Receptor Coding Sequence Research Articles

Olfactory receptor coding sequences cause silencing of episomal constructs in multiple cell lines

The mammalian olfactory system consists of sensory neurons with specialized odorant-binding capability accomplished by mutually exclusive odorant receptor (OR) expression. Mutually exclusive OR expression is a complex multi-step process regulated by a number of cis and trans factors, including pan-silencing of all OR genes preceding the robust and stable expression of the one OR selected in each sensory neuron. We transfected two olfactory-placode-derived cell lines modeling immature odorant sensory neurons, as well as the GD25 fibroblast cell line, with episomes containing CMV-driven GFP and TK-driven hygromycin reporter genes. We inserted various coding sequences, along with an IRES, immediately upstream of the GFP gene to produce bicistronic mRNAs driven from the local CMV promoter. We found that the presence of several OR coding sequences resulted in significantly diminished episomal expression of GFP in all three cell lines. These findings suggest that OR coding sequences have intrinsic self-silencing capability that might facilitate mutually exclusive OR expression in olfactory sensory neurons by making it less likely that multiple ORs acquire an above-threshold level of expression at once.

P1629Crispr/Cas9 to elucidate cardiac specific effects of Gdf11

Abstract Background Heart failure with preserved ejection fraction (HFpEF) is a prevalent clinical condition in the aging population. HFpEF lacks specific therapies with a significant impact on survival, different therapeutic approaches have indeed failed, indicating a specific need in understanding the pathophysiology of the disease. Cardiac hypertrophy is one of the main features of HFpEF that contributes to impair diastolic function. Reducing ventricular stiffness associated with cardiomyocyte hypertrophy may indeed result in improved diastolic filling. Growth differentiation factor 11 (Gdf11), a TGF-β family factor, has been identified as a circulating factor able to reduce cardiac hypertrophy. Purpose Similar to myostatin (Gdf8), Gdf11 promotes the activation of atrophy pathways that induce ubiquitination of sarcomeric proteins. Gdf11 and Gdf8 activate both type I and II Tgf-β receptors, specifically by interacting with Acvr2a and Acvr2b (type II) and Alk 4/5/7 (type I), they activate Smad 3/4 pathway. Our data indicate a specific and more potent effect of Gdf11 in reducing cardiomyocytes size that is not recapitulated by Gdf8. Understanding the specific effect of Gdf11 on cardiomyocytes is crucial to develop therapeutic strategies to target the hypertrophic phenotype. Methods To investigate the effects of specific type I receptor KO on Smad signaling cascade we performed our preliminary experiment on HL-1 cells, a cardiac muscle cell line carrying a doxycycline inducible Cas9 transgene. Cell sensitivity of HL-1 to Gdf11 and Gdf8 was tested by performing a dose-response curve using a luciferase reporter for Smad 3/4 pathway activation (CAGA12). Selective Tgf-β type I receptor KO was induced using, for each receptor, two sgRNAs that have been designed to cleave the receptor coding sequence creating INDEL mutations and disrupt proper translation of the protein and confirmed by western blotting. Smad 3/4 activity was measured using a CAGA12-luciferase assay on HL-1-Cas9 transduced cells. Results Our data in HL-1 cells confirm a more potent effect of Gdf11 in activating Smad 3/4 pathway when compared to Gdf8 (Fig. 1A). Selective Alk4 and Alk5 KO induced a similar reduction in Smad 3/4 activation for both Gdf11 and Gdf8. Interestingly, Alk7 KO significantly reduced Gdf11 signaling that was not recapitulated when using Gdf8, suggesting that Alk7 receptor is crucial for Gdf11-dependent Smad 3/4 activation in HL-1 cells (Fig. 1B). Figure 1 Conclusions Our preliminary results indicate that part of Gdf11 cardiac specificity when compared to Gdf8 may reside in the usage of Alk7 for signaling. Alk7 has proposed as a protective factor for pathological cardiac hypertrophy by negatively regulating Mek-Erk1/2 signalling. Our results are now under investigation in vitro using mouse neonatal cardiomyocytes expressing Cas-9 and in vivo using cardiac-specific-Cas9-expressing C57 transgenic mice, in resting condition and after induction of pathological hypertrophy.

Retroviral UNC13D Gene Transfer Restores Cytotoxic Activity of T Cells Derived from Familial Hemophagocytic Lymphohistiocytosis Type 3 Patients In Vitro.

Familial hemophagocytic lymphohistiocytosis (FHL) is a group of life-threatening, autosomal recessive disorders of severe hyperinflammation. FHL type 3 (FHL-3) accounts for about 30% of FHL cases. It is characterized by mutations in the UNC13D gene that give rise to functionally impaired or absent Munc13-4 protein, resulting in impaired secretion of lytic granules by cytotoxic lymphocytes. Etoposide-based therapy is currently used as the standard of care that results in around 60% 5-year survival, illustrating the need for novel treatment approaches. Key problems include treatment toxicity and failure to induce or maintain remission of the hyperinflammation. Instead of immunosuppression, transplantation of autologous gene-corrected T cells can be envisaged as an approach to restore the impaired immune reaction. This study established a protocol that enabled hyperactivated, FHL-3 patient-derived T cells to be cultured and a codon-optimized UNC13D expression cassette to be delivered by either alpha- or gamma-retroviral gene transfer. The data demonstrate that the established protocol can be applied to FHL-3 patient cells with various genetic backgrounds and that gamma-retroviral UNC13D transfer restored expression of functional Munc13-4, as well as degranulation capacity and cell-mediated cytotoxicity of those patient-derived CD8+ T cells. Furthermore, the study shows that the co-introduction of a truncated low-affinity nerve growth factor receptor coding sequence enabled the therapeutic effect to be optimized by enriching transduced cells in a Good Manufacturing Practice-compliant manner. In conclusion, this study lays the foundation for an adaptive immune cell therapy approach aiming at immunological stabilization of FHL-3 patients with autologous, immune-competent T cells prior to hematopoietic stem-cell transplantation.

Mapping odorant receptors in neural space

The olfactory system represents chemical information as spatial and temporal activity patterns across glomeruli in the olfactory bulb. The degree to which chemical features are mapped systematically in the glomerular array has remained controversial. Using mouse genetics, we have begun to elucidate the factors influencing the organization of glomeruli in the olfactory bulb. We find that the three broad classes of olfactory receptor genes (Class I, Class II, and TAARs) are roughly organized into distinct domains of the dorsal olfactory bulb. Despite the fact that odorant receptors are involved in axon guidance, this mechanism is receptor-independent and derives from the axon guidance identities of sensory neurons in the epithelium. Consequently, the same receptor (i.e. a given response specificity) can be mapped to different bulb domains when the receptor is expressed by different cell types. Our overall results show that odorant responsiveness can be decoupled from glomerular position, and that glomerular position is not necessarily determined by stimulus specificity. In this view, the glomerular map may be shaped by the selection of receptor coding sequences (which determine receptor identity) and their corresponding promoters (which determines cell type expression) over evolutionary time. The precision of potential chemotopic maps that could form in the glomerular array is limited by the precision of this “evolutionary mapping” mechanism.

Obese Locus in WNIN/Obese Rat Maps on Chromosome 5 Upstream of Leptin Receptor

WNIN/Obese (WNIN/Ob) rat a new mutant model of metabolic syndrome was identified in 1996 from an inbred Wistar rat strain, WNIN. So far several papers are published on this model highlighting its physical, biochemical and metabolic traits. WNIN/Ob is leptin resistant with unaltered leptin or its receptor coding sequences - the two well-known candidate genes for obesity. Genotyping analysis of F2 progeny (raised from WNIN/Ob × Fisher - 344) in the present study localized the mutation to a recombinant region of 14.15cM on chromosome 5. This was further corroborated by QTL analysis for body weight, which narrowed this region to 4.43 cM with flanking markers D5Rat256 & D5Wox37. Interval mapping of body weight QTL shows that the LOD score peak maps upstream of leptin receptor and shows an additive effect suggesting this as a novel mutation and signifying the model as a valuable resource for studies on obesity and metabolic syndrome.

A common gene exclusion mechanism used by two chemosensory systems

Sensory coding strategies within vertebrates involve the expression of a limited number of receptor types per sensory cell. In mice, each vomeronasal sensory neuron transcribes monoallelically a single V1R pheromone receptor gene, chosen from a large V1R repertoire. The nature of the signals leading to this strict receptor expression is unknown, but is apparently based on a negative feedback mechanism initiated by the transcription of the first randomly chosen functional V1R gene. We show, in vivo, that the genetic replacement of the V1rb2 pheromone receptor coding sequence by an unrelated one from the odorant receptor gene M71 maintains gene exclusion. The expression of this exogenous odorant receptor in vomeronasal neurons does not trigger the transcription of odorant receptor-associated signalling molecules. These results strongly suggest that despite the different odorant and vomeronasal receptor expression sites, function and transduction cascades, a common mechanism is used by these chemoreceptors to regulate their transcription.

Molecular cloning and pharmacological characterization of rat melatonin MT 1 and MT 2 receptors

In order to interpret the effects of melatonin ligands in rats, we need to determine their activity at the receptor subtype level in the corresponding species. Thus, the rat melatonin rMT 1 receptor was cloned using DNA fragments for exon 1 and 2 amplified from rat genomic DNA followed by screening of a rat genomic library for the full length exon sequences. The rat rMT 2 receptor subtype was cloned in a similar manner with the exception of exon 1 which was identified by screening a rat genomic library with exon 1 of the human hMT 2 receptor. The coding region of these receptors translates proteins of 353 and 364 amino acids, respectively, for rMT 1 and rMT 2. A 55% homology was observed between both rat isoforms. The entire contiguous rat MT 1 and MT 2 receptor coding sequences were cloned, stably expressed in CHO cells and characterized in binding assay using 2-[ 125I]-Iodomelatonin. The dissociation constants ( K d) for rMT 1 and rMT 2 were 42 and 130 pM, respectively. Chemically diverse compounds previously characterized at human MT 1 and MT 2 receptors were evaluated at rMT 1 and rMT 2 receptors, for their binding affinity and functionality in [ 35S]-GTPγS binding assay. Some, but not all, compounds shared a similar binding affinity and functionality at both rat and human corresponding subtypes. A different pharmacological profile of the MT 1 subtype has also been observed previously between human and ovine species. These in vitro results obtained with the rat melatonin receptors are thus of importance to understand the physiological roles of each subtype in animal models.

Cis-Regulatory Elements within the Odorant Receptor Coding Region

Complex regulatory mechanisms lead to the expression in each olfactory neuron of one allele of only one of the 1000 odorant receptor (OR) genes. In this issue, Nguyen et al. (2007) provide evidence that regulatory elements residing within the coding region of OR genes are involved in the singularity of OR gene expression.

The C57BL/6NCrl‐lb Mouse: A New Model for Metabolic Syndrome

A novel mutation, lb in the leptin receptor gene, has been identified in C57 BL/6NCrl mice from a barrier production colony at Charles River Laboratories. Animals heterozygous for the mutation exhibit a wild-type phenotype. The mutant lb/lb mice have been characterized by metabolic, genetic, and phenotypic methods. The lb/lb mutant mice display key features of the metabolic syndrome; they are markedly obese, and hyperinsulinemic by 8 weeks of age, with insulin levels exceeding 200 ng/ml by 18 weeks of age. When compared to other models of metabolic syndrome, mutant lb mice also had hyperglycemia greater than that of the B6.V-Lepob/J (ob) mouse, but less than the BKS.Cg-m+/+Leprdb/J (db). Like db mice, the lb mice had increased leptin levels, higher in fact, than db mice at 17–18 weeks of age. Genetic complementation studies crossing mice heterozygous for the lb mutation with Leprdb heterozygous mice produced 25% fat and 75% lean offspring, confirming that the mutations are in the same gene. DNA sequence analysis of the leptin receptor coding sequence in lb mice has not revealed any mutations. These mice have characteristics of the ob and the db mice. This distinct phenotype makes these mice an additional model for drug discovery studies of metabolic syndrome.

Mapping and characterization of the dominant black colour locus in sheep.

Dominant black pigment synthesis in sheep is caused by alterations of the melanocortin-1 receptor (MC1-R) coding sequence. Using five bovine microsatellite markers we have mapped the sheep MC1-R gene to chromosome 14, corresponding to the location in other mammalian species. The existence of two independent mutations, both causing an amino acid substitution, in distantly related breeds of sheep, support the hypothesis that the observed black pigment synthesis is caused by a mutual effect of the two mutations. As similar mutations are found separately at both locations in dominant black variants of other animal species, it is also possible that any of the two mutations could be sufficient for a partial pigment switch.

Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54.

Hypogonadotropic hypogonadism is defined as a deficiency of the pituitary secretion of follicle-stimulating hormone and luteinizing hormone, which results in the impairment of pubertal maturation and of reproductive function. In the absence of pituitary or hypothalamic anatomical lesions and of anosmia (Kallmann syndrome), hypogonadotropic hypogonadism is referred to as isolated hypogonadotropic hypogonadism (IHH). A limited number of IHH cases are due to loss-of-function mutations of the gonadotropin-releasing hormone receptor. To identify additional gene defects leading to IHH, a large consanguineous family with five affected siblings and with a normal gonadotropin-releasing hormone receptor coding sequence was studied. Homozygosity whole-genome mapping allowed the localization of a new locus within the short arm of chromosome 19 (19p13). Sequencing of several genes localized within this region showed that all affected siblings of the family carried a homozygous deletion of 155 nucleotides in the GPR54 gene. This deletion encompassed the splicing acceptor site of intron 4-exon 5 junction and part of exon 5. The deletion was absent or present on only one allele in unaffected family members. GPR54 has been initially identified as an orphan G protein-coupled receptor with 40% homology to galanin receptors. Recently, a 54-aa peptide derived from the KiSS1 protein was identified as a ligand of GPR54. The present study shows that loss of function of GPR54 is a cause of IHH, and it identifies GPR54 and possibly KiSS1 protein-derived peptide as playing a major and previously unsuspected role in the physiology of the gonadotropic axis.

Analysis of the human growth hormone receptor and IGF-I coding sequences in children with growth disorders.

Analysis of GHR and IGF-I coding sequences in 47 children with normal serum levels of GH, low IGF-I and growth disorders generally did not show mutation in the genes studied. Only one boy had a mutation located in the fifth exon of the GHR gene (C-->T in codon 88). This suggests that the growth disorders in this group of children might be due to a defect in a DNA region regulating expression of the GHR and IGF1 genes or genes involved in their regulation.

Structure and transcription of the human m3 muscarinic receptor gene.

We have isolated and characterized the human m3 muscarinic receptor gene and its promoter. Using 5' rapid amplification of cDNA ends (RACE), internal polymerase chain reaction (PCR), and homology searching to identify EST clones, we determined that the cDNA encoding the m3 receptor comprises 4,559 bp in 8 exons, which are alternatively spliced to exclude exons 2, 4, 6, and/or 7; the receptor coding sequence occurs within exon 8. Analysis of P1 artificial chromosome (PAC) and bacterial artificial chromosome (BAC) clones and of PCR- amplified genomic DNA, and homology searching of human chromosome 1 sequence provided from the Sanger Centre (Hinxton, Cambridge, UK) revealed that the m3 muscarinic receptor gene spans at least 285 kb. A promoter fragment containing bp -1240 to +101 (relative to the most 5' transcription start site) exhibited considerable transcriptional activity during transient transfection in cultured subconfluent, serum-fed canine tracheal myocytes, and 5' deletion analysis of promoter function revealed the presence of positive transcriptional regulatory elements between bp -526 and -269. Sequence analysis disclosed three potential AP-2 binding sites in this region; five more AP-2 consensus binding motifs occur between bp -269 and +101. Cotransfection with a plasmid expressing human AP-2alpha substantially increased transcription from m3 receptor promoter constructs containing 526 or 269 bp of 5' flanking DNA. Furthermore, m3 receptor promoter activity was enhanced by long-term serum deprivation of canine tracheal myocytes, a treatment that is known to increase AP-2 transcription-promoting activity in these cells. Together, these data suggest that expression of the human m3 muscarinic receptor gene is regulated in part by AP-2 in airway smooth muscle.

Screening for insulin-like growth factor-I receptor mutations in patients with Silver-Russell syndrome.

Individuals with a deletion of 15q26.1-->qter which contains the insulin-like growth factor-I (IGF-I) receptor gene exhibit phenotypical similarities to patients with Silver-Russell syndrome (SRS) who represent a group of short children affected by pre- and postnatal growth failure and several dysmorphic features. The genetic basis of SRS is presumably heterogeneous. The IGF-I receptor gene is a good SRS candidate gene because the biological effects of the major growth factors IGF-I and -II are transmitted through the IGF-I receptor during pre- and postnatal growth. All 12 patients studied exhibited the classical SRS phenotype with low birth weight, connatal macrocephaly, typical facial features and short stature at presentation. Klinodactyly of the fifth finger was found in ten patients, and skeletal asymmetry in seven. Chromosomal analysis gave normal results. Uniparental disomy of chromosome 7 was excluded. After reverse transcription of RNA, 19 overlapping cDNA fragments encompassing the whole IGF-I receptor coding sequence were PCR-amplified and screened for mutations by single strand conformation analysis (SSCP). In eight of 12 patients studied, we detected four SSCP polymorphisms which were directly sequenced. All single base exchanges (C948A, C2343T, G3174A and G3915A) were silent variants of the IGF-I receptor gene, two of which were also present in control individuals. In conclusion, for the first time, the complete coding region of the IGF-I receptor gene was screened for mutations in patients with SRS. Our data do not support the suggestion that the IGF-I receptor is frequently involved in the pathogenesis of SRS.

Cloning and characterization of the mouse alpha1C/A-adrenergic receptor gene and analysis of an alpha1C promoter in cardiac myocytes: role of an MCAT element that binds transcriptional enhancer factor-1 (TEF-1).

alpha1-Adrenergic receptor (AR) subtypes in the heart are expressed by myocytes but not by fibroblasts, a feature that distinguishes alpha1-ARs from beta-ARs. Here we studied myocyte-specific expression of alpha1-ARs, focusing on the subtype alpha1C (also called alpha1A), a subtype implicated in cardiac hypertrophic signaling in rat models. We first cloned the mouse alpha1C-AR gene, which consisted of two exons with an 18 kb intron, similar to the alpha1B-AR gene. The receptor coding sequence was >90% homologous to that of rat and human. alpha1C-AR transcription in mouse heart was initiated from a single Inr consensus sequence at -588 from the ATG; this and a putative polyadenylation sequence 8.5 kb 3' could account for the predominant 11 kb alpha1C mRNA in mouse heart. A 5'-nontranscribed fragment of 4.4 kb was active as a promoter in cardiac myocytes but not in fibroblasts. Promoter activity in myocytes required a single muscle CAT (MCAT) element, and this MCAT bound in vitro to recombinant and endogenous transcriptional enhancer factor-1. Thus, alpha1C-AR transcription in cardiac myocytes shares MCAT dependence with other cardiac-specific genes, including the alpha- and beta-myosin heavy chains, skeletal alpha-actin, and brain natriuretic peptide. However, the mouse alpha1C gene was not transcribed in the neonatal heart and was not activated by alpha1-AR and other hypertrophic agonists in rat myocytes, and thus differed from other MCAT-dependent genes and the rat alpha1C gene.

Molecular cloning, sequence analysis and pharmacological properties of the porcine 5-HT(1D) receptor.

A cDNA encoding the full-length 5-HT(1D) receptor derived from porcine cerebral cortex was amplified, cloned and sequenced, using guinea-pig 5-HT(1D) receptor coding sequence oligonucleotide primers in reverse transcription-polymerase chain reaction (RT - PCR). The 5' and 3' ends of the porcine 5-HT(1D) receptor cDNA were verified by inverse PCR. Sequence analysis of porcine 5-HT(1D) receptor cDNA revealed an open reading frame of 1134 nucleotides encoding a polypeptide of 377 amino acids having 92% homology with the human 5-HT(1D) receptor and 88 - 90% homology with other species homologues. The porcine 5-HT(1D) receptor cDNA was further subcloned into a mammalian expression vector pcDNA3 and expressed in monkey Cos-7 cells. Radioligand binding assays using either [(3)H]-5-CT or [(3)H]-GR125743 on Cos-7 cell membranes showed that pK(i) values of 14 serotonin ligands were highly correlated with those obtained with the human 5-HT(1D) receptor. Nonetheless, a selective antagonist at the human 5-HT(1D) receptor, BRL15572, only poorly recognized the porcine homologue. Using membranes from cells co-expressing the porcine 5-HT(1D) receptor and rat G(alphail)Cys(351) Ile protein, it was shown that 5-HT and zolmitriptan increased, while ketanserin decreased basal [(35)S]-GTPgammaS binding. The potency of zolmitriptan in the [(35)S]-GTPgammaS binding assay (pEC(50): 8. 46+/-0.08) agreed with its affinity in displacing the radioligands [(3)H]-5-CT and [(3)H]-GR125743 (pK(i): 8.38+/-0.15 and 8.67+/-0.08, respectively). In conclusion, we have established the cDNA sequence and pharmacology of the cloned porcine 5-HT(1D) receptor. This information would be useful in exploring the role of divergent amino acid residues in the receptor-ligand interaction as well as the role of 5-HT(1D) receptor in pathophysiological processes relevant for novel drug discovery in diseases such as migraine.

Cloning, expression and pharmacological characterisation of the mouse somatostatin sst 5 receptor

The mouse somatostatin (somatotropin release inhibiting factor, SRIF) sst 5 receptor coding sequence was cloned from a mouse BALB/c genomic library. It shows 97% and 81% homology with the corresponding rat and human receptors, respectively. The msst 5 receptor messenger RNA (mRNA) is present at low levels in the adult mouse brain, with significant expression in a few nuclei only, e.g. in the septum (lateral septal nuclei) or the amygdala (medial amygdaloid nucleus); very few signals were observed in the mesencephalon, metencephalon, and myelencephalon (except the dorsal motor nucleus of the vagus nerve). The msst 5 receptor was stably expressed in the hamster fibroblast cell line CCL39-SRE-Luci, which harbours the luciferase reporter gene driven by the serum responsive element. [ 125I]LTT-SRIF-28 ([Leu 8, D-Trp 22, 125I-Tyr 25]-SRIF-28), [ 125I]Tyr 10-CST, [ 125I]CGP 23996, and [ 125I]Tyr 3-octreotide labelled msst 5 receptors with high affinity (pK d values: 11.0, 10.15, 9.75 and 9.43) and in a saturable manner, but defined different Bmax values: 697, 495, 540 and 144 fmoles/mg, respectively. [ 125I]LTT-SRIF-28-labelled sites displayed the following rank order: SRIF-28> rCST-14> somatuline > CGP-23996= SRIF-14= octreotide, whereas [ 125I]Tyr 3-octreotide-labelled sites displayed a different profile: octreotide > SRIF-28> rCST-14= somatuline > SRIF-14> CGP-23996. The pharmacological profiles determined with [ 125I]LTT-SRIF-28, [ 125I]CGP 23996 and [ 125I]Tyr 10-CST correlated highly significantly (r 2 =0.88–0.99), whereas [ 125I]Tyr 3-octreotide binding was rather divergent (r 2 =0.77). Also, human and mouse sst 5 receptor profiles are very different, e.g. r 2 =0.385 for [ 125I]Tyr 10-CST and r 2 =0.323 for [ 125I]LTT-SRIF-28-labelled sites. Somatostatin induces expression of luciferase reporter gene in CCL39-SRE-Luci cells. The profile was consistent with a msst 5 receptor-mediated effect although apparent potency in the luciferase assay was much reduced compared to radioligand binding data: Octreotide = SRIF-28> rCST-14= SRIF-14= CGP-23996. Octreotide, SRIF-28, BIM23052 and D Tyr Cyanamid 154806 behaved as full or nearly full agonists in comparison to SRIF-14, whereas the other compounds had relative efficacies of 40 to 70%. The present study shows that agonists radioligands define apparently different receptor populations in terms of number of sites and pharmacological profile in cells expressing a single recombinant receptor. These variations suggest that the conformation of the ligand receptor complex may vary depending on the agonist. Further, the msst 5 receptor, although primarily coupled to Gi/Go proteins, is able to stimulate luciferase gene expression driven by the serum responsive element. Finally, it is suggested that putative sst 2 selective agonists e.g. octreotide, RC160 or BIM23027 show similar or higher potency at msst 5 receptors than SRIF-14.

Cloning of partial putative gonadotropin hormone receptor sequence from fish.

A search for the presence of mariner-like elements in the Labeo rohita genome by polymerase chain reaction led to the amplification of a partial DNA sequence coding for a putative transmembrane domain of gonadotropin hormone receptor. The amplified DNA sequence shows a high degree of homology to the available turkey and human luteinizing and follicle stimulating hormone receptor coding sequences. This is the first report on cloning such sequences of piscine origin.

Stable amino-acid sequence of the mannose-6-phosphate/insulin-like growth-factor-II receptor in ovarian carcinomas with loss of heterozygosity and in breast-cancer cell lines.

The mannose-6-phosphate/insulin-like growth factor 2 receptor (Man-6-P/IGFII receptor) is involved in lysosomal enzyme sorting, IGFII degradation and pro-TGFbeta activation. Genetic alterations in hepatocarcinomas and a few breast cancers suggest that this receptor behaves as a tumor suppressor. Moreover, hypersecretion and Man-6-P-independent targeting of cathepsins in breast and ovarian carcinomas also suggest alterations in this receptor. We studied the Man-6-P/IGFII receptor gene in 8 ovarian carcinomas, and 4 breast- and ovarian-cancer cell lines. The results confirmed a frequent loss of heterozygosity (LOH) in the 6q27-qter region in 5 out of 8 ovarian carcinomas. We used 23 overlapping RT-PCR fragments to sequence the whole coding region of the Man-6-P/IGFII receptor. The 2491 amino-acid sequence of this receptor was perfectly conserved in 9 out of 10 of our samples, including MCF7 and MDA-MB231 cells and 5 ovarian carcinomas with LOH. This allowed us to rectify the 2 previously published sequences which differed in several bases, and to propose a consensus amino-acid sequence. The only amino-acid change (Thr --> Ala) was in BG1 ovarian-cancer cells, and was due to an A-to-G substitution on one allele at nucleotide 2561. We found no bi-allelic alterations in the 9 ovarian carcinomas, but 3 silent nucleotide substitutions leading to a lower cordon usage in 2 ovarian carcinomas with LOH. No mutation of the Man-6-P/IGFII receptor coding sequence was found in breast-cancer cell lines to explain the cathepsin-D hypersecretion and Man-6-P-independent trafficking described. We propose that, in breast and ovarian cancers, the frequent loss of one allele, associated with over-expression of some of its ligands, might be sufficient to saturate the receptor protein, displace the ligands to other sites, and consequently facilitate tumor progression.

Mutational analysis of three tumor suppressor genes in two models of rat hepatocarcinogenesis.

An albumin-simian virus 40 (SV40) large T-antigen (T-Ag) transgenic model and a chemically induced model of multistage hepatocarcinogenesis were created in our laboratory to study the molecular mechanisms involved in the genesis and progression of neoplasia in the rat liver. In the study presented here, these two models of rat hepatocarcinogenesis were used to perform a comparative mutational analysis of three tumor suppressor genes involved in hepatic neoplastic growth. By using polymerase chain reaction-single strand conformation polymorphism analysis and sequencing, exons 5-8 of the p53 tumor suppressor gene and a region between nt 4325 and 4479 of the rat mannose 6-phosphate/insulin-like growth factor 2 receptor (M6p/Igf2r) coding sequence were screened. The latter is homologous to the human M6P/IGF2r coding sequence which is mutated in human hepatocellular carcinoma. A complete single strand conformation polymorphism analysis of the entire coding region of the rat adenomatous polyposis coli (Apc) gene was also performed for the first time in rat tumorigenic samples. Twenty-six chemically induced rat hepatocellular carcinomas, 21 neoplasms from the livers of SV40 T-Ag animals, and five immortalized hepatic cell lines from the transgenic rats were evaluated. None of the hepatic tumors exhibited mutations in the regions analyzed. The albumin-SV40 T-Ag transgenic cell line L-60, derived from normal hepatic tissue, had two mutations in contiguous codons of exon 5 of the p53 gene: a GGT --> GTT missense transversion in codon 183 and a silent mutation in codon 184. The transversion, which may affect the DNA binding domain of the p53 protein, probably originated during cell culture and may have been positively selected because it gave a growth advantage to the mutated cells. The studied region of the M6p/Igf2r gene was not found to be mutated in these two models of rat hepatocarcinogenesis. Although M6p/Igf2r, Apc, and p53 have been shown to be mutated in a variety of human hepatic proliferative diseases, our results indicate that aberrations in these genes may not be necessary for liver carcinogenesis in the rat.