Testatin: A cystatin-related gene expressed during early testis development

To isolate genes involved in morphogenic aspects of testis development, and which may act in cell signaling pathways downstream of the testis-determining gene Sry, we have developed a modified mRNA differential display method named signal peptide differential display. It was used to target those genes that encode proteins having a signal peptide sequence. By using this method, we isolated a gene named testatin. This gene was found to be related to a group of genes that encodes cysteine protease inhibitors known as cystatins. Cystatins and their target proteases have been associated with tumor formation and metastasis, but also are involved in natural tissue remodeling events such as bone resorption and embryo implantation. We show that testatin expression is restricted to fetal gonads and adult testis. Furthermore, testatin is expressed during testis cord formation in pre-Sertoli cells, believed to be the site of Sry action, at a time immediately after the peak of Sry expression. This finding suggests that testatin might be activated by transcription factors that are known to orchestrate the early testis development pathway. This gene therefore represents one of the putative downstream targets likely to have an essential role in tissue reorganization during early testis development.

Regulation of the human ether-a-gogo related gene (HERG) K+ channels by reactive oxygen species

Human ether-a-gogo related gene (HERG) K+ channels are key elements in the control of cell excitability in both the cardiovascular and the central nervous systems. For this reason, the possible modulation by reactive oxygen species (ROS) of HERG and other cloned K+ channels expressed in Xenopus oocytes has been explored in the present study. Exposure of Xenopus oocytes to an extracellular solution containing FeSO4 (25–100 μM) and ascorbic acid (50–200 μM) (Fe/Asc) increased both malondialdehyde content and 2′,7′-dichlorofluorescin fluorescence, two indexes of ROS production. Oocyte perfusion with Fe/Asc caused a 50% increase of the outward K+ currents carried by HERG channels, whereas inward currents were not modified. This ROS-induced increase in HERG outward K+ currents was due to a depolarizing shift of the voltage-dependence of channel inactivation, with no change in channel activation. No effect of Fe/Asc was observed on the expressed K+ currents carried by other K+ channels such as bEAG, rDRK1, and mIRK1. Fe/Asc-induced stimulation of HERG outward currents was completely prevented by perfusion of the oocytes with a ROS scavenger mixture (containing 1,000 units/ml catalase, 200 ng/ml superoxide dismutase, and 2 mM mannitol). Furthermore, the scavenger mixture also was able to reduce HERG outward currents in resting conditions by 30%, an effect mimicked by catalase alone. In conclusion, the present results seem to suggest that changes in ROS production can specifically influence K+ currents carried by the HERG channels.

AF5q31, a newly identified AF4-related gene, is fused to MLL in infant acute lymphoblastic leukemia with ins(5;11)(q31;q13q23)

Infant acute lymphoblastic leukemia (ALL) with MLL gene rearrangements is characterized by early pre-B phenotype (CD10−/CD19+) and poor treatment outcome. The t(4;11), creating MLL-AF4 chimeric transcripts, is the predominant 11q23 chromosome translocation in infant ALL and is associated with extremely poor prognosis as compared with other 11q23 translocations. We analyzed an infant early preB ALL with ins(5;11)(q31;q13q23) and identified the AF5q31 gene on chromosome 5q31 as a fusion partner of the MLL gene. The AF5q31 gene, which encoded a protein of 1,163 aa, was located in the vicinity of the cytokine cluster region of chromosome 5q31 and contained at least 16 exons. The AF5q31 gene was expressed in fetal heart, lung, and brain at relatively high levels and fetal liver at a low level, but the expression in these tissues decreased in adults. The AF5q31 protein was homologous to AF4-related proteins, including AF4, LAF4, and FMR2. The AF5q31 and AF4 proteins had three homologous regions, including the transactivation domain of AF4, and the breakpoint of AF5q31 was located within the region homologous to the transactivation domain of AF4. Furthermore, the clinical features of this patient with the MLL-AF5q31 fusion transcript, characterized by the early pre-B phenotype (CD10−/CD19+) and poor outcome, were similar to those of patients having MLL-AF4 chimeric transcripts. These findings suggest that AF5q31 and AF4 might define a new family particularly involved in the pathogenesis of 11q23-associated-ALL.

MRG1, the product of a melanocyte-specific gene related gene, is a cytokine-inducible transcription factor with transformation activity

Identification of cytokine-inducible genes is imperative for determining the mechanisms of cytokine action. A cytokine-inducible gene, mrg1 [melanocyte-specific gene (msg1) related gene], was identified through mRNA differential display of interleukin (IL) 9-stimulated and unstimulated mouse helper T cells. In addition to IL-9, mrg1 can be induced by other cytokines and biological stimuli, including IL-1α, -2, -4, -6, and -11, granulocyte/macrophage colony-stimulating factor, interferon γ, platelet-derived growth factor, insulin, serum, and lipopolysaccharide in diverse cell types. The induction of mrg1 by these stimuli appears to be transient, with induction kinetics similar to other primary response genes, implicating its role in diverse biological processes. Deletion or point mutations of either the Box1 motif (binds Janus kinase 1) or the signal transducer and activator of transcription 3 binding site-containing region within the intracellular domain of the IL-9 receptor ligand binding subunit abolished or greatly reduced mrg1 induction by IL-9, suggesting that the Janus kinase/signal transducer and activator of transcription signaling pathway is required for mrg1 induction, at least in response to IL-9. Transfection of mrg1 cDNA into TS1, an IL-9-dependent mouse T cell line, converted these cells to IL-9-independent growth through a nonautocrine mechanism. Overexpression of mrg1 in Rat1 cells resulted in loss of cell contact inhibition, anchorage-independent growth in soft agar, and tumor formation in nude mice, demonstrating that mrg1 is a transforming gene. MRG1 is a transcriptional activator and may represent a founding member of an additional family of transcription factors.

Expression of the mouse cerberus-related gene, Cerr1, suggests a role in anterior neural induction and somitogenesis

The Xenopus cerberus gene encodes a secreted factor that is expressed in the anterior endomesoderm of gastrula stage embryos and can induce the formation of ectopic heads when its mRNA is injected into Xenopus embryos [Bouwmeester, T., Kim, S., Lu, B. & De Robertis, E. M. (1996) Nature (London) 382, 595–601]. Here we describe the existence of a cerberus-related gene, Cerr1, in the mouse. Cerr1 encodes a putative secreted protein that is 48% identical to cerberus over a 110-amino acid region. Analysis of a mouse interspecific backcross panel demonstrated that Cerr1 mapped to the central portion of mouse chromosome 4. In early gastrula stage mouse embryos, Cerr1 is expressed in the anterior visceral endoderm and in the anterior definitive endoderm. In somite stage embryos, Cerr1 expression is restricted to the most recently formed somites and in the anterior presomitic mesoderm. Germ layer explant recombination assays demonstrated that Cerr1-expressing somitic-presomitic mesoderm, but not older Cerr1-nonexpressing somitic mesoderm, was able to mimic the anterior neuralizing ability of anterior mesendoderm and maintain Otx2 expression in competent ectoderm. In most Lim1−/− headless embryos, Cerr1 expression in the anterior endoderm was weak or absent. These results suggest that Cerr1 may play a role in anterior neural induction and somite formation during mouse development.

A yeast artificial chromosome-based map of the region of chromosome 20 containing the diabetes-susceptibility gene, MODY1, and a myeloid leukemia related gene.

We have generated a physical map of human chromosome bands 20q11.2-20q13.1, a region containing a gene involved in the development of one form of early-onset, non-insulin-dependent diabetes mellitus, MODY1, as well as a putative myeloid tumor suppressor gene. The yeast artificial chromosome contig consists of 71 clones onto which 71 markers, including 20 genes, 5 expressed sequence tags, 32 simple tandem repeat DNA polymorphisms, and 14 sequence-tagged sites have been ordered. This region spans about 18 Mb, which represents about 40% of the physical length of 20q. Using this physical map, we have refined the location of MODY1 to a 13-centimorgan interval (approximately equal to 7 Mb) between D20S169 and D20S176. The myeloid tumor suppressor gene was localized to an 18-centimorgan interval (approximately equal to 13 Mb) between RPN2 and D20S17. This physical map will facilitate the isolation of MODY1 and the myeloid tumor suppressor gene.

Identification of a new mammalian centrin gene, more closely related to Saccharomyces cerevisiae CDC31 gene

Among the numerous centrin isoforms identified by two-dimensional gel electrophoresis in human cells, an acidic and slow-migrating isoform is particularly enriched in a centrosome fraction. We report here that this isoform specifically reacts with antibodies raised against Saccharomyces cerevisiae Cdc31p and is present, as other centrin isoforms, in the distal lumen of centrioles. It is encoded by a new centrin gene, which we propose to name HsCEN3 (Homo sapiens centrin gene 3). This gene is more closely related to the yeast CDC31 gene, and shares less identity with algae centrin than HsCEN1 and HsCEN2. A murine CDC31-related gene was also found that shows 98% identity and 100% similarity with HsCEN3, demonstrating a higher interspecies conservation than the murine centrin gene MmCEN1 (Mus musculus centrin gene 1) with either HsCEN1, or HsCEN2. Finally, immunological data suggest that a CDC31-related gene could exist in amphibians and echinoderms as well. All together, our data suggest the existence of two divergent protein subfamilies in the current centrin family, which might be involved in distinct centrosome-associated functions. The possible implication of this new mammalian centrin gene in centrosome duplication is discussed.

Gene-for-gene disease resistance without the hypersensitive response in Arabidopsis dnd1 mutant

The cell death response known as the hypersensitive response (HR) is a central feature of gene-for-gene plant disease resistance. A mutant line of Arabidopsis thaliana was identified in which effective gene-for-gene resistance occurs despite the virtual absence of HR cell death. Plants mutated at the DND1 locus are defective in HR cell death but retain characteristic responses to avirulent Pseudomonas syringae such as induction of pathogenesis-related gene expression and strong restriction of pathogen growth. Mutant dnd1 plants also exhibit enhanced resistance against a broad spectrum of virulent fungal, bacterial, and viral pathogens. The resistance against virulent pathogens in dnd1 plants is quantitatively less strong and is differentiable from the gene-for-gene resistance mediated by resistance genes RPS2 and RPM1. Levels of salicylic acid compounds and mRNAs for pathogenesis-related genes are elevated constitutively in dnd1 plants. This constitutive induction of systemic acquired resistance may substitute for HR cell death in potentiating the stronger gene-for-gene defense response. Although cell death may contribute to defense signal transduction in wild-type plants, the dnd1 mutant demonstrates that strong restriction of pathogen growth can occur in the absence of extensive HR cell death in the gene-for-gene resistance response of Arabidopsis against P. syringae.

Targeting a SWI/SNF-related chromatin remodeling complex to the β-globin promoter in erythroid cells

Chromatin remodeling complexes such as the SWI/SNF complex make DNA accessible to transcription factors by disrupting nucleosomes. However, it is not known how such complexes are targeted to the promoter. For example, a SWI/SNF1-like chromatin remodeling complex erythroid Krüppel-like factor (EKLF) coactivator-remodeling complex 1 (E-RC1) disrupts the nucleosomes over the human β-globin promoter in an EKLF-dependent manner. However, it is not known whether E-RC1 is targeted specifically to the β-globin promoter or whether E-RC1 is randomly targeted, but its activity is evident only at the β-globin promoter. Because E-RC1 cannot remodel chromatin over the β-globin promoter without EKLF in vitro, it has been proposed that SWI/SNF1-like complexes such as E-RC1 are targeted specifically to the promoter by selectively interacting with promoter-associated transcription factors such as EKLF. In this report, we test this hypothesis in the cellular context by using the ProteIN POsition Identification with Nuclease Tail (PIN*POINT) assay. We find that the Brahma-related gene (BRG) 1 and BRG1-associated factor (BAF) 170 subunits of E-RC1 are both recruited near the transcription initiation site of the β-globin promoter. On transiently transfected templates, both the locus control region and the EKLF-binding site are important for their recruitment to the β-globin promoter in mouse erythroleukemia cells. When the β-globin promoter was linked to the cytomegalovirus enhancer, the E-RC1 complex was not recruited, suggesting that recruitment of the E-RC1 complex is not a general property of enhancers.

t(11;22)(q23;q11.2) in acute myeloid leukemia of infant twins fuses MLL with hCDCrel, a cell division cycle gene in the genomic region of deletion in DiGeorge and velocardiofacial syndromes

We examined the MLL genomic translocation breakpoint in acute myeloid leukemia of infant twins. Southern blot analysis in both cases showed two identical MLL gene rearrangements indicating chromosomal translocation. The rearrangements were detectable in the second twin before signs of clinical disease and the intensity relative to the normal fragment indicated that the translocation was not constitutional. Fluorescence in situ hybridization with an MLL-specific probe and karyotype analyses suggested t(11;22)(q23;q11.2) disrupting MLL. Known 5′ sequence from MLL but unknown 3′ sequence from chromosome band 22q11.2 formed the breakpoint junction on the der(11) chromosome. We used panhandle variant PCR to clone the translocation breakpoint. By ligating a single-stranded oligonucleotide that was homologous to known 5′ MLL genomic sequence to the 5′ ends of BamHI-digested DNA through a bridging oligonucleotide, we formed the stem–loop template for panhandle variant PCR which yielded products of 3.9 kb. The MLL genomic breakpoint was in intron 7. The sequence of the partner DNA from band 22q11.2 was identical to the hCDCrel (human cell division cycle related) gene that maps to the region commonly deleted in DiGeorge and velocardiofacial syndromes. Both MLL and hCDCrel contained homologous CT, TTTGTG, and GAA sequences within a few base pairs of their respective breakpoints, which may have been important in uniting these two genes by translocation. Reverse transcriptase-PCR amplified an in-frame fusion of MLL exon 7 to hCDCrel exon 3, indicating that an MLL-hCDCrel chimeric mRNA had been transcribed. Panhandle variant PCR is a powerful strategy for cloning translocation breakpoints where the partner gene is undetermined. This application of the method identified a region of chromosome band 22q11.2 involved in both leukemia and a constitutional disorder.

msg1, a novel melanocyte-specific gene, encodes a nuclear protein and is associated with pigmentation.

Messenger RNA transcripts of the highly pigmented murine melanoma B16-F1 cells were compared with those from their weakly pigmented derivative B16-F10 cells by differential display. A novel gene called msg1 (melanocyte-specific gene) was found to be expressed at high levels in B16-F1 cells but at low levels in B16-F10 cells. Expression of msg1 was undetectable in the amelanotic K1735 murine melanoma cells. The pigmented murine melanocyte cell line melan-a expressed msg1, as did pigmented primary cultures of murine and human melanocytes; however, seven amelanotic or very weakly pigmented human melanoma cell lines were negative. Transformation of murine melanocytes by transfection with v-Ha-ras or Ela was accompanied by depigmentation and led to complete loss of msg1 expression. The normal tissue distribution of msg1 mRNA transcripts in adult mice was confined to melanocytes and testis. Murine msg1 and human MSG1 genes encode a predicted protein of 27 kDa with 75% overall amino acid identity and 96% identity within the C-terminal acidic domain of 54 amino acids. This C-terminal domain was conserved with 76% amino acid identity in another protein product of a novel human gene, MRG1 (msg1-related gene), isolated from normal human melanocyte cDNA by 5'-rapid amplification of cDNA ends based on the homology to msg1. The msg1 protein was localized to the melanocyte nucleus by immunofluorescence cytochemistry. We conclude that msg1 encodes a nuclear protein, is melanocyte-specific, and appears to be lost in depigmented melanoma cells.

A close relative of the adrenoleukodystrophy (ALD) gene codes for a peroxisomal protein with a specific expression pattern.

Adrenoleukodystrophy (ALD), a severe demyelinating disease, is caused by mutations in a gene coding for a peroxisomal membrane protein (ALDP), which belongs to the superfamily of ATP binding cassette (ABC) transporters and has the structure of a half transporter. ALDP showed 38% sequence identity with another peroxisomal membrane protein, PMP70, up to now its closest homologue. We describe here the cloning and characterization of a mouse ALD-related gene (ALDR), which codes for a protein with 66% identity with ALDP and shares the same half transporter structure. The ALDR protein was overexpressed in COS cells and was found to be associated with the peroxisomes. The ALD and ALDR genes show overlapping but clearly distinct expression patterns in mouse and may thus play similar but nonequivalent roles. The ALDR gene, which appears highly conserved in man, is a candidate for being a modifier gene that could account for some of the extreme phenotypic variability of ALD. The ALDR gene is also a candidate for being implicated in one of the complementation groups of Zellweger syndrome, a genetically heterogeneous disorder of peroxisome biogenesis, rare cases of which were found to be associated with mutations in the PMP70 (PXMP1) gene.

A novel amplicon at 8p22–23 results in overexpression of cathepsin B in esophageal adenocarcinoma

Cathepsin B (CTSB) is overexpressed in tumors of the lung, prostate, colon, breast, and stomach. However, evidence of primary genomic alterations in the CTSB gene during tumor initiation or progression has been lacking. We have found a novel amplicon at 8p22–23 that results in CTSB overexpression in esophageal adenocarcinoma. Amplified genomic NotI–HinfI fragments were identified by two-dimensional DNA electrophoresis. Two amplified fragments (D4 and D5) were cloned and yielded unique sequences. Using bacterial artificial chromosome clones containing either D4 or D5, fluorescent in situ hybridization defined a single region of amplification involving chromosome bands 8p22–23. We investigated the candidate cancer-related gene CTSB, and potential coamplified genes from this region including farnesyl-diphosphate farnesyltransferase (FDFT1), arylamine N-acetyltransferase (NAT-1), lipoprotein lipase (LPL), and an uncharacterized expressed sequence tag (D8S503). Southern blot analysis of 66 esophageal adenocarcinomas demonstrated only CTSB and FDFT1 were consistently amplified in eight (12.1%) of the tumors. Neither NAT-1 nor LPL were amplified. Northern blot analysis showed overexpression of CTSB and FDFT1 mRNA in all six of the amplified esophageal adenocarcinomas analyzed. CTSB mRNA overexpression also was present in two of six nonamplified tumors analyzed. However, FDFT1 mRNA overexpression without amplification was not observed. Western blot analysis confirmed CTSB protein overexpression in tumor specimens with CTSB mRNA overexpression compared with either normal controls or tumors without mRNA overexpression. Abundant extracellular expression of CTSB protein was found in 29 of 40 (72.5%) of esophageal adenocarcinoma specimens by using immunohistochemical analysis. The finding of an amplicon at 8p22–23 resulting in CTSB gene amplification and overexpression supports an important role for CTSB in esophageal adenocarcinoma and possibly in other tumors.

WISP genes are members of the connective tissue growth factor family that are up-regulated in Wnt-1-transformed cells and aberrantly expressed in human colon tumors

Wnt family members are critical to many developmental processes, and components of the Wnt signaling pathway have been linked to tumorigenesis in familial and sporadic colon carcinomas. Here we report the identification of two genes, WISP-1 and WISP-2, that are up-regulated in the mouse mammary epithelial cell line C57MG transformed by Wnt-1, but not by Wnt-4. Together with a third related gene, WISP-3, these proteins define a subfamily of the connective tissue growth factor family. Two distinct systems demonstrated WISP induction to be associated with the expression of Wnt-1. These included (i) C57MG cells infected with a Wnt-1 retroviral vector or expressing Wnt-1 under the control of a tetracyline repressible promoter, and (ii) Wnt-1 transgenic mice. The WISP-1 gene was localized to human chromosome 8q24.1–8q24.3. WISP-1 genomic DNA was amplified in colon cancer cell lines and in human colon tumors and its RNA overexpressed (2- to >30-fold) in 84% of the tumors examined compared with patient-matched normal mucosa. WISP-3 mapped to chromosome 6q22–6q23 and also was overexpressed (4- to >40-fold) in 63% of the colon tumors analyzed. In contrast, WISP-2 mapped to human chromosome 20q12–20q13 and its DNA was amplified, but RNA expression was reduced (2- to >30-fold) in 79% of the tumors. These results suggest that the WISP genes may be downstream of Wnt-1 signaling and that aberrant levels of WISP expression in colon cancer may play a role in colon tumorigenesis.

Identification of transforming growth factor-β- regulated genes in Caenorhabditis elegans by differential hybridization of arrayed cDNAs

Members of the transforming growth factor-β family play critical roles in body patterning, in both vertebrates and invertebrates. One transforming growth factor-β-related gene, dbl-1, has been shown to regulate body length and male ray patterning in Caenorhabditis elegans. We screened arrayed cDNAs to identify downstream target genes for the DBL-1 signaling by using differential hybridization. C. elegans cDNAs representing 7,584 independent genes were arrayed on a nylon membrane at high density and hybridized with 33P-labeled DNA probes synthesized from the mRNAs of wild-type, dbl-1, sma-2, and lon-2 worms. Signals for all the spots representing hybridized DNA were quantified and compared among strains. The screening identified 22 and 2 clones, which were positively and negatively regulated, respectively, by the DBL-1 signal. Northern hybridization confirmed the expression profiles of most of the clones, indicating good reliability of the differential hybridization using arrayed cDNAs. In situ hybridization analysis revealed the spatial and temporal expression patterns of each clone and showed that at least four genes, including the gene for the type I receptor for DBL-1, sma-6, were transcriptionally regulated by the DBL-1 signal.