Existence of Pink1 antisense RNAs in mouse and their localization.

PTEN-induced kinase 1 (PINK1), which is identified as the gene transactivated by the tumor suppressor PTEN, has been found to be one of the causative genes in Parkinson's disease (PD). In order to understand PD, rodent models containing affected Pink1 such as loss-of-function mutations have been exploited. Recently, natural antisense RNA of PINK1 has been demonstrated to be involved in the regulation of the PINK1 locus. However, no antisense RNAs of Pink1 except for human have been reported so far. Therefore, in the present study, while searching for the Pink1 antisense RNAs in mouse, we found that the antisense RNAs are transcribed from a mouse genomic region corresponding to the human region from which the antisense RNAs are produced. Further, we investigated the localization of the antisense RNAs in mouse brain using in situ hybridization; this demonstrated that the antisense RNAs were localized in the regions of brain where the Pink1 mRNA was found. In addition, the mRNA and antisense RNAs were found more densely in the hippocampus than in the other brain regions in newborn and 1-week-old mice, while those RNAs were found uniformly in the mouse brain regions of embryo day (E) 14, E17, and 8-weeks-old.

Localization of sense and antisense transcripts of Prdx2 gene in mouse tissues.

Recently, it has been reported that antisense RNAs are transcribed from a large number of genes in various species including human and mouse. The Prdx2 gene, which is indicated to be involved in signal transduction related to platelet-derived growth factor as well as to protection from oxidizing agents, has been shown to produce sense and antisense transcripts. To obtain clues for possible roles of Prdx2 antisense transcripts, we have performed Northern blot analysis and in situ hybridization on tissues of 8-week-old C57BL/6J mice. The Northern blot analysis revealed that major parts of sense and antisense transcripts were poly(A-)-RNA. The analysis of the fractionated RNA of fibroblasts indicated that the poly(A-)-RNA would be localized in the cytoplasm of cells. The in situ hybridization demonstrated that the sense and antisense transcripts were localized in almost the same limited areas of brain, testis, and spleen. It also revealed that the sense and antisense transcripts coexisted in Purkinje cells. In thymus and stomach, the antisense transcripts were detected, but sense transcripts were not. When tissues of BALB/c mice were examined by in situ hybridization, the observations were essentially the same as those of C57BL/6J except that it appeared that the amounts of sense and antisense transcripts in testis of BALB/c were greater than those in C57BL/6J, and that the amounts of antisense transcripts in stomach of BALB/c were much smaller than those in C57BL/6J.

Systematic genome-wide approach to positional candidate cloning for identification of novel human disease genes.

BACKGROUND: Recent large-scale genome projects afford a unique opportunity to identify many novel disease genes and thereby better understand the genetic basis of human disease. Functional Annotation of Mouse (FANTOM) 2, the largest mouse transcriptome project yet, provides a wealth of data on novel genes, splice variants and non-coding RNA, and provides a unique opportunity to identify novel human disease genes. AIMS: To demonstrate the power of combining the FANTOM 2 cDNA dataset with a positional candidate approach and bioinformatics analysis to identify genes underlying human genetic disease. RESULTS: By mapping all FANTOM 2 cDNA to the human genome, we were able to identify mouse clones that co-localised on the human genome with mapped but uncloned human disease loci. By this method we identified mouse and corresponding human genes mapping within the loci of 100 different human genetic diseases (mapped interval of <5 cM). Of particular interest was the elucidation through FANTOM 2 novel mouse gene data of candidate human genes for the following: (i) developmental -disorders: neural tube defect, Meckel syndrome, Wolf--Hirschhorn syndrome and keratosis follicularis spinulosa decalvans cum ophiasi; (ii) neurological disorders: benign familial infantile convulsions 3, early-onset cerebellar ataxia with retained tendon reflexes, infantile-onset spinocerebellar ataxia and vacuolar neuro-myopathy and (iii) cancer-related syndromes: tylosis with oesophageal cancer and low-grade B-cell chronic lymphatic leukaemia. CONCLUSIONS: The FANTOM 2 data will dramatically accelerate efforts to identify genes underlying human disease. It will also facilitate the creation of transgenic mouse models to help elucidate the function of potential human disease genes.

Expression of the GLI2 oncogene and its isoforms in human basal cell carcinoma.

BACKGROUND: Mutations of the patched (Ptc) gene, a developmental regulator implicated in the signalling pathway via sonic hedgehog (Shh) and smoothened (Smo), play an essential pathogenic role in the development of basal cell carcinomas (BCCs). We previously reported the upregulation of Shh signal transducers, including Ptc, Smo and hedgehog-interacting protein, in BCCs. In vertebrates, specific downstream effectors in the Shh signalling pathway include three zinc-finger transcription factors, Gli1, Gli2 and Gli3. Gli1 possesses only an activation domain, while Gli2 and Gli3 contain both activation and repression domains. It remains unclear which of these transcription factors are responsible for the development of BCCs. OBJECTIVES: To examine the expression pattern of Gli2 mRNA by human BCCs in comparison with those by normal human skin and various skin tumours. METHODS: We performed quantitative reverse transcriptase-polymerase chain reaction analyses with a series of samples from BCCs, other skin tumours and normal skin. RESULTS: We found that Gli2 mRNA expression was enhanced in the BCCs we examined, whereas there was no significant increase in other skin tumours or normal skin. Of four spliced Gli2 isoforms designated Gli2alpha, beta, gamma and delta, the expression of Gli2beta mRNA was increased the most in BCCs. CONCLUSIONS: As Gli2beta is an isoform spliced at the first splicing site containing a repression domain and consists of an intact activation domain, its overexpression may lead to the upregulation of the Shh signalling pathway, thereby inducing BCCs.

Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs.

Only a small proportion of the mouse genome is transcribed into mature messenger RNA transcripts. There is an international collaborative effort to identify all full-length mRNA transcripts from the mouse, and to ensure that each is represented in a physical collection of clones. Here we report the manual annotation of 60,770 full-length mouse complementary DNA sequences. These are clustered into 33,409 'transcriptional units', contributing 90.1% of a newly established mouse transcriptome database. Of these transcriptional units, 4,258 are new protein-coding and 11,665 are new non-coding messages, indicating that non-coding RNA is a major component of the transcriptome. 41% of all transcriptional units showed evidence of alternative splicing. In protein-coding transcripts, 79% of splice variations altered the protein product. Whole-transcriptome analyses resulted in the identification of 2,431 sense-antisense pairs. The present work, completely supported by physical clones, provides the most comprehensive survey of a mammalian transcriptome so far, and is a valuable resource for functional genomics.

Expression of a sonic hedgehog signal transducer, hedgehog-interacting protein, by human basal cell carcinoma.

BACKGROUND: Aberrant activation of the hedgehog pathway has been identified in various human tumours, including familial and sporadic basal cell carcinomas (BCCs). It has been postulated that binding of sonic hedgehog protein (SHH) to its receptor, patched protein (PTC), releases the inhibitory effect of PTC against smoothened protein (SMO), another protein of the SHH signalling pathway. The positive SMO signalling is not downregulated in BCCs because of the mutational inactivation of PTC. Recently, hedgehog-interacting protein (HIP) was found to bind to SHH directly and attenuate SHH signalling like PTC, while its expression was induced by SHH signals. OBJECTIVES: To examine the expression patterns of HIP, SHH and PTC gene mRNA by human BCCs, in comparison with those by normal human skin and various skin tumours. METHODS: We performed quantitative reverse transcriptase-polymerase chain reaction analyses with a series of samples from BCCs, other skin tumours and normal skin. RESULTS: We found that the mRNA expression of both HIP and PTC genes was enhanced in all samples of BCCs, whereas none of the other skin tumours tested exhibited an increased level of such mRNAs as compared with normal skin. The transcription of the SHH gene, however, was at a baseline level in most BCCs. CONCLUSIONS: These results indicate that both HIP and PTC gene expression are specifically involved in the development of BCCs, and that the production of HIP is linked with the expression of the PTC gene but not the SHH gene.

Speculations on the role of natural antisense transcripts in mammalian X chromosome evolution.

Recent comprehensive transcriptome analyses in mice have revealed tremendous numbers of natural antisense transcripts in a hitherto ignored category of genes in eukaryotes. We discuss the possible biological roles of these transcripts and their relationships with mammalian sex chromosome evolution. Of 60,770 full-length cDNA sequences, as many as 2,500 pairs of sense-antisense transcripts (SATs) with the potential to form RNA duplex via their complementary sequences have been identified. This high number of antisense transcripts indicates their generic roles in gene expression regulation. These SATs are almost evenly distributed along the chromosomes, with the exception of the X chromosome. The rate of occurrence of SATs on the X chromosome is one-third to one-half that on the autosomes, and this under-representation must be related to a property intrinsic to the X chromosome. Here we hypothesize that monoallelically expressed antisense RNA regulates its sense partner, but that this regulatory system cannot operate on the mammalian X chromosome, as the mammalian X chromosome is effectively in a hemizygous state in both sexes. Loss of such regulation may be involved in the evolution of the X chromosome itself.

Computational analysis of full-length mouse cDNAs compared with human genome sequences.

Although the sequencing of the human genome is complete, identification of encoded genes and determination of their structures remain a major challenge. In this report, we introduce a method that effectively uses full-length mouse cDNAs to complement efforts in carrying out these difficult tasks. A total of 61,227 RIKEN mouse cDNAs (21,076 full-length and 40,151 EST sequences containing certain redundancies) were aligned with the draft human sequences. We found 35,141 non-redundant genomic regions that showed a significant alignment with the mouse cDNAs. We analyzed the structures and compositional properties of the regions detected by the full-length cDNAs, including cross-species comparisons, and noted a systematic bias of GENSCAN against exons of small size and/or low GC-content. Of the cDNAs locating the 35,141 genomic regions, 3,217 did not match any sequences of the known human genes or ESTs. Among those 3,217 cDNAs, 1,141 did not show any significant similarity to any protein sequence in the GenBank non-redundant protein database and thus are candidates for novel genes.

Functional annotation of a full-length mouse cDNA collection.

The RIKEN Mouse Gene Encyclopaedia Project, a systematic approach to determining the full coding potential of the mouse genome, involves collection and sequencing of full-length complementary DNAs and physical mapping of the corresponding genes to the mouse genome. We organized an international functional annotation meeting (FANTOM) to annotate the first 21,076 cDNAs to be analysed in this project. Here we describe the first RIKEN clone collection, which is one of the largest described for any organism. Analysis of these cDNAs extends known gene families and identifies new ones.

Anti-tumor immunity against CT26 colon tumor in mice immunized with plasmid DNA encoding beta-galactosidase fused to an envelope protein of endogenous retrovirus.

Endogenous retroviral gene products have been recognized as being expressed in human cancerous tissues. However, these products have not been shown to be antigenic targets for T-cells, possibly due to immune tolerance. Since carcinogen-induced colon tumor CT26 expresses an envelope protein, gp70, of an endogenous ecotropic murine leukemia virus that is comparable to human tumor-associated antigens, we examined whether a DNA vaccine containing the gp70 gene induces protective immunity against CT26 cells. Injection of mice with plasmid DNA (pDNA) encoding gp70 alone failed to induce anti-gp70 antibody (Ab) or anti-CT26 cytotoxic T lymphocyte (CTL) responses. However, immunization with pDNA encoding the beta-galactosidase (beta-gal)/gp70 fusion protein induced anti-gp70 Ab and anti-CT26 CTL responses and conferred protective immunity against CT26 cells. These results indicate that beta-gal acts as an immunogenic carrier protein that helps in the induction of immune responses against the poorly immunogenic gp70. Considering these results, it is possible that potential tolerance to the endogenous retroviral gene products expressed by human tumors may be overcome by DNA vaccines that contain an endogenous retroviral gene fused to genes encoding immunogenic carrier proteins.

YAC/BAC-based physical and transcript mapping around the gracile axonal dystrophy (gad) locus identifies Uchl1, Pmx2b, Atp3a2, and Hip2 genes.

We generated a yeast artificial chromosome (YAC)/bacterial artificial chromosome (BAC)-based physical and transcript map of a region containing the gracile axonal dystrophy (gad) locus on mouse chromosome 5. The YAC/BAC contig consists of 13 YAC and 49 BAC clones onto which 4 genes, 40 expressed sequence tags, and 7 new DNA polymorphisms were ordered. Using this physical map, we mapped Uchl1 encoding ubiquitin carboxyl-terminal hydrolase I, whose deletion has been determined to cause the gad mutation. We also mapped three other recently identified genes: Hip2, encoding Huntingtin interacting protein 2; Atp3a2, encoding a P-type ATPase; and Pmx2b, encoding PHOX2b.

X-Linked syndrome of polyendocrinopathy, immune dysfunction, and diarrhea maps to Xp11.23-Xq13.3.

We describe genetic analysis of a large pedigree with an X-linked syndrome of polyendocrinopathy, immune dysfunction, and diarrhea (XPID), which frequently results in death during infancy or childhood. Linkage analysis mapped the XPID gene to a 17-cM interval defined by markers DXS8083 and DXS8107 on the X chromosome, at Xp11. 23-Xq13.3. The maximum LOD score was 3.99 (recombination fraction0) at DXS1235. Because this interval also harbors the gene for Wiskott-Aldrich syndrome (WAS), we investigated mutations in the WASP gene, as the molecular basis of XPID. Northern blot analysis detected the same relative amount and the same-sized WASP message in patients with XPID and in a control. Analysis of the WASP coding sequence, an alternate promoter, and an untranslated upstream first exon was carried out, and no mutations were found in patients with XPID. A C-->T transition within the alternate translation start site cosegregated with the XPID phenotype in this family; however, the same transition site was detected in a normal control male. We conclude that XPID maps to Xp11.23-Xq13.3 and that mutations of WASP are not associated with XPID.

Expression of sonic hedgehog signal transducers, patched and smoothened, in human basal cell carcinoma.

In basal cell nevus syndrome (BCNS) patients, mutations of a gene, patched (ptc), which encodes a putative signal transducer of sonic hedgehog protein (SHH), were found and are thought to be one of the major causes of BCNS. The SHH signaling pathway is an important developmental pathway, and ptc protein (PTC) is a suppressive component serving as a receptor for the secreted SHH. Another transmembrane protein, smoothened (SMO), forms a complex with PTC and regulates this signaling pathway. Recent transgenic studies have strengthened the importance of the SHH signaling system in the etiology of basal cell carcinoma (BCC). In this study, we examined the expression patterns of mRNA for ptc and smo in two different BCC subtypes and normal skin. We found that the expressions of ptc and smo mRNA were enhanced in the tumor nests of the nodular BCC, especially at the advancing portions, but were under the detectable level in the superficial BCC cases examined, indicating that ptc and smo mRNA expressions might be associated with BCC tumor progression and divide the BCC histologic types into two subtypes, superficial and nodular types. In addition, no obvious signals for ptc and smo mRNA were detected in the normal human epidermis, appendages, or seborrheic keratosis, indicating that the abnormal proliferation of follicular epithelial cells caused by ptc, smo and/or other genetic changes, which also cause ptc and smo overexpressions, might result in BCC tumor formation.

Intragenic deletion in the gene encoding ubiquitin carboxy-terminal hydrolase in gad mice.

The gracile axonal dystrophy (gad) mouse is an autosomal recessive mutant that shows sensory ataxia at an early stage, followed by motor ataxia at a later stage. Pathologically, the mutant is characterized by 'dying-back' type axonal degeneration and formation of spheroid bodies in nerve terminals. Recent pathological observations have associated brain ageing and neurodegenerative diseases with progressive accumulation of ubiquitinated protein conjugates. In gad mice, accumulation of amyloid beta-protein and ubiquitin-positive deposits occur retrogradely along the sensory and motor nervous systems. We previously reported that the gad mutation was transmitted by a gene on chromosome 5 (refs 10,11). Here we find that the gad mutation is caused by an in-frame deletion including exons 7 and 8 of Uchl1, encoding the ubiquitin carboxy-terminal hydrolase (UCH) isozyme (Uch-l1) selectively expressed in the nervous system and testis. The gad allele encodes a truncated Uch-l1 lacking a segment of 42 amino acids containing a catalytic residue. As Uch-l1 is thought to stimulate protein degradation by generating free monomeric ubiquitin, the gad mutation appears to affect protein turnover. Our data suggest that altered function of the ubiquitin system directly causes neurodegeneration. The gad mouse provides a useful model for investigating human neurodegenerative disorders.

Identification of a novel gene, OASIS, which encodes for a putative CREB/ATF family transcription factor in the long-term cultured astrocytes and gliotic tissue.

Gliosis is a characteristic response of astrocytes to inflammation and trauma of the central nervous system (CNS). To study the mechanisms underlying gliosis, we performed differential display screening for genes specifically induced in long-term cultured astrocytes used as an in vitro gliosis model. We identified and characterized a gene (named OASIS, for old astrocyte specifically-induced substance) expressed in long-term cultured mouse astrocytes, or 'old astrocytes (OA)'. The OASIS gene encoded a putative transcription factor belonging to the cyclic AMP responsive element binding protein/activating transcription factor (CREB/ATF) gene family, with homology to box B-binding factor-2 (BBF-2), a Drosophila transcription factor. Its expression was developmentally regulated; OASIS mRNA was primarily expressed in the salivary gland and cartilage in the mouse embryo and it was transiently upregulated in the brain during postnatal two weeks. The expression became weaker in the adult brain. We also demonstrated that an expression of the OASIS mRNA was induced in response to the cryo-injury of the mouse cerebral cortex. The distribution pattern of the OASIS-positive cells in the injured cortex was very similar to that of the glial fibrillary acidic protein (GFAP)-positive cells. These results suggest that OASIS protein may play a role in gliotic events.

Eos: a novel member of the Ikaros gene family expressed predominantly in the developing nervous system.

We identified a novel member of the Ikaros gene family, which has critical roles in the development of lymphoid lineages. This gene, which we named Eos, was expressed predominantly in the developing central and peripheral nervous system. Eos protein could interact with itself and Ikaros protein through its C-terminal portion in the yeast two hybrid assay. These findings suggested that Eos may have important roles in neural development similarly to the Ikaros family in the development of hemolymphoid tissue.

Dictyostelium discoideum nuclear plasmid Ddp5 is a chimera related to the Ddp1 and Ddp2 plasmid families.

The 14,955-bp Dictyostelium discoideum nuclear plasmid Ddp5 contains six transcribed open reading frames. One of these is related to the rep gene of the Ddp2 plasmid, and the other five are related to genes present on the Ddp1 plasmid. The absence of a homolog of the Ddp1 G1 gene, coupled with the presence of the Ddp2 rep gene homolog and of a 1.6-kb inverted repeat analogous to the inverted repeats on members of the Ddp2 plasmid family, suggests that Ddp5 uses Ddp2-like replication and copy number control mechanisms and that it should be assigned to the Ddp2 plasmid family. Ddp5 carries genes homologous to the D1/D3 and D2 genes of the Ddp1 plasmid as well as the Ddp1 G2/G3/D4, G5/D6, and G6/G4/D5 genes. The products of the Ddp5 G2-like, G5-like, and G6-like genes are likely to be transcription factors regulating the expression of themselves and of the other Ddp5 genes. The D1-like and D2-like genes may confer a selective advantage to plasmid-bearing cells, because they can be deleted from plasmid-based shuttle vectors with no apparent effect on vector maintenance. Updated sequence information for the Ddp1 G5/D6, D1/D3, and D2 genes as well as the Dmp1 and Dmp2 G5-like genes is presented. The locations of introns in the G5-like and D1-like genes of Ddp5 and in the homologous genes of the Ddp1, Dmp1, and Dmp2 plasmids were identified. These introns all have GU at the 5' intron border and AG at the 3' intron border, are short (59 to 71 nucleotides), and are AT-rich. A conserved HHCC domain was identified in the G5 proteins; this is a putative zinc binding domain and may be involved in protein-DNA interaction.

Ornithine decarboxylase overexpression in mouse 10T1/2 fibroblasts: cellular transformation and invasion.

BACKGROUND: Ornithine decarboxylase (ODC) plays a pivotal role in the synthesis of polyamines, a group of chemical compounds that are essential for cell growth. Recent reports have shown that ODC overexpression may be involved in malignant transformation of immortalized NIH 3T3 cells. We have demonstrated that ODC-overproducing mouse breast cancer cells are more invasive in vitro than control cells. However, little information is available concerning the relationship between ODC overexpression, tumor invasion, and metastasis and the signal transduction pathways involved in ODC-induced transformation and invasion. PURPOSE: Our purpose was twofold: 1) to determine whether ODC overexpression is directly involved in tumor cell invasion and 2) to determine whether ODC overexpression induces mitogen-activated protein (MAP) kinase activities that are associated with cell growth and transformation. METHODS: We transfected C3H clone 8 mouse 10T1/2 fibroblasts with an expression vector that carries a complementary DNA encoding rat ODC. Neomycin-resistant cells that overproduced ODC (4-6.5 times the control levels) were isolated. The transformed phenotype of these cells was determined by assessing colony formation and anchorage-independent growth in soft agar. The invasiveness of the cells was studied by means of an invasion assay that used Matrigel-coated filters in Boyden chambers. The MAP kinase activity of the cells was assayed by an in-gel kinase assay, using myelin basic protein as the substrate. RESULTS: Overexpression of ODC induced not only cell transformation and anchorage-independent growth in soft agar but also invasiveness through a Matrigel-coated filter. The ODC-overproducing transfectants showed enhanced MAP kinase activity that paralleled the magnitude of cell invasiveness. CONCLUSIONS: ODC plays a pivotal role not only in cell transformation but also in cancer cell invasion. ODC overexpression enhanced MAP kinase activity. IMPLICATIONS: Our results demonstrate a connection between the polyamine/ODC and the MAP kinase signal transduction pathways and suggest that MAP kinase may play a pivotal role in ODC-induced cell transformation and invasion.

Locations of crossover breakpoints within the CMT1A-REP repeat in Japanese patients with CMT1A and HNPP.

The crossover breakpoints for Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) are located in the CMT1A-REP repeat flanking a 1.5-Mb region of chromosome 17p11.2-12. The precise locations of the breakpoints are heterogeneous, and we analyzed the relative frequency distribution of breakpoints in 33 unrelated Japanese CMT1A and 3 unrelated HNPP families. The CMT1A-REP repeat region was divided into five regions, A, B, C, D and E, based on restriction site differences between the proximal and distal CMT1A-REP repeats. The frequency distribution of breakpoints within the CMT1A-REP repeat in the Japanese patients was 3% in region A, .78% in B/C and 19% in D, which is similar to that in Caucasian patients. This result also indicates that an 8-kb region defined by region B/C is a recombinational hotspot within the CMT1A-REP repeat in Japanese patients.