Hideo Fujitani

The Complete Sequence of Mitochondrial Genome from a Gynogenetic Triploid “Ginbuna” (Carassius auratus langsdorfi)

Abstract The complete mitochondrial (mt) genome of the gynogenetic triploid ginbuna (Carassius auratus langsdorfi, AZ3 line) has been cloned and sequenced. The genome consisted of 16,578 bp and encoded the same set of genes (13 proteins, 2 rRNAs and 22 tRNAs) in addition to a D-loop region, as described for other vertebrate mtDNAs. Comparison with other teleost mtDNAs demonstrated that the protein/rRNA-coding regions of the ginbuna were highly homologous both in length and nucleotide composition to those of the carp, indicating fairly close relationship between the triploid ginbuna and the carp. Although the size of the ginbuna D-loop was almost the same as that of the carp, the nucleotide sequence showed a moderate variation. More comprehensive sequence data of the D-loop regions will lead to the elucidation of phylogenetic relationships among Carassius auratus subspecies.

Nucleotide sequence and polymerase chain reaction/restriction fragment length polymorphism analyses of Aleutian disease virus in ferrets in Japan

Two ferrets with spontaneous Aleutian disease (AD) were found in Japan. The diagnosis was verified by polymerase chain reaction (PCR) amplification of part of the capsid gene specific to AD virus (ADV). The nucleotide sequences (365 bp in length) of the amplified fragments from the 2 ferrets differed by a single nucleotide, producing an amino acid alteration. Compared with other types of ADV, these isolates had 96% sequence similarity to a published ferret ADV (FADV) in contrast to <91% homology to various types of mink ADV (MADV). The phylogenetic tree of ADVs indicates that these 2 isolates and the published FADV belong to the same genetic group and definitely are divergent from MADVs. The predicted amino acid sequence of the hypervariable segment in the capsid gene was conserved among the 3 types of FADV. These results indicated that the 2 isolates found in Japan were new DNA types of FADV and could have been derived from FADV(s). A restriction fragment length polymorphism (RFLP) method to distinguish the ferret types of ADV from the mink types of ADV was developed on the basis of differences in their nucleotide sequences. Digestion of the PCR products with AfaI or ScaI provided different cleavage patterns for FADV and MADV. This PCR/RFLP analysis of the ADV capsid gene will be a valuable asset for diagnosis of this virus infection in ferrets.

Development of Species-Specific PCR Techniques for the Detection of Tortoise Herpesvirus

Previously, a nested polymerase chain reaction (PCR) was employed with consensus degenerate primers targeting highly conserved motifs within herpesviral DNA polymerase genes to detect a newly described tortoise herpesvirus. However, nucleotide sequence information obtained from the final amplified fragment was restricted to a small region of 181 bp. In the present study, additional sequences flanking this segment were determined from a PCR product successfully amplified using a set of known degenerate primers, which covered a 692-bp region within the tortoise herpesviral DNA polymerase gene. Polymerase chain reaction primers for specific amplification of the tortoise herpesviral DNA were designed on the basis of these nucleotide sequences and successfully amplified tortoise herpesviral DNA from the tissues of tortoises that were well characterized histopathologically with herpesviral infection. The lower limit of detection was 1,000 herpesviral DNA equivalents in the presence of normal tortoise genomic DNA. Furthermore, a more sensitive and specific PCR technique for the identification of herpesviral infections in tortoises was developed employing a heminested form, which will enable the detection of latent infections or herpesvirus carriers in tortoises.

Nucleotide sequence and expression of a cDNA encoding canine carbonic anhydrase VI (CA-VI).

A full-length cDNA clone of a canine carbonic anhydrase VI (CA-VI) was generated from the canine parotid gland by using a reverse transcription-polymerase chain reaction (RT-PCR) technique with degenerate primers designed from conserved regions of the same locus in humans and bovines employing RACE (rapid amplification of cDNA ends) techniques. The cDNA sequence was 1351 base pairs (bp) long and was predicted to encode a 320-amino-acid polypeptide containing a putative signal peptide of 17 amino acids. The deduced amino acid sequence of mature CA-VI showed the highest similarity of 74% to that of human CA-VI. RT-PCR analysis with primers specific to the canine CA-VI demonstrated strong signals in the major salivary glands and weak signals in the minor salivary glands and esophagus of a healthy dog. No CA-VI mRNA was detected in the pancreas, liver or the digestive tract except the esophagus.

Characterization of DNA markers isolated from the gynogenetic triploid ginbuna (Carassius auratus langsdorfi) by representational difference analysis

Representational difference analysis (RDA) was carried out to isolate genetic markers for gynogenetic triploid ginbuna (Carassius auratus langsdorfi) suitable for clarifying its genomic makeup. Four polymorphic DNA fragments were obtained by seven series of RDA, in which amplicons prepared from the diploid ginbuna or the goldfish (C. a. auratus) as drivers were subtracted from those prepared from the triploid ginbuna as testers. These fragments detected presence- and absence-type in the amplicons prepared from C. auratus fishes. Hybridization analyses with each of these markers to genomic DNA revealed that these sequences were present in the DNA of all tested populations of C. auratus fishes, but were absent from the DNA of other cyprinid fishes. Thus, these markers could detect restriction fragment length polymorphisms among C. auratus fishes. Three out of four RDA markers were identified in the amplicons mainly from the triploid ginbuna and the goldfish. One remaining marker was shared between a clonal line of the triploid ginbuna and several individuals of the diploid ginbuna. These data provide additional evidence for the genomic contribution made by the ancestor(s) of the goldfish and the diploid ginbuna to the triploid hybrid ginbuna.

Cloning and Sequencing of Wee 1 from the Ovary of the Gynogenetic Japanese Silver Crucian Carp (ginbuna) [ Carassius auratus langsdorfi ]

Wee 1 is one of the genes which regulates the cell cycle. cDNA encoding a Wee1-homolog has been isolated from Japanese silver crucian carp (ginbuna) by using polymerase chain reaction (PCR) techniques. The cDNA obtained from the ovary tissue encoded a protein of 526 amino acids and the deduced amino acid sequence showed high homology (69.9%) with Xenopus laevis Wee1 in the protein kinase domain. A phylogenetic analysis among Wee1 amino acid sequences from various organisms showed that the crucian carp Wee1 was closely related to Xenopus Wee1 and human Wee1B. The vertebrate Wee1s were classified into two types: the meiotic type and the mitotic type. The crucian carp Wee1 described here belonged to the meiotic type. Furthermore, a long-PCR technique allowed us to isolate the wee 1 gene of almost full length. The wee 1 gene was about 6.3 kbp in length and contained 12 exons, with the open reading frame starting at the second exon.