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Hitoshi Sakano

Publications and source records attributed to Hitoshi Sakano.

At least 19 recordsLinked to original sources
PubMed2006

Antigen-receptor genes of the agnathan lamprey are assembled by a process involving copy choice.

Jawless vertebrates have acquired immunity but do not have immunoglobulin-type antigen receptors. Variable lymphocyte receptors (VLRs) have been identified in lamprey that consist of multiple leucine-rich repeat (LRR) modules. An active VLR gene is generated by the assembly of a series of variable gene segments, including many that encode LRRs. Stepwise assembly of the gene segments seems to occur by replacement of the intervening DNA between the 5' and 3' constant-region genes. Here we report that lamprey (Lethenteron japonicum) assemble their VLR genes by a process involving 'copy choice'. Regions of short homology seemed to prime copying of donor LRR-encoding sequences into the recipient gene. Those LRR-encoding germline sequences were abundant and shared extensive sequence homologies. Such genomic organization permits initiation of copying anywhere in an LRR-encoding module for the generation of various hybrid LRRs. Thus, a vast repertoire of recombinant VLR genes could be generated not only by copying of various LRR segments in diverse combinations but also by the use of multiple sites in an LRR gene segment for priming.

Alleles↗
PubMed2006

A neuronal identity code for the odorant receptor-specific and activity-dependent axon sorting.

In the mouse, olfactory sensory neurons (OSNs) expressing the same odorant receptor (OR) converge their axons to a specific set of glomeruli in the olfactory bulb. To study how OR-instructed axonal fasciculation is controlled, we searched for genes whose expression profiles are correlated with the expressed ORs. Using the transgenic mouse in which the majority of OSNs express a particular OR, we identified such genes coding for the homophilic adhesive molecules Kirrel2/Kirrel3 and repulsive molecules ephrin-A5/EphA5. In the CNGA2 knockout mouse, where the odor-evoked cation influx is disrupted, Kirrel2 and EphA5 were downregulated, while Kirrel3 and ephrin-A5 were upregulated, indicating that these genes are transcribed in an activity-dependent manner. Mosaic analysis demonstrated that gain of function of these genes generates duplicated glomeruli. We propose that a specific set of adhesive/repulsive molecules, whose expression levels are determined by OR molecules, regulate the axonal fasciculation of OSNs during the process of glomerular map formation.

Animals↗
PubMed2006

Odorant receptor-derived cAMP signals direct axonal targeting.

In mammals, odorant receptors (ORs) direct the axons of olfactory sensory neurons (OSNs) toward targets in the olfactory bulb. We show that cyclic adenosine monophosphate (cAMP) signals that regulate the expression of axon guidance molecules are essential for the OR-instructed axonal projection. Genetic manipulations of ORs, stimulatory G protein, cAMP-dependent protein kinase, and cAMP response element-binding protein shifted the axonal projection sites along the anteriorposterior axis in the olfactory bulb. Thus, it is the OR-derived cAMP signals, rather than direct action of OR molecules, that determine the target destinations of OSNs.

Animals↗
PubMed2006

Olfactory sensory neurons expressing class I odorant receptors converge their axons on an antero-dorsal domain of the olfactory bulb in the mouse.

Vertebrate odorant receptor (OR) genes are divided phylogenetically into two distinct classes: the fish-like class I and the terrestrial-specific class II. In the present study, we systematically analysed mouse class I OR genes (42 subfamilies) to elucidate the expression profiles in the olfactory epithelium (OE) and the projection sites of their olfactory sensory neurons (OSNs) in the olfactory bulb (OB). In situ hybridization (ISH) revealed that most class I OR genes (36 subfamilies) were expressed in the dorso-medial zone (zone 1) of the OE. Furthermore, there appeared to be no significant differences in the distributions of OSNs expressing class I genes within zone 1. These results indicate that there is a clear boundary between zone 1 and non-zone 1 areas in the OE. Some class I ORs are known to possess ligand specificity for aliphatic acids, aldehydes and alcohols. Our ISH analysis has revealed that OSNs expressing the class I ORs in zone 1 tend to converge their axons on a cluster of glomeruli in an antero-dorsal domain that is assumed to be involved in responses to the aliphatic compounds on the OB.

Animals↗
PubMed2005

Continuous and overlapping expression domains of odorant receptor genes in the olfactory epithelium determine the dorsal/ventral positioning of glomeruli in the olfactory bulb.

In mammals, olfactory signals received by odorant receptors (ORs) in the olfactory epithelium (OE) are converted to a topographical map of activated glomeruli in the olfactory bulb (OB). It has been reported that the OE can be divided into four topographically distinct zones and that olfactory sensory neurons (OSNs) expressing a particular OR gene are randomly distributed within one zone. Here, we analyzed 80 different class II OR genes for their expression patterns in the OE by in situ hybridization. It was found that the expression area in the OE does not always fit into one of the four conventional zones. Expression areas are specific to each OR gene and are arranged in an overlapping and continuous manner in the OE. We also analyzed a spatial relationship between the OE and the OB for OSN projection. Our transgenic as well as DiI retrograde staining experiments demonstrated that the dorsal/ventral arrangement of glomeruli in the OB is correlated with the expression areas of corresponding ORs along the dorsomedial/ventrolateral axis in the OE. The present study indicates that the OR gene choice may be more restricted by the OSN location in the OE than what has been thought.

Animals↗
PubMed2004

Joining mutants of RAG1 and RAG2 that demonstrate impaired interactions with the coding-end DNA.

In V(D)J joining of antigen receptor genes, two recombination signal sequences (RSSs), 12- and 23-RSSs, form a complex with the protein products of recombination activating genes, RAG1 and RAG2. DNaseI footprinting demonstrates that the interaction of RAG proteins with substrate RSS DNA is not just limited to the signal region but involves the coding sequence as well. Joining mutants of RAG1 and RAG2 demonstrate impaired interactions with the coding region in both pre- and postcleavage type complexes. A possible role of this RAG coding region interaction is discussed in the context of V(D)J recombination.

Base Sequence↗
PubMed2004

One neuron-one receptor rule in the mouse olfactory system.

Similar to the expression of antigen receptor genes in lymphocytes, the mammalian odorant receptor (OR) genes are expressed in a mutually exclusive and monoallelic manner in olfactory sensory neurons (OSNs). DNA rearrangement has long been regarded as a possible mechanism for the allelic exclusion of the OR genes. However, mice cloned from mature OSN nuclei expressed the full repertoire of ORs, and the possibility of irreversible gene translocation was excluded as a mechanism to activate a single OR gene in each OSN. How is allelic exclusion achieved in the olfactory system? Recent transgenic experiments indicated an inhibitory role of the OR protein in preventing further activation of other OR genes. Stochastic activation of an OR gene and negative-feedback regulation by the OR gene product might ensure the maintenance of the one neuron-one receptor rule in the mammalian olfactory system.

Animals↗
PubMed2004

In vitro processing of the 3'-overhanging DNA in the postcleavage complex involved in V(D)J joining.

The postcleavage complex involved in V(D)J joining is known to possess a transpositional strand transfer activity, whose physiological role is yet to be clarified. Here we report that RAG1 and RAG2 proteins in the signal end (SE) complex cleave the 3'-overhanging structure of the synthetic coding-end (CE) DNA in two successive steps in vitro. The 3'-overhanging structure is attacked by the SE complex imprecisely, near the double-stranded/single-stranded (ds/ss) junction, and transferred to the SE. The transferred overhang is then resolved and cleaved precisely at the ds/ss junction, generating either the linear or the circular cleavage products. Thus, the blunt-end structure is restored for the SE and variably processed ends are generated for the synthetic CE. This 3'-processing activity is observed not only with the core RAG2 but also with the full-length protein.

Animals↗
PubMed2003

Pub, a novel PU.1 binding protein, regulates the transcriptional activity of PU.1.

PU.1 is a member of the Ets family of transcription factors and plays critical roles in the development of hematopoietic cells such as macrophages and B cells. To elucidate the molecular mechanism(s) underlying the regulation of PU.1 function, we screened for PU.1 interacting proteins using a yeast two-hybrid approach. As a result, a novel PU.1 binding factor, which we termed Pub, was isolated. The Pub protein has one B-box zinc finger domain, followed by a coiled-coil region and a B30.2-like domain, these features being characteristic of the tripartite motif (TRIM) family of protein. The PEST domain of PU.1 was found to interact with the N-terminal portion of Pub, a region that includes the TRIM which is considered to mediate protein-protein interactions. Northern blot and RT-PCR analyses demonstrated that Pub is predominantly expressed in hematopoietic tissues and cells where PU.1 is also expressed. Using a luciferase-based assay, we showed that Pub inhibited the transcriptional activity of PU.1. Moreover, the B-box zinc finger domain of Pub was critical for this inhibitory activity. These data suggest that Pub may be important in regulating the transcriptional activity of PU.1.

Amino Acid Sequence↗
PubMed2003

Negative feedback regulation ensures the one receptor-one olfactory neuron rule in mouse.

In the mouse olfactory system, each olfactory sensory neuron (OSN) expresses only one odorant receptor (OR) gene in a monoallelic and mutually exclusive manner. Such expression forms the genetic basis for OR-instructed axonal projection of OSNs to the olfactory bulb of the brain during development. Here, we identify an upstream cis-acting DNA region that activates the OR gene cluster in mouse and allows the expression of only one OR gene within the cluster. Deletion of the coding region of the expressed OR gene or a naturally occurring frame-shift mutation allows a second OR gene to be expressed. We propose that stochastic activation of only one OR gene within the cluster and negative feedback regulation by that OR gene product are necessary to ensure the one receptor-one neuron rule.

Alleles↗
PubMed2003

O-MACS, a novel member of the medium-chain acyl-CoA synthetase family, specifically expressed in the olfactory epithelium in a zone-specific manner.

In rodents, the olfactory epithelium (OE) can be divided into four topographically distinct zones, and each member of the odorant receptor (OR) gene family is expressed only in one particular zone. To study the functional significance of the zonal structure of the OE, we searched for genes expressed in a zone-specific manner by using the differential display method. Among the clones isolated from the rat OE, we characterized a novel olfactory protein termed O-MACS, a member of the medium-chain acyl-CoA synthetase family. The o-macs gene encodes a protein of 580 amino acids, sharing 56-63% identity with other MACS family proteins. RT-PCR analysis demonstrated that the o-macs gene is expressed only in the OE, unlike other MACS family genes. In situ hybridization revealed that the o-macs transcripts are present in the neuronal cell layer of olfactory sensory neurons (OSNs) as well as in the supporting and basal cell layers in the most dorso-medial area (zone 1) of the OE. Developmental analysis revealed that the o-macs gene is already expressed on embryonic day 11.5, before the onset of the OR gene expression, in a restricted area within the rat olfactory placode. Recombinant O-MACS protein tagged with c-Myc and His6 demonstrated an acyl-CoA synthetase activity for fatty acid activation, and protein localization to mitochondria like other MACS family proteins. The present study indicates that this novel protein may play important roles in processing odorants in a zone-specific manner, or the zonal patterning of the OE during development.

Amino Acid Sequence↗
PubMed2003

Developmental elimination of ectopic projection sites for the transgenic OR gene that has lost zone specificity in the olfactory epithelium.

In rodents, olfactory receptor (OR) genes are expressed in one of four zones in the olfactory epithelium (OE), and olfactory sensory neurons (OSNs) expressing the same OR project their axons to a specific set of glomeruli on the olfactory bulb (OB). Using the yeast artificial chromosome (YAC) transgenic system, we have analysed the expression of the murine OR gene MOR29A of the MOR28 cluster located on chromosome 14. Although expression of the endogenous MOR29A was restricted to the most dorsomedial zone, the transgenic MOR29A (Tg MOR29A) was expressed in all four zones of the OE. When the OB of the transgenic mouse was analysed, the axons of the OSNs expressing Tg MOR29A were found to project not only to the dorsal side but also to the ventral side of the OB as well. The ectopic projection sites on the ventral side gradually disappear during postnatal development. Naris occlusion prevents this elimination, suggesting that odorant stimulation is involved in eliminating the ectopic projection sites.

Animals↗
PubMed2003

The high-mobility-group box protein SSRP1/T160 is essential for cell viability in day 3.5 mouse embryos.

The high-mobility-group (HMG) SSRP1 protein is a member of a conserved chromatin-remodeling complex (FACT/DUF/CP) implicated in DNA replication, basal and regulated transcription, and DNA repair. To assist in the functional analysis of SSRP1, the Ssrp1 gene was targeted in murine embryonic stem cells, and the mutation was introduced into the germ line. Embryos homozygous for the targeted allele die soon after implantation, and preimplantation blastocysts are defective for cell outgrowth and/or survival in vitro. The Ssrp1 mutation was also crossed into a p53 null background without affecting growth and/or survival defects caused by loss of Ssrp1 function. Thus, Ssrp1 appears to encode nonredundant and p53-independent functions that are essential for cell viability.

Alleles↗
PubMed2002

Stomatin-related olfactory protein, SRO, specifically expressed in the murine olfactory sensory neurons.

We identified a stomatin-related olfactory protein (SRO) that is specifically expressed in olfactory sensory neurons (OSNs). The mouse sro gene encodes a polypeptide of 287 amino acids with a calculated molecular weight of 32 kDa. SRO shares 82% sequence similarity with the murine stomatin, 78% with Caenorhabditis elegans MEC-2, and 77% with C. elegans UNC-1. Unlike other stomatin-family genes, the sro transcript was present only in OSNs of the main olfactory epithelium. No sro expression was seen in vomeronasal neurons. SRO was abundant in most apical dendrites of OSNs, including olfactory cilia. Immunoprecipitation revealed that SRO associates with adenylyl cyclase type III and caveolin-1 in the low-density membrane fraction of olfactory cilia. Furthermore, anti-SRO antibodies stimulated cAMP production in fractionated cilia membrane. SRO may play a crucial role in modulating odorant signals in the lipid rafts of olfactory cilia.

Adenylyl Cyclases↗
PubMed2002

Genomic analysis of the murine odorant receptor MOR28 cluster: a possible role of gene conversion in maintaining the olfactory map.

Genomic analysis was performed for the murine odorant receptor (OR) genes. The MOR28 cluster on chromosome 14 was extensively studied. It contains six OR genes, MOR28, 10, 83, 29A, 29B and 30. The human homolog of this cluster is located on the human chromosome 14, and contains five OR genes, HOR28/10, 83, 29A, 29B and 30. Sequence comparison of these OR gene paralogs and orthologs suggests that the coding homologies are accounted for not only by recent gene duplication, but also by gene conversion among the coding sequences within the cluster. A possible role of gene conversion in the olfactory system is discussed in the context of the olfactory map.

Animals↗
PubMed2002

BET, a novel neuronal transmembrane protein with multiple EGF-like motifs.

Using the signal sequence trap method, we have identified a cDNA clone coding for a type I transmembrane protein, BET, with 10 epidermal growth factor (EGF) motifs in the extracellular domain. In situ hybridization revealed that the bet mRNA is specifically expressed in the mitral/tufted cells in the olfactory bulb, Purkinje cells in the cerebellum, and pyramidal cells in the hippocampus. Using polyclonal antibodies, we have demonstrated that the BET protein is highly glycosylated and is localized in patches in the dendrites and in the somata of neurons. Since the predicted structure of BET shares many similarities with the Notch ligands, this novel protein may play crucial roles in establishing the neuronal networks in the olfactory system, cerebellum, and hippocampus. BET is the first transmembrane protein containing only multiple EGF-like repeats specifically expressed in the brain.

Animals↗