Sex determination by ultrasonography prior to artificial breeding of the pufferfish Arothron manilensis (Tetraodontiformes, Tetraodontidae).

Artificial breeding was induced in the pufferfish Arothron manilensis following ultrasonographic sex determination. Hormonal treatment of mature male and female specimens followed the collection (and measurement) of fully developed eggs by cannulation. Fertilized eggs (0.85 ± 0.02 mm diameter) were spherical, demersal and individually adhesive. Hatching occurred 5 days after fertilization, larvae being 2.23 ± 0.15 mm in total length and 2.08 ± 0.14 mm in notochord length. The larvae had all died within 14 days of hatching. To improve artificial breeding techniques for A. manilensis, it is necessary to determine more appropriate timing for hormone injection, as well as feeding nutrient-enhanced SS type Brachionus sp. to newly hatched larvae.

Molecular phylogenetics of the fresh water sleepers Odontobutis (Gobiiformes: Odontobutidae) and its implications on biogeography of freshwater ichthyofauna of East Asia.

The genus Odontobutis is a group of freshwater fishes endemic to East Asia. Phylogenetic relationships among the Odontobutis species have never been fully tested due to incomplete taxon sampling and that molecular data have not been collected in many Odontobutis species. In the present study, we sampled 51 specimens from all known eight Odontobutis species with two outgroups (Perccottus glenii and Neodontobutis hainanensis). We collected sequence data of 4434 single-copy nuclear coding loci using gene capture and Illumina sequencing. A robust phylogeny of the Odontobutis with many individuals for each species was built, supporting the current taxonomy that all extant Odontobutis species are valid. The two species from Japan (O. hikimius + O. obscurus) formed an independent clade sister to the "continental odontobutids", whereas the species from southern China (O. sinensis + O. haifengensis) separated from the rest species of the genus. Surprisingly species from the lower reaches of the Yangtze River (O. potamophilus) was more closely related to species from the Korean Peninsula and northeastern China than to the middle reaches of the Yangtze River, such that their relationship was ((O. sinensis + O. haifengensis)(O. platycephala + (O. yaluensis + (O. potamophilus + O. interruptus)))). Divergence time among the Odontobutis was estimated using 100 most clock-like loci and three fossil calibration points. The crown group of the Odontobutis was estimated at 9.0 Ma during the late Miocene (5.6-12.7 Ma, 95% HPDs). Ancestral range of the genus was reconstructed using Reconstruct Ancestral States in Phylogenies (RASP) and BioGeoBEARS. The result suggested that the common ancestor of modern Odontobutis probably was distributed in Japan, southern China or the Korean Peninsula. A series of geographical events in East Asia since the late Miocene, such as the opening of the Japan/East Sea, rapid uplift of the Tibetan Plateau and climate change in the northern region of the Yellow River might account for diversification and current distribution pattern of the Odontobutis.

Tandem spawning and development in the African freshwater pufferfish Tetraodon schoutedeni.

To advance breeding techniques for the African freshwater pufferfish Tetraodon schoutedeni and observe tandem spawning closely, we monitored the reproduction of captive individuals. Eight spawning sessions (stable water temperature 24-25°C; daily light period 07:00-19:00) occurred between May 2016 and November 2017. After 65-150 min of tandem swimming (the male biting and clinging to the female's abdomen), 3-50 spherical, weakly adhesive eggs were spawned, being scattered onto the sandy substrate or water plants. The removal of cohabitants (potentially eating spawned eggs) and provision of small initial food items, such as small-type Brachionus spp., for larval fish were essential for successful breeding.

Species phylogeny and diversification process of Northeast Asian Pungitius revealed by AFLP and mtDNA markers.

Pungitius is a highly diversified genus of sticklebacks (Gasterosteidae) occurring widely in northern parts of the Northern Hemisphere. Several ecologically and genetically divergent types that are largely isolated reproductively but occasionally hybridize in sympatry have been discovered in Northeast Asia, although the taxonomy and evolutionary relationships among them remain unclear. We used amplified fragment length polymorphism (AFLP) and mitochondrial DNA (mtDNA) markers to infer phylogenies among individuals collected from sympatric and allopatric populations, including the type localities of the described species. Phylogenetic analyses based on 2683 polymorphic AFLP loci confirmed seven species, each of which (except for one entirely allopatric species P. platygaster) was clearly differentiated from one or two other sympatric species and constituted a highly supported monophyletic clade with conspecific allopatric populations. The phylogeny showed that two lineages arose early; one gave rise to two species (circumpolar species P. pungitius and Paratethys species P. platygaster) and the other to five species endemic to Northeast Asia (P. sinensis, P. tymensis, P. polyakovi, P. kaibarae, and P. bussei). The brackish-water, freshwater, and Omono types previously discovered in Japan were reidentified as P. pungitius, P. sinensis, and P. kaibarae, respectively. A marked incongruence was noted between the phylogenies of AFLP and mtDNA markers, suggesting the occasional occurrence of hybridization and mtDNA introgression among distinct species. Our results highlight that the marginal seas of Northeast Asia played a key role as barriers to or facilitators of gene flow in the evolution of species diversity of Pungitius concentrated in this region.

The shortened spinal cord in tetraodontiform fishes.

In teleosts, the spinal cord generally extends along the entire vertebral canal. The Tetraodontiformes, in which the spinal cord is greatly reduced in length with a distinct long filum terminale and cauda equina, have been regarded as an aberration. The aims of this study are: 1) to elucidate whether the spinal cord in all tetraodontiform fishes shorten with the filum terminale, and 2) to describe the gross anatomical and histological differences in the spinal cord among all families of the Tetraodontiformes. Representative species from all families of the Tetraodontiformes, and for comparison the carp as a common teleost, were investigated. In the Triacanthodidae, Triacanthidae, and Triodontidae, which are the more ancestral taxa of the Tetraodontiformes, the spinal cord extends through the entire vertebral canal. In the Triacanthidae and Triodontidae, the caudal half or more spinal segments of the spinal cord, however, lack gray matter and consist largely of nerve fibers. In the other tetraodontiform families, the spinal cord is shortened forming a filum terminale with the cauda equina, which is prolonged as far as the last vertebra. The shortened spinal cord is divided into three groups. In the Ostraciidae and Molidae, the spinal cord tapers abruptly at the cranium or first vertebra forming a cord-like filum terminale. In the Monacanthidae, Tetraodontidae, and Diodontidae, it abruptly flattens at the rostral vertebrae forming a flat filum terminale. The spinal cord is relatively longer in the Monacanthidae than that in the other two families. It is suggested by histological features of the flat filum terminale that shortening of the spinal cord in this group progresses in order of the Monacanthidae, Tetraodontidae, and Diodontidae. In the Balistidae and Aracanidae, the cord is relatively long and then gradually decreased in dorso-ventral thickness.

Multiple invasions into freshwater by pufferfishes (teleostei: tetraodontidae): a mitogenomic perspective.

Pufferfishes of the Family Tetraodontidae are the most speciose group in the Order Tetraodontiformes and mainly inhabit coastal waters along continents. Although no members of other tetraodontiform families have fully discarded their marine lives, approximately 30 tetraodontid species spend their entire lives in freshwaters in disjunct tropical regions of South America, Central Africa, and Southeast Asia. To investigate the interrelationships of tetraodontid pufferfishes and thereby elucidate the evolutionary origins of their freshwater habitats, we performed phylogenetic analysis based on whole mitochondrial genome sequences from 50 tetraodontid species and closely related species (including 31 newly determined sequences). The resulting phylogenies reveal that the family is composed of four major lineages and that freshwater species from the different continents are independently nested in two of the four lineages. A monophyletic origin of the use of freshwater habitats was statistically rejected, and ancestral habitat reconstruction on the resulting tree demonstrates that tetraodontids independently entered freshwater habitats in different continents at least three times. Relaxed molecular-clock Bayesian divergence time estimation suggests that the timing of these invasions differs between continents, occurring at 0-10 million years ago (MA) in South America, 17-38 MA in Central Africa, and 48-78 MA in Southeast Asia. These timings are congruent with geological events that could facilitate adaptation to freshwater habitats in each continent.

Unique patterns of pelvic fin evolution: a case study of balistoid fishes (Pisces: Tetraodontiformes) based on whole mitochondrial genome sequences.

Balistoid fishes have a unique and reduced pelvic fin structure, which does not exhibit paired structures. The pelvic complex exhibits reductive trends, but its rudimentary structure was retained among balistoids, and its unidirectional and parsimonious reduction in more derived lineages has been hypothesized based on morphology. We investigated the evolution of pelvic complex reduction in balistoids using whole mitochondrial genome (mitogenome) data from 33 species (27 newly determined during the study) that represent the entire morphological diversity of balistoids. Partitioned maximum likelihood and Bayesian analyses were conducted with two datasets that comprised concatenated nucleotide sequences from 13 protein-coding genes (all positions included; third codon positions converted into purine [R] and pyrimidine [Y] [RY-coding]) plus 22 transfer RNA and two ribosomal RNA genes. The resultant trees were well resolved and largely congruent, with most internal branches having high support values. The mitogenomic datasets strongly supported monophylies of both balistids and monacanthids, but rejected previous hypotheses on the intra-relationships in each family. The present tree topology revealed that highly reduced pelvic complexes had multiple origins, and optimization of the traits on the resultant tree strongly suggested the non-unidirectional and independent reduction of pelvic complexes in balistoids. The evolution of balistoid pelvic structure is very different among fishes that exhibit its reductive trends, and this uniqueness in pelvic evolution may be a link to their reproductive behaviors.

Explosive speciation of Takifugu: another use of fugu as a model system for evolutionary biology.

Although the fugu Takifugu rubripes has attracted attention as a model organism for genomic studies because of its compact genome, it is not generally appreciated that there are approximately 25 closely related species with limited distributions in the waters of East Asia. We performed molecular phylogenetic analyses and constructed a time tree using whole mitochondrial genome sequences from 15 Takifugu species together with 10 outgroups to examine patterns of diversification. The resultant time tree showed that the modern Takifugu species underwent explosive speciation during the Pliocene 1.8-5.3 Ma, which is comparable with that of the Malawi cichlids and tropheine cichlids in Lake Tanganyika. Considering their limited distributions and remarkable variations in coloration, morphology, and behavior, the results of the present study strongly suggest that Takifugu species are strong candidates as a model system for evolutionary studies of speciation mechanisms in marine environments where few such organisms are available.

A new perspective on phylogeny and evolution of tetraodontiform fishes (Pisces: Acanthopterygii) based on whole mitochondrial genome sequences: basal ecological diversification?

BACKGROUND: The order Tetraodontiformes consists of approximately 429 species of fishes in nine families. Members of the order exhibit striking morphological diversity and radiated into various habitats such as freshwater, brackish and coastal waters, open seas, and deep waters along continental shelves and slopes. Despite extensive studies based on both morphology and molecules, there has been no clear resolution except for monophyly of each family and sister-group relationships of Diodontidae + Tetraodontidae and Balistidae + Monacanthidae. To address phylogenetic questions of tetraodontiform fishes, we used whole mitochondrial genome (mitogenome) sequences from 27 selected species (data for 11 species were newly determined during this study) that fully represent all families and subfamilies of Tetraodontiformes (except for Hollardinae of the Triacanthodidae). Partitioned maximum likelihood (ML) and Bayesian analyses were performed on two data sets comprising concatenated nucleotide sequences from 13 protein-coding genes (all positions included; third codon positions converted into purine [R] and pyrimidine [Y]), 22 transfer RNA and two ribosomal RNA genes (total positions = 15,084). RESULTS: The resultant tree topologies from the two data sets were congruent, with many internal branches showing high support values. The mitogenomic data strongly supported monophyly of all families and subfamilies (except the Tetraodontinae) and sister-group relationships of Balistidae + Monacanthidae and Tetraodontidae + Diodontidae, confirming the results of previous studies. However, we also found two unexpected basal splits into Tetraodontoidei (Triacanthidae + Balistidae + Monacanthidae + Tetraodontidae + Diodontidae + Molidae) and Triacanthodoidei (Ostraciidae + Triodontidae + Triacanthodidae). CONCLUSION: This basal split into the two clades has never been reported and challenges previously proposed hypotheses based on both morphology and nuclear gene sequences. It is likely that the basal split had involved ecological diversification, because most members of Tetraodontoidei exclusively occur in shallow waters (freshwater, brackish and coastal waters, and open seas), while those of Triacanthodoidei occur mainly in relatively deep waters along continental shelves and slopes except for more derived ostraciids. This suggests that the basal split between the two clades led to subsequent radiation into the two different habitats.

Rapid characterization of drug-drug interaction in plasma protein binding using a surface plasmon resonance biosensor.

High-throughput characterization of drug-drug interactions in plasma protein binding was demonstrated by using a surface plasmon resonance (SPR) biosensor. The method used in this study enabled the discrimination between the two modes of binding inhibition, direct competition and negative allosteric effect, which was difficult in conventional SPR approaches. Two theoretical equations were used representing SPR binding response for directly competitive binding or for independent binding. The experimental binding data for human serum albumin was processed by non-linear least squared regression of the equations. By this approach, drug-drug interactions were classified into three modes, direct competition, independent binding, and allosteric interaction, which were almost consistent with previous reports. In addition, dissociation constants were also estimated roughly for direct competition and for independent binding. The analytical throughput was almost as high as in the previous reports; three minutes per injection. This method is a powerful tool for the characterization of drug-drug interaction at an early stage of new drug development.

Phylogenetic position of tetraodontiform fishes within the higher teleosts: Bayesian inferences based on 44 whole mitochondrial genome sequences.

Tetraodontiformes includes approximately 350 species assigned to nine families, sharing several reduced morphological features of higher teleosts. The order has been accepted as a monophyletic group by many authors, although several alternative hypotheses exist regarding its phylogenetic position within the higher teleosts. To date, acanthuroids, zeiforms, and lophiiforms have been proposed as sister-groups of the tetraodontiforms. The monophyly and sister-group status was investigated using whole mitochondrial genome (mitogenome) sequences from 44 purposefully-chosen species (26 sequences newly-determined during the study) that fully represent the major tetraodontiform lineages plus all the groups that have been hypothesized as being close relatives. Partitioned Bayesian analyses were conducted with the three datasets that comprised concatenated nucleotide sequences from 13 protein-coding genes (with and without, or with RY-coding, 3rd codon positions), plus 22 transfer RNA and two ribosomal RNA genes. The resultant trees were well resolved and largely congruent, with most internal branches being supported by high posterior probabilities. Mitogenomic data strongly supported the monophyly of tetraodontiform fishes, placing them as a sister-group of either Lophiiformes plus Caproidei or Caproidei only. The sister-group relationship between Acanthuroidei and Tetraodontiformes was statistically rejected using Bayes factors. These results were confirmed by a reanalysis of the previously published nuclear RAG1 gene sequences using the Bayesian method. Within the Tetraodontiformes, however, monophylies of the three superfamilies were not recovered and further taxonomic sampling and subsequent efforts should clarify these relationships.

Phylogenetic and taxonomic relationships of northern Far Eastern phoxinin minnows, Phoxinus and Rhynchocypris (Pisces, Cyprinidae), as inferred from allozyme and mitochondrial 16S rRNA sequence analyses.

Analyses of allozyme (18 loci) and partial mitochondrial DNA (mtDNA) sequences (1295 bp, 16S rRNA) support the classification of phoxinin minnows from the northern Far East into 2 genera of 8 species: Phoxinus phoxinus, Rhynchocypris oxycephalus, R. perenurus, R. czekanowskii, R. kumgangensis, R. semotilus, R. lagowskii and R. sp. (bergi ?). Although R. lagowskii from Japan and the Amur basin and R. sp. from Vladivostok region to Korea have been classified into a single species by many authors as R. lagowskii, they form separate clusters in both analyses, suggesting different specific status. Some R. oxycephalus and R. perenurus had the mtDNA haplotypes of R. lagowskii and R. czekanowskii, respectively, which probably indicates that local introgression of mtDNA occurred through inter-specific hybridization. Rhynchocypris forms a monophyletic cluster with dace genera Tribolodon and Pseudaspius, not with Phoxinus. Eurasian and American Phoxinus are suggested to be paraphyletic.

Takifugu obscurus is a euryhaline fugu species very close to Takifugu rubripes and suitable for studying osmoregulation.

BACKGROUND: The genome sequence of the pufferfish Takifugu rubripes is an enormously useful tool in the molecular physiology of fish. Euryhaline fish that can survive both in freshwater (FW) and seawater (SW) are also very useful for studying fish physiology, especially osmoregulation. Recently we learned that there is a pufferfish, Takifugu obscurus, common name "mefugu" that migrates into FW to spawn. If T. obscurus is indeed a euryhaline fish and shares a high sequence homology with T. rubripes, it will become a superior animal model for studying the mechanism of osmoregulation. We have therefore determined its euryhalinity and phylogenetic relationship to the members of the Takifugu family. RESULTS: The following six Takifugu species were used for the analyses: T. obscurus, T. rubripes, T. niphobles, T. pardalis, T. poecilonotus, and T. porphyreus. When transferred to FW, only T. obscurus could survive while the others could not survive more than ten days in FW. During this course of FW adaptation, serum Na+ concentration of T. obscurus decreased only slightly, but a rapid and large decrease occurred even in the case of T. niphobles, a peripheral fresh water species that is often seen in brackish river mouths. Phylogenetic analysis using nucleotide sequences of the mitochondrial 16S ribosomal RNA gene of each species indicated that the six Takifugu species are very closely related with each other. CONCLUSION: T. obscurus is capable of adapting to both FW and SW. Its genomic sequence shares a very high homology with those of the other Takifugu species such that the existing Takifugu genomic information resources can be utilized. These properties make "mefugu", which has drawn little attention from animal physiologists until this study, a useful model animal for studying the molecular mechanism of maintaining body fluid homeostasis.

A dopamine D4 receptor antagonist attenuates ischemia-induced neuronal cell damage via upregulation of neuronal apoptosis inhibitory protein.

Neuronal apoptosis inhibitory protein (NAIP/BIRC1), the inhibitor of apoptosis protein (IAP) family member, suppresses neuronal cell death induced by a variety of insults, including cell death from ischemia and stroke. The goal of the present study was to develop an efficient method for identification of compounds with the ability to upregulate endogenous NAIP and to determine the effects on these compounds on the cellular response to ischemia. A novel NAIP-enzyme-linked immunosorbent assay (ELISA)-based in vitro drug-screening system is established. Use of this system identified an antagonist of dopamine D4 receptor, termed L-745,870, with a potent NAIP upregulatory effect. L-745,870-mediated NAIP upregulation in neuronal and nonneuronal cultured cells resulted in decreased vulnerability to oxidative stress-induced apoptosis. Reducing NAIP expression via RNA interference techniques resulted in prevention of L-745,870-mediated protection from oxidative stress. Further, systemic administration of L-745,870 attenuated ischemia-induced damage of the hippocampal CA1 neurons and upregulated NAIP expression in the rescued hippocampal CA1 neurons in a gerbil model. These data suggest that the NAIP upregulating compound, L-745,870, has therapeutic potential in acute ischemic disorders and that our NAIP-ELISA-based drug screening may facilitate the discovery of novel neuroprotective compounds.

Verification of components of acellular pertussis vaccines that have been distributed solely, been in routine use for the last two decades and contributed greatly to control of pertussis in Japan.

An ideal acellular pertussis vaccine is now under investigation worldwide. We have had acellular pertussis vaccines available for the last 22 years, which contributed greatly to the control of pertussis in Japan, although it has not been known whether they are one of ideal acellular pertussis vaccines or not. Moreover, the formulations of acellular pertussis vaccines that we have been using have not been widely recognized. Serum samples were taken from recipients of the T type, B type, and two-component acellular pertussis vaccine and assayed by ELISA for anti-PT, anti-FHA, and anti-69 kD OMP antibody levels and by the agglutination test. Although it was shown that T type vaccine contained four components (PT, FHA, 69 kD OMP, agglutingen), B type vaccine contained three components (PT, FHA, 69 kD OMP) and the two-component vaccine contained PT and FHA, it was concluded that PT and FHA were essential and common antigens contained in all three acellular pertussis vaccines in Japan. The national monitoring system for adverse effects of routine immunization demonstrated low reactogenicity of DTaP in Japan. This resulted in high acceptance rates of DTaP and in virtual control of pertussis.

Safety and efficacy of acellular pertussis vaccine in Japan, evaluated by 23 years of its use for routine immunization.

BACKGROUND: Real evaluation of any vaccine can only be done after the vaccine has been in routine use for a substantially long period of time. In Japan, acellular pertussis vaccine was introduced and totally replaced whole cell pertussis vaccine in 1981. From 1982 to 1988 40.3 million doses of acellular pertussis vaccine were given to 2-year-olds and from 1989 to 2001 59.3 million doses of acellular pertussis vaccine were given to 3-month-olds. It is now time to evaluate the efficacy and safety of acellular pertussis vaccine by the use of national data officially supplied by the Government. METHODS: Government national surveillance of pertussis, which began in 1981, was used to analyze epidemiology of pertussis. Official Government reports on acceptance rates of pertussis were analyzed. A peer review has been made on all severe neurological illnesses/death occurring after pertussis immunization which have been applied for through the Vaccine Injury Compensation System, Ministry of Health Labor and Welfare, Japan. RESULTS: High acceptance rates of acellular pertussis vaccine combined with diphtheria and tetanus toxoids (DTaP) has been maintained and a dramatic decrease in pertussis was noted over the past 23 years. Neurological illnesses temporally associated both with whole cell and with acellular pertussis vaccination has been a rare phenomenon. However, incidences of encephalopathy/encephalitis and status epileptics/frequent convulsions, febrile seizures/provocation of convulsions, and sudden deaths were significantly lower with acellular pertussis vaccination than with whole cell pertussis vaccination. CONCLUSION: With the use of acellular pertussis vaccine which has been accepted by the public, pertussis has been well controlled in Japan.

Removal of gelatin from live vaccines and DTaP-an ultimate solution for vaccine-related gelatin allergy.

From the early 1990s infants started to receive acellular pertussis vaccine combined with diphtheria and tetanus toxoids (DTaP) before live vaccines such as measles, rubella, and mumps vaccines, which contained gelatin as a stabilizer. Then, an increasing number of cases of anaphylactic/allergic reactions to those live vaccines were reported. Almost all these cases had a previous history of receiving three or four doses of DTaP containing gelatin.Anaphylactic/allergic reactions to live measles vaccine were analyzed using information obtained from the Reporting System, a retrospective study, as well as from the Monitoring System, a prospective study. Dramatic decreases in anaphylactic/allergic reactions to live measles vaccines were observed immediately after each manufacturer marketed gelatin-free or gelatin (hypo-allergic)-containing live measles vaccine, and since the end of 1998 reports on anaphylactic/allergic reactions to live measles vaccine have almost ceased.

Speciation and dispersal of Tribolodon species (Pisces, Cyprinidae) around the Sea of Japan.

Gene products of 22 protein coding loci from 29 populations of a uniquely sea-going cyprinid genus Tribolodon, T. hakonensis (anadromous and/or river-resident), T. brandtii (anadromous), T. ezoe (freshwater) and T. nakamurai (freshwater), were analyzed in order to infer the genetic divergence and dispersal of Tribolodon species around the Sea of Japan. According to the genetic distance, T. brandtii is located near the center between T. hakonensis and T. ezoe-T. nakamurai cluster. The outgroup root (Pseudaspius leptocephalus, Rhynchocypris lagowskii and Leuciscus waleckii) is attached between T. hakonensis-T. brandtii and the T. ezoe-T. nakamurai clusters. The origin of the genus Tribolodon and its salinity tolerance may date back to the Miocene Sea-of-Japan Lake and the Pliocene Paleo-Sea-of-Japan, respectively, as judged from the divergence time between Tribolodon and the outgroups. The genetic relationships and distribution pattern of Tribolodon species suggest they speciated on the Sakhalin-Japan side, and the continental populations of T. hakonensis and T. brandtii originated from anadromous colonizers. A new faunal element of Japanese freshwater fishes, the Sakhalin-Japan sub-element, is proposed for Tribolodon and some other fishes.

Functional human NAIP promoter transcription regulatory elements for the NAIP and PsiNAIP genes.

Neuronal apoptosis inhibitory protein (NAIP) has been shown to inhibit apoptosis in vitro and in vivo with an expression which is regulated in a variety of cells and tissues and may be modulated by a variety of external stimuli. To understand the molecular basis of the transcriptional regulation of the NAIP gene, we have analyzed the 5'-flanking region and transcription of the human NAIP gene. The functional promoter and silencer elements were identified by luciferase reporter constructs in transient transfection experiments using four different human cells. Although the location of the functional elements were shared among the different cells used, the activities for the NAIP promoter varied. Further, cell type-specific protein binding activities were observed by an electrophoretic mobility shift assay (EMSA). EMSA analysis with specific antibodies and DNA sequence analysis identified the POU domain transcription factor Brn-2 as a candidate transcriptional regulator of the NAIP gene. The DNA sequence of the promoter region of the PsiNAIP gene, a copy gene for NAIP, was nearly identical to that of the NAIP gene, indicating a common regulatory mechanism for transcription of the NAIP and PsiNAIP genes. Indeed, the transcript of the PsiNAIP gene was identified. These results provided the first evidence for the functional promoter and candidate transcriptional factor for the NAIP gene and transcription of the PsiNAIP gene.