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 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 Afal 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.

Reliable sex identification of dogs by modified PCR/RFLP analysis.

To find definitive RFLP sites for canine sex determination, DNA segments corresponding to parts of the canine ZFX and ZFY genes were amplified by PCR and were directly sequenced. According to the newly defined sequence data, the combination of Haelll and Cfr13I sites was found to be useful for not only identifying the sex of the canine DNA samples but also distinguishing them from the human DNA. Conveniently, these two enzymes worked simultaneously in the same single buffer. The double-digestion of the ZFX/ZFY PCR products with HaeIII and Cfr13I showed banding patterns unique to males and females in Canis familialis. This PCR/RFLP method was confirmed to be applicable to various breeds of dog.

The maternal origins of the triploid ginbuna (Carassius auratus langsdorfi): phylogenetic relationships within the C. auratus taxa by partial mitochondrial D-loop sequencing.

The hyper-variable segments (323-327 bp) of the mitochondrial D-loop for 169 Carassius auratus fishes in Japan were amplified by the polymerase chain reaction and the amplified products were sequenced directly and compared. A dendrogram showing three major clusters was generated with the sequence data for 37 haplotypes at 66 polymorphic sites. One cluster (cluster I) exclusively consisted of the gengorobuna, which was regarded as an independent (sub) species. The triploid ginbuna belonged to two remaining clusters, mainly in the diploid ginbuna cluster (cluster III) and partially in the goldfish cluster (cluster II). This finding suggests that the triploid ginbuna has been derived from two different maternal lineages. The triploid ginbuna was considered to have come into existence during the last ice age on the basis of this phylogenetic data. No geographic differentiation was observed with respect to the triploid ginbuna sampled at three different localities in Japan; the Shibuta River in Kanagawa, Lake Imba in Chiba and Lake Biwa in Shiga. The phylogenetic tree also demonstrated a monophyletic relationship amongst the nigorobuna, the nagabuna and the ginbuna, sharing cluster III. The nigorobuna and nagabuna populations have most likely arisen from geographic and temporal variations within the ginbuna populations. We also discuss the evolutionary origin of the triploid in view of its paternal ancestors.

Polymerase chain reaction (PCR) for the detection of herpesvirus in tortoises.

A polymerase chain reaction (PCR)-based method using a herpesvirus consensus primer was assessed for the identification of herpesviral infections in tortoises. A single band of about 230 bp was detected in PCR products from two out of twenty swabs taken from the oral cavity, three out of three paraffin-embedded tissue sections from the liver (two cases) and oral mucosa (one case), and one out of two fresh tissue samples from the oral mucosa. Nucleotide sequencing of these PCR products indicated that the herpesvirus present in these tortoises might belong to the alphaherpesvirinae. PCR using swabs and biopsy tissues was a sensitive and highly specific method for the diagnosis of herpesviral infections in tortoises.

Characterization of repetitive DNA sequences carrying 5S rDNA of the triploid ginbuna (Japanese silver crucian carp, Carassius auratus langsdorfi).

Repetitive DNA sequences (Hi-b; 209 bp in length) were isolated from the HindIII digests of the genomic DNA of the triploid ginbuna, Carassius auratus langsdorfi. Sequence analyses revealed that the Hi-b repetitive units were comprised of the complete coding regions of 5S rDNA (120 bp in size) and their 5'flanking regions. The sequences of the Hi-b units from the same individual were highly homogeneous. Southern blot hybridization to the Hi-b probe displayed intricate patterns that represented the presence of other repetitive units containing the Hi-b related sequences. A major family of repetitive sequences related to the Hi-b was then obtained by the polymerase chain reaction using asymmetry primers for the 5S coding regions. These 331-bp sequences (AZ5S's) contained 5S pseudogenes as well as the almost entire Hi-b sequences, and seemed to be the true 5S rDNAs. The tandem arrangements of the AZ5S sequences explained most of the complex results of Southern blots. Another class of intriguing repeat units (Hi-b-beta and Hi-b-gamma) were also isolated. Fluorescence in situ hybridization data revealed two major signals on a pair of homologous chromosomes and several minor signals on other chromosomes in the triploid ginbuna, indicating the existence of the 5S related sequences as several separate clusters. The major spots were shared with the tetraploid ginbuna and goldfish, but not with the diploid ginbuna. When the genomic organization of the Hi-b related sequences in other cyprinid fishes was examined, the hybridization patterns of the ginbuna were very similar to those of the goldfish, but were clearly different from those of the gengorobuna. The carp genome showed less complex patterns. Thus, the present 5S rDNA-related sequences could be candidates for phylogenetic molecular markers for the crucian carp.

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

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.

Polyploid-specific repetitive DNA sequences from triploid ginbuna (Japanese silver crucian carp, Carassius auratus langsdorfi).

Repetitive DNA sequences (Cal3nDr) in the genome of a triploid ginbuna (Carassius auratus langsdorfi) were isolated from the DraI digests of the genomic DNA. This AT-rich (61%) Cal3nDr monomer was 137 bp in length. The nucleotide similarity among the monomers from the same individual was considerably high (above 97%). Hybridization analyses revealed that the Cal3nDr sequences were organized into tandem arrays. These DNA sequences were present only in triploid and tetraploid ginbunas and were absent from diploid ginbuna, gengorobuna, goldfish, and other cyprinid fishes, and therefore appeared to be specific to polyploid ginbunas. In situ hybridization data showed their localization on one to four out of a total of 150 to 156 chromosomes, depending on the individuals or clonal lines, of the triploid ginbuna. The origin of the Cal3nDr sequences is also discussed on the basis of observation of the artificial triploid ginbuna produced by crossing a diploid female with a tetraploid male.

A family of highly repetitive DNAs from "ginbuna" (Carassius auratus langsdorfi) genome common to Carassius auratus populations.

A family of highly repetitive DNAs (Hi-a) in the genome from a population of the crucian carp, tentatively identified as the ginbuna (Carassius auratus langsdorfi), was isolated from the HindIII digests and characterized. The Hi-a monomer (268 or 269 bp) was AT-rich (64.9%) with internal repetitive oligomers. The nucleotide similarity among monomers within the same individual was 88-98%, whose sequence alterations occurred mostly at restricted sites. Hybridization analyses revealed that the Hi-a family was organized into tandem array(s) representing satellite DNAs, and that its variants that share certain restriction sites of the repeat unit were dispersed into the tandem array(s) of Hi-a DNAs at varying periodicities. The lack of sequences homologous to the ginbuna Hi-a in the genomes from cyprinid fishes other than gengorobuna and goldfish suggests that Hi-a DNAs are specifically present in the species C. auratus. The genome of goldfish differed somewhat in the quantity or the genomic organization of Hi-a DNAs from that of the ginbuna.

Comparison of primary and secondary 26S rRNA structures in two Tetrahymena species: evidence for a strong evolutionary and structural constraint in expansion segments.

We have determined the nucleotide sequence of the 26S large subunit (LSU) rRNA genes for two Tetrahymena species, T. thermophila and T. pyriformis. The inferred rRNA sequences are presented in their most probable secondary structures based on compensatory mutations, energy, and conservation criteria. The majority of the nucleotide changes between the two Tetrahymena LSU rRNAs and the positions of a relatively large deletion and of the processing cleavage sites resulting in the generation of the hidden break are all located within the so-called divergent domains or expansion segments. These are regions within the common core of secondary structure where expansions have taken place during the evolution of the rRNA of higher eukaryotes. The dispensable nature of some of the expansion segments has been taken as evidence of their non-functionality. However, our data show that a considerable selective constraint has operated to preserve the secondary structure of these segments. Especially in the case of the D2 and D8 segments, the presence of a considerable number of compensatory base changes suggests that the secondary structure of these regions is of functional importance. Alternatively, these expansion segments may have maintained characteristic folding patterns because only such structures are being tolerated within otherwise functionally important regions.

Effects of quaternary ammonium compounds on the degradation of lipids in lysosomes.

We have examined effects of quaternary ammonium compounds on the in vitro degradation of endogenous lipids in isolated lysosomes. The degree of lipid degradation was assessed by determining hydrolysis products of labeled lipid. Lipolysis was inhibited by quaternary ammonium compounds. The degrees of inhibition were as follows: ethidium bromide greater than N-methylatropinium bromide (NMA) greater than tubocurarine. The inhibition of lipolysis by these quaternary ammonium compounds is not necessarily correlated with the differences in their polarities, molecular weight or structures. The degradation of three phospholipid classes was inhibited by NMA with phosphatidylcholine the most vulnerable. The effect of NMA on the hydrolysis of [14C]dipalmitoylphosphatidylcholine (phospholipid) by lysosomal soluble proteins was also examined. The effect of NMA on phospholipase A1 was assessed by the formation of lysophosphatidylcholine, and that on phospholipase C was assessed as the sum of mono- and diglyceride formations. The action of NMA on the phospholipid degradation was similar to that of cationic amphiphilic drugs, but it differed somewhat from that of chloroquine for each enzyme. From these results, it was concluded that one of the inhibitory mechanisms of phospholipid degradation by NMA was the direct interaction between NMA and phospholipase A1 or C.

Inhibitory effects of quaternary ammonium compounds on lysosomal degradation of endogenous proteins.

The effects of quaternary ammonium compounds on the degradation of endogenous proteins in isolated lysosomes were studied. Proteolysis was assayed by measuring the trichloroacetic acid-soluble radioactivity in the lysosomes which had been labeled in vivo with 14C-leucine. p-Biphenylmethyl-(dl-tropyl-alpha-tropinium)bromide (BTTB), at concentrations higher than 0.85 mM, was found to inhibit the lysosomal proteolysis stoichiometrically. Other quaternary ammonium compounds, such as N-methylatropinium bromide (NMA), cetyltrimethylammonium bromide (CTAB), tubocurarine and gallamine, were also examined in the presence or absence of Triton X-100 which is able to destroy the lysosomal membranes. Of these four compounds, NMA was the most effective proteolysis inhibitor, showing 25% inhibition for intact lysosomes at a concentration of 1 mM of the compound. When these compounds were assayed after Triton X-100 treatment of the lysosomes, the effects of all the drugs were augmented, suggesting that they, after accumulation in the lysosomes, act through direct interactions with lysosomal proteases. This was supported by a kinetic analysis of the action of NMA on cathepsin B, a typical lysosomal protease.

Characterization of argentosomes, a type of secondary lysosomes, and the sub-organellar distribution of a lysosomotropic basic compound.

Secondary lysosomes (argentosomes) were isolated by centrifugation in a discontinuous sucrose density gradient from livers of rats administered colloidal silver. Compared to the crude homogenate, the purities of the argentosome preparations were 17.4- and 18.5-fold in terms of acid phosphatase and N-acetyl-beta-glucosaminidase activities, respectively. By lipid analysis, the argentosomes were shown to have intermediate properties between normal lysosomes and tritosomes with regard to the contents of triglyceride and cholesterol. The phospholipid content in the argentosomes was also different from that in these two organelles. The cross-point of argentosomes shifted more to the acidic side than that of normal lysosomes. The data on the binding of tritiated p- biphenylmethyl-(dl-tropyl-alpha-tropiniun)bromide [( 3H]BTTB) to argentosomes indicated that the degree of binding and/or incorporation of this basic compound to the organelles was much higher than that to normal lysosomes. These results suggested that the distribution of BTTB on or within argentosomes might be under the control of the surface charge of the argentosomal membranes.

Hypolipidemic effect and enhancement of peroxisomal beta-oxidation in the liver of rats by sodium-(E)-3-(4-(3-pyridylmethyl)phenyl)-2-methyl propenoate (OKY-1581), a potent inhibitor of TxA2 synthetase.

The effects of sodium-(E)-3-(4-(3-pyridylmethyl)phenyl)-2-methyl propenoate (OKY-1581) and (E)-3-(4-(imidazolylmethyl)phenyl)-2-propenoic acid (OKY-046), potent inhibitors to thromboxane A2 synthetase, on peroxisomal beta-oxidation and on lipid levels of liver and serum in the rat were studied. When the animals were administered with OKY-1581 at the dose levels of 100 and 500 mg/kg body weight for 2 weeks, the activity of peroxisomal beta-oxidation increased 2.2- and 6.3-fold respectively. Catalase activity increased 1.3-fold, whereas D-amino acid oxidase (DAAO) and urate oxidase activities did not change. Carnitine acetyltransferase and carnitine palmitoyltransferase activities also increased 2.2- - 4.1-fold and 2.7- - 4.2-fold respectively. These changes of the enzymes related to lipid metabolism were also confirmed by the results of a cell fractionation study. Moreover, the induction of peroxisome proliferation-associated polypeptide having a molecular weight of 80000, which is a bifunctional enzyme in the peroxisomal beta-oxidation system was also observed electrophoretically in the light mitochondrial fraction of the liver of OKY-1581-treated rat. The contents of triglyceride and cholesterol in the serum decreased. These results indicated that the action of OKY-1581 in enhancing hepatic peroxisomal-oxidation is similar to that of a potent hypolipidemic peroxisome proliferator such as clofibrate. On the other hand, differing from OKY-1581, OKY-046 at the dose level of 500 mg/kg for 2 weeks showed no effect on serum and liver lipid levels and on the activities of the peroxisomal enzymes, including a cyanide-insensitive fatty acyl-CoA oxidizing system and carnitine acetyl transferase.