Mechanism for transcriptional gain of function resulting from chromosomal translocation in alveolar rhabdomyosarcoma.

The t(2;13) translocation of alveolar rhabdomyosarcoma results in tumor-specific expression of a chimeric transcription factor containing the N-terminal DNA-binding domain of PAX3 and the C-terminal transactivation domain of FKHR. Here we have tested the hypothesis that PAX3-FKHR gains function relative to PAX3 as a consequence of switching PAX3 and FKHR transactivation domains, which were previously shown to have similar potency but distinct structural motifs. In transient cotransfection assays with human expression constructs, we have demonstrated the increased ability of PAX3-FKHR to activate transcription of a reporter gene located downstream of multimerized e5, PRS-9, or CD19 DNA-binding sites in three cell lines. For example, PAX3-FKHR was 100-fold more potent than PAX3 as an activator binding to e5 sites in NIH 3T3 cells. To compare transactivation potency independent of PAX3-specific DNA binding, we tested GAL4 fusions of full-length PAX3 and PAX3-FKHR or their respective C-terminal transactivation domains on a reporter with GAL4 DNA-binding sites. In this context, full-length PAX3-FKHR was also much more potent than PAX3. Additionally, the activity of each full-length protein was decreased relative to its C-terminal domain, demonstrating that N-terminal sequences are inhibitory. By deletion analysis, we mapped a bipartite cis-acting inhibitory domain to the same subregions within the DNA-binding domains of both PAX3 and PAX3-FKHR. We have shown, however, that the structurally distinct transactivation domains of PAX3 and PAX3-FKHR differ 10- to 100-fold in their susceptibility to inhibition, thus elucidating a mechanism by which PAX3 gains enhanced function during oncogenesis.

Characterization of the transforming activity of p80, a hyperphosphorylated protein in a Ki-1 lymphoma cell line with chromosomal translocation t(2;5).

We have molecularly cloned a cDNA encoding a protein uniquely expressed and hyperphosphorylated at tyrosine residues in a Ki-1 lymphoma cell that contained chromosomal translocation t(2;5). The encoded protein p80 was shown to be generated by fusion of a protein-tyrosine kinase and a nucleolar protein B23/nucleophosmin (NPM). The coding sequence of this cDNA turned out to be virtually identical to that of the fusion cDNA for NPM-anaplastic lymphoma kinase (ALK) previously cloned from the transcript of the gene at the breakpoint of the same translocation. Overexpression of p80 in NIH 3T3 cells induced neoplastic transformation, suggesting that the p80 kinase is aberrantly activated. The normal form of p80 was predicted to be a receptor-type tyrosine kinase on the basis of its sequence similarity to the insulin receptor family of kinases. However, an immunofluorescence study using COS cells revealed that p80 was localized to the cytoplasm. Thus, subcellular translocation and activation of the tyrosine kinase presumably by its structural alteration would cause the malignant transformation. We also showed that a mutant p80 lacking the NPM portion was unable to transform NIH 3T3 cells. Thus, the NPM sequence is essential for the transforming activity, suggesting that the chromosomal translocation is responsible for the oncogenesis. Finally, Shc and insulin receptor substrate 1 (IRS-1) were tyrosine-phosphorylated and bound to p80 in p80-transformed cells. However, mutants of p80 that were defective for binding to and phosphorylation of Shc and insulin receptor substrate 1 could transform NIH 3T3 cells. Association of these mutants with GRB2 was still observed, suggesting that interaction of p80 with GRB2 but not with Shc or IRS-1 was relevant for cell transformation.

Chromosomal localization of the mammalian peptide-methionine sulfoxide reductase gene and its differential expression in various tissues.

Peptide methionine sulfoxide reductase (MsrA; EC 1.8.4.6) is a ubiquitous protein that can reduce methionine sulfoxide residues in proteins as well as in a large number of methyl sulfoxide compounds. The expression of MsrA in various rat tissues was determined by using immunocytochemical staining. Although the protein was found in all tissues examined, it was specifically localized to renal medulla and retinal pigmented epithelial cells, and it was prominent in neurons and throughout the nervous system. In addition, blood and alveolar macrophages showed high expression of the enzyme. The msrA gene was mapped to the central region of mouse chromosome 14, in a region of homology with human chromosomes 13 and 8p21.

cDNA structure, tissue distribution, and chromosomal localization of rat PC7, a novel mammalian proprotein convertase closest to yeast kexin-like proteinases.

By using reverse transcription-coupled PCR on rat anterior pituitary RNA, we isolated a 285-bp cDNA coding for a novel subtilisin/kexin-like protein convertase (PC), called rat (r) PC7. By screening rat spleen and PC12 cell lambda gt11 cDNA libraries, we obtained a composite 3.5-kb full-length cDNA sequence of rPC7. The open reading frame codes for a prepro-PC with a 36-amino acid signal peptide, a 104-amino acid prosegment ending with a cleavable RAKR sequence, and a 747-amino acid type I membrane-bound glycoprotein, representing the mature form of this serine proteinase. Phylogenetic analysis suggests that PC7 represents the most divergent enzyme of the mammalian convertase family and that it is the closest member to the yeast convertases krp and kexin. Northern blot analyses demonstrated a widespread expression with the richest source of rPC7 mRNA being the colon and lymphoid-associated tissues. In situ hybridization revealed a distinctive tissue distribution that sometimes overlaps with that of furin, suggesting that PC7 has widespread proteolytic functions. The gene for PC7 (Pcsk7) was mapped to mouse chromosome 9 by linkage analysis of an interspecific backcross DNA panel.

Hypothesis: "Rogue cell"-type chromosomal damage in lymphocytes is associated with infection with the JC human polyoma virus and has implications for oncopenesis.

The hemagglutination inhibition antibody titers against the JC and BK polyoma viruses (JCV and BKV, respectively) are significantly elevated in individuals exhibiting "rogue" cells among their cultured lymphocytes. However, the elevation is so much greater with respect to JCV that the BKV elevation could readily be explained by cross reactivity to the capsid protein of these two closely related viruses. The JCV exhibits high sequence homology with the simian papovavirus, simian virus 40 (SV40), and inoculation of human fetal brain cells with JCV produces polyploidy and chromosomal damage very similar to that produced by SV40. We suggest, by analogy with the effects of SV40, that these changes are due to the action of the viral large tumor antigen, a pluripotent DNA binding protein that acts in both transcription and replication. The implications of these findings for oncogenesis are briefly discussed.

Regulation of meiotic chromatin loop size by chromosomal position.

At meiotic prophase, chromatin loops around a proteinaceous core, with the sizes of these loops varying between species. Comparison of the morphology of sequence-related inserts at different sites in transgenic mice demonstrates that loop size also varies with chromosomal geography. Similarly, chromatin loop lengths differ dramatically for interstitially and terminally located hamster telomeric sequences. Sequences, telomeric or otherwise, located at chromosome termini, closely associate with the meiotic proteinaceous core, forming shorter loops than identical interstitial sequences. Thus, we present evidence that different chromatin packaging mechanisms exist for interstitial versus terminal chromosomal regions, which act separately from those operating at the level of the DNA sequence. Chromosomal position plays the dominant role in chromatin packaging.

Olfactory neuroblastoma is a peripheral primitive neuroectodermal tumor related to Ewing sarcoma.

Olfactory neuroblastoma (ONB) is a malignant tumor of the nasal mucosa whose histogenesis is unclear. A relationship to neuroblastoma (NB), a pediatric tumor of the sympathetic nervous system, is based on morphologic similarities and the expression of similar neural antigens. However, the clinical presentation of ONB differs from that of NB, and MYCN amplification characteristic of NB is not observed. We have therefore examined the relationship of this malignancy to other classes of neural tumors. In previous studies, two ONB cell lines demonstrated cytogenetic features and patterns of protooncogene expression suggestive of a relationship to the Ewing sarcoma family of childhood peripheral primitive neuroectodermal tumors (pPNETs). The pPNETs show t(11;22)(q24;q12) or t(21;22)(q22;q12) chromosomal translocations fusing the EWS gene from 22q12 with either the FL11 gene on 11q24 or the ERG gene on 21q22. We therefore analyzed ONBs for the presence of pPNET-associated gene fusions. Both cell lines showed rearrangement of the EWS gene, and fluorescence in situ hybridization (FISH) of each case demonstrated fusion of EWS and FL11 genomic sequences. Moreover, both lines expressed EWS/FL11 fusion transcripts with in-frame junctions between exon 7 of EWS and exon 6 of FL11 as described for pPNETs. We identified similar gene fusions in four of six primary ONB cases. None of the cases expressed tyrosine hydroxylase, a catecholamine biosynthetic enzyme widely expressed in NB. Our studies indicate that ONB is not a NB but is a member of the pPNET family.

DNA rearrangement mediated by inverted repeats.

Inverted repeats of DNA are widespread in the genomes of eukaryotes and prokaryotes and can mediate genome rearrangement. We studied rearrangement mediated by plasmid-borne inverted repeats in Escherichia coli. We show that inverted repeats can mediate an efficient and recA-independent recombination event. Surprisingly, the product of this recombination is not that of simple inversion between the inverted repeats, but almost exclusively an unusual head-to-head dimer with complex DNA rearrangement. Moreover, this recombination is dramatically reduced by increasing the distance separating the repeats. These results can be readily explained by a model involving reciprocal switching of the leading and lagging strands of DNA replication within the inverted repeats, which leads to the formation of a Holliday junction. Reciprocal strand switching during DNA replication might be a common mechanism for genome rearrangement associated with inverted duplication.

The chromosomal distribution of mouse odorant receptor genes.

Odorant receptors (ORs) on nasal olfactory sensory neurons are encoded by a large multigene family. Each member of the family is expressed in a small percentage of neurons that are confined to one of several spatial zones in the nose but are randomly distributed throughout that zone. This pattern of expression suggests that when the sensory neuron selects which OR gene to express it may be confined to a particular zonal gene set of several hundred OR genes but select from among the members of that set via a stochastic mechanism. Both locus-dependent and locus-independent models of OR gene choice have been proposed. To investigate the feasibility of these models, we determined the chromosomal locations of 21 OR genes expressed in four different spatial zones. We found that OR genes are clustered within multiple loci that are broadly distributed in the genome. These loci lie within paralogous chromosomal regions that appear to have arisen by duplications of large chromosomal domains followed by extensive gene duplication and divergence. Our studies show that OR genes expressed in the same zone map to numerous loci; moreover, a single locus can contain genes expressed in different zones. These findings raise the possibility that OR gene choice may be locus-independent or involve consecutive stochastic choices.

Influence of alkyltransferase activity and chromosomal locus on mutational hotspots in Chinese hamster ovary cells.

High-density mutational spectra have been established for exon 3 of the gene encoding adenine phosphoribosyltransferase (APRT) of the Chinese hamster ovary (CHO) cell line derivative D422 and closely related and/or modified lines by using the mutagen ethyl methanesulfonate (EMS). The total number of selectable sites (GC-->AT transitions yielding a selectable APRT- phenotype) was estimated at 31 based on our own accumulated data base of 136 sequenced exon 3 mutations and on literature reports. D422 and two other APRT hemizygous lines each yielded very similar spectra and showed two populations of mutable sites: (i) 24 "baseline" sites that followed the Poisson distribution and therefore were equally susceptible to mutation and (ii) two hotspots, one comprising a cluster at nucleotides 1293-1309 and the other at nucleotide 1365. Collectively, the latter sites were about 10-fold more frequently mutated than the others. CHO cells are mer- as they lack the repair enzyme O6-methylguanidine methyltransferase (EC 2.1.1.63). In modified repair-proficient CHO cells, the distribution of mutations among all of the 31 sites was random, with only 3 of the 19 GC-->AT transitions in the above hotspots. To determine whether the distribution was locus-dependent, two independent lines carrying single copies of transfected APRT genes were generated from a derivative of D422 carrying a deletion in the endogenous APRT gene. Nucleotides 1293-1309 were again no longer preferentially mutated, but the site at nucleotide 1365 was still a hotspot. We conclude that mutational spectra in mer- cells are at least in part locus dependent and that some sequences are particularly susceptible to EMS mutagenesis and perhaps also to methyltransferase repair.

Enterococcus faecalis pheromone binding protein, PrgZ, recruits a chromosomal oligopeptide permease system to import sex pheromone cCF10 for induction of conjugation.

Conjugative transfer of the plasmid pCF10 by Enterococcus faecalis donor cells occurs in response to a peptide sex pheromone, cCF10, secreted by recipients. The plasmid-encoded cCF10 binding protein, PrgZ, is similar in sequence to binding proteins (OppAs) encoded by oligopeptide permease (opp) operons. Mutation of prgZ decreased the sensitivity of donor cells to pheromone, whereas inactivation of the chromosomal E. faecalis opp operon abolished response at physiological concentrations of pheromone. Affinity chromatography experiments demonstrated the interaction of the pheromone with several putative intracellular regulatory molecules, including an RNA molecule required for positive regulation of conjugation functions. These data suggest that processing of the pheromone signal involves recruitment of a chromosomal Opp system by PrgZ and that signaling occurs by direct interaction of internalized pheromone with intracellular effectors.

Alignment of a 1.2-Mb chromosomal region from three strains of Rhodobacter capsulatus reveals a significantly mosaic structure.

High-resolution physical maps of the genomes of three Rhodobacter capsulatus strains, derived from ordered cosmid libraries, were aligned. The 1.2-Mb segment of the SB1003 genome studied here is adjacent to a 1-Mb region analyzed previously [Fonstein, M., Nikolskaya, T. & Haselkorn, H. (1995) J. Bacteriol. 177, 2368-2372]. Probes derived from the ordered cosmid set of R. capsulatus SB1003 were used to link cosmids from the St. Louis and 2.3.1 strain libraries. Cosmids selected this way did not merge into a single contig but formed several unlinked groups. EcoRV restriction maps of the ordered cosmids were then constructed using lambda terminase and fused to derive fragments of the chromosomal map. In order to link these fragments, their ends were transcribed to produce secondary probes for hybridization to gridded cosmid libraries of the same strains. This linking reduced the number of subcontigs to three for the St. Louis strain and one for the 2.3.1 strain. Hybridization of the same probes back to the ordered cosmid set of SB1003 positioned the subcontigs on the high-resolution physical map of SB1003. The final alignment of the restriction maps shows numerous large and small translocations in this 1.2-Mb chromosomal region of the three Rhodobacter strains. In addition, the chromosomes of the three strains, whose fine-structure maps can now be compared over 2.2 Mb, are seen to contain regions of 15-80 kb in which restriction sites are highly polymorphic, interspersed among regions in which the positions of restriction sites are highly conserved.

Site-directed mutagenesis of Azotobacter vinelandii ferredoxin I: cysteine ligation of the [4Fe-4S] cluster with protein rearrangement is preferred over serine ligation.

The [4Fe-4S] cluster of Azotobacter vinelandii ferredoxin I receives three of its four ligands from a Cys-Xaa-Xaa-Cys-Xaa-Xaa-Cys sequence at positions 39-45 while the fourth ligand, Cys20, is provided by a distal portion of the sequence. Previously we reported that the site-directed mutation of Cys20 to Ala (C20A protein) resulted in the formation of a new [4Fe-4S] cluster that obtained its fourth ligand from Cys24, a free cysteine in the native structure. That ligand exchange required significant protein rearrangement. Here we report the conversion of Cys20 to Ser (C20S protein), which gives the protein the opportunity either to retain the native structure and use the Ser20 O gamma as a ligand or to rearrange and use Cys24. X-ray crystallography demonstrates that the cluster does not use the Ser20 O gamma as a ligand; rather it rearranges to use Cys24. In the C20S protein the [4Fe-4S] cluster has altered stability and redox properties relative to either C20A or the native protein.

Nodulating strains of Rhizobium loti arise through chromosomal symbiotic gene transfer in the environment.

Rhizobia were isolated from nodules off a stand of Lotus corniculatus established with a single inoculant strain, ICMP3153, 7 years earlier in an area devoid of naturalized Rhizobium loti. The isolates showed diversity in growth rate, Spe I fingerprint of genomic DNA, and hybridization pattern to genomic DNA probes. The 19% of isolates that grew at the same rate as strain ICMP3153 were the only isolates that had the same fingerprint as strain ICMP3153. Sequencing of part of the 16S rRNA gene of several diverse isolates confirmed that they were not derived from the inoculant strain. Nevertheless, all non-ICMP3153 strains gave EcoRI and Spe I hybridization patterns identical to ICMP3153 when hybridized to nodulation gene cosmids. Hybridization of digests generated by the very rare cutting enzyme Swa I revealed that the symbiotic DNA region (at least 105 kb) was chromosomally integrated in the strains. The results suggest that the diverse strains arose by transfer of chromosomal symbiotic genes from ICMP3153 to nonsymbiotic rhizobia in the environment.

kappa-Opioid receptor in humans: cDNA and genomic cloning, chromosomal assignment, functional expression, pharmacology, and expression pattern in the central nervous system.

Using the mouse delta-opioid receptor cDNA as a probe, we have isolated genomic clones encoding the human mu- and kappa-opioid receptor genes. Their organization appears similar to that of the human delta receptor gene, with exon-intron boundaries located after putative transmembrane domains 1 and 4. The kappa gene was mapped at position q11-12 in human chromosome 8. A full-length cDNA encoding the human kappa-opioid receptor has been isolated. The cloned receptor expressed in COS cells presents a typical kappa 1 pharmacological profile and is negatively coupled to adenylate cyclase. The expression of kappa-opioid receptor mRNA in human brain, as estimated by reverse transcription-polymerase chain reaction, is consistent with the involvement of kappa-opioid receptors in pain perception, neuroendocrine physiology, affective behavior, and cognition. In situ hybridization studies performed on human fetal spinal cord demonstrate the presence of the transcript specifically in lamina II of the dorsal horn. Some divergences in structural, pharmacological, and anatomical properties are noted between the cloned human and rodent receptors.