Differential Display Technique Research Articles

Candidate-gene cloning and targeted marker enrichment of wheat chromosomal regions using RNA fingerprinting - differential display

The usefulness of the RNA fingerprinting – differential display technique in gene cloning and targeted marker enrichment in wheat is demonstrated. A small region of chromosome 5BL was targeted that contains Ph1, a chromosome-pairing regulator gene. The cultivar Chinese Spring (CS) and mutant ph1b are almost identical except for chromosome 5BL, which, in the mutant line, carries an interstitial deletion encompassing the Ph1 gene. Poly(A)+ RNA of the two lines from anthers at developmental stages ranging from pre-meiotic mitosis to anaphase II was PCR-amplified using 38 pairwise combinations of 19 primers. The 35S-labeled amplified products were size-separated on denaturing polyacrylamide-urea gels. A total of 3154 fragment bands were observed, of which 43 were present in CS but absent in the ph1b mutant. These 43 fragment bands were eluted, re-amplified, and used as probes in gel-blot DNA analyses of wheat group 5 nullisomic-tetrasomic lines and the ph1b mutant. Twenty-four of these 43 probes were single- or few-copy sequences. Eight of the 24 probes mapped to wheat group 5 and five mapped to the deletion of the ph1b mutant. Three of these five probes were further localized to the submicroscopic region containing the Ph1 gene, by using two deletion lines flanking the region. Northern-blot analysis revealed that the gene corresponding to one of these three probes expresses mainly during meiosis and is from the B genome.Key words: RNA fingerprinting – differential display, wheat, gene cloning, marker enrichment, Ph1 gene.

Detection of Differentially Expressed Genes in Esophageal Carcinoma Using Non-RI Differential Display with High Specificity.

Differentially expressed genes in human esophageal carcinoma/normal tissue pairs were identified by means of a modified differential display technique to overcome the limitations of the conventional technique. Out of the eight cDNAs isolated, three were novel. The other five clones consisted of cDNA encoding cytokeratin 13, complement 7, KIAA1160, expression sequence tag (EST)-Hs110855 and EST-Hs13662. Quantitative reverse transcription-PCR (RT-PCR) for 10 carcinoma/normal tissue pairs confirmed down-regulation of cytokeratin 13, complement 7 and KIAA1160 mRNAs in carcinoma tissue and up-regulation of EST-Hs110855 mRNA. EST-Hs13662 mRNA also seemed to increase in carcinoma tissues but not to a statistically significant extent. In situ hybridization confirmed that cytokeratin 13 mRNAs localized in differentiating keratinocytes of normal epithelia and had disappeared in carcinomas, suggesting that tihs down-regulation reflects de-differentiation during carcinogenesis. The functions of complement 7 and KIAA1160 mRNAs are unclear in normal tissue but their down-regulation in carcinoma tissue may help the development of esophageal carcinoma. The expression of EST-Hs110855 mRNA reportedly observed in carcinomas of different origins suggests that tihs EST is a carcinogenesis-related gene. Our modified technique, which eliminates the source of false-positives, reduces the screening time, and dispense with the radioisotope, was found to be useful for isolating differentially expressed genes from clinical specimens with apparently genetically distant cellular populations and a very limited mass.

Cytokinin-induced gene expression in cultured green cells of Nicotiana tabacum identified by fluorescent differential display.

The cell growth and plastid development of cultured green tobacco cells were maintained by the phytohormone cytokinin. After subculture into cytokinin-free medium, when cytokinin treatment was resumed, physiological changes induced by cytokinin were analyzed. Changes in chlorophyll biosynthesis and photosynthetic gene expression were observed 1 week after cytokinin induction, and changes in cell growth were observed 2 weeks after cytokinin induction. Two cytokinin-induced genes (cig) were isolated from these cells using the fluorescent differential display technique. Northern analysis confirmed that expression of these cig was induced by both natural and synthetic cytokinins. The expression of cig1 was also induced by abscisic acid, and its cDNA sequence was similar to the proline dehydrogenase gene. The expression of cig2 is specific to cytokinin and is not induced by other phytohormones. The amino acid sequence encoded by cig2 is similar to the GDP/GTP exchange factor eIF2B, which regulates translation initiation. The expression of these cig suggests a complex induction system involving cytokinin and other phytohormones.

TENB2, a proteoglycan identified in prostate cancer that is associated with disease progression and androgen independence.

TENB2 encodes a putative transmembrane proteoglycan, related to the EGF/heregulin family of growth factors and follistatin, which has been identified through the application of a differential display technique to a xenograft model of prostate cancer. Northern analysis and competitive PCR were used to demonstrate significantly increased TENB2 expression (p = 0.0003) on the acquisition of androgen independence in the model system. TENB2 is also overexpressed in clinical prostate carcinoma vs. its benign counterpart (p < 0.0001), with particular prominence in high-grade tumours, and shows a high degree of tissue specificity, being detected on a multitissue Northern array exclusively in brain and prostate material. Studies of recombinant protein expression demonstrate that TENB2 is a chondroitin sulphate proteoglycan. The presence of an EGF and 2 follistatin domains suggests a role in the regulation of growth factor signalling either as a ligand precursor, a membrane-bound receptor or as a binding protein for growth factors. These data are indicative of a significant role for TENB2 in the progression of poorly differentiated tumour types, with implications for prostate cancer detection, prognosis and therapy.

SFRP2 expression in rabbit myogenic progenitor cells and in adult skeletal muscles.

Satellite cells derived from fast- and slow-twitch muscles have different properties in culture. We have used the differential display technique to retrieve genes differentially expressed in fast- and slow-twitch muscle satellite cell cultures. Amongst these genes we have identified, cloned, sequenced and studied the expression in muscle of rabbit secreted frizzled related protein 2 (SFRP2) mRNA, whose importance in cell fate determination has been well documented. It has been shown that SFRP2 is widely expressed in the developing embryo but its expression in the adult is much more restricted. We show that primary cultures of satellite cells from adult rabbit fast- and slow-twitch muscles strongly and differentially express SFRP2 mRNA. Embryonic rabbit muscle cell primary cultures also strongly express SFRP2 mRNA whereas the myoblast C2.7 cell line shows little expression. We also studied SFRP2 mRNA expression in growing, regenerating and denervated muscles. Embryonic rabbit muscles express SFRP2 mRNA but this rapidly falls off after birth. In adult rabbit muscles SFRP2 mRNA is detected within 1 day of either muscle damage or denervation. Thereafter the SFRP2 mRNA expression profiles are different for fast- and slow-twitch muscle. The function of SFRP2 in muscle is unknown but its putative activity as a Wnt antagonist and its precocious expression after muscle damage suggest a role in satellite cell activation.

Changes in expression of mRNAs in the gerbil hippocampus following transient forebrain ischemia

The phenomenon of delayed neuronal death in CA 1 neurons following brief duration of global ischemia has eluded definitive explanation. Using a differential display technique, we examined changes in expression ofmRNAs in the hippocampus following 5-min cerebral ischemia in Mongolian gerbils.. Under pentobarbital anesthesia, gerbils were sacrificed by decapitation at 6 h (n = 20) and 2 days (n = 20) after ischemia, and sham-operated gerbils (n = 20) were sacrificed at 6 h after surgery. Total RNA was isolated from the hippocampal samples in each group for the differential display analysis. The mRNAs were classified into three patterns; gradual disappearance, decrease and recovery, and new appearance. Representative mRNAs in three patterns were subcloned and sequenced partly. An mRNA in the gradual disappearance pattern showed homologous with neuronal pentraxin. In situ hybridization and Northern blot analyses of neuronal pentraxin revealed the gradual disappearance pattern. An mRNA in the decrease and recovery pattern showed homologous with 14-3-3 protein y-subtype, and an mRNA in the new e appearance pattern showed no homology in the data base. The differential display analysis is a usefultechnique with which to investigate changes in expression ofmRNAs following transient cerebral ischemia. The novel mRNA may be involved in the treatment of cerebral ischemia. Further studies are necessary for this point. [Neural Res 2000; 22: 825-831]

Decreased expression of SPRR3 in Chinese human oesophageal cancer.

cDNA fragments that were differentially expressed between human oesophageal carcinomas and matched normal adjacent mucosa were isolated using an improved mRNA differential display technique. One of them was identified as the 3'-untranslated region of SPRR3 and was homologous to the esophagin cDNA. Northern blot, dot blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that SPRR3 expression was lost in three cell lines of oesophageal carcinoma and was dramatically decreased in 54 out of 57 primary oesophageal carcinomas compared with adjacent normal mucosa. Esophagin has been shown to be down-regulated in western oesophageal carcinomas. The data suggest that esophagin is probably the protein product of the gene SPRR3 and that altered mRNA expression of SPRR3/esophagin is a frequent event in the development of Chinese oesophageal cancer.

Fungal metabolism of environmentally persistent compounds: Substrate recognition and metabolic response

Mechanism of lignin biodegradation caused by basidiomycetes and the history of lignin biodegradation studies were briefly reviewed. The important roles of fungal extracellular ligninolytic enzymes such as lignin and manganese peroxidases (LiP and MnP) were also summarized. These enzymes were unique in their catalytic mechanisms and substrate specificities. Either LiP or MnP system is capable of oxidizing a variety of aromatic substrates via a one-electron oxidation. Extracellular fungal system for aromatic degradation is non-specific, which recently attracts many people working in a bioremediation field. On the other hand, an intracellular degradation system for aromatic compounds is rather specific in the fungal cell. Structurally similar compounds were prepared and metabolized, indicating that an intracellular degradation strategy consisted of the cellular systems for substrate recognition and metabolic response. It was assumed that lignin-degrading fungi might be needed to develop multiple metabolic pathways for a variety of aromatic compounds caused by the action of non-specific ligninolytic enzymes on lignin. Our recent results on chemical stress responsible factors analyzed using mRNA differential display techniques were also mentioned.

Identification of argininosuccinate lyase as a hypoxia-responsive gene in rat hepatocytes

Background/Aims: The differential oxygenation of periportal and perivenous hepatocytes has been demonstrated as a major determinant in the zonated expression of certain metabolic pathways in the liver. We have searched for novel genes whose expression could be modulated by hypoxia in cultured rat hepatocytes. Methods: Primary cultures of rat hepatocytes were incubated under normoxic (21% oxygen) or hypoxic (3% oxygen) conditions for 6 h. Differences in gene expression under both conditions were analyzed using the technique of differential display by means of PCR. Results: We have identified the enzyme argininosuccinate lyase (ASL) as being downregulated by hypoxia. ASL is a cytosolic protein which participates in urea metabolism. ASL expression was time-dependently reduced in hypoxia. Hypoxia modulated the responses of this gene to the two main hormonal signals which induce ASL mRNA: glucocorticoids and cAMP. ASL mRNA levels decreased in response to ATP-reducing agents. CoCl 2 mimicked the effect of hypoxia, suggesting the implication of a hemoprotein in this response. Hypoxia did not affect ASL mRNA stability, indicating that this effect occurs at the transcriptional level. Conclusions: Our observations suggest that differences in oxygen levels across the hepatic parenchyma could participate in the zonated expression of ASL.

Differential Expression of Intracisternal A-Particle Transcripts in Immunogenic versus Tumorigenic S49 Murine Lymphoma Cells

Tumorigenic S49 mouse lymphoma cells (T-25) were compared to their nontumorigenic (immunogenic) substrate-adherent descendants (T-25-Adh), using the differential display technique. A 784-bp fragment with 92% sequence homology to the intracisternal A-particle (IAP) element family was isolated from the latter cells. IAP sequences are endogenous, noninfectious retroviral elements that can undergo transpositions and act as mutagens. Expression of IAP transcripts (as detected by the isolated fragment) was 5- to 10-fold higher in T-25-Adh cells than in T-25 cells. IAP RT-PCR cDNA clones derived from the immunogenic T-25-Adh cells, but not from T-25 cells, contain two distinctive motifs: (i) a motif characteristic of IAP elements expressed in lymphoid cells (lymphocyte specific, LS); (ii) a nonapeptide sequence known to stimulate cytotoxic T lymphocytes in a leukemia cell line expressing IAP sequences. In addition, expression of transcripts containing these motifs is enhanced in the immunogenic cells as opposed to the tumorigenic cells. Furthermore, one of the IAP elements (belonging to the LS1 subfamily) is specifically hypomethylated in the DNA of the immunogenic cells. The above-mentioned relationship was strengthened when tumorigenic revertants derived from T-25-Adh cells, as well as independently selected tumorigenic and immunogenic S49 sublines, were studied. In all cases, enhanced immunogenicity was linked to the up-regulation of specific IAP elements. No transpositions of LS1 elements were observed among the different sublines studied. These findings suggest that, in the S49 lymphoma, selectively expressed IAP retroviral elements may function in a tumor suppressive capacity by affecting the immunogenic potential of these cells.

Cloning and characterization of a receptor-like protein kinase gene associated with senescence.

Senescence-associated genes are up-regulated during plant senescence and many have been implicated in encoding enzymes involved in the metabolism of senescing tissues. Using the differential display technique, we identified a SAG in bean (Phaseolus vulgaris) leaf that was exclusively expressed during senescence and was designated senescence-associated receptor-like kinase (SARK). The deduced SARK polypeptide consists of a signal peptide, a leucine-rich repeat in the extracellular region, a single membrane-spanning domain, and the characteristic serine/threonine protein kinase domain. The mRNA level for SARK increased prior to the loss of chlorophyll and the decrease of chlorophyll a/b-binding protein mRNA. Detached mature bean leaves, which senesce at an accelerated rate compared with leaves on intact plants, showed a similar temporal pattern of SARK message accumulation. Light and cytokinin, which delayed the initiation of leaf senescence, also delayed SARK gene expression; in contrast, darkness and ethylene, which accelerated senescence, advanced the initial appearance of the SARK transcript. SARK protein accumulation exhibited a temporal pattern similar to that of its mRNA. A possible role for SARK in the regulation of leaf senescence was considered.

Heterogeneous expression of mRNA in rat liver lobules as detected by differential display.

Although liver hepatocytes appear to be uniform histologically, they are considerably heterogeneous with respect to their individual physiological capacities. In order to find still unknown genes that are heterogeneously expressed and with the aim of evaluating the usefulness of the differential display technique for this purpose, we performed differential displays with mRNA isolated from hepatocytes from the periportal and pericentral zone of the rat liver. In this way we identified at least two mRNAs exclusively expressed in the pericentral fraction. Sequence analysis revealed that the corresponding genes encode proteins with proline-glutamate dipeptide repeats similar to ones previously identified in rat pheochromocytoma and brain. In situ hybridization confirmed the heterogeneous distribution of the mRNA. Only one to two cell lines surrounding the terminal hepatic venules were positive, strongly resembling the heterogeneous expression of the enzyme glutamine synthetase. Our work demonstrates that the differential display method is a useful tool for the identification of genes that are differentially expressed in individual parenchymal cells. In fact, our results prove that differential display technology can be used for the identification of cellular markers for distinct subpopulations of cells in a given tissue.

Retrieving parasite specific liver stage gene products in Plasmodium yoelii infected livers using differential display

Differential display (DD) has been routinely used to identify genes whose expression pattern is altered by changes in the cellular environment and/or at different stages of development. Most reports utilizing DD contain conventional DD primers that have high guanine and cytosine content and would not be expected to be optimal for Plasmodium which has approximately 30–40% G+C. In an attempt to accommodate the high adenine and thymidine rich genome of Plasmodium yoelii, we utilized PCR primers containing 40, 50 and 60% G+C and modified the existing DD technique. Thus 40% G+C appeared to be the most suitable to amplify Plasmodium genome. Gene specific primers were generated from the sequences of selected DD bands amplified using the 40% G+C primers and were used to verify that the DD clones were of parasite origin by PCR and sequence alignment. Additional data on five of the selected DD clones, designated P2T1L5, P2T1L6, P2T6L11, P2T7L12 and P2T7L13, suggested that all are expressed during the P. yoelii liver stage infection. Interestingly, P2T1L5 is also expressed during the sporozoite stage of the life cycle and both P2T1L6 and P2T6L11 are present as blood stage antigens. The results of this study suggest that DD incorporating primers with low G+C content allows the identification of P. yoelii messages from infected mouse livers.

Identification of STKA-dependent genes in Dictyostelium discoideum

During culmination of Dictyostelium aggregates, prespore and prestalk cells undergo terminal differentiation to form spores and a cellular stalk. Disruption of the cell-fate gene stkA leads to a phenotype in which all the cells destined to become spores end up as stalk cells. ‘Stalky’ mutants express normal levels of prespore cell transcripts but fail to produce the culmination-stage spore transcript spiA. The stkA gene encodes a putative GATA-type transcription factor (STKA). In order to identify possible downstream targets of STKA we used the technique of mRNA differential display and isolated four cDNA fragments that hybridise to mRNAs present during the later stages of development. All four gene tags were cloned and sequenced. mRNAs represented by these four sequence tags do not accumulate during culmination of ‘stalky’ cells and therefore must be specific to the spore pathway. By screening a cDNA library, longer cDNAs for all four were cloned and sequenced. Three of these contained complete protein-coding regions while only a partial cDNA was recovered for the fourth. One of the corresponding proteins has significant homology to a surface zinc metalloproteinase (GP63) of the protozoan parasite Leishmania, while another is closely related to a human pre-RNA binding protein (hnRNP R).

Glutathione and a UV light-induced glutathione S-transferase are involved in signaling to chalcone synthase in cell cultures.

UV irradiation stimulates expression of the gene encoding the key enzyme chalcone synthase (CHS), which leads to the generation of protective flavonoids in parsley cell cultures. CHS transcripts increase after 3 to 4 hr, and early genes are involved in the signal transduction to the CHS promoter. By using the fluorescent differential display technique in a large-scale screening, several early UV light-induced genes were isolated. Of these, a novel glutathione S-transferase (PcGST1) is induced within 2 hr and precedes CHS expression. Overexpression of PcGST1 in transformed cell lines containing a CHS promoter/luciferase reporter (CHS-LUC) affected the onset of LUC transcription. Supplementing these cell lines with glutathione immediately stimulated CHS-LUC expression within 2 hr in dark-incubated cells and resulted in a biphasic induction profile in UV-irradiated cells. Our data indicate the involvement of glutathione and PcGST1 in early events of a UV light-dependent signal transduction pathway to CHS. In this context, the oxidative status of a cell acts as a central regulating element.

Estrogen Targets Genes Involved in Protein Processing, Calcium Homeostasis, and Wnt Signaling in the Mouse Uterus Independent of Estrogen Receptor-α and -β

Estrogen actions in target organs are normally mediated via activation of nuclear estrogen receptors (ERs). By using mRNA differential display technique, we show, herein, that estradiol-17beta (E(2)) and its catechol metabolite 4-hydroxy-E(2) (4OHE(2)) can modulate uterine gene expression in ERalpha(-/-) mice. Whereas administration of E(2) or 4OHE(2) rapidly up-regulated (4-8-fold) the expression of immunoglobulin heavy chain binding protein (Bip), calpactin I (CalP), calmodulin (CalM), and Sik similar protein (Sik-SP) genes in ovariectomized wild-type or ERalpha(-/-) mice, the expression of secreted frizzled related protein-2 (SFRP-2) gene was down-regulated (4-fold). Bip, CalP, and CalM are calcium-binding proteins and implicated in calcium homeostasis, whereas SFRP-2 is a negative regulator of Wnt signaling. Bip and Sik-SP also possess chaperone-like functions. Administration of ICI-182,780 or cycloheximide failed to influence these estrogenic responses, demonstrating that these effects occur independent of ERalpha, ERbeta, or protein synthesis. In situ hybridization showed differential cell-specific expression of these genes in wild-type and ERalpha(-/-) uteri. Although progesterone can antagonize or synergize estrogen actions, it had minimal effects on these estrogenic responses. Collectively, the results demonstrate that estrogens have a unique ability to influence specific genes in the uterus not involving classical nuclear ERs.

Simultaneous identification of two cyclohexanone oxidation genes from an environmental Brevibacterium isolate using mRNA differential display.

The technique of mRNA differential display was used to identify simultaneously two metabolic genes involved in the degradation of cyclohexanone in a new halotolerant Brevibacterium environmental isolate. In a strategy based only on the knowledge that cyclohexanone oxidation was inducible in this strain, the mRNA population of cells exposed to cyclohexanone was compared to that of control cells using reverse transcription-PCR reactions primed with a collection of 81 arbitrary oligonucleotides. Three DNA fragments encoding segments of flavin monooxygenases were isolated with this technique, leading to the identification of the genes of two distinct cyclohexanone monooxygenases, the enzymes responsible for the oxidation of cyclohexanone. Each monooxygenase was expressed in Escherichia coli and characterized. This work validates the application of mRNA differential display for the discovery of new microbial metabolic genes.

Analysis of genes under the downstream control of the t(8;21) fusion protein AML1-MTG8: overexpression of the TIS11b(ERF-1, cMG1) gene induces myeloid cell proliferation in response to G-CSF

The AML1-MTG8 fusion transcription factor generated by t(8;21) translocation is thought to dysregulate genes that are crucial for normal differentiation and proliferation of hematopoietic progenitors to cause acute myelogenous leukemia (AML). Although AML1-MTG8 has been shown to repress the transcription of AML1 targets, none of the known targets of AML1 are probably responsible for AML1-MTG8-mediated leukemogenesis. In this study, 24 genes under the downstream control of AML1-MTG8 were isolated by using a differential display technique. Analysis with deletion mutants of AML1-MTG8 demonstrated that the regulation of the majority of these genes requires the region of 51 residues (488-538) containing the Nervy homology region 2 (NHR2), through which AML1-MTG8 interacts with MTGR1. Among the 24 genes identified, 10 were considered to be genes under the control of AML1, because their expression was altered by AML1b or AML1a or both. However, the other 14 genes were not affected by either AML1b or AML1a, suggesting the possibility that AML1-MTG8 regulates a number of specific target genes that are not normally regulated by AML1. Furthermore, an up-regulated gene, TIS11b (ERF-1,cMG1), was highly expressed in t(8;21) leukemic cells, and the overexpression of TIS11b induced myeloid cell proliferation in response to granulocyte colony-stimulating factor. These results suggest that the high-level expression of TIS11b contributes to AML1-MTG8-mediated leukemogenesis.

Analysis of genes under the downstream control of the t(8;21) fusion protein AML1-MTG8: overexpression of the TIS11b(ERF-1, cMG1) gene induces myeloid cell proliferation in response to G-CSF

AbstractThe AML1-MTG8 fusion transcription factor generated by t(8;21) translocation is thought to dysregulate genes that are crucial for normal differentiation and proliferation of hematopoietic progenitors to cause acute myelogenous leukemia (AML). Although AML1-MTG8 has been shown to repress the transcription of AML1 targets, none of the known targets of AML1 are probably responsible for AML1-MTG8-mediated leukemogenesis. In this study, 24 genes under the downstream control of AML1-MTG8 were isolated by using a differential display technique. Analysis with deletion mutants of AML1-MTG8 demonstrated that the regulation of the majority of these genes requires the region of 51 residues (488-538) containing the Nervy homology region 2 (NHR2), through which AML1-MTG8 interacts with MTGR1. Among the 24 genes identified, 10 were considered to be genes under the control of AML1, because their expression was altered by AML1b or AML1a or both. However, the other 14 genes were not affected by either AML1b or AML1a, suggesting the possibility that AML1-MTG8 regulates a number of specific target genes that are not normally regulated by AML1. Furthermore, an up-regulated gene, TIS11b (ERF-1,cMG1), was highly expressed in t(8;21) leukemic cells, and the overexpression of TIS11b induced myeloid cell proliferation in response to granulocyte colony-stimulating factor. These results suggest that the high-level expression of TIS11b contributes to AML1-MTG8-mediated leukemogenesis.

Molecular cloning and characterization of a human metalloprotease disintegrin— a novel marker for dendritic cell differentiation

The 1,25-dihydroxyvitamin D3(1,25- [OH]2VD3) modulates the differentiation of monocytic cell lines and monocytes (MOs) in vitro. Up to now several target genes of 1,25(OH)2VD3have been described in monocytic cell lines; however, little is known about target genes in primary MOs. With the Differential Display technique, we found a transcript up-regulated by 1,25(OH)2VD3 in short-term cultured human blood MOs, which we called MADDAM (metalloprotease and disintegrin dendritic antigen marker; EMBL/GenBank/DDBJ accession no. Y13786). Northern blot analysis confirmed this result and revealed a signal of MADDAM messenger RNA (mRNA) at about 7.5 kilobases (kb). Long-term culture (more than 20 hours) of MOs during macrophage (MAC) differentiation led to a rapid and complete down-regulation of MADDAM expression. In contrast, MADDAM expression was maintained in MOs differentiated along the dendritic cell (DC) pathway and induced in CD34+-derived DCs. In addition, in situ hybridization revealed signals of MADDAM mRNA in follicles of human lymph nodes and MADDAM mRNA was detected in freshly isolated human blood-DCs by reverse transcription-polymerase chain reaction (RT-PCR). By means of a database search, we found that MADDAM is a member of the ADAM (a metalloprotease and disintegrin) family, the human homologue to murine meltrin-β (ADAM 19). From these data, we conclude that MADDAM is an important marker for the differentiation and characterization of DCs and the distinction between MACs and DCs.