Catherine L. Keck

TrnR2, a novel receptor that mediates neurturin and GDNF signaling through Ret

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) comprise a family of TGF-beta-related neurotrophic factors (TRNs), which have trophic influences on a variety of neuronal populations. A receptor complex comprised of TrnR1 (GDNFR alpha) and Ret was recently identified and found to be capable of mediating both GDNF and NTN signaling. We have identified a novel receptor based on homology to TrnR1, called TrnR2, that is 48% identical to TrnR1, and is located on the short arm of chromosome 8. TrnR2 is attached to the cell surface via a GPI-linkage, and can mediate both NTN and GDNF signaling through Ret in vitro. Fibroblasts expressing TrnR2 and Ret are approximately 30-fold more sensitive to NTN than to GDNF treatment, whereas those expressing TrnR1 and Ret respond equivalently to both factors, suggesting the TrnR2-Ret complex acts preferentially as a receptor for NTN. TrnR2 and Ret are expressed in neurons of the superior cervical and dorsal root ganglia, and in the adult brain. Comparative analysis of TrnR1, TrnR2, and Ret expression indicates that multiple receptor complexes, capable of mediating GDNF and NTN signaling, exist in vivo.

In silico-initiated cloning and molecular characterization of a novel human member of the L1 gene family of neural cell adhesion molecules

To discover genes contributing to mental retardation in 3p- syndrome patients we have used in silico searches for neural genes in NCBI databases (dbEST and UniGene). An EST with strong homology to the rat CAM L1 gene subsequently mapped to 3p26 was used to isolate a full-length cDNA. Molecular analysis of this cDNA, referred to as CALL (cell adhesion L1-like), showed that it is encoded by a chromosome 3p26 locus and is a novel member of the L1 gene family of neural cell adhesion molecules. Multiple lines of evidence suggest CALL is likely the human ortholog of the murine gene CHL1: it is 84% identical on the protein level, has the same domain structure, same membrane topology, and a similar expression pattern. The orthology of CALL and CHL1 was confirmed by phylogenetic analysis. By in situ hybridization, CALL is shown to be expressed regionally in a timely fashion in the central nervous system, spinal cord, and peripheral nervous system during rat development. Northern analysis and EST representation reveal that it is expressed in the brain and also outside the nervous system in some adult human tissues and tumor cell lines. The cytoplasmic domain of CALL is conserved among other members of the L1 subfamily and features sequence motifs that may involve CALL in signal transduction pathways.

Nonrandom Breakpoints of Unbalanced Chromosome Translocations in Human Hepatocellular Carcinoma Cell Lines

In the search for specific chromosomal alterations in human hepatocellular carcinomas (HCC), we analyzed two new HCC cell lines and identified nonrandom changes by combined G-banding and fluorescence in situ hybridization (FISH). Cell line 7703 was established from an HCC deriving from a patient in the Qidong region of China, where the incidence of HCC is very high and is associated with hepatitis-B virus infection and exposure to aflatoxin. This line has a highly rearranged karyotype eliciting complex rearrangements involving the majority of chromosomes. The second line, SK-Hep-1, derived from a liver adenocarcinoma, is less heterogeneous, having few altered chromosomes. We have characterized the majority of structural and numerical alterations and identified in both lines unbalanced translocations with the breakpoints nonrandomly involving regions 1p36 and 3p14 and gain of chromosome 6p and 8q. While gain of 6p and 8q are recurrent in HCC, translocations of 1p and 3p are described for the first time. Damage and recombination at the breakpoint sites on chromosomes 1 and 3 might have resulted in activation of proto-oncogene, formation of new oncogenic chimeric genes, or loss of tumor suppressor genes.

Gene structure and chromosomal localization of mouse cyclin G2 (Ccng2)

Cyclins are essential activators of cyclin-dependent kinases (Cdk) which, in turn, play pivotal roles in controlling transition through cell-cycle checkpoints. Cyclin G2 is a recently discovered second member of the G-type cyclins. The two members of the G-type cyclins, cyclin G1 and cyclin G2, share high structural similarity but their function remains to be defined. Here we characterize the structure of the mouse cyclin G2 gene by first cloning and sequencing the full-length mouse cyclin G2 cDNA. The cyclin G2 cDNA was used to isolate the cyclin G2 gene from a BAC library and to establish that the gene was transcribed from eight exons spanning a total of 8604bp. The cyclin G2 gene was mapped by fluorescence in situ hybridization (FISH) to mouse chromosome 5E3.3.-F1.3. This region is syntenic to a region on human chromosome 4. The expression of cyclins G1 and G2 was examined in various tissues, but no correlation between expression patterns of the two genes was observed. However, during hepatic ontogenesis the cyclin G2 expression level decreased with age, whereas cyclin G1 expression increased. Transient expression of cyclin G2-green fluorescent protein (GFP) fusion protein in NIH3T3 cells showed that cyclin G2 is essentially a cytoplasmic protein, in contrast to the largely nuclear localization of cyclin G1. Our data suggest that, despite the close structural similarity between mouse cyclins G1 and G2, these proteins most likely perform distinct functions.

Palmitoyl-protein thioesterase gene expression in the developing mouse brain and retina: implications for early loss of vision in infantile neuronal ceroid lipofuscinosis.

Mutations in the palmitoyl-protein thioesterase (PPT) gene cause infantile neuronal ceroid lipofuscinosis (INCL), the clinical manifestations of which include the early loss of vision followed by deterioration of brain functions. To gain insight into the temporal onset of these clinical manifestations, we isolated and characterized a murine PPT (mPPT)-cDNA, mapped the gene on distal chromosome 4, and studied its expression in the eye and in the brain during development. Our results show that both cDNA and protein sequences of the murine and human PPTs are virtually identical and that the mPPT expression in the retina and in the brain is temporally regulated during development. Furthermore, the retinal expression of mPPT occurs much earlier and at a higher level than in the brain at all developmental stages investigated. Since many retinal and brain proteins are highly palmitoylated and depalmitoylation by PPT is essential for their effective recycling in the lysosomes, our results raise the possibility that inactivating mutations of the PPT gene, as occur in INCL, are likely to cause cellular accumulation of lipid-modified proteins in the retina earlier than in the brain. Consequently, the loss of vision occurs before the deterioration of brain functions in this disease.

Molecular Characterization of Murine Pancreatic Phospholipase A2

The pancreatic secretory phospholipase A(2) (sPLA(2)IB) is considered to be a digestive enzyme, although it has several important receptor-mediated functions. In this study, using the newly isolated murine sPLA(2)IB cDNA clone as a probe, we demonstrate that in addition to the pancreas, the sPLA(2)IB mRNA was expressed in extrapancreatic organs such as the liver, spleen, duodenum, colon, and lungs. We also demonstrate that sPLA(2)IB mRNA expression was detectable from the 17(th) day of gestation in the developing mouse fetus, coinciding with the time of completion of differentiation of the pancreas. Furthermore, the mRNA expression pattern of sPLA(2)IB was distinct from those of sPLA(2)IIA and cPLA(2) in various tissues examined. The murine sPLA(2)IB gene structure is well conserved, consistent with findings in other mammalian species, and this gene mapped to the region of mouse chromosome 5F1-G1.1. Taken together, our results suggest that sPLA(2)IB plays important roles both in the pancreas and in extrapancreatic tissues and that in the mouse, its expression is developmentally regulated.

Localization of the rat M1 muscarinic receptor gene to Chromosome 1q43-51

Muscarinic acetyl choline receptors are widely distributed in the brain (Levy et al. 1991). Five different subtypes have been identified that play important roles in pyramidal function, parasympathetic regulation, and in the learning process (Wei et al. 1994). Central muscarinic receptors, and specifically the M1 muscarinic receptor, have further been hypothesized as contributory to differences in a complex startle response in the spontaneously hypertensive rat (SHR) (Casto and Printz 1990) and to the development of hypertension (Buccafusco 1996). In humans a comparable startle response to mild stressors was reported (Valls-Sole et al. 1997). In the SHR, the expression of the M1 subtype is increased in most brain stem areas with critical function for blood pressure regulation. Therefore, the M1 muscarinic receptor gene may be considered as a candidate gene for hypertension. At present, however, it is not clear whether the M1 muscarinic receptor has a genetic linkage with hereditary hypertension. In an initial approach to evaluate such a potential role, we have in our present study established the position of the M1 muscarinic receptor gene in the rat genome. The cDNAs of all five muscarinic receptor subtypes have recently been cloned and sequenced in human, mouse, and rat (Bonner et al. 1987). The M1 muscarinic receptor gene consists of 1 single exon containing all of the coding and 3 8 untranslated sequence, and several exons containing only 5 8 untranslated sequences. In the human genome it has been mapped to the long arm of Chr 11 in the 11q13 region (Bonner et al. 1991). Receptorautoradiography in brainstem sections of normotensive (WKY) or hypertensive (SHR) rats demonstrated considerable overexpression of the M1 muscarinic receptor in several brainstem areas of the SHR such as nucleus tractus solitarii and rostroventrolateral medulla, areas with critical function for blood pressure regulation (Klett, submitted). Chromosomal assignment of the M1 muscarinic receptor gene was achieved by PCR and SSCP analysis of the DNA samples derived from a somatic cell hybrid panel of rat/mouse cell lines (Szpirer et al. 1984). A polymorphism between a laboratory mouse (strain C57BL/C) and the Wistar Kyoto (WKY) rat strain was established for a 329-b fragment of the M1 muscarinic receptor gene from near the poly(A) site corresponding to base 2140–2568 of Genbank Accession M16406. This fragment was PCR amplified by a specific primer set (forward primer: 5 8-ACAAATATCCAAGCGTGAGCAGG-38; backward primer: 5 8-GCTGAGGATGGAAAGAAAGAACAG-3 8) and confirmed by sequence analysis. This fragment showed length polymorphism between DNA of the rat and the mouse species on a 7% polyacrylamide gel and a clearly distinctive migration pattern on a 7% single-stranded conformation polymorphism (SSCP) polyacrylamide gel (Fig. 1). For SSCP analysis, 5ml of the PCR product was denatured in the presence of 80 mM methylmercury hydroxide, then heated to 85°C for 4 min and immediately quenched on ice before being loaded onto the gel. Under these conditions, SSCP analysis of the DNA fragments derived from the rat/mouse somatic cell hybrid panel DNAs revealed that the M1 muscarinic receptor gene is located on rat Chr 1. Only three clones were positive for the specific rat PCR product, that is, the clones containing rat Chr 1. For the other chromosomes, at least two discordant clones were counted. In the rat several polymorphic markers have been identified that show an association with the phenotype high blood pressure (Kovacs et al. 1997). The SA gene reported in a different study to be associated with the elevated blood pressure in the rat is also located on Chr 1 (Szpirer et al. 1993). There is, therefore, mounting evidence that polymorphisms in certain regions of Chr 1 may be linked to the increased blood pressure in the SHR. To confirm the chromosomal location and to further address a putative pathogenic role of the M1 muscarinic receptor in the pathogenesis of hypertension, we examined the exact position of the M1 receptor gene by fluorescence in situ hybridization (FISH). Chromosomes derived from rat fetal fibroblast cultures were used for (FISH). As a probe we used an 8.7-kb BamH1 fragment of the M1 muscarinic gene (7 kb of intron and 1.7 kb of coding exon sequences) ligated into pUC18, resulting in plasmid RR3p2. The probes were labeled for nick translation with biotinor digoxigenin-11-dUTP. The FISH protocol as described in detail elsewhere (Zimonjic et al. 1994) was followed, including pretreatment with RNase, denaturation in formamide, and hybridization with 200 ng of the DNA probe. Biotinand digoxigenin-labeled DNA was detected by fluorescein isothiocyanate (FITC)-conjugated avidin DVS (Vector Laboratories) and rhodamine-conjugated antidigoxigenin (Boehringer Mannheim, Inc.), respectively. Chromosomes were counterstained with propidium iodide (PI) or diaminophylindole (DAPI). For each chromosomal spread, two consecutive 8-bit grayscale images (FITC and propidium iodide, rhodamine and DAPI) were recorded by intensified CCD camera, enhanced for sharpness and contrast, merged (Genejoin MaxPix), and digitally printed on Tektronic’s Phaser II SDX dyesublimation printer. The location of fluorescent signals to specific chromosome bands was determined as previously described (Zimonjic et al. 1995). In two independent experiments, 70–80% of the metaphases hybridized with biotinylated or digoxigenin-labeled probes had symmetrical fluorescent signal on the largest chromosome readily identifiable as Chr 1. Seventy-five percent of the labeled spreads exhibited a signal at the same site on two homologous Chrs 1 (Fig. 2). A negligible nonspecific background level consisting of randomly distributed single fluorescent spots was observed in both experiments. The specific signal was directly localized on DSAPI Correspondence to: C. Klett Mammalian Genome 9, 476–478 (1998).

Assignment of the Bog gene (RBBP9) to syntenic regions of mouse chromosome 2G1-H1 and human chromosome 20p11.2 by fluorescence in situ hybridization.

We have isolated a rat cDNA encoding a novel protein called Bog from a rat liver epithelial cell line. Bog is a retinoblastoma (pRb) binding protein which can bind all of the retinoblastoma family members. Overexpression of Bog can displace E2F-1 from pRb rendering the cell insensitive to the growth inhibitory effects of TGF-ß1. Further, overexpression of Bog can lead to and is associated with a transformed phenotype (Woitach et al., 1998). Since overexpression of Bog is associated with transformation, the Bog gene was mapped to mouse chromosome 2G1–H1 and to human chromosome 20p11.2 to gain insight into the possible relationship with the transformed phenotype in other cell types. These are syntenic in mouse and human genomes. In the human, the region of 20p11.2 has been reported to be a recurrent region of rearrangement, demonstrating both amplification in several cancers (Knuutila et al., 1998) and deletion in Wilm’s tumors (Altura et al., 1996).