gapdh cDNA probe. Figure 4; Determination of the amounts of fibronectin and LIF fragments in PCR-cDNA libraries. Relative amount of fibronectin and LIF fragments in PCR libraries was determined by comparison of the signal intensity of fibronectin and LIF in specified cDNA libraries (Dpn-sLib and EcoR-sLib) with the signal intensity of defined amounts of the cDNA fragments by Southern ^'blot hybridization. Lane 1, 100 ng PCR-cDNA library from untreated cells; lane 2, 100 ng PCR-cDNA library from IFN-b + MEZ treated cells; lane 3 to 7, increasing amounts of cDNA fragments of fibronectin and LIF (0.001, 0.01, 0.1, 1 and 10 ng, respectively). Phosphorl ager (Molecular Dynamics) scanning determined hybridized signal intensity.

Figure 5: Nucleotide sequence and corresponding amino acid sequence of HuUBP43. Three AU-rich sequences (AUUUA) are underlined and poly (A) signal (AATAAA) is bold-faced.

Figure 6: Effect if various compounds effecting growth and differentiation in HO-1 human melanoma cells on HuUBP43 expression. RNA samples were extracted from HO-1 cells treated as indicated for 24 hr. Northern hybridizations were performed as described in Materials and Methods. Northern blots were probed with a ³²P-labeled HuUBP43 cDNA, the blots were stripped and reprobed with a ³²P-labeled GAPDH cDNA.

Abbreviations and concentrations of the indicated reagents are as follows: CTL, control; DMSO, 0.1% dimethyl sulfoxide; MEZ, mezerein 10 ng/ml; IFN-β, 2,000 U/ml interferon-β; IFN- β + MEZ, 2,000 U/ml interferon-β plus 10 ng/ml mezerein; IFN-γ, interferon-γ 100 U/ml ; IFN-γ + MEZ, 100 U/ml interferon-g plus 10 ng/ml mezerein; RA, all-trans-retinoic acid 2.5 mM; MPA, mycophenolic acid 3 mM; D-0, serum-free media; TPA, 12-0-tetradecanoylphorbol-13-acetate 16 nM; cAMP, 3 ' -5 ' cyclic adenosine monophosphate 1 mM; 8-Br-cAMP, 8-bromo-3 ' -5 ' cyclic adenosine monophosphate 1 mM; MMS, methylmethane sulfonate 10 ng/ml.

Figure 7 : Effect of ligands for various membrane receptors on HuUBP43 expression. RNA samples were extracted from HO-1 cells treated as indicated for 24 hr . Northern hybridizations were performed as described in Materials and Methods. Northern blots were probed with a ³²P-labeled HuUBP43 cDNA, the blots were stripped and reprobed with a ³²P-labeled GAPDH cDNA. Abbreviations and concentrations of indicated reagents are as follows: CTL, control; IFN- , 1,000 U/ml interferon-αi; IFN-β, 1,000 U/ml interferon-β ; IFN-γ, 1,000 U/ml interferon-γ; IL-6, 1 ng/ml interleukin- 6; EGF, 10 ng/ml epidermal growth factor; TGF-α, 10 ng/ml transforming growth factor- ; TGF-β, 2.5 ng/ml transforming growth factor β; TNF-α, 10 ng/ml tumor necrosis factor α,- PDGF, 10 ng/ml platelet-derived growth factor.

Figure 8: Effect of various IFN-α subtypes on HuUBP43expression. RNA samples were extracted from HO-1 cells treated with 100 U/ml of the indicated IFN-α subtypes for 24 hr. For comparison, the induction of HuUBP43 expression by the same dose of IFN-β is included. Northern blot hybridizations were performed as in Materials and- Methods. Northern blots were probed with a ³²P-labeled HuUBP43 cDNA, the blots were stripped and reprobed with a ³²P-labeled GAPDH cDNA.

Figure 9: Kinetics and dose response of HuUBP43 expression in HO-1 cells treated with IFN-β. (A) Time course of HuUBP43 expression. RNA samples were extracted from HO-1 cells treated with 2,000 U/ml IFN-β or 2,000 U/ml IFN-β plus 10 ng/ml mezerein and harvested at the indicated time. (B) IFN-β dose-response of HuUBP43 expression. RNA samples were extracted from HO-1 cells treated with indicated amount of IFN-β for 24 hr. (C) Northern blot analyses of HuUBP43 expression following incubation with actinomycin D (ActD) . HO-1 melanoma cells were treated with 5 mg/ml actinomycin D after 24 hr incubation with 2,000 U/ml IFN-β or 2,000 U/ml IFN-β + 10 ng/ml MEZ. Cells were harvested at the indicated time after actinomycin D treatment. (D) Northern blot analysis of HuUBP43 expression after blocking protein synthesis by cycloheximide (CHX) . RNA samples were extracted from HO-1 melanoma cells pretreated with 50 mg/ml cycloheximide for 30 min and treated with the indicated reagents for 8 hr . Northern blot hybridizations were performed as described in Materials and Methods. Northern blots were probed with a ³²P-labeled HuUBP43 cDNA, the blots were stripped and reprobed with a ³²P-labeled GAPDH cDNA.

Figure 10: Effect of IFN-β on H UBP43 expression in various human normal and tumor cell lines. RNA samples were extracted from the indicated cells treated with 2,000 U/ml IFN-β for 24 hr. Northern blot hybridizations were performed as described in Materials and Methods. Northern blots were probed with a ³²P-labeled HuUύP43 cDNA, the blots were stripped and reprobed with a ³²P-labeled GAPDH cDNA.

Figure 11: Effect of IFN-β on HuUBP43 expression in human cells containing an intact or mutated components of the interferon signaling pathway. The various cell types were treated with IFN-β for 6 hr and harvested for RNA extraction. Northern blot hybridizations were performed as described in Materials and Methods. Northern blots were probed with a ³²P- labeled HuUBP43 cDNA, the blots were stripped and reprobed with a ³²P-labeled GAPDH cDNA. The complementing genes for the mutant human cells are as follows: 2fTGH, wild-type; U1A, tyk2; U3A, STAT1; U4A, jakl; U5A, IFNAR2.

Figure 12: Northern blot analysis of HuUBP43 expression in different human organs. Multiple tissue Northern blots containing 2 mg of poly A⁺ RNA from various organs was probed with a ³²P-labeled HuUBP43 cDNA, the blots were stripped and reprobed with a ³P-labeled β-actin cDNA. Northern blot hybridizations were performed as described in Materials and Methods .

Detailed Description of the Invention

(d) with an appropriate amount of a double-stranded oligonucleotide; wherein the oligonucleotide sequence comprises (i) an internal recognition site for a second restriction enzyme and (ii) a ligatable cohesive end at one end which is capable of hybridizing with a ligatable cohesive end resulting from digestion of the first pool of cDNA with the first restriction enzyme, wherein the second restriction enzyme recognizes a restriction site at about every 4000 bases of DNA, under suitable conditions for ligation of the oligonucleotide to the cDNA; (g) admixing the digested second pool of cDNA (tester) from step (e) with an appropriate amount of a double-stranded,, nucleotide; wherein the oligonucleotide sequence comprises (i) an internal recognition site for a second restriction enzyme and (ii) a ligatable cohesive end at one end which is capable of hybridizing with a ligatable cohesive end resulting from digestion of the second pool of cDNA with the 5 first restriction enzyme, wherein the second restriction enzyme recognizes a restriction site at about every 4000 bases of DNA, under suitable conditions for ligation of the oligonucleotide to the cDNA; (h) performing a polymerase chain reaction (PCR) using an aliquot of the ligation

IC mixture resulting from step (f) (tester cDNA) admixed with a PCR primer, wherein the first PCR primer has a sequence which allows it to anneal to the oligonucleotide-derived region of the resulting ligation product from step (f) ; (i) performing a polymerase chain reaction (PCR) using an

15 aliquot of the ligation mixture resulting from step (g) (driver cDNA) mixed with the PCR primer; (j) digesting the PCR products from the PCR reaction performed in step (h) (tester cDNA) with the second restriction enzyme under appropriate digestion conditions; (k) admixing an aliquot of

20 the digestion product (tester cDNA) from step (j) with an excess of PCR products from step (i) (driver cDNA) under hybridization conditions; and (1) ligating the hybridization mixture from step (k) with a sufficient amount of a vector previously digested with the second restriction enzyme, so

25 as to isolate a differentially expressed gene from among the two pools of double-stranded DNA. This method is also known as the "RaSH" method.

In one embodiment of the invention, the driver cells are 3^'C untreated cells and the tester cells are cells treated with a compound .

In another embodiment of the invention, the driver cells are cells with a normal phenotype and the tester cells are cancer cells.

In another embodiment of the invention, the driver cells are cells susceptible to infection by Human Immunodeficiency Virus (HIV) and the tester cells are cells resistant to infection by HIV.

In another embodiment of the invention, the driver cells are in one state of differentiation and the tester cells are identical to the driver cells except that they are in a different state of differentiation.

In another embodiment of the invention, the driver cells are stem^' cells and the tester cells are cells which have undergone differentiation.

In another embodiment of the invention, the compound is a carcinogen, a pollutant, or a differentiation agent.

In another embodiment of the invention, the first restriction enzyme is DpnII, TaqL , Rsal , or EcoRII .

In another embodiment of the invention, the second restriction enzyme is Xhol .

In another embodiment of the invention, the excess is about 5-fold excess to more than 1, 000-fold excess.

In another embodiment of the invention, the method is carried out so that the driver cDNA and the tester cDNA are reversed so as to isolate genes which are highly expressed in the driver cells and wherein expression of such genes is in less in the tester cells.

The present invention provides for a previously unknown nucleic acid identified by the RaSH method described herein. The present invention also provides for an interferon inducible gene identified by the RaSH method described herein. The invention provides for a nucleic acid which is induced in a cell following viral infection of the cell identified by the RaSH method described herein. The invention provides for a nucleic acid encoding a human UBP43 protein identified by the RaSH method.

The term "driver" is used herein to describe either the cell population, the mRNA or the cDNA which is derived from such a cell population which is the cell population which is used as the source of excess background cDNAs .

The term "tester" is used herein to describe either the cell population, the mRNA or the cDNA derived from such cell population, which is the cell population used as the source of the differentially modulated (or expressed) cDNAs .

For example, "driver cDNA" subtracted from "tester cDNA" will result in a population of cDNAs which are differentially expressed in the tester cDNA pool (i.e. more highly expressed in the tester cDNA pool than in the driver cDNA pool) .

The present invention provides for a method wherein the use of the driver cDNAs and the tester cDNAs are reversed so that one can isolate cDNAs representing (or corresponding to) mRNAs which are either increasing or decreasing between driver and tester cell populations.

For example, the present invention provides for methods for rapid subtraction hybridization of two pools of double- stranded cDNA to isolate a differentially expressed gene which comprises: (a) isolating RNA from a first population of cells (driver cells) and separately from a second population of cells (tester cells) ; (b) producing a first double- stranded cDNA pool (driver cDNA) from mRNA isolated from the first population of cells; (c) producing a second double-stranded cDNA pool (tester cDNA) from mRNA isolated from the second population of cells; (d) admixing the first pool of cDNA (driver cDNA) with a first restriction enzyme which recognizes a restriction site at about every 200 to 250 bases of DNA under appropriate conditions for restriction digestion of the second pool of cDNA; (e) admixing the second pool of cDNA (tester cDNA) with an amount of the first restriction enzyme under appropriate conditions for restriction digestion of the second pool of cDNA; (f) admixing the digested second pool of cDNA (tester) from step (d) with an appropriate amount of each of two oligonucleotides; wherein each oligonucleotide sequence comprises (i) a restriction enzyme recognition site which would be recognized by the first restriction enzyme and (ii) a restriction enzyme site which would be recognized by a second restriction enzyme, wherein the second restriction enzyme recognizes a restriction sites at about every 500 to 800 bases of DNA, under suitable conditions for ligation of the oligonucleotides to ends of the cDNAs; (g) admixing the digested first pool of cDNA (control) from step (e) with an appropriate amount of each of two oligonucleotides; wherein each oligonucleotide sequence comprises (i) a restriction enzyme recognition site which would be recognized by the first restriction enzyme and (ii) a restriction enzyme site which would be recognized by a second restriction enzyme, wherein the second restriction enzyme recognizes a restriction site at about every 500 to 800 bases, under suitable conditions for ligation of the oligonucleotides to ends of the cDNAs; (h) performing a polymerase chain reaction (PCR) using an aliguot of the ligation mixture resulting from step (f) (control cDNA) admixed with a PCR primer, wherein the first PCR primer has a sequence which allows it to anneal to the oligonucleotide-derived region of the resulting ligation product from step (f) ; (i) performing a polymerase chain reaction (PCR) using an aliquot of the ligation mixture resulting from step (g) (tester cDNA) mixed with the PCR primer; (j) digesting the PCR products from the PCR reaction performed in step (h) (control cDNA) with the second restriction enzyme under appropriate digestion conditions; (k) admixing an excess of the digestion product (control cDNA) from step (j) with an aliquot of PCR products from step (i) (tester cDNA) under hybridization conditions; (1) ligating the hybridization mixture from step (k) with a sufficient amount of a vector previously digested with the first restriction enzyme, so as to isolate a differentially expressed gene among the two pools of double-stranded DNA.

In one embodiment of invention, the control cells are cells untreated cells and the tester cells are cells treated with a compound.

In one embodiment of the invention, the control cells are cells with a normal phenotype and the tester cells are cancer cells .

In another embodiment of the invention, the control cells are cells susceptible to infection by Human Immunodeficiency Virus (HIV) and the tester cells are cells resistant to infection by HIV.

In another embodiment of the invention, the control cells are in one state of differentiation and the tester cells are identical to the control cells except that they are in a different state of differentiation.

In another embodiment of the invention, the control cells are stem cells and the tester cells are cells which have undergone differentiation.

The restriction enzymes which are used in the methods presented herein are of two types: (1) "frequent cutters" are restriction enzymes which recognize its restriction site sequence in DNA about every 200-500 bases. Again, this range is an approximation and the difference between these two types of restriction enzymes would be known to one of skill in the art. It is well known which restriction enzymes are frequent cutters and which are infrequent cutters. One other characteristic of a frequent cutter restriction enzyme is that the number of bases which make up its restriction site sequence is often four (4) or five (5) nucleotides in length.

In contrast, the second type of restriction enzyme used in the methods presented herein are restriction enzymes which are "infrequent cutters" which are restriction enzymes which recognize their restriction site sequence only once about every 4000 bases within any given piece of DNA. This size range is an approximation and can vary somewhat. This type of restriction enzyme is also characterized by the number of bases which make up its restriction site sequence. Often, 5 this type of restriction enzyme recognizes a sequence which is six (6) , seven (7) , eight (8) or more bases in length.

In a further embodiment of the invention, the first restriction enzyme is DpnII , Taql , Rsal , or -EcoRII. These 0 are examples of frequent cutters.

In another embodiment of the invention, the second restriction enzyme is Xhol . This is an example of a infrequent cuttei . There are many other examples of both 5 infrequent cutters and frequent cutters which would be known to one of skill in the art. They are not listed herein, but can easily be found in any of the many molecular biological general references or catalogs, e.g. PROMEGA™, NEW ENGLAND BIOLABS™. C

In another embodiment of the invention, the method is b carried out so that the driver cDNA and the tester cDNA are reversed so as to isolate genes which are highly expressed in the control cells and wherein expression in less in the tester cells.

C One advantage of the methods presented herein .is that one can utilize a smaller number of cells as the starting cell populations than would be necessary using previous methods. The smaller quantities of cells would only provide small quantities of mRNA and cDNA. Polymerase chain reactions (PCR) may be used in conjunction with this method in order to allow one to use these smaller tissue/cell preparations.

In one embodiment of the invention, first pool of cDNA is isolated from cells at a first stage of differentiation and the second pool of cDNA is isolated from identical cells as the first pool except that the cells are at a later stage of differentiation than the first stage.

In another embodiment of the invention, the first pool of cDNA is isolated from cells treated with a chemotherapeutic agent or agents and the second pool of cDNA is isolated from identical cells as the first pool except that the cells are not treated with a chemotherapeutic agent or agents.

In another embodiment of the invention, the first pool of cDNA is isolated from cells derived from an embryo and the second pool of cDNA is isolated from identical cells as the first pool except that the cells are derived from an adult.

In another embodiment of the invention, the first pool of cDNA is isolated from cancerous cells and the second pool of cDNA is isolated from identical cells as the first pool except that the cells are normal.

In another embodiment of the invention, the first pool of cDNA is isolated from cells treated with an infectious agent and the second pool of cDNA is isolated from identical cells as the first pool except that the cells are not treated with an infectious agent .

In another embodiment of the invention, the first pool of cDNA is isolated from cells treated to induce a specific cellular change or pathway and the second pool of cDNA is isolated from identical cells as the first pool except that the cells are untreated.

The two pools of cDNA are isolated from two different cell populations which differ in a specific way. For example, one pool of cDNA may differ from the second pool of cDNA due to differences in growth regulation, terminal differentiation, apoptosiε, senescence, neurodegeneration, cardiac dysfunction, angiogenesis or muscular degeneration. In addition, the difference between the two populations of cells may be due to a difference in a biochemical pathway. The difference between the two populations of cells is induced by environmental changes or therapeutic changes in one embodiment of the invention.

In another embodiment, the cells are taken from a tissue sample, a blood sample, a saliva sample, an embryonic sample o ^■ a tumor biopsy.

In another embodiment, the cells are taken from an embryonic sample, a tissue sample, an animal sample or a plant sample.

In one embodiment, the cells are derived or obtained from any animal species, any plant species or any bacterial species. For example, the cells may be obtained or derived from a human, a mouse, a rat, a dog, a fowl, a reptile, a horse, a bovine, a fish, a primate, a feline, a bacterial cell, a yeast, a fungi, a seed, or a plant.

In one embodiment of the invention, the vector is a phage vector, a phagemid vector, a retroviral vector or a plasmid vector. In one preferred embodiment, the vector is suicide vector which is characterized by only surviving and being able to be detected and propagated if the vector contains an insert. In another embodiment, the vector is a pSK vector, a pKS vector, a pPCRII vector, or a pGEM vector.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology and recombinant DNA technology which 0 are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook,

Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual,

Second Edition (1989); DNA Cloning, Vols . I and II (D. N.

Glover ed. 1985) ; Oligonucleotide Synthesis (M. J. Gait ed. 5 1984); Nucleic Acid Hybridization (B. D. Hames & S. J.

Higgins eds. 1984); Animal Cell Culture (R. K. Freshney ed.

1986); Immobilized Cells and Enzymes (IRL press, 1986);

Perbal, B., A Practical Guide to Molecular Cloning (1984); the series, Methods In Enzymology (S. Colowick and N. Kaplan C eds., Academic Press, Inc.); and Handbook of Experimental

Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell eds.,

1986, Blackwell Scientific Publications).

As used in this specification and the appended claims, the 5 singular forms "a, " "an" and "the" include plural references unless the content clearly dictates otherwise.

As used herein "nucleic acid molecule" includes both DNA and RNA and, unless otherwise specified, includes both double- C stranded and single-stranded nucleic acids. Also included are hybrids such as DNA-RNA hybrids. Reference to a nucleic acid sequence can also include modified bases as long as the modification does not significantly interfere either with binding of a ligand such as a protein by the nucleic acid or Watson-Crick base pairing.

A cell has been "transformed" by exogenous DNA when such exogenous DNA has been introduced inside the cell membrane. Exogenous DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In procaryotes and yeasts, for example, the exogenous DNA may be maintained on an episomal element, such as a plasmid. In eucaryotic cells, a stably transformed cell is generally one in which the exogenous DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication, or one which includes stably maintained extrachromosomal plasmids. This stability is demonstrated by the ability of the eucaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the exogenous DNA.

The transformation procedure used depends upon the host to be transformed. Mammalian cells can conveniently be transformed using, for example, DEAE-dextran based procedures, calcium phosphate precipitation (Graham, F. L. and Van der Eb, A. J. (1973) Virology 52:456-467), protoplast fusion, liposome-mediated transfer, polybrene- mediated transfection and direct microinjection of the DNA into nuclei. Bacterial cells will generally be transformed using calcium chloride, either alone or in combination with other divalent cations and DMSO (Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) ) . DNA can also be introduced into bacterial cells by electroporation. Methods of introducing exogenous DNA into yeast hosts typically include either the transformation of spheroplasts or transformation of intact yeast cells treated with alkali cations.

Site-specific DNA cleavage is performed by treating with the suitable restriction enzyme (or enzymes) under conditions which are generally understood in the art, and the particulars of which are specified by the manufacturer of these commercially available restriction enzymes (See, e.g. New England Biolabs Product Catalog) . In general, about 1 μg of plasmid or DNA sequences is cleaved by one unit of enzyme in about 20 μl of buffer solution. Typically, an excess of restriction enzyme is used to insure complete digestion of the DNA substrate. Incubation times of about one hour to two hours at about 37° C. are workable, although variations can be tolerated. After each incubation, protein is removed by extraction with phenol/chloroform, and may be followed by ether extraction, and the nucleic acid recovered from aqueous fractions by precipitation with ethanol. If desired, size separation of the cleaved fragments may be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A general description of size separations is found in Methods in Enzymology 65:499-560 (1980).

Restriction cleaved fragments may be blunt ended by treating with the large fragment of E. coli DNA polymerase I (Klenow) in the presence of the four deoxynucleotide triphosphates

(dNTPs) using incubation times of about 15 to 25 min at 20°C to 25oC in 50 mM Tris (pH 7.6) 50 mM NaCI , 6 mM MgCl₂, 6 mM

DTT and 5-10 μM dNTPε . The Klenow fragment fills in at 5 ' sticky ends but chews back protruding 3' single strands, even though the four dNTPs are present. If desired, selective repair can be performed by supplying only one of the dNTPs, or with selected dNTPs, within the limitations dictated by the nature of the sticky ends. After treatment with Klenow, the mixture is extracted with phenol/chloroform and ethanol precipitated. Treatment under appropriate conditions with Si nuclease or Bal-31 results in hydrolysis of any single-stranded portion.

Ligations are performed in 10-50 μl volumes under the following standard conditions and temperatures using T4 DNA ligase. Ligation protocols are standard (D. Goeddel (ed.) Gene Expression Technology: Methods in Enzymology (1991) ) .

In vector construction employing "vector fragments", the vector fragment is commonly treated with bacterial alkaline phosphatase (BAP) or calf intestinal alkaline phosphatase

(CIP) in order to remove the 5' phosphate and prevent religation of the vector. Alternatively, re-ligation can be prevented in vectors which have been double digested by additional restriction enzyme digestion of the unwanted fragments .

This invention is illustrated in the Experimental Details section which follows. These sections are set forth to aid in an understanding of the invention but are not intended to, and should not be construed to, limit in any way the invention as set forth in the claims which follow thereafter.

EXPERIMENTAL DETAILS

Example 1: RaSH, a rapid subtraction hybridization approach for identifying and cloning differentially expressed σenes

Human melanoma cells growth arrest irreversibly and terminally differentiate upon treatment with a combination of fibroblast interferon (IFN-β) and the protein kinase C activator mezerein (MEZ) . This experimental protocol also results in a loss of tumorigenic potential and profound changes in gene expression. Various cloning and cDNA microarray strategies are being used to determine the complete spectrum of gene expression changes underlying these alterations in human melanoma cells. An efficient and rapid subtraction hybridization approach, RaSH, has been developed that is permitting the identification of genes of potential relevance to cancer growth control and terminal cell differentiation. RaSH cDNA libraries are prepared from double-stranded cDNAs that are enzymatically digested into small fragments, ligated to adapters, PCR amplified followed by incubation of tester and driver PCR fragments. This subtraction hybridization scheme is technically simple and results in the identification of a high proportion of differentially expressed sequences, including known genes and those not described in current DNA databases. The RaSH approach represents an efficient methodology for identifying and cloning genes displaying differential expression that associate with and potentially regulate complex biological processes .

Orderly temporal changes in gene expression are major determinants of normal physiological processes and they represent primary mediators of altered cellular properties that define various disease states (1-3) . In these contexts, the ability to elucidate the molecular causes of normal and abnormal cellular changes requires the identification and clarification of function of the spectrum of differentially expressed genes. Attaining this goal continues to represent a major effort in both academic and industrial research laboratories. This task is of high priority and offers promise for discovering target genes regulating specific disease states. Once appropriate genes are recognized, methods such as combinatorial chemistry and high throughput screening can be used to identify and develop new molecules with broad ranges of therapeutic potential .

Several approaches are providing an initial description of potentially important and possibly relevant genes involved in or associated with normal processes and specific disease states, including aging, differentiation, development, cancer, cardiovascular disease and neurodegeneration (1-4) . Specific molecular approaches that have proven especially informative in identifying differentially expressed genes include differential RNA display (4,5), serial analysis of gene expression (6,7), subtraction hybridization (8-11), reciprocal subtraction differential RNA display (12) , representational difference analysis (13), RNA fingerprinting by arbitrarily primed PCR (14), electronic subtraction (15) and combinatorial matrix gene analysis (16) . Subtraction hybridization represents a particularly attractive method for isolating genes that are differentially expressed in diverse target cells, without prior knowledge of their functional or biochemical characteristics. A limitation of this scheme can often be attributed to technical difficulties, encountered in performing this procedure (11,17). The traditional subtraction hybridization method involves hybridization of first strand cDNAs generated from tester mRNAs with mRNAs obtained from drivers . Single stranded unhybridized cDNAs are then selected by hydroxylapatite column chromotography or biotin-avidin extraction and are used as templates for the second strand cDNA synthesis. These approaches can only analyze a fraction of the overall changes in gene expression, require large amounts of mRNA, and are lengthy and labor intensive procedures. To circumvent some of these problems, cDNA libraries in phage plasmid vectors have been used as both testers and drivers leading to successful construction of subtracted cDNA libraries by a number of investigators (8,18,19). However, constructing cDNA libraries and preparation of cDNA fragments for hybridization are laborious, some times difficult, processes. PCR-based cDNA subtraction considerably accelerates the procedures for cDNA library preparation and provides a new direction for subtraction, although it also involves several tedious steps during or after hybridization (13,20) .

A new protocol, Rapid Subtraction Hybridization (RaSH) , is presently described that significantly simplifies the process of cDNA subtraction in comparison with other methodologies. Additional advantages of the RaSH scheme include efficiency of subtraction and significant reductions in cost. Proof-of-principle for the RaSH approach is presently provided using a well-established human melanoma cell culture model of terminal differentiation (21,22). In human melanoma cells, treatment with the combination of IFN- β + MEZ results in irreversible growth arrest, loss of tumorigenic properties and terminal cell differentiation (21,22). These changes in melanoma physiology involve specific temporally regulated modifications in gene expression (22-24) . Potentially relevant genes associated with this process have been identified from a temporally spaced subtracted IFN-β + MEZ treated HO-1 human melanoma cDNA library using several molecular approaches including random screening of cDNAs, high throughput microchip cDNA microarrays and random cDNA clonal analysis and screening by reverse Northern hybridization (8,23,24). These approaches are providing a molecular snapshot of genes regulated during terminal differentiation and they have produced a useful database for determining the utility and efficacy of the RaSH approach. In the present study the RaSH scheme has identified a high proportion of genes (-45%) displaying differential expression as a function of treatment with IFN- β + .MEZ. These include genes previously identified as differentially expressed ESTs in the HO-1 melanoma system as well as additional previously unidentified known genes and genes without sequence homology to reported genes in DNA databases. In these contexts, the efficacy of the RaSH approach as a strategy for identifying differentially expressed genes, as now applied to the process of cellular differentiation, has been confirmed.

Materials and Methods

Cell Lines and Culture Conditions. The HO-1 human melanoma cell line was established from a metastatic inguinal node lesion from a 49 year-old female and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (100 U/100 ug/ml) (21) . Cultures were seeded at 1.5 X 10⁶ cells per 10- cm plate, and 24 hr later the medium was changed without inducers or with IFN-β (2000 U/ml) , MEZ (10 ng/ml) or with IFN-β plus MEZ (2000 U/ml + 10 ng/ml) . For library construction and subtraction, HO-1 cells were untreated or treated with IFN-β plus MEZ (2000 U/ml + 10 ng/ml) for 2, 4, 8, 16 and 24 hr.

RNA Isolation and Northern Blot Analysis. Total RNA was isolated by the guanidinium/phenol procedure from untreated or IFN-β, MEZ, or IFN-β plus MEZ treated cells. Northern blotting was performed as described previously (8-10) . Northern blots were quantitated by densitometric analysis using a Molecular Dynamics densitometer (Sunnyvale, CA) . Poly (A) RNA was purified using Oligo(dT) cellulose columns (Gibco BRL, Gaithersburg, MD) .

Primer Designs. Sequences of oligonucleotides were as follows: XEA-18 TGATCACTCGAGACCAGG (SEQ ID NO: ), XET-18

TGATCACTCGAGACCTGG (SEQ ID NO: ), XE-14 CTGATCAC TCGAG^'A

(SEQ ID NO: ), XEA-13 CCAGGTCTCGAG (SEQ ID NO: ), XET-13

CCTGGTCTCGAG (SEQ ID NO: ); XDPN-18 CTGA TCACTCGAGAGATC (SEQ ID NO: ), XDPN-14 CTGATCACTCGAGA (SEQ ID NO: ),

XDPN-12 GATCTCTCGAGT (SEQ ID NO: ) . The adapters formed from the two sets of oligonucleotides contain an Xho I recognition site.

Preparation of PCR-Based cDNA Libraries. One microgram of poly (A) RNA from control cells (driver) or IFN-β + MEZ treated cells (tester) was used for double-stranded cDNAs synthesis using standard protocols (8,17,25). The cDNAs were then digested with EcoR II (Sigma) or Dpn II (New England Biolab) at 37°C for 3 hr followed by phenol/chloroform extraction and ethanol precipitation. The digested cDNAs were mixed with primers XE-14/XEA-13/XET-13 (final concentration 20 mM) or XDPN-1 /XDPN-12 (final concentration 20 mM) in 30 ml of IX ligation buffer (Gibco BRL) , heated at 55°C for 1 min, and cooled down to 14°C within 1 hr. After adding 3 ml of T4 ligase (5 U/ml) to the mixtures individually, ligation was carried out at 14°C overnight. The mixtures were diluted to 100 ml with TE buffer (pH 7.0), and at least 40 ml of the mixtures were used for PCR amplification. The PCR mixtures were set up as following: 1 ml of the cDNA mixture, 10 ml 10X PCR buffer, 1 mM dNTPs, 10 mM XEA-18/XET-18 or XDPN-18, and 1 U Taq polymerase (Gibco BRL) . The parameters for PCR were one cycle for 5 min at 72°C followed by 25 cycles for 1 min at 94°C, 1 min at 55°C 1, 1 min at 72°C followed by one cycle for 3 min at 72°C. The PCR products were pooled and purified using Centricon columns (A icon, Bedford, MA) . Ten mg of the tester PCR products were digested with Xho I followed by phenol/ chloroform extraction and ethanol precipitation. Two sets of cDNA libraries were thus prepared: the libraries amplified with XEA-18/XET-18 and the libraries amplified with XDPN-18.

Subtraction Hybridization and Generation of Subtracted Libraries . One hundred ng of the tester cDNA was mixed with

3 mg of the driver cDNA in 10 ml of a hybridization solution (0.5 M NaCI, 50 mM Tris pH 7.5, 0.2% SDS, 40% formamide) , and after boiling for 5 min, incubated at 42°C for 48 hr. The hybridization mixture was phenol/chloroform extracted, ethanol precipitated, and dissolved in 20 ml of TE buffer. One ml of the mixture was ligated with 1 mg of Xho I- digested, CIP-treated pCRII plasmids, overnight at 14°C, and transformed into Shot-1 bacteria.

Colony Screening. Bacterial colonies were randomly picked and PCR amplified. The PCR products were blotted onto filters and reverse Northern blotting was performed to identify cDNAs displaying differential expression in control and differentiated cells (12,24) . The results were confirmed by Northern blotting. The sequences of these clones were determined using automated cycle sequencing at the DNA facility of Columbia University.

Results and Discussion

Melanoma Cell Culture Model System for Defining the Molecular Determinants of Growth Control and Differentia ion.

Abnormalities in differentiation are common properties in many diverse cancers (26,27) . Treatment of human melanoma cells with the combination of IFN-β + MEZ results in a rapid cessation of growth, an induction of melanogenesis, production of profound morphological changes including the development of dendrite-like processes, changes in cell surface antigens and alterations in gene expression (8,9,21- 24,26,28) . Kinetic studies designed to define the temporal relationship between inducer treatment and induction of terminal differentiation in HO-1 human melanoma cells demonstrate that the first 24 hr of inducer treatment is critical for irreversibly committing the majority of treated cells to terminal differentiation (21,22) .

In order to define the spectrum of gene expression changes occurring . during commitment to differentiation and maintenance of terminal differentiation in human melanoma cells^' a modified subtraction hybridization technique was employed (8) . Since differentiation is a temporal process, our approach to producing subtracted libraries involved the isolation of temporally spaced mRNAs (encompassing the first 24 hr of treatment) from IFN-β + MEZ and control untreated cells and subtracting control (driver) from differentiation inducer treated, cells (tester) (8) . Initial screening of this differentiation inducer treated HO-1 subtracted library included 70 random clones of which 23 clones (-33%) displayed differential expression as a consequence of treatment with the inducing agents and 7 of these differentially expressed clones initially represented novel sequences not reported in then current DNA databases (8,26). The originally novel melanoma differentiation associated (mda) genes included mda-2 which is homologous to the germ- cell-specific transcription repressor Tctex-1 (29,30), -m a-4 a member of the human interferon-inducible gene family associated with control of tumorigenicity in a model of human melanoma (31) , -mda-6 the universal cyclin dependent kinase inhibitor p21 (8,32,33), mda-1 a novel ubiquitous cancer growth suppressor • gene (34,35) , mda-9 a differentiation-associated and interferon gamma inducible gene (36,37) and mda-1 and mda-5 (8) still without representation in the DNA database and without a defined function. Employing the same subtracted library and screening of 1000 immobilized cDNAs on glass chips an additional 112 cDNAs (26 known and 11 novel) (-11%) displaying elevated expression as a function of differentiation in HO-1 cells have been identified (23) . Moreover, screening of an additional 400 random cDNA clones from the subtracted library by reverse Northern hybridization identified an additional 65 differentially expressed cDNAs (30 known and 26 novel) (-16%) , some of which had also been identified in the first library screen or by the high density cDNA microarray approach (24) . These analyses provide base-line data for specific gene expression changes occurring during the processes of induction and maintenance of differentiation in human melanoma cells .

RaSH Protocol and Its Application for Identifying Genes Differentially Expressed During HO-1 Differentiation. Subtraction hybridization provides a general methodology for directly selecting for unique cDNA species and removing common expressed sequences between cellular genomes (8-11) .

The application of the original approach was often not straightforward and success required a high degree of t technical competence (11) . The ability to use subtraction hybridization for identifying differentially expressed genes has been improved by the development of PCR-based subtraction hybridization approaches (13,20). However, this innovation is not trouble free, since this approach is laborious and often ineffective in identifying differentially expressed genes. To simplify the subtraction hybridization approach and make this methodology more amenable to diverse research laboratories we have developed a simple, efficient and affordable rapid subtraction hybridization approach, RaSH.

A schematic of the RaSH approach is shown in Fig. 1. For the present study, pooled RNAs (2, 4, 8, 16 and 24 hr) extracted from control and IFN-β + MEZ treated cells were used in the RaSH protocol to identify genes differentially expressed in the inducer treated cells . The cDNA libraries were constructed by synthesizing double-stranded cDNAs, digesting the cDNAs into small fragments, ligating the fragments to adapters, and amplifying with PCR. Purifying the PCR products by means of Centricon columns produces reproducible results. Experiments were performed using a 4-bp restriction enzyme Dpn II and the 4 1/2-bp restriction enzyme BcoR II to compare the effects of cDNA fragment size on redundancy of subtracted cDNA species. Compared to the average size of 256 bp generated by Dpn II, EcoR II digested cDNAs into fragments of 512 bp on average, which reduced the redundancy of large gene species while at the same time decreasing the representation of- small gene species in the RaSH cDNA libraries .

Subtraction hybridization during the RaSH approach is performed by incubating the tester and driver PCR fragments without the need for any additional PCR amplification steps. Instead of utilizing the PCR-based subtraction approach, which can result in amplification bias, RaSH uses a different strategy based on mass driven subtraction by altering the ratio of input tester to driver. In the RaSH scheme, subtracted cDNAs are selected simply by matching the ends of the cDNA fragments to the ends of the plasmid vectors during ligation, from which subtracted libraries are constructed. This simple step of subtraction makes RaSH different from any other cDNA subtraction protocol. The RaSH approach incorporates a reverse Northern analysis which offers the ability to rapidly pretest large numbers of cDNA clones for differential expression (12,24). The reverse Northern step permits the first tier identification and elimination of possible false positive clones. This procedure can theoretically also be used for subsequent rounds of RaSH clone identification using previously identified RaSH sequences to eliminate redundant clonal selection. Moreover, it is possible to scale up this approach and use high-throughput screening platforms containing RaSH-derived clones for monitoring temporal and distinct patterns of gene expression of multiple cDNAs within one assay. Once identified as a potential positive clone, confirmation of differential expression can be verified by Northern blotting analyses (12,24) .

To construct subtracted cDNA libraries, tester cDNAs were digested with Dpn II or EcόR II, ligated to Dpn II- or EcoR II -end adapters, amplified with specific primers, digested with Xho I (which recognizes the Xho I site in the primers) , and subtracted with driver cDNAs treated in an identical manner without enzymatic digestion (Fig. 1) . In this manner, two cDNA libraries (Dpn-sLib and Bco-sLib) were constructed. Colonies from the two libraries were randomly isolated and the PCR amplified products were used for reverse Northern analysis. This analysis resulted in the identification of -50% differentially expressed clones. The results of screening of 32 representative clones from each library are shown in Fig. 2 indicating differential expression in the majority of clones from both libraries when one compares control and differentiation-inducer treated HO-1 cells. Clones from both libraries were sequenced and compared with previously identified genes deposited in GenBank and EMBO databases. Both libraries resulted in the identification of known and unidentified gene sequences. Although the majority of clones contained single gene inserts, as with other cloning strategies some colonies were isolated that contained more than one insert (usually two) ligated in tandem.

The reverse Northern analysis results were confirmed with Northern blots using both known' and unidentified sequences from each library (Tables 1 and 2) . This resulted in a high degree of concordance (-89%) between positive signals identified using the reverse Northern screen and true differential expression as indicated by Northern blotting (Figs. 2 and 3) . It should be noted however, because equal amounts of mRNA at 2 , 4, 8, 16 and 24 hr after treatment of HO-1 cells with the differentiation inducers were pooled and used for poly (A) RNA selection and tester cDNA synthesis, the results of reverse Northern blots in the current experiments should be interpreted as a comparison between the expression level of control and the average level of the differentiation-induced cells at different time points. If a single time point had been used the proportion and types of differentially expressed genes identified would in all likelihood be different, unless they displayed elevated expression over a broad time period (22-24) . Defined temporal expression kinetics appear to be the rule rather than the exception during a 72 hr evaluation of the expression of a subset of microarrayed subtracted HO-1 cDNAs using temporally spaced RNAs isolated from HO-1 cells treated with IFN-β + MEZ (23) . This is also apparent after inspection of the various mda genes identified using RaSH as indicated by temporal kinetics of expression over a 72 -hr time course, 2, 4, 8, 24 and 72 h (Fig. 3) . For example, genes such as mda-D-57, mda-D-55, mda-E-64, mda-E-61 and mda-D-42 are inducible within 2 or 4 h exposure to IFN-β + MEZ, whereas other mda genes such as, mda-D-33, mda-D-47, mda-D- 66, mda-D-56, mda-D-27, mda-E-47 and mda-D-34 display low or no induction within this time frame (Fig. 3) . In fact, all of the mda genes analyzed exhibit defined temporal kinetics confirming similar results obtained using high throughput microarray analyses (23) .

The genes identified using RaSH in the melanoma differentiation model can be classified into four mda gene subtypes based on their pattern of induction as confirmed by Northern blotting (8) . These include, Type I mda genes (upregulated by IFN-β and IFN-β + MEZ) , Type II mda genes (upregulated by MEZ and IFN-β + MEZ) , type III mda genes (upregulated by IFN-β, MEZ and IFN-β + MEZ) and Type IV mda gene (upregulated primarily by IFN-β + MEZ) (8) (Fig. 3) . Excluding redundant gene identification, in the initial RaSH analyses (-10% of the subtracted libraries) using the enzymes Dpn II and BcoR II a total of 17 Type I, 14 Type II,

I Type III and 7 type IV mda genes were identified based on Northern blotting analyses (Tables 1 and 2) . Examples of the four types of mda genes initially identified by reverse Northern blotting and then classified into specific mda subtypes by Northern blotting is shown in Fig. 3. Using the enzyme Dpn II in the RaSH approach, a comparable number of Type I and II genes were identified, 10 and 11 respectively, whereas only 1 Type III and 3 Type IV genes were identified

(Table 1) . With the enzyme EcoR II' in the RaSH approach, 8

Type I genes and only 4 Type II genes were identified, whereas no Type III and 5 Type IV genes were cloned (Table

2) . As found in previous screenings of temporally spaced IFN-β + MEZ subtracted HO-1 cDNA libraries several categories of known genes were identified, including IFN- inducible genes (HLA, Cig-5, 1-8U, GBP I), MEZ-inducible genes (prolactin receptor-associated protein) , a differentiation factor (LIF) , genes involved in growth inhibition or apoptosis (mda-6, HuGADD34) and cytoskeleton and extracellular matrix genes (fibronectin, integrin a5)

(Tables 1 and 2) . In addition, 7 sequences without representation in current DNA databases were also identified

^• using RaSH. Of the 25 distinct genes identified from the Dpn-sLib and 17 distinct genes cloned from the EcoR-sLib, only one Type I (2' -5' oligoadenylate synthetase) , one Type

II (leukemia inhibitory factor, LIF) and one Type IV (fibronectin) gene were common to both libraries (Tables 1 and 2) .

It is well established that the induction of many interferon stimulated genes occur through activation of the JAK/STAT signaling pathway (38,39) and MEZ can induce activation of specific protein kinase C subtypes (40) . Based on the relative proportion of mda gene subtypes identified from subtracted libraries using RaSH and other gene identification strategies (23,24), it is probable that the ability of IFN-β + MEZ to induce terminal differentiation may involve the combined activation of both of these pathways . Further studies to address the role of each respective pathway, i.e., JAK/STAT signaling and PKC activation, in induction of growth arrest and terminal differentiation in human melanoma cells by IFN-β + MEZ should prove informative and these investigations are currently in progress .

As with previous subtraction hybridization approaches

(8,23,24) clonal redundancy was apparent in both RaSH libraries (Tables 1 and 2) . As examples, fibronectin (-8 kb) was isolated 5 times from the Dpn-sLib and 7 times from the EcoR-sLib and leukemia inhibitory factor (LIF) (-7.6 kb) was isolated 2 times from the Dpn-sLib and 3 times from the EcoR-sLib. In contrast, redundancy in the isolation of smaller cDNAs, including a₅ integrin (2 isolates) (4.1 kb) and the interferon responsive gene 1-8U (3 isolates) (0.8 kb) occurred only in the Dpn-sLib and these sequences were not detected in the EcoR-sLib. Size may not be the only factor resulting in differential abundance of clones identified in libraries prepared with the two different enzymes, since interleukin-11 was isolated 3 times from the Dpn-sLib, -6.9 kb in size, and was not present in the 10% of EcoR-sLib evaluated clones. It is apparent, however, from this limited analysis of the two RaSH-derived libraries that using EcoR II to digest cDNAs may result in reduction of sequence redundancy compared with Dpn II. However, the use of Dpn II in the RaSH approach may facilitate the isolation of smaller sized cDNAs than using the enzyme EcoR II. The RaSH approach successfully identified genes previously recognized as those differentially expressed during induction of terminal differentiation in human melanoma cells (8,22-24). This includes genes identified: during the initial random screening of the subtracted cDNA library (mda-6 (p21) and vimentin) ; using high density microchip cDNA arrays (fibronectin, HLA-B, Mn superoxide dismutase and 2 '5' oligoadenylate synthetase) ; and random clonal isolation and screening by reverse Northern hybridization (HLA-B) .

Moreover, previous studies have demonstrated changes in the expression of intercellular adhesion molecule 1 (ICAM-1)

(28) and a_s integrin (22) during induction of differentiation in HO-1 cells and both of these gene changes were identified using the RaSH approach. In addition, RaSH has identified novel sequences not detected using the previous protocols (Tables 1 and 2) . In contrast, genes such as the transcription factor ISGF-3 and mitochondrial-associated genes that were identified 11 and 52 times, respectively, using microarrays of subtracted cDNA clones (23) were not identified using the RaSH approach. The reason for the differences in the types of genes identified using the dissimilar methodologies is not known. One possibility is that all of the approaches, including RaSH, have not evaluated the entire spectrum of cDNAs present in the subtracted libraries that have been generated. In the case of the high-density cDNA microarray analysis only 1000 of the -10,000 clones in the subtracted cDNA library were evaluated (23) . Similarly, using the RaSH approach only 10% of either the Dpn-sLib or the EcoR-sLib have been evaluated. Alternatively, it is possible that specific cloning and identification biases are present in the different approaches, thereby resulting in only a subset of genes being identified with any specific subtraction approach. This may be true with respect to the microchip array approach, since only random cDNAs of >. 500 bp could be formatted for evaluation on chips (23) , whereas smaller subtracted cDNAs were employed for the reverse Northern approach (24) .

Efficiency of subtraction in RaSH was determined by comparing the percentage of specific gene fragments present in the PCR-amplified cDNA library versus those in the subtracted library. The fibronectin and LIF fragments were used for this purpose because of their large cDNA size, -8 and -7.6 kb, respectively, which resulted in redundant copies of these genes in both subtracted libraries (Dpn-sLib and EcoR-sLib) (Tables 1 and 2) . The relative amounts of each of these cDNA fragments were estimated by Southern blotting of the PCR cDNA libraries and in comparison with different quantities of the cDNA fragments (Fig. 4) . Enrichment was then calculated as the percentage of the fragment in the PCR libraries divided by the percentage of the clones in the subtracted library. Enrichment of LIF was 544-fold in the Dpn-sLib and 96-fold in the EcoR-sLib; while enrichment of fibronectin was 75-fold in the Dpn-sLib and 6- fold in the EcoR-sLib. The representation of cDNA species was reduced in the EcoR II PCR amplified library because larger cDNA fragments were produced by EcoR II digestion. The presence of fewer cDNA species in this library might explain the "lower efficiency" in relative copy number. The present data also indicates that PCR amplification in the RaSH procedure does not significantly alter the proportion of expressed genes. Based on this consideration it is possible that RaSH could be performed without a PCR amplification step and this possibility is currently being tested. However, it is also probable that low abundance messages may be lost if PCR amplification is not used in the RaSH approach.

The differences between RaSH and other PCR-based subtraction protocols are readily apparent. Except for the use of PCR for cDNA amplification, the experimental approaches are distinct since RaSH includes different primer designs, subtraction approaches, and subtracted sequence selection. Although both cDNA populations from control and differentiated HO-1 cells are amplified using the same primers, only the tester cDNA fragments are digested with Xho I which recognizes the internal Xho I site in the primer. Therefore, the cDNA fragments from the differentiated cells have clonable Xho I sticky-ends, which distinguishes them from those of driver cDNAs . Only one round of hybridization is necessary in the RaSH protocol, since the subtraction stringency can be adjusted by simply changing the ratio of tester and driver during subtraction. Unlike other PCR-based protocols, no PCR amplification is employed in RaSH during subtraction. The absence of these additional steps also significantly simplifies the subtraction procedures. The use of EcoR II (4.5 bp cutter) as opposed to Dpn II in producing RaSH subtracted libraries can be used to reduce redundant gene identification. However, the application of either restriction enzyme in the RaSH approach results in the efficient identification of both similar and distinct genes. It should also be emphasized that the efficiency of the RaSH approach in identifying differentially expressed genes is superior to previous approaches, i.e., 45% versus -20% for the combination of random cDNA clone isolation, high throughput screening of microarrayed subtracted cDNAs and random cDNA isolation and analysis by reverse Northern blotting (8,23,24) .

In conclusion, we have developed a rapid and simple cDNA subtraction protocol, RaSH, and presently applied this procedure to isolate melanoma differentiation associated

(mda) genes. The novelty of this method is reflected in the manner in which cDNA fragments are prepared, the steps used in subtraction and the method of selecting subtracted sequences . The mda genes isolated using RaSH represent both known and previously unidentified sequences of potential relevance to growth control and differentiation. Based on the ease of performance and the efficiency of differential gene isolation embodied in the RaSH approach, this methodology should find wide application for facilitating the identification of relevant genes associated with and potentially causative of important biological phenomena.

Table 1.

Clones isolated using RaSH with the restriction enzyme Dpn II Nomenclature¹ Identity² Mda Type³

mda-O-25 HLA cw6 Type I mda-D-26 novel, human homolog of Mus Type I musculus small GTP-binding protein mda-O-21, D-49, fibronectin Type IV D-53, D-58, D-69 mda-D-28 vascular endothelial growth factor Type IV

(VEGF) (165) mda-D-33 Cig5 (interferon-responsive RNA) Type I mda-D-34, D-36 interieukin- 11 (IL-11) Type IV D-46 mda-D-35 prolactin receptor-associated protein (PRA) Type II mda-O-39 interleukin-1 (IL-1) Type II mda-D-40 plasminogen activator inhibitor type II Type II mda-D-41 G-binding protein I Type I mda-D-42, D-54 leukemia inhibitory factor (LIF) Type II mda-O-45 KIAA0439 from human brain Type II mda-D-48, D-52 α₅ integrin Type II mda-O-50 mac-2 binding protein 90 Kd product Type I mda-O-51, D-61 urokinase-type plasminogen Type II activator receptor mda- D-70 monocyte activation antigen Type I mda-D-55 2 ' -5 ' oligoadenylate synthetase Type I

7nda-D-56 Mn superoxide dismutase Type HI mda-O-51, D-59,' 1-8U from the interferon-inducible Type I D-62 gene family mda-O-65 ICAM-1 (intercellular adhesion molecule l)Type I mda-D-68 snRNP protein B Type I mda-O-32, D-47, novel Type II

D-63, D-66

Clones are designated as melanoma differentiation associated (mda) and the D designation refers to the fact that the restriction enzyme Dpn II was used for isolation by the RaSH approach. Sequences were searched against various DNA databases to determine sequence identities. Novel, no similar sequence reported in current DNA databases.

³Type I mda gene: inducbile by IFN-β and IFN-β + MEZ; Type II mda gene: inducible by MEZ and IFN-β + MEZ; Type III mda gene: inducible by IFN-β, MEZ and IFN-β + MEZ; Type IV mda gene: inducible predominantly by IFN-β + MEZ.

Table 2. Clones isolated using RaSH with the restriction enzyme EcoR II

Nomenclature¹ Identity² Mda Type³ mda-E-28, E-34, E-38, fibronectin Type IV E-41, E-45, E-49, E-50 mda-E-31 2 ' -5 ' oligoadenylate synthetase Type I mda-E-32, E-57, E-58 leukemia inhibory factor (LIF) Type II mda-E-35 transporter 1 , ABC Type I mda-E-36, E-42, E-52 HLA-B Type I mda-E-39 KIAA0180 Type II mda-E-40 vimentin Type I mda-E-44 TNF-α Type II mda-E-46, E-55 calcium-activated potassium channelType IV mda-E-41 ninjurin 1 Type IN mda-E-51 HMG-Y protein isoform Type I mda-E-54 proteasome 26S subunit Type IN mda-E-61 human GADD34 Type II mda-E-68 mda-6 (p21, afl, CIP1, SDH) Type IV mda- E-43, E-63, E-64 novel Type I

Clones are designated as melanoma differentiation associated (mda) and the D designation refers to the fact that the restriction enzyme EcoR II was used for isolation by the RaSH approach.

Sequences were searched against various DNA databases to determine sequence identities. Novel, no similar sequence reported in current DNA databases.

³Type I mda gene: inducbile by IFN-β and IFN-β + MEZ; Type II mda gene: inducible by MEZ and IFN-β + MEZ; Type III mda gene: inducible by IFN-β, MEZ and IFN-β + MEZ; Type IV mda gene: inducible predominantly by IFN-β + MEZ .

Nucleotide sequence (SEQ ID NO: ) and corresponding amino acid sequence (SEQ ID NO: ) of HuUBP43.

AAAGCGAGGGCTGGAAGTCTGGAGCAGGTGCGCGGCTGCAACGGCAGCCGCGGGAAGCTC 60 GGGCCGGCAGGGTTTCCCCGCACGCTGGCGCCCAGCTCCCGGCGCGGAGGCCGCTGTAAG 120 TTTCGCTTTCCATTCAGTGGAAAACGAAAGCTGGGCGGGGTGCCACGAGCGCGGGGCCAG 180 ACCAAGGCGGGCCCGGAGCGGAACTTCGGTCCCAGCTCGGTCCCCGGCTCAGTCCCGACG 240 TGGAACTCAGCAGCGGAGGCTGGACGCTTGCATGGCGCTTGAGAGATTCCATCGTGCCTG 300 GCTCACATAAGCGCTTCCTGGAAGTGAAGTCGTGCTGTCCTGAACGCGGGCCAGGCAGCT 360 GCGGCCTGGGGGTTTTGGAGTGATCACGAATGAGCAAGGCGTTTGGGCTCCTGAGGCAAA 420

M S K A F G L R Q TCTGTCAGTCCATCCTGGCTGAGTCCTCGCAGTCCCCGGCAGATCTTGAAGAAAAGAAGG 480

I C Q S I L A E S s Q S P A D E Ξ K K

AAGAAGACAGCAACATGAAGAGAGAGCAGCCCAGAGAGCGTCCCAGGGCCTGGGACTACC 5 0 E E D S N M K R E Q P R E R P R A W D Y CTCATGGCCTTGTTGGTTTACACAACATTGGACAGACCTGCTGCCTTAACTCCTTGATTC 600 P H G L V G L H N I G Q T C C L N S L I AGGTGTTCGTAATGAATGTGGACTTCACCAGGATATTGAAGAGGATCACGGTGCCCAGGG 660 Q V F V M N V D F T R I L K R I T V P R GAGCTGACGAGCAGAGGAGAAGCGTCCCTTTCCAGATGCTTCTGCTGCTGGAGAAGATGC 720 G A D E Q R R S V P F Q M L L L L E K M AGGACAGCCGGCAGAAAGCAGTGCGGCCCCTGGAGCTGGCCTACTGCCTGCAGAAGTGCA 780 Q D S R Q K A V R P E L A Y C Q C ACGTGCCCTTTTTTGTCCAACATGATGCTGCCCAACTGTACCTCAAACTCTGGAACCTGA 840 N V P F F V Q H D A A Q L Y L K N TTAAGGACCAGATCACTGATGTGCACTTGGTGGAGAGACTGCAGGCCCTGTATATGATCC 900 I K D Q I T D V H V E R Q A L Y M I GGGTGAAGGACTCCTTGATTTGCGTTGACTGTGCCATGGAGAGTAGCAGAAACAGCAGCA 960 R V K D S L I C V D C A M E S S R N S S TGCTCACCCTCCCACTTTCTCTTTTTGATGTGGACTCAAAGCCCCTGAAGACACTGGAGG 1020 M L T P S L F D V D S K P L K T L E ACGCCCTGCACTGCTTCTTCCAGCCCAGGGAGTTATCAAGCAAAAGCAAGTGCTTCTGTG 1080 D A L H C F F Q P R E L S S K S K C F C AGAACTGTGGGAAGAAGACCCGTGGGAAACAGGTCTTGAAGCTGACCCATTTGCCCCAGA 1140 E N C G K K T R G K Q V L K L T H L P Q CCCTGACAATCCACCTCATGCGATTCTCCATCAGGAATTCACAGACGAGAAAGATCTGCC 1200 T T I H M R F S I R N S Q T R K I C ACTCCCTGTACTTCCCCCAGAGCTTGGATTTCAGCCAGATCCTTCCAATGAAGCGAGAGT 1260 H S L Y F P Q S D F S Q I L P M K R E CTTGTGATGCTGAGGAGCAGTCTGGAGGGCAGTATGAGCTTTTTGCTGTGATTGCGCACG 1320 S C D A E E Q S G G Q Y Ξ L- F A V I A H TGGGAATGGCAGACTCCGGTCATTACTGTGTCTACATCCGGAATGCTGTGGATGGAAAAT 1380 V G M A D S G H Y C V Y I R N A V D G K GGTTCTGCTTCAATGACTCCAATATTTGCTTGGTGTCCTGGGAAGACATCCAGTGTACCT 14 0 W F C F N D S N I C V S W E D I Q C T ACGGAAATCCTAACTACCACTGGCAGGAAACTGCATATCTTCTGGTTTACATGAAGATGG 1500 Y G N P N Y H W Q E T A Y V Y M M AGTGCTAATGGAAATGCCCAAAACCTTCAGAGATTGACACGCTGTCATTTTCCATTTCCG 1560 E C *

TTCCTGGATCTACGGAGTCTTCTAAGAGATTTTGCAATGAGGAGAAGCATTGTTTTCAAA 1620 CTATATAACTGAGCCTT IT ATAATTAGGGATATTATCAAAATATGTAACCATGAGGCC 1680 CCTCAGGTCCTGATCAGTCAGAATGGATGCTTTCACCAGCAGACCCGGCCATGTGGCTGC 1740 TCGGTCCTGGGTGCTCGCTGCTGTGCAAGACATTAGCCCTTTAGTTATGAGCCTGTGGGA 1800 ACTTCAGGGGTTCCCAGTGGGGAGAGCAGTGGCAGTGGGAGGCATCTGGGGGCCAAAGGT 1860 CAGTGGCAGGGGGTATTTCAGTATTATACAACTGCTGTGACCAGACTTGTATACTGGCTG 1920 AATATCAGTGCTGTTTGTAATTTTTCACTTTGAGAACCAACATTAATTCCATATGAATCA 1980 AGTGTTTTGTAACTGCTATTC _T_rATTCAGCAAATAT_I_TATTGATCATCTCTTCTCCAT 2040 AAGATAGTGTGATAAACACAGTCATGAATAAAGTTATTTTCCAC 2084

Example 2 ; Cloning and characterization of a human ubiquitin-processing protease, HuUBP43 . from terminally differentiated human melanoma cells using a rapid subtraction hybridization protocol RaSH

Defects in growth control and differentiation occur frequently in human cancers. In the case of human melanoma cells, treatment with a combination of fibroblast interferon (IFN-β) and the protein kinase C activator mezerein (MEZ) results in an irreversible loss of proliferative^ potential and tumorigenic properties with a concomitant induction of terminal differentiation. These changes in cellular properties are associated with an induction and suppression in specific subsets of genes that occur in a temporal manner. To identify the complete repertoire of gene changes occurring during melanoma reversion to a more differentiated state a number of molecular approaches are being used. These include,^' subtraction hybridization using temporally spaced cDNA libraries, random cDNA isolation and evaluation by reverse Northern blotting and high throughput microarray analysis of subtracted cDNA clones. In the present study we have used a novel approach, rapid subtraction hybridization (RaSH) , to identify and clone an additional gene of potential relevance to cancer growth control and terminal cell differentiation. RaSH has identified a human ubiquitin-processing protease gene, HuUBP43 , that is differentially expressed in melanoma cells as a function of treatment with IFN-β or IFN-β + MEZ. HuUBP43 is a type I interferon inducible gene that is upregulated in a diverse panel of normal and tumor cells when treated with IFN-β via the JAK/STAT kinase pathway. This gene may contribute to the phenotypic changes induced by IFN-β during growth arrest and differentiation in human melanoma cells and other cell types as well as the antiviral and growth inhibitory effects of interferon.

Introduction

A potentially less toxic form of cancer therapy involves the- use of agents that alter the program of differentiation in cancer cells, 'differentiation therapy' , resulting in a loss of proliferative and tumorigenic potential and acquisition of a more differentiated phenotype (Sachs, 1978; Jimenez and Yunis, 1987; Fisher et al . , 1985; Waxman et al . , 1991; Jiang et al . , 1994a; Waxman, 1995). The underlying principle of differentiation therapy is that cancer cells display reversible defects in differentiation, which may result from a reduced level of or an extinction in expression of specific genes negatively regulating the cancerous state

(Sachs, 1978; Jiang and Fisher, 1993; Jiang et al . , 1993, 1994a; Waxman, 1995). With human melanoma, it is possible to reprogram these tumor cells to a more normal state by treatment with IFN-β and the protein kinase C activating agent MEZ (Fisher et al . , 1985; Jiang et al . , 1993). In this context, human melanoma is providing an important model for systematically identifying consequential genes involved in cancer cell growth and differentiation (Jiang and Fisher, 1993; Jiang et al . , 1994b, 1995a, b, 1996; Su et al . , 1998; Huang et al . , 1999a, b; Madireddi et al . , 2000a, b) . Previously identified genes of relevance to cancer cell physiology cloned using this model include, melanoma differentiation associated gene-2, mda-2, which exhibits homology to the germ-cell- specific transcription repressor Tctex-1 (Roux et ^■ al., 1994; O'Neill and Artz, 1995), mda-4 a component of the human interferon-inducible gene family that is implicated in the control of tumorigenicity in a model of human melanoma (DeYoung et al . , 1997), mda-6 which is the universal cyclin dependent kinase inhibitor p21 (Jiang and Fisher, 1993; Xiong et al . , 1993; Jiang et al . , 1995b), mda- 7 a ubiquitous cancer growth suppressing gene (Jiang et al., 1995a, 1996; Su et al . , 1998; Madireddi et al . , 2000b) and mda- 9 a gamma interferon inducible differentiation-associated gene (Lin et al . , 1996, 1998) . In addition, recent studies using the differentiation induction subtraction hybridization (DISH) approach combined with high throughput microchip cDNA array screening (1000 cDNAs) (Huang et al . , 1999a) and random cDNA clone isolation and reverse Northern blotting (400 cDNAs) (Huang et al . , 1999b) , have identified 56 known and 37 novel cDNAs that display differential expression as a function of induction of terminal differentiation in HO-1 human melanoma cells.

A number of approaches are currently available for identifying and cloning differentially expressed genes. These include, subtraction hybridization

(Jiang and Fisher, 1993; Jiang et al . , 1995a, b; Su et al., 1997; Sagerstrom et al . , 1997; Wan and Erlanger, 1997) , differential RNA display (Liang and Pardee, 1992; Shen et al . , 1995; Wan and Erlanger, 1997) , reciprocal subtraction differential RNA display (Kang et al . , 1998), RNA fingerprinting by arbitrarily primed PCR (Ralph et al . , 1993; McClelland et al . , 1995), representational difference analysis (Hubank and Schatz, 1994, 1999), electronic subtraction (Adams et al . , 1993; Wan et al . , 1996), serial analysis of gene expression (Velculescu et al . , 1995; Zhang et al . , 1997) and combinatorial gene matrix analysis (Schena et al . , 1995) . Subtraction hybridization has proven to be a particularly robust methodology for identifying and cloning genes displaying altered expression as a function of changes in cellular physiology without prior insight into the functionality of the differentially expressed genes (Jiang and Fisher, 1993; Jiang et al . , 1995a, b; Su et al . , 1997; Sagerstrom et al . , 1997; Wan and Erlanger, 1997). Significant drawbacks to this approach include the laborious and tedious nature of conventional subtraction approaches, technical difficulties in producing high quality randomized cDNA libraries for subtraction and the difficulty in insuring adequate removal of common sequences and enrichment in differentially expressed sequences. Commercial approaches, often PCR-based, are available and these have resulted in variable success in identifying relevant differentially expressed genes.

A new procedure, Rapid Subtraction Hybridization (RaSH) (Jiang et al . , 2000), has been developed that substantially simplifies the cDNA subtraction approach when compared with other technologies . In the present study we have used the RaSH methodology in combination with the human melanoma terminal differentiation model (Fisher et al . , 1985; Jiang et al., 1993) to identify a differentially expressed gene associated with loss of proliferative control and terminal differentiation in human melanoma cells. RaSH identified a gene not previously detected using other subtraction hybridization or high throughput cDNA microarray analysis approaches

(Jiang and Fisher, 1993; Huang et al . , 1999a, b) that encodes a human ubiquitin-processing protease,

HuUBP43. Expression of this gene is rapidly upregulated, within 2 hr, in human melanoma cells after treatment with IFN-β or IFN-β + MEZ. HuUBP43 is also induced in additional melanomas and other cell types predominantly by IFN-β with lower induction by leukocyte interferon (IFN-α) and poly I:C and barely detectable induction by gamma interferon (IFN-g) , indicating that this ubiquitin- processing protease is a Type I interferon- responsive gene. Analysis of mutant human cell lines defective in the JAK/STAT signaling pathway (Darnell et al . , 1994; Stark et al . , 1998) indicate that HuUBP43 requires these archetypal interferon signal molecules for activation. Based on the important and well-documented biochemical effects of this enzyme on the fate of cellular proteins (Hershko and Ciechanover, 1992; Hershko, 1996; D' ndrea and Pell an, 1998; Laney and Hochstrasser, 1999), HuUBP43 may profoundly influence human cancer cell growth and differentiation, as well as interferon- mediated processes. Materials and methods

2.1. Cell lines and culture conditions

The HO-1 human melanoma cell line was derived from a metastatic inguinal node lesion from a 49-yr old woman and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (100 U/100 mg/ml) in a 5% C02 95% air-humidified incubator

(Fisher et al . , 1985). Additional human cell types included, melanoma (MeWo, LO-1, C8161, SK-MEL wtp53 ,

SK-MEL mtp53, WM35, WM239, WM278, SH-1, FO-1) ,

^• normal SV40-T-antigen immortalized melanocyte (FM516-SV) , normal mammary epithelial (HMEC and HBL-

100) , human normal skin fibroblast (HSF) , breast ^• carcinoma (MCF-7, T47D) , colorectal carcinoma

(SW613), cervical carcinoma (HeLa) , glioblastoma multiforme (T98G, GBM-18) , prostate carcinoma (DU- 145) , endometrial carcinoma (HONE-l) , normal cerebellum (NC) and osteosarcoma (Saos-2) (Jiang et al . , 1995b, 1996; Su et al . , 1998; Madireddi et al . , 2000b) . All cancer cells and normal cells, except HMEC, were grown in DMEM-10. HMEC was grown in medium provided by Clonetics (Su et al . , 1998) . 2fTGH, U1A, U3A, U4A, and U5A cells were grown in DMEM-10 supplemented with 250 mg/ml hygromycin (McKendry et al . , 1991).

2.2 RNA isolation and Northern blot analysis. Total RNA was isolated by the guanidinium/phenol procedure and analyzed by Northern blotting as described previously (Jiang et al . , 1994b). Northern blots were quantitated by densitometric analysis using a Molecular Dynamics densitometer (Sunnyvale, CA) . Poly (A) RNA was purified using Oligo(dT) cellulose columns (Gibco BRL, Gaithersburg, MD) .

2.3. RaSH procedure

Primer Design. Sequences of oligonucleotides were as follows: XDPN-18 CTGA TCACTCGAGAGATC, XDPN-14 CTGATCACTCGAGA, XDPN-12 GATCTCTCGAGT . The adapters formed from the two sets of oligonucleotides contained an Xho I recognition site.

Preparation of PCR-based cDNA libraries. One microgram of poly (A) RNA from control cells (driver) or IFN-β + MEZ treated cells (tester) was used for double-stranded ^: cDNA synthesis using standard protocols (Gubler and Hoffman, 1983) . The cDNAs were then digested with Dpn II (New England Biolab) at 37oC for 3 hr followed by phenol/chloroform extraction and ethanol precipitation. The digested cDNAs were mixed with primers XDPN-14/XDPN-12 (final concentration 20 mM) in 30 ml of IX ligation buffer (Gibco BRL) , heated at 55oC for 1 min, and cooled down to 14oC within 1 hr. After adding 3 ml of T4 ligase (5 U/ml) to the mixtures individually, ligation was carried out at 14oC overnight. The mixtures were diluted to 100 ml with TE buffer (pH 7.0), and at least 40 ml of the mixtures were used for PCR amplification. The PCR mixtures were set up as following: 1 ml of the cDNA mixture, 10 ml 10X PCR buffer, 1 mM dNTPs, 10 mM XDPN-18, and 1 U Taq polymerase (Gibco BRL) . The parameters for PCR were one cycle for 5 min at 72oC followed by 25 cycles for 1 min at 94oC, 1 min at 55oC 1, 1 min at 72oC followed by one cycle for 3 min at 72oC. The PCR products were pooled and purified using Centricon columns (Amicon, Bedford, MA) . Ten mg of the tester PCR products were digested with Xho I followed by phenol/ chloroform extraction and ethanol precipitation .

Subtraction hybridization and generation of subtracted libraries. One hundred ng of the tester cDNA was mixed with 3 mg of the driver cDNA in 10 ml of a hybridization solution (0.5 M NaCI, 50 mM Tris pH 7.5, 0.2% SDS, 40% formamide) , and after boiling for 5 min, incubated at 420C for 48 hr. The hybridization mixture was phenol/chloroform extracted, ethanol precipitated, and dissolved in 20 ml of TE buffer. One ml of the mixture was ligated with 1 mg of Xho I-digested, CIP-treated pCRII plasmids, overnight at 14oC, and transformed into Shot-1 bacteria.

Colony screening. Bacterial colonies were randomly picked and PCR amplified. The PCR products were blotted onto filters and reverse Northern blotting was performed to identify cDNAs displaying differential expression in control and differentiated cells (Huang, et al . , 1999b). The results were confirmed by Northern blotting. The sequences of these clones were determined using automated cycle sequencing at the DNA facility of Columbia University.

2.4'. Cloning of the HuUBP43 full-length cDNA

The full-length cDNA for HuUSP43 was obtained by the SMART cDNA RACE procedure (Clontech, CA) . DNA and protein sequence analyses were performed using the GCG sequence analysis software package.

Results and discussion

3.1. Cloning of HuUBP43 as an IFN-β and IFN-β + MEZ inducible gene in human melanoma cells

A potentially significant gene, HuUBP43, was identified in IFN-β + MEZ treated HO-1 cells using the RaSH approach and a Dpn II subtracted library (Dpn-sLib) . RACE cloning produced a full-length mda- D-15 (HuUBP43) clone (Fig. 5) . The full-length HuUBP43 consists of 2084 nt, not including the poly (A) track, with an open reading frame starting at position 390 and terminating at position 1508. The open reading frame is flanked by 5'- and 3'- untranslated regions of 389 and 626 nt, respectively. The 5'-UTR contains 2 in-frame stop codons at position 119 and 283. An additional ATG is present at position 272, which codes for 12 amino acids out of frame. In the 3'-UTR, there are three consensus ATTTA elements at position 1638, 2002, and 2015, which are involved in mRNA stability, and a canonical polyadenylation signal AATAAA upstream of the poly (A) track. The derived polypeptide displays a high degree of homology to the murine ubiquitin specific protease (UBP43) , with 70% amino acid identity (Liu et al . , 1999). Almost complete identity is apparent in six conserved domains: Cys box (15/16) , QQDAQEF motif (7/7) , LPQILVIHLKRF consensus sequence (12/12), and His boxes (17/18, 7/9, 3/4) . In two recent reports, HuUBP43 (mda-D-15) has been identified as the human homologue of murine UBP43 (Schwer et al . , 2000) and by differential RNA display as the interferon inducible gene ISG43 (Li et al . , 2000) . The sequences reported in the Schwer et al. (2000) and Li et al . (2000) papers differ from those reported in this paper in two significant ways . Both of the previously reported sequences are shorter than HuUBP43 (225-bp in UBP43 and 380-bp in ISG43) . Secondly, the first 39-bp of UBP43 and 11-bp of ISG43 do not match the reported genomic sequence or the HuUBP43 sequence suggesting potential cloning artifacts in the two previous studies.

HuUBP43 expression is induced in both IFN-β only and IFN-β + MEZ treated HO-1 cells (Fig. 6) . In contrast, no induction is observed with MEZ and the combination of IFN-β + MEZ does not result in greater induction than observed with only IFN-β, suggesting that HuUBP43 is responding directly to interferon. In HO-1 cells, IFN-β induces growth suppression and the induction of melanin synthesis, cellular and biochemical changes that are completely reversible when this inducing agent is removed

(Jiang et al . , 1993) . To determine if other differentiation and growth modulating agents effect HuUBP43 expression in HO-1 cells, cultures were treated with DMSO, mycophenolic acid (MPA) , 12-0- tetradecanoyl-phorbol-13 acetate JTPA) , retinoic acid (RA) , cAMP and 8-Br-cAMP (Fig. 6) . None of these treatment protocols resulted in significant induction of this human ubiquitin specific protease. Since at the doses tested MPA, TPA and RA can reversibly induce specific components of the differentiation and growth arrest program, but not terminal differentiation as observed with IFN-β + MEZ, these findings provide support for a link between IFN-β treatment and HuUBP43 induction in HO- 1 cells rather than an effect resulting from changes in the state of growth or differentiation in HO-1 cells. Moreover, other agents which can exert physiological changes in specific cancer cells, including melanoma, such as interleukin-6 (IL-6) , epidermal growth factor (EGF) , transforming growth factor-α (TGF-α) , transforming growth factor-β (TGF- β) , tumor necrosis factor-α (TNF-α) and platelet derived growth factor (PDGF) , did not induce HuUBP43 expression (Fig. 7) . Similarly, growth in medium devoid of serum (DMEM-0), which suppresses growth in HO-1 cells, or treatment with the DNA damaging agent methyl methanesulfonate (MMS) , do not affect HuUBP43 expression in HO-1 cells (Fig. 6) . Taken together, these data provide support for the conclusion that a primary inducer of HuUBP43 in HO-1 cells is IFN-β.

HuUBP43 is a type I interferon inducible gene HuUBP43 is induced to a similar level in HO-1 cells treated with recombinant IFN-β, natural human fibroblast interferon (Hb) or IFN-w (Fig. 8) . HuUBP43 induction is also apparent in HO-1 cells treated with peripheral blood leukocyte (PBL) derived interferon, various IFN-α subtypes, including IFN-αA, -αB, -αC, -αF, -αG, -αH, -αl and - αJ, albeit to lesser extents than with IFN-β (Fig. 7) . Of the IFN-α subtypes, induction is greatest using INF-αB, IFN-αC and IFN-αl . In contrast, no induction of HuUBP43 occurs in HO-1 cells treated with IFN-t (Fig. 8) . In addition, HuUBP43 is marginally induced by treatment of HO-1 cells with recombinant immune interferon (IFN-γ) (Fig. 6) . These data indicate that INF-β is the most active inducer of HuUBP43 with IFN-α inducing significant but lower induction (~4-fold less with IFN-αA than with IFN-β) and IFN-γ inducing only a minor induction of HuUBP43 (~10-fold less than IFN-β) . In contrast, Li et al . (2000) demonstrate that ISG43 (HuUBP43) is induced maximally by IFN-β, in 2fTGH cells, whereas IFN-γ (200 units/ml) is a better inducer of this gene than IFN-α (500 units/ml) . The difference between our study and that of Li et al . (2000) may result from variations in the response of HO-1 versus 2fTGH cells to the three types of interferon. The present data provides direct evidence that HuUBP43 is a type I, IFNα/β, inducible gene in HO-1 cells.

Temporal expression analysis indicates that HuUBP43 is an IFN-β early response gene in HO-1 cells, as shown for ISG43 (Li et al . , 2000) and the murine DUB-1 and DUB-2 deubiquitinating genes (Zhu et al . , 1996, 1997), since induction was observed as early as 2 hr after IFN-β treatment (Fig. 9) . Induction of HuUBP43 is apparent when HO-1 cells are treated with as little as 0.1 unit/ml of IFN-β (Fig. 9). As indicated previously, HuUBP43 is also induced by treatment of HO-1 cells with the combination of IFN- β + MEZ, but this induction is similar to that found with IFN-β alone (Fig. 5) . Similarly, the half-life of HuUBP43 mRNA in HO-1 cells treated with only IFN- β or IFN-β + MEZ is similar, i.e., -4 hr (Fig. 9). To determine if HuUBP43 induction in HO-1 cells is a primary response following IFN-β and IFN-β + MEZ treatment and if protein synthesis is required, experiments were performed using the protein synthesis inhibitor cycloheximide (Fig. 9) . These studies demonstrate that HuUBP43 induction by IFN-β does not require new protein synthesis, indicating that HuUBP43 is a primary interferon response gene in HO-1 cells.

To determine if IFN-β could induce HuUBP43 in additional cell types, a panel of normal and cancer- derived human cells were evaluated (Fig. 10) . Induction of HuUBP43 by IFN-β was observed in normal human cells, including immortalized melanocyte (FM516-SV) , early passage mammary epithelial cells (HMEC) , human skin fibroblasts (HSF) and cerebellum- derived cells (NC) , and histologically diverse human ^' cancer cell lines, including melanoma (HO-1, LO-1, C8161, SKMEL-p53wt, WM35, WM239, WM278, SH-1, FO-1) , glioblastoma multiforme (T98G, GBM-18) , osteosarcoma (Saos-2) and carcinomas of the breast (MCF-7, T47D) , cervix (HeLa) , endometrium (HONE-1) , colon (SW613) , and prostate (DU-145) (Fig. 10) . In contrast, IFN-β did not induce HuUBP43 in SKMEL-mtp53 or MeWo cells, both of which contain a mutated p53 , whereas IFN-β induced or increased HuUBP43 expression in human melanoma cells with confirmed or presumptive wild- type p53 genes. However, since induction of HuUBP43 by IFN-β occurs in Saos-2 cells, it is clear that induction of this gene by IFN-β can occur independent of expression of the tumor suppressor genes p53 and RB . In this context, SKMEL-mtp53 and MeWo may contain additional defects preventing induction of the appropriate biochemical changes necessary for induction of HuUBP43 by IFN-β. In the endometrial carcinoma cell line HONE-1 and the glioblastoma multiforme cell line T98G, induction of HuUBP43 is significantly less than in the majority of normal or cancer cell lines tested. The mechanism underlying the differential induction of HuUBP4J by IFN-β in these specific cell lines and the high de novo level of HuUBP43 in SKMEL-wtp53 cells versus the majority of treated cell types is not presently known .

It is well established that the induction of many interferon stimulated genes occur through activation of the JAK/STAT signaling pathway (Darnell et al . , 1994; Stark et al . , 1998). To explore this possibility with respect of the ability of IFN-β to induce HuUBP43 expression, a series of mutant cell types either containing a functional JAK/STAT pathway (2fTGH) or lacking a functional Tyk2 (U1A) , STAT1 (U3A) , JAK1 (U4A) or the IFNAR2 (U5A) were analyzed (Darnell et al . , 1994; Lutfalla et al . , 1995) . As can be seen in Fig. .12, IFN-β induces HuUBP43 in parental 2fTGH cells, but induction does not occur in cells defective in STAT1 or JAK1 or in cells lacking a functional type I interferon receptor (IFNAR2) . In the case of U1A cells, which are defective in Tyk2 signaling, some induction of HuUBP43 is apparent. Previous studies indicate that U1A cells are leaky with respect to their response to IFN-β (Darnell et al . , 1994). With the exception of U1A cells, which were not analyzed in the Lin et al . (2000) study, the present results provide similar evidence for a biochemical pathway, i.e., induction of JAK/STAT, by which IFN-β induces HuUBP43 (ISG43) in target cells. In this context, it will be informative to determine if the JAK/STAT or Tyk2 pathways are modified in cells resistant to maximal induction of HuUBP43 by IFN-β, such as SKMEL-mutp53 , MeWo, HONE-1 and T98G, or if defects in other signaling pathways mediate the attenuated response to IFN-β in these cells.

Expression of HuUBP43 in normal human tissues

To determine which normal tissues express HuUBP43 Multiple Tissue Northern blots (Clontech) containing poly (A) RNA from different human organs and tissues were probed with HuUBP43 (Fig. 12) . Maximum expression was observed in the liver, with elevated levels also apparent in the ovary and thyroid.

Significant expression of HuUBP43 was also evident in the heart, placenta, lung, skeletal muscle, kidney, pancreas, spleen, thymus and prostate, with lower level expression in the lung, small intestine, lymph node, trachea and colon. Small but detectable levels of HuUBP43 were also apparent in the adrenal gland, testis and peripheral blood leukocytes. In contrast, expression was not detected, after a similar exposure time, in the brain, stomach, spinal cord or bone marrow. Schwer et al . (2000) analyzed tissue expression of the same gene, a human homologue of murine UBP43 (Uspl8) , by dot-blot using a human RNA Master Blot (Clontech) . Their data indicates both similarities and differences in expression of this human ubiquitin-specific protease. The lowest levels of expression of UBP43 were also found in brain tissues and the testis. Increased expression was apparent in the liver, similar to the present observation. In contrast, differences in the relative expression of HuUBP43 in specific tissues was apparent, including higher relative expression of UBP43 in the adrenal gland, spinal cord, bone marrow and stomach, and lower relative expression in the thymus. The reasons for these quantitative differences in expression may relate to the different methodologies used to evaluate mRNA levels, i.e., dot-blots versus Northern blots.

3.4. HuUBP43 : role in interferon response and differentiation

Protein deubiquitination is a noteworthy process implicated in many important cellular events, including cell cycle progression, signal transduction and transcriptional activation (Hochstrasser, 1996; Moazed and Johnson, 1996; D'Andrea and Pellman, 1998) . Two distinct families of deubiquitinating enzymes are recognized- ubiquitin carboxyl-terminal hydrolases (UCHs) and ubiquitin-processing proteases (UBPs) . The UBP enzymes are relatively large proteins, varying in size between 526 to 2691 amino acids, with the capacity to cleave a wide spectrum of protein substrates (Hershko, 1996; Laney and Hochstrasser, 1999) . A hallmark of these enzymes is the presence of six short consensus conserved regions, referred to as DHI through DHVI (for deubiquitinating enzyme homology domain I to VI) , with wide disparity in their remaining amino acid sequences (Wilkinson et al . 1995) . The conserved regions are critical for generating the active site of the enzyme (Huang et al . , 1995; Papa and Hochstrasser, 1993) and the high level of divergence in the remaining parts of the molecule are believed to contribute to the unique biochemical properties, substrate specificities and cellular localizations of the different UBPs (D'Andrea and Pellman, 1998; Laney and Hochstrasser, 1999) .

Previous studies have implicated murine UBPs in monocytic differentiation, response to cytokines

(specifically IL-2 and IL-3) and growth control in an IL-3 -dependent murine pro-B cell line (Zhu et al., 1996,1997; Liu et al . , 1999). From the present study it is clear that HuUBP43 is induced as a function of IFN-β treatment of human melanoma and other cell types. Exposure of specific human melanoma cells to IFN-β induces transient growth suppression and a reversible induction of specific differentiation phenotypes, such as melanin synthesis (Fisher et al . , 1985; Jiang et al . , 1993, 1995c) . In these contexts, it is possible that HuUBP43 contributes to growth^* control and differentiation in human melanoma cells, possibly by regulating ubiquitin-dependent proteolysis or the ubiquitination state of an unknown growth regulatory factor (s). In the case of terminal differentiation induced by IFN-β + MEZ it is possible that HuUBP43 assists in the differentiation process by cooperating with a gene(s) induced by- MEZ to effect the terminal differentiation process. Previous studies have identified 4 classes of mda genes, including genes regulated by IFN-β and IFN-β + MEZ (type I mda genes) , MEZ and IFN-β + MEZ (type II mda genes) , IFN-β, MEZ and IFN-β + MEZ (type III mda genes) or preferentially by IFN-β + MEZ (type IV mda genes) . In this context, HuUBP43 (mda-D-15) is a type I mda gene that is induced by IFN-β as well as the combination of IFN-β + MEZ. Studies are in progress to define the relationship between the four different classes of mda genes and their contributions to growth control, reversible differentiation and terminal differentiation.

Based on its ability to be selectively induced by interferons, particularly IFN-β, in diverse cell types, HuUBP43 may play an important role in processes initiated by these cytokines, including growth suppression and antiviral activity. It does not appear that HuUBP43 displays direct growth suppressing properties in HO-1 cells, since ectopic expression of this gene in this melanoma cell line only marginally inhibits colony formation, i.e., <10% reduction in comparison with vector transfected control cells (data not shown) . However, further studies are required to determine if ectopic expression of HuUBP43 in HO-1 cells can potentiate the effect of MEZ, thereby resulting in either enhanced growth suppression or induction of terminal differentiation .

A relationship between HuUBP43 and antiviral activity is suggested by the cloning of ISG43

(HuUBP43) by differential display as a function of

RNase-L expression (Li et al . 2000) . RNase-L functions in IFN-mediated growth inhibition and apoptosis (Hassel et al . , 1993; Castelli et al . , 1997, 1998; Zhou et al . , 1997). During viral infection, RNase-L appears to target viral RNA for destruction, potentially through the local activation of the 2-5A system by viral double stranded RNA (Baglioni et al . , 1984; Li et al . , 1998) . It has been suggested that ISG43 may be a substrate for RNase-L degradation, by a posttranscriptional mechanism, thereby attenuating the interferon response in cells (Li et al . , 2000), which if constitutive can be detrimental to cells (Lee and Estaban, 1994; Castelli et al . , 1997). In this context, ISG43 (HuUBP423) induction by interferon is negatively regulated by RNase-L, thereby assisting in fine-tuning the regulation of interferon stimulated gene expression in virally infected or interferon treated cells .

HuUBP43 has been mapped to chromosome 22qll.2 (Schwer et al . , 2000) . This area of the genome contains a region referred to as the DiGeorge syndrome critical region (DSCR) , including a minimal area of 2 Mb which is consistently deleted in DiGeorge syndrome and related disorders, including velocardiofacial syndrome (Goldberg et al . , 1993). DiGeorge syndrome is associated with thymic aplasia or hypoplasia, parathyroid hypoplasia or congenital cardiac abnormalities. In addition to containing the HuUBP43 gene, the DSCR also contains a putative transcription factor, an adhesion molecule receptor, a serine-threonine kinase and several additional proteins with unknown functions (Sirotkin et al . , 1997) . It is not presently known if HuUBP43 directly or indirectly contributes to the development of DiGeorge syndrome. However, based on the potential importance of ubiquitin-specific proteases in regulating normal_^ cellular physiology, HuUBP43 may be considered a candidate gene for DiGeorge syndrome .

Conclusions

We have developed a rapid and simple cDNA subtraction protocol, RaSH (Jiang et al . , 2000), which is easy to perform and efficiently identifies differentially expressed genes. The RaSH approach has been applied to study the process of terminal differentiation in human melanoma cells. This approach resulted in the identification and cloning of a novel human ubiquitin-specific protease, HuUBP43 (Li et al . , 2000; Schwer et al . , 2000). HuUBP43 is shown to be a type I interferon (IFN- α/b) , predominantly IFN-β, inducible early response gene that is dependent on the JAK/STAT kinase pathway for expression. The location of HuUBP43 is within a critical region of deletion of DiGeorge syndrome (Schwer et al . , 2000), which suggests that this gene may be necessary for normal development and when expression is absent can contribute to the development of DiGeorge syndrome and related pathological conditions.

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Claims

What is claimed is.

1. A method for rapid subtraction hybridization of two pools of double-stranded ^• cDNA to isolate a differentially expressed gene which comprises:

(a) isolating RNA from a first population of cells

(driver cells) and separately from a second population of cells (tester cells) ;

(b) producing a first double- stranded cDNA pool (driver cDNA) from mRNA isolated from the first population of cells;

(c) producing a second double-stranded cDNA pool

(tester cDNA) from mRNA isolated from the second population of cells;

(d) admixing the first pool of cDNA (driver cDNA) with a first restriction enzyme which recognizes a restriction site at about every 200 to 500 bases of DNA under appropriate conditions for restriction digestion of the first pool of cDNA;

(e) admixing the second pool of cDNA (tester cDNA) with an amount of the first restriction enzyme under appropriate conditions for restriction digestion of the second pool of cDNA; (f) admixing, the digested first pool of cDNA (driver) from step (d) with an appropriate amount of a double-stranded oligonucleotide; wherein the oligonucleotide sequence comprises (i) an internal recognition site for a second restriction enzyme and (ii) a ligatable cohesive end at one end which is capable of hybridizing with a ligatable cohesive end resulting from digestion of the first pool of cDNA with the first restriction enzyme, wherein the second restriction enzyme recognizes a restriction site at about every 4000 bases of DNA, under suitable conditions for ligation of the oligonucleotide to the cDNA;

(g) admixing the digested second pool of cDNA (tester) from step (e) with an appropriate amount of a double- stranded nucleotide; wherein the oligonucleotide sequence comprises (i) an internal recognition site for a second restriction enzyme and (ii) a ligatable cohesive end at one end which is capable of hybridizing with a ligatable cohesive end resulting from digestion of the second pool of cDNA with the first restriction enzyme, wherein the second restriction enzyme recognizes a restriction site at about every 4000 bases of DNA, under suitable conditions for ligation of the oligonucleotide to the cDNA;

(h) performing a polymerase chain reaction (PCR) using an aliquot of the ligation mixture resulting from step (f) (tester cDNA) admixed with a PCR primer, wherein the first PCR primer has a sequence which allows it to anneal to the oligonucleotide-derived region of the resulting ligation product from step ⁽f⁾;

(i) performing a polymerase chain reaction (PCR) using an aliquot of the ligation mixture resulting from step (g) (driver cDNA) mixed with the PCR primer;

(j) digesting the PCR products from the PCR reaction performed in step (h) (tester cDNA) with the second restriction enzyme under appropriate digestion conditions;

(k) admixing an aliquot of the digestion product

(tester cDNA) from step (j) with an excess of PCR products from step (i) (driver cDNA) under hybridization conditions;

(1) ligating the hybridization mixture from step (k) with a sufficient amount of a vector previously digested with the second restriction enzyme, so as to isolate a differentially expressed gene from among the two pools of double-stranded DNA.

2. The method of claim 1, wherein the driver cells are untreated cells and the tester cells are cells treated with a compound.

3. The method of claim 1, wherein the driver cells are cells with a normal phenotype and the tester cells are cancer cells.

4. The method of claim 1, wherein the driver cells are cells susceptible to infection by Human Immunodeficiency Virus (HIV) and the tester cells are cells resistant to infection by HIV.

5. The method of claim 1, wherein the driver cells are in one state of differentiation and the tester cells are identical to the driver cells except that they are in a different state of differentiation.

6. The method of claim 1, wherein the driver cells are stem cells and the tester cells are cells which have undergone differentiation.

7. The method of claim 2, wherein the compound is a carcinogen, a pollutant, or a differentiation agent.

8. The method of claim 1, wherein the first restriction enzyme is Dpnll, Tagl, -Rsal, or -Eco-RII.

9. The method of claim 1, wherein the second restriction enzyme is Xhol .

10. The method of claim 1, wherein the excess is about 5- fold excess to more than 1, 000-fold excess.

11. The method of claim 1, wherein the method is carried out so that the driver cDNA and the tester cDNA are reversed so as to isolate genes which are highly expressed in the driver cells and wherein expression of such genes is in less in the tester cells.

12. A previously unknown nucleic acid identified by the method of claim 1.

13. An interferon inducible gene identified by the method of claim 1.

14. A nucleic acid which is induced in a cell following viral infection of the cell identified by the method of claim 1.

15. A nucleic acid encoding a human UBP43 protein identified by the method of claim 1.