Detection of new diagnostic markers in pathology by focus on growth-regulatory endogenous lectins. The case study of galectin-7 in squamous epithelia.

Lectins represent one of pivotal regulators of the cell proliferation The potential of galectin-7 as a new prognostic marker was studied in normal and transformed squamous epithelia of both ectodermal (epidermis, cornea vs. trichoepithelioma, basal and squamous cell carcinoma) and endodermal (vocal fold epithelium vs. carcinoma) origin. Studies on the cultured cells were also performed. Expression of galectin-7 seems to be connected to the process of stratification, no matter of origin of epithelium. Its expression is significantly reduced in malignant cells, thus galectin-7 might be a differentiation marker of epithelial malignancies.

Glycoprotein 90K/MAC-2BP interacts with galectin-1 and mediates galectin-1-induced cell aggregation.

The glycoprotein 90K was originally described as a tumor-secreted antigen and subsequently found to have immunostimulatory activity as well as other possible functions. This protein interacts with an endogenous lectin, galectin-3, and may play a role in tumor metastasis through this interaction. Because 90K is heavily glycosylated, it may also interact with other members of the galectin family, which would contribute to the multifunctionality of 90K. To test this possibility, we studied the recognition of 90K by galectin-1, which, like galectin-3, has been associated with neoplastic transformation. In a solid-phase binding assay, human recombinant galectin-1 bound immobilized human recombinant 90K in a fashion that was inhibitable by lactose. Galectins 1 and 3 appeared to bind to separate sites on 90K because they did not affect the binding of each other. The dissociation constant of galectin-1 to 90K was on the order of 10(-7) M. Galectin-1 also induced aggregation of a human melanoma cell line, A375, in a carbohydrate-dependent manner, and this appeared to be mediated, at least in part, by 90K expressed on A375 cells, since it was inhibitable by a specific anti-90K monoclonal antibody. We conclude that 90K interacts with both galectin-1 and galectin-3 and both interactions contribute to the formation of multicell aggregates. Because both of these galectins as well as 90K are often over-expressed in neoplasm, these interactions may occur in the setting of various carcinomas and contribute to their progression and metastasis.

Wedgelike glycodendrimers as inhibitors of binding of mammalian galectins to glycoproteins, lactose maxiclusters, and cell surface glycoconjugates.

Galectins are mammalian carbohydrate-binding proteins that are involved in cell-cell and cell-matrix adhesion, cell migration, and growth regulation with relevance to inflammation and tumor spread. These important functions account for the interest to design suitable low molecular weight inhibitors that match the distinct modes of presentation of the carbohydrate recognition domains of the different galectin subfamilies. Using 3,5-di-(2-aminoethoxy)benzoic acid as the branching unit, wedgelike glycodendrimers with two, four, and eight lactose moieties (G1-G3) were synthesized. They were tested in solid-phase competition assays with lactose maxiclusters and various N-glycan branching profiles (miniclusters) as the matrix and also in cell assays. Prototype galectins-1 and -7, chimera-type galectin-3, a plant (AB)(2) toxin, and a lactose-binding immunoglobulin G fraction from human serum were the carbohydrate-binding targets. Potent inhibition and remarkable cluster effects were seen for the homodimeric galectin-1, especially in combination with biantennary N-glycans as the matrix. Remarkably, for the tetravalent G2 glycodendrimer, the inhibitory potency of each lactose unit reached a maximum value of 1667 relative to free lactose. In haemagglutination experiments as a model for cell adhesion, galectin-3 was markedly sensitive to increased sugar valency and a relative potency per lactose of 150 was reached. The spatial orientation of the carbohydrate recognition domains of the endogenous lectins and the branching pattern of the carbohydrates of the glycoprotein matrices used are both important factors in the design and synthesis of glycodendrimers with galectin-selective properties.

Human galectin-3 is a novel chemoattractant for monocytes and macrophages.

Galectin-3 is a beta-galactoside-binding protein implicated in diverse biological processes. We found that galectin-3 induced human monocyte migration in vitro in a dose-dependent manner, and it was chemotactic at high concentrations (1.0 microM) but chemokinetic at low concentrations (10-100 nM). Galectin-3-induced monocyte migration was inhibited by its specific mAb and was blocked by lactose and a C-terminal domain fragment of the protein, indicating that both the N-terminal and C-terminal domains of galectin-3 are involved in this activity. Pertussis toxin (PTX) almost completely blocked monocyte migration induced by high concentrations of galectin-3. Galectin-3 caused a Ca2+ influx in monocytes at high, but not low, concentrations, and both lactose and PTX inhibited this response. There was no cross-desensitization between galectin-3 and any of the monocyte-reactive chemokines examined, including monocyte chemotactic protein-1, macrophage inflammatory protein-1alpha, and stromal cell-derived factor-1alpha. Cultured human macrophages and alveolar macrophages also migrated toward galectin-3, but not monocyte chemotactic protein-1. Finally, galectin-3 was found to cause monocyte accumulation in vivo in mouse air pouches. These results indicate that galectin-3 is a novel chemoattractant for monocytes and macrophages and suggest that the effect is mediated at least in part through a PTX-sensitive (G protein-coupled) pathway.

Rapid growth of a retroperitoneal rhabdomyosarcoma following right hemicolectomy for ascending colon cancer: report of a case.

We report herein the case of a 69-year-old man in whom rapid growth of a retroperitoneal rhabdomyosarcoma occurred following hemicolectomy for ascending colon cancer. On his first admission for surgery, a small lesion, 1.5 cm in diameter, was detected adjacent to the inner side of the left kidney by abdominal axial computed tomography (CT), which was initially suspected to be a benign lesion; however, a postoperative follow-up CT scan done 5 months later revealed that the lesion had enlarged remarkably to 8 cm in diameter. Thus, total resection was performed under the presumed diagnosis of a malignant retroperitoneal tumor. The tumor was found to be adjacent to the inner portion of the left kidney and covered by Gerota's fascia. As it involved the ileolumbar muscle and had a metastatic lymph node, complete resection was performed. The resected specimen was 8.5 x 6.5 x 5 cm in size and was histologically confirmed as a retroperitoneal rhabdomyosarcoma of embryonal type. Two courses of adjuvant chemotherapy with adriamycin, vincristine, and cyclophosphamide were given, and the patient has shown no signs of recurrence for 2 years since his second operation.

Requirement of divalent galactoside-binding activity of ecalectin/galectin-9 for eosinophil chemoattraction.

We have previously isolated and cloned a novel eosinophil chemoattractant (ECA) from a human T-cell-derived expression library. This ECA, termed ecalectin, is a variant of human galectin-9, a member of a beta-galactoside binding animal lectin family, which contains two conserved carbohydrate recognition domains (CRDs). In the present study, we addressed whether carbohydrate binding activity is required for the ECA activity of ecalectin and whether both CRDs are essential for this activity. Recombinant full-length wild-type ecalectin (ecalectin-WT) and N-terminal and C-terminal CRD (ecalectin-NT and -CT, respectively) were generated. All of these recombinant proteins exhibited affinity for lactose, a property shared by galectins, but ecalectin-WT exhibited substantially higher hemagglutination activities than ecalectin-NT and -CT. Furthermore, ecalectin-WT showed over 100-fold higher ECA activity than ecalectin-NT and -CT; combination of recombinant domain fragments did not reconstitute the ECA and hemagglutination activities of the full-length protein. ECA activity of ecalectin-WT was inhibited by lactose in a dose-dependent manner. Site-directed mutation of positions Arg(65) of ecalectin-NT and Arg(239) of ecalectin-CT to an aspartic acid residue resulted in the loss of both lactose-binding and ECA activities. We conclude that divalent galactoside-binding activity is required for eosinophil chemoattraction by ecalectin.

Protein domain mapping by lambda phage display: the minimal lactose-binding domain of galectin-3.

Mapping of protein domains having a distinct function is essential to understanding the protein's structure-function relationship. We used a bacteriophage lambda surface expression vector, lambdafoo, in order to determine the minimal carbohydrate-binding domain of human galectin-3 (Gal-3). Gal-3 cDNA was randomly digested by DNase I and cloned into the phage vector. The library generated was screened by affinity selection using lactose immobilized on agarose beads. DNA sequence analysis of a set of isolated clones defined the minimal folding domain of Gal-3 required for lactose binding, which consisted of 136 amino-acid residues. Using the phage clones isolated, we also determined relative dissociation constants in solution between lactose and the minimal domain expressed on the phage surface. This technique does not require either purified or labeled proteins, and bacteriophage lambda surface display may, therefore, be useful for protein domain mapping and in vitro studies of various macromolecular interactions.

Mapping of the minimal domain encoding a conformational epitope by lambda phage surface display: factor VIII inhibitor antibodies from haemophilia A patients.

Haemophilia A patients who receive repeated transfusion of fVIII concentrates often develop inhibitor alloantibodies, resulting in reduced efficacy of the therapy. Determination of fVIII epitopes for the alloantibodies is essential for an understanding of their inhibitory effect on blood coagulation. Random fragments of fVIII displayed on lambda phage particles were selected using two patient plasmas immobilized onto the surface of a microtiter plate. A set of clones defined the minimal domain that consisted of 157 amino acid residues including cysteine at both boundaries. The minimal domain absorbed most of the binding activities of the plasmas to fVIII, suggesting that the domain contains a major determinant for the plasmas. Site-directed mutagenesis and chemical denaturation of the domain confirmed that a tertiary structure formed by the disulfide bridge was recognized by the antibodies. The epitope domain defined overlaps with fVIII binding sites to vWf and phospholipid, and may play an important role in blood coagulation. Thus, the bacteriophage lambda surface display may be useful for mapping the minimal folding domain of various protein antigens that contain a conformational epitope.

Efficient epitope mapping by bacteriophage lambda surface display.

A bacteriophage lambda surface expression system, lambda foo, was used for epitope mapping of human galectin-3. We constructed random epitope and peptide libraries and compared their efficiencies in the mapping. The galectin-3 cDNA was randomly digested by DNase 1 to make random epitope libraries. The libraries were screened by affinity selection using a microtiter plate coated with monoclonal antibodies. Direct DNA sequencing of the selected clones defined two distinct epitope sites consisting of nine and 11 amino-acid residues. Affinity selection of random peptide libraries recovered a number of sequences that were similar to each other but distinct from the galectin-3 sequence. These results demonstrate that a single affinity selection of epitope libraries with antibodies is able to define an epitope determinant as small as nine residues long and is more efficient in epitope mapping than random peptide libraries.

Human T lymphotropic virus-I infection of human T lymphocytes induces expression of the beta-galactoside-binding lectin, galectin-3.

Animal lectins play important roles in a variety of biological processes via their recognition of glycoconjugates. Galectin-3 is a beta-galactoside-binding lectin previously designated as epsilon BP (IgE-binding protein), CBP35, Mac-2, L-29, and L-34, and its expression has been associated with various physiological and pathological processes, including cell growth, tumor transformation, and metastasis. Galectin-3 is widely distributed in various tissues and cell types and is expressed in many leukocytes, with the notable exception of B and T lymphocytes. We now report that galectin-3 is abundantly expressed in a number of human T lymphotropic virus (HTLV)-I-infected human T cell lines, including F6T, HUT 102, K3T, MT-2, and SLB-I, but is not expressed in non-HTLV-I-infected T cell lines such as Jurkat, CEM, and MOLT-4. In addition, the galectin-3 level was markedly increased in human thymocytes after infection with HTLV-I as compared with uninfected thymocytes. The up-regulation of galectin-3 expression appeared to correlate well with HTLV-I gene expression, as undetectable or very low levels of galectin-3 were found in the S1T and ATL-1K cell lines, which are nonproductively infected with HTLV-I. In co-transfection experiments, the galectin-3 promoter was significantly up-regulated by expression vectors encoding the 40-kd Tax protein, a potent transactivator in HTLV-I. Analysis of various Tax mutants suggested that galectin-3 promoter induction is dependent on activation of the cyclic-AMP-responsive element binding protein/activation transcription factor family of transcription factors and, to a lesser extent, nuclear factor-kappa B/Rel induction. Transfection of human promonocytic U-937 cells with an HTLV-I Tax expression vector induced galectin-3 expression in this cell line. Functionally, galectin-3 was shown to activate interleukin-2 production in Jurkat T cells. Together, these findings raise the possibility that HTLV-I Tax production induces the transcription and subsequent synthesis and secretion of galectin-3, which in turn may further activate these T cells and contribute to the altered properties of cell growth found in adult T cell leukemia induced by HTLV-I.

Galectin-3 promotes adhesion of human neutrophils to laminin.

Galectin-3 is a member of a growing family of animal lectins composed of three domains, with the amino-terminal half consisting of a short segment followed by tandem repeats, and the carboxyl-terminal half representing the carbohydrate-recognition domain. Previously, we have shown that galectin-3 binds to the surface of human neutrophils and is capable of activating these cells. We have now studied the effect of exogenous galectin-3 on adhesion of human neutrophils to laminin-coated microtiter plates and found that this lectin promotes the adhesion in a dose-dependent manner. The effect was dependent on the lectin's carbohydrate-binding function, as well as its amino-terminal region. The galectin-3-induced adhesion was reduced significantly in the presence of EDTA, even though Ca2+ and Mg2+ are not required for the lectin binding, and the adhesion was significantly less at 4 degrees C, as compared with 37 degrees C. Galectin-3 also induced neutrophil adhesion to fibronectin, which is not recognized by the lectin, but much higher concentrations of the lectin were required, and the effect is completely dependent on Ca2+ and Mg2+. We conclude that galectin-3 induces neutrophil adhesion to laminin through a combination of two distinct mechanisms: 1) the lectin bridges neutrophils to laminin, in a carbohydrate-dependent and Ca2+-, Mg2+-independent manner, and 2) the lectin induces activation of neutrophils, in the presence of the divalent cations, resulting in the positive regulation of other cell adhesion molecules and enhanced adhesion to laminin. The results suggest that galectin-3 may play a role in the traversing of neutrophils through the basement membrane at inflammation sites.

Modulation of functional properties of galectin-3 by monoclonal antibodies binding to the non-lectin domains.

Galectin-3 is a member of a newly defined family of animal lectins, which is composed of three domains: a small amino-terminal domain, a domain containing repeating elements, and a carboxyl-terminal domain containing the carbohydrate-recognition site. Various functions have been described or proposed for this lectin, and it appears that galectin-3 has diverse roles. Murine monoclonal antibodies (MAbs) have been generated from mice hyperimmunized with recombinant human galectin-3 or galectin-3C (the carboxyl-terminal domain), and seven MAbs have been characterized in detail. All MAbs generated against the intact galectin-3 recognize the amino-terminal region of the molecule, as demonstrated by ELISA and immunoblotting using recombinant galectin-3C and galectin-3NR, which contains the amino-terminal domain and all the repeating elements. Their epitopes were all found to be within the first 45 amino acids of galectin-3, as determined by using galectin-3 mutants with a truncated amino-terminal region. However, these MAbs were found to profoundly modulate the lectin activities of galectin-3. The MAb B2C10 inhibited (i) the binding of 125I-labeled galectin-3 to IgE coated on microtiter plates; (ii) the galectin-3's hemagglutination activity; and (iii) galectin-3-induced superoxide production by human neutrophils. Other MAbs, especially A3A12, caused marked potentiation of these activities. The results support our model that the lectin function of galectin-3 is influenced by protein homodimerization resulting from self-association of the amino-terminal region of the molecule. The potentiating activities of some MAbs are probably due to facilitation of dimerization galectin-3, and the inhibitory activity of MAb B2C10 is probably the result of its disruption of the self-association process.

Evidence for IgG autoantibodies to galectin-3, a beta-galactoside-binding lectin (Mac-2, epsilon binding protein, or carbohydrate binding protein 35) in human serum.

Galectin-3 is a beta-galactoside-binding animal lectin formerly called epsilon protein, Mac-2, carbohydrate binding protein 35, CBH 30, L-29, or L34. The possible occurrence of autoantibodies to galectin-3 was investigated because crosslinking of galectins bound to IgE or Fc epsilon RI might produce mediator release from mast cells or basophils. Unexpectedly, a control serum from an individual free of current allergic symptoms was found to have a significantly elevated level of IgG anti-galectin-3 by ELISA employing galectin-3-coated wells incubated with test serum followed by HRPO-conjugated goat anti-human IgG. The reaction was not inhibitable by lactose, suggesting that it is not a result of binding of IgG by galectin-3 through lectin-carbohydrate interactions. The antibody activity was specifically adsorbed by galectin-3 and protein A-conjugated Sepharose and was associated primarily with subclass IgG1. The presence of the antibodies was confirmed by immunoblotting showing binding of IgG to the 30-kD galectin-3 band. The relevant epitopes were in the galectin-3 N-terminal domain. The propositus was subsequently found to have adenocarcinoma of the colon, and titers of IgG anti-galectin-3 were found to be sharply elevated after hemicolectomy. Similar antibody titers have not been found in family members, but small numbers of normal persons and patients with malignant neoplasms have been found to have evidence of IgG anti-galectin-3 antibodies at lower titers than the propositus. The pathogenesis of this autoimmune reaction is unclear, though there is a trend for it to occur in older persons.

Expression and function of galectin-3, a beta-galactoside-binding lectin, in human monocytes and macrophages.

A family of beta-galactoside-binding animal lectins has recently been designated as galectins. One member of this family, galectin-3, has been known as epsilon BP for its IgE-binding activity and as Mac-2, a macrophage surface antigen, CBP35, CBP30, L-29, and L-34. Although much information has accumulated on the expression of this lectin in murine macrophages and human monocytic cell lines, little is known about the expression and function of this protein in normal human monocytes/macrophages. We now report that galectin-3 is expressed in normal human peripheral blood monocytes and its level increases dramatically as human monocytes differentiate into macrophages upon culturing in vitro. Immunoblot analysis showed that there was a 5-fold increase in the level of galectin-3 after 1 day of culture and greater than a 12-fold increase after 5 days. Immunocytochemical analysis confirmed this progressive increase of galectin-3 expression in cultured monocytes. Immunogold cytochemistry/electron microscopy analysis revealed that galectin-3 was expressed on the surface of human monocytes and that the level of cell surface galectin-3 increased progressively as these cells differentiated into macrophages. The level of galectin-3 in human monocytes/macrophages was modulated by stimuli such as lipopolysaccharide and interferon-gamma, and galectin-3 was secreted when monocytes were stimulated by calcium ionophore A23187 Soluble galectin-3 caused superoxide release from human monocytes; this activity was dependent on the lectin property of galectin-3, as it was inhibitable by lactose. Thus, galectin-3 may modulate the function of this cell type in an autocrine or paracrine fashion through binding to cell surface glycoconjugates.

A human lectin, galectin-3 (epsilon bp/Mac-2), stimulates superoxide production by neutrophils.

A family of soluble animal lectins, galectins, with beta-galactoside-binding activity, is gaining increased attention. One member of this family, galectin-3, has been previously designated by this group as epsilon bp, for its IgE-binding activity. On the basis of the saccharide specificity and other biochemical characteristics of epsilon bp, it is possible that this lectin could have an important extracellular modulatory role, functioning through recognition of critical cell surface glycoproteins on many cell types. We present evidence here that recombinant human epsilon bp activates human neutrophils in a dose-dependent manner as demonstrated by superoxide production. The observed activity is dependent on the lectin property of epsilon bp intrinsic to its carboxyl-terminal domain, as it could be inhibited effectively by lactose, a known saccharide ligand of epsilon bp. However, the amino-terminal domain is also necessary for the observed activity, as epsilon bp-C (the carboxyl-terminal domain fragment) is devoid of neutrophil-activating activity, even though it retains the carbohydrate-binding property. Affinity purification of lysates from cell surface-radio-iodinated neutrophils revealed two major protein bands of M(r) 115,000 and M(r) 180,000 that are recognized by epsilon bp and preliminary data suggested that one of these proteins is NCA-160, a human carcinoembryonic Ag-related glycoprotein. This study thus lends further support to our view of an extracellular function for epsilon bp and suggests that this protein has an important role in inflammation and host defense through modulating the function of neutrophils.

Transition from TCR-beta dimer to TCR-alpha beta-expressing cells by introduction of an alpha-chain in an immature thymocyte cell line.

The molecular basis for the surface expression of TCR-beta chain in the absence of association with TCR-alpha, -gamma, or -delta chain by an immature thymocyte cell line was investigated. The TCR-beta chain expressed by this cell line was not encoded by any unique DNA sequence, nor was it inserted into the membrane via a glycosyl-phosphatidylinositol linkage. Transfection of two other beta-chains derived from mature T cell clones resulted in the surface expression of dimers of the transfected beta-chains in both cases. Immunoprecipitation of the beta-dimer-CD3 complex demonstrated that the association of the beta-dimer with the CD3 complex, especially the CD3 zeta chain, was so weak that they dissociated under the detergent conditions in which the TCR-CD3 complex of mature T cells is kept intact. Transfection of TCR-alpha chain resulted in the expression of a TCR-alpha beta-CD3 complex and the disappearance of beta-dimers. In accordance with the changes in TCR complex components, the association between TCR-alpha beta and CD3 complex became stable and the cells transduced signals more efficiently. The results demonstrate that the expression of TCR-beta as part of an incomplete TCR-CD3 complex is developmentally regulated and the expression of TCR-alpha chain results in normal configuration and function of TCR complex.

Molecular cloning and characterization of the gene encoding mouse melanoma antigen by cDNA library transfection.

We have isolated a cDNA (H52) of 2.8-kb-long encoding an 80-kDa mouse melanoma Ag that is defined by a syngeneic anti-B16 melanoma mAb with an ability to block anti-melanoma cytotoxic T cell responses. H52 transfectants were brightly stained with the antibody, and the 80-kDa molecule was immunoprecipitated from the transfectants. Northern blot analysis showed that this transcript was detected in mouse melanoma cells of C57BL/6 and DBA/2 origin, C1300 A/J neuroblastoma, L cell (C3H) and EL-4 T lymphoma (C57BL/6), faintly in BW5147 (AKR) T lymphoma, but not in other tumors, such as S913 fibrosarcoma (C57BL/10), NIH3T3, 70 Z/3 pre-B lymphoma, and P3U1 plasmacytoma (BALB/c). Since the transcripts were not found in normal C57BL/6 tissues of fetus, newborn, and adult origin, the H52 expression is associated with transforming phenotypes. However, no tissue- or cell type-specific expression was observed. Nucleotide sequence analysis has clearly demonstrated that H52 cDNA encodes the full length of the env gene and long terminal repeat region of endogenous ecotropic murine leukemia provirus of AKV-type, which is defective in C57BL/6. The H52 envelope protein has several amino acid changes compared to those of AKV, one of which is in the env 14 peptide region preferentially associated with MHC molecule, suggesting the possible reason for the difference of antibody reactivity even in H52-positive tumors. We also demonstrate that CTL against H52 transfectant kills B16 melanoma. Thus, the above results are direct evidence that even the endogenous self molecule, when constitutively expressed, does act as a tumor Ag.

Properties of mouse melanoma antigen and its secretion mechanism from the cell surface.

We analyzed the biochemical properties and biological significance of the melanoma antigen secreted in the culture supernatants of B16 melanoma cells. The 80 kilodalton (kd) molecule bearing the epitopes of mouse melanoma antigen was found to associate noncovalently with an 18 kd moiety in the culture supernatants as well as on the cell surface. Tunicamycin treatment of B16 cells did not affect the expression of the 69 kd nonglycosylated form of the 80 kd molecule but did abolish the association between the two molecules on the cell surface. We could not detect this antigen as a soluble form when the N-linked glycosylation was inhibited. Therefore, the glycosylation of the 80 kd molecule is essential for the formation of the 80 kd/18 kd complex and also for the secretion. Moreover, the affinity-purified melanoma antigen from the supernatants could induce anti-melanoma suppressor cells which block the generation of cytotoxic T lymphocytes against melanoma cells. Thus, the 80 kd glycoprotein as a soluble melanoma antigen performed a pivotal function in the escape mechanisms of melanoma cells from the host immune surveillance system.

Characterization of a melanoma antigen with a mouse-specific epitope recognized by a monoclonal antibody with antimetastatic ability.

We have characterized properties of a melanoma antigen with a mouse-specific melanoma epitope expressed on B16 melanoma by using syngeneic monoclonal antibodies with antimetastatic ability. The molecule recognized by the antibody is a membrane glycoprotein with a molecular weight of 80,000. Studies on tunicamycin treatment indicated that the core size of the molecule appeared to have a molecular weight of 69,000 and also suggested that the carbohydrate moiety was greatly responsible for the conformation of the mouse melanoma epitope. The antigen was released or shed into the culture medium from the cell surface, and the turnover rate of the antigen was within 1.5 h.

Method of genomic DNA cloning by the combination of cosmid shuttle vector and monoclonal antibody.

We report here the strategy to isolate the DNA fragment of any species origin which encodes cell surface antigen by using cosmid library transfection and cell sortings with a monoclonal antibody. We took the mouse melanoma antigen defined by monoclonal antibody as a model system and rescued the genomic DNA by in vitro packaging, showing the feasibility of this procedure.