Isolation and characterization of 19 polymorphic microsatellite DNA markers in the Japanese brown frog (Rana japonica)

We report the isolation and characterization of 19 polymorphic microsatellite DNA markers in the Japanese brown frog (Rana japonica). These markers were tested in 24 individuals each collected from three distinct populations in Ichikai-machi, Tochigi Prefecture. The number of observed alleles per locus ranged from 3 to 24 across all populations, and the values of observed and expected heterozygosities ranged from 0.130 to 1 and from 0.125 to 0.941, respectively. These microsatellite loci will be useful for investigating the intraspecific genetic variation and population structure of this species.     

Antimicrobial peptides with atypical structural features from the skin of the Japanese brown frog Rana japonica

Japonicin-1 (FFPIGVFCKIFKTC) and japonicin-2 (FGLPMLSILPKALCILLKRKC), two peptides with differential growth-inhibitory activity against the Gram-negative bacterium, Escherichia coli and the Gram-positive bacterium Staphylococcus aureus, were isolated from an extract of the skin of the Japanese brown frog Rana japonica. Both peptides show little amino acid sequence similarity to previously characterized antimicrobial peptides isolated from the skins of Ranid frogs. Circular dichroism studies, however, demonstrate that japonicin-2 adopts an alpha-helical conformation in 50% trifluoroethanol in common with many other cationic antimicrobial peptides synthesized in amphibian skin. Peptides belonging to the brevinin-1, brevinin-2, and tigerinin families, previously identified in the skins of Asian Ranid frogs, were not detected but a temporin-related peptide (ILPLVGNLLNDLL.NH(2); temporin-1Ja), that atypically bears no net positive charge, was isolated from the extract. The minimum inhibitory concentrations (MICs) of the peptides against E. coli were japonicin-1, 30 microM; japonicin-2, 12 microM; and temporin-1Ja > 100 microM. The MICs against S. aureus were japonicin-1, > 100 microM; japonicin-2, 20 microM; and temporin-1Ja, > 100 microM.     

Function of the Mos/MAPK pathway during oocyte maturation in the Japanese brown frog Rana japonica

Fully grown immature oocytes acquire the ability to be fertilized with sperm after meiotic maturation, which is finally accomplished by the formation and activation of the maturation-promoting factor (MPF). MPF is the complex of Cdc2 and cyclin B, and its function in promoting metaphase is common among species. The Mos/mitogen-activated protein kinase (MAPK) pathway is also commonly activated during vertebrate oocyte maturation, but its function seems to be different among species. We investigated the function of the Mos/MAPK pathway during oocyte maturation of the frog Rana japonica. Although MAPK was activated in accordance with MPF activation during oocyte maturation, MPF activation and germinal vesicle breakdown (GVBD) was not initiated when the Mos/MAPK pathway was activated in immature oocytes by the injection of c-mos mRNA. Inhibition of Mos synthesis by c-mos antisense RNA and inactivation of MAPK by CL100 phosphatase did not prevent progesterone-induced MPF activation and GVBD. However, continuous MAPK activation and MAPK inhibition through oocyte maturation accelerated and delayed MPF activation, respectively. Furthermore, Mos induced a low level of cyclin B protein synthesis in immature oocytes without the aid of MAPK. These results suggest that the general function of the Mos/MAPK pathway, which is not essential for MPF activation and GVBD in Rana oocytes, is to enhance cyclin B translation by Mos itself and to stabilize cyclin B protein by MAPK during oocyte maturation.     

Amino acid sequence of a lectin from Japanese frog (Rana japonica) eggs

The complete amino acid sequence and the location of disulfide bonds of a lectin from Japanese frog (Rana japonica) eggs, which specifically agglutinates transformed cells, are presented. The sequence was determined by analysis of peptides generated by digestion of the S-carboxyamidomethylated protein with Achromobacter protease I, or chymotrypsin, and by chemical cleavage with BNPS-skatole or cyanogen bromide. The lectin is a single-chain protein consisting of 111 residues, with a pyroglutamyl residue at the amino terminus. Four disulfide bonds link half-cystinyl residue 19 to 72, 34 to 82, 52 to 97, and 94 to 111. The sequence and the location of the disulfide bonds are highly homologous to those of bull frog (Rana catesbeiana) egg S-lectin. They are also homologous to human angiogenin, a tumor angiogenesis factor, and a family of pancreatic ribonucleases.     

Fluctuation in abundance and age structure of a breeding population of the Japanese brown frog,Rana japonica gunther (Amphibia,anura)

A breeding population of Rana japonica was studied at a marsh on the campus of Hiroshima University in Higashi-Hiroshima during the five years 1995-1999. The mark-recapture study showed that the size of the breeding population varied from year to year, and increased more than twofold in 1999 in comparison with the preceding years. The sex ratio of the breeding population (male/female) was from nearly 1.0 to 1.6. Frogs of both sexes were estimated to breed for the first time at the age of one or two years, and their maximum age was four years according to skeletochronology using phalanges and mark-recapture. Modes of the estimated ages were one year for males during the study years except 1997, but one or two years for females. Two thirds of breeding frogs, irrespective of their sex, were estimated to breed only once throughout their lives.     

Twitch potentiation of frog (Rana japonica) skeletal muscle by antipyrine

1. The effects of antipyrine on single muscle fibres from the frog Rana japonica were examined. Antipyrine potentiated the twitch tension, but did not affect the tetanus response or induce any contracture by itself. 2. The duration of the action potential was prolonged in a dose-dependent manner. 3. Antipyrine did not affect the mechanical threshold, the uptake of Ca2+, or the Ca2+ induced release of Ca2+ from the sarcoplasmic reticulum. 4. Ca2+-channel blockers did not affect the twitch potentiation by antipyrine.     

Antimicrobial peptides from the skin of the Japanese mountain brown frog, Rana ornativentris.

Six peptides with antimicrobial activity were isolated from an extract of freeze-dried skin of the Japanese mountain brown frog Rana ornativentris. Two structurally related peptides (brevinin-20a GLFNVFKGALKTAGKHVAGSLLNQLKCKVSGGC, 11 nmol/g dried tissue, and brevinin-20b GIFNVFKGALKTAGKHVAGSLLNQLKCKVSGEC, 170 nmol/g) belong to the brevinin-2 family, previously identified in Asian and European, but not North American, Ranid frogs. Four peptides (temporin-10a FLPLLASLFSRLL.NH2, 13 nmol/g; temporin-10b FLPLIGKILGTI L.NH2, 350 nmol/g; temporin-10c FLPLLASLFSRLF.NH2, 14 nmol/g; and temporin-10d FLPLLASLFSGLF.NH2, 8 nmol/g) are members of the temporin family first identified in the European common frog Rana temporaria but also found in the skins of North American Ranids. The brevinin-2 peptides showed broad-spectrum activity against the gram-positive bacterium, Staphylococcus aureus, the gram-negative bacterium, Escherichia coli and the yeast Candida albicans, whereas the temporins showed potent activity only against S. aureus. The brevinins and temporins belong to the class of cationic antimicrobial peptides that adopt an amphipathic alpha-helical conformation but it is significant that temporin-10d, which lacks a basic amino acid residue, is still active against S. aureus (minimum inhibitory concentration=13 microM compared with 2 microM for temporin-10a). This suggests that strong electrostatic interaction between the peptide and the negatively charged phospholipids of the cell membrane is not an absolute prerequisite for antimicrobial activity.     

Taxon-specific zeta -crystallin in Japanese tree frog (Hyla japonica) lens

The present study demonstrated that the 38-kDa protein, instead of rho-crystallin (36 kDa), is expressed taxon specifically in the lens of Japanese tree frog (Hyla japonica). The 38-kDa protein was distinguished from rho-crystallin expressed in the lenses of bullfrog (Rana catesbeiana) and European common frog (Rana temporaria) immunochemically. Although the N terminus of the 38-kDa protein was blocked, the analyses of partial amino acid sequences showed that the protein was zeta-crystallin. Analysis of cDNA sequence encoding zeta-crystallin of the tree frog lens demonstrated that the deduced protein consisted of 329 amino acids including initial methionine and having 62.2 and 62.9% identity with zeta-crystallin of camel and guinea pig lenses, respectively. The molecular mass of the deduced structure was calculated to be 35,564 Da. zeta-Crystallin of the tree frog lens exhibited the intrinsic enzymatic activity of quinone reductase (EC, NADPH:quinone oxidoreductase). The crystallin specifically catalyzed the reduction of 9,10-phenanthrenequinone (Km, 42 microm) using NADPH (Km, 60 microm) as a cofactor. The enzymatic activity was inhibited by dicumarol, anti-coagulant drug, with IC50 of 4 microm. On gel filtration chromatography, the crystallin was recovered as 150-kDa molecular mass complex, indicating that the crystallin was homotetramer consisting of 38-kDa subunits. The crystallin gene was expressed specifically in the lens. These results show that taxon-specific crystallins such as zeta- and rho-crystallins may be available for the biochemical discrimination of Hyla- and Rana groups among frogs.     

Molecular cloning of a cDNA encoding atypical antimicrobial and cytotoxic Brevinin-1Ja from the skin of the Japanese brown frog, Rana japonica

Antimicrobial peptides (AMPs) are important components of the host innate defense system against pathogenic microbial invasion in many organisms. In the present study, we cloned by RT-PCR a cDNA from total RNA prepared from the skin of the Japanese brown frog Rana japonica. The cDNA directs the synthesis of a novel, non-C-terminally alpha-amidated peptide composed of 21 amino acid residues (FLGSLIGAAIPAIKQLLGLKK). The putative peptide showed limited sequence similarity to atypical acyclic brevinin-1OK family AMPs originally isolated from the skin of the Ryukyu brown frog (R. okinavana), which lacks the COOH-terminal cyclic domain commonly observed in typical brevinin-1 groups, but that contains a C-terminally alpha-amidated residue. Although it is unclear whether the peptide, designated brevinin-1Ja, is produced in R. japonica skin, a synthetic replicate of the peptide showed differential growth-inhibiting activity against the Gram-positive bacterium Staphylococcus aureus and Gram-negative bacterium Escherichia coli (minimal inhibitory concentrations: 15 microM and 119 microM, respectively), and produced cell death of mammalian COS7 cells (LD50=28 microM).     

Inheritance and linkage analysis of ten enzyme and blood protein loci in the Japanese brown frogRana japonica

In order to clarify the genetic linkage groups of the Japanese brown frogRana japonica, and compare them with those of other vertebrates, the inheritance of 10 enzyme and blood protein loci was examined in 267 offspring derived from 18 crosses using 10 males heterozygous at these loci. Most of the segregation tests exhibited no significant deviations from the expected normal Mendelian ratios. Of 32 pairs of loci tested for linkage, 29 pairs showed independent assortment in all crosses examined. In the three other locus pairs, betweenADH-1 andAlb, IDH-1 andHb-1, andLDH-B andMPI, all offspring analyzed were parental, and there were no recombinants. These three linkage groups comprising six loci were thus established inR. japonica, whereas no linkage between the other four loci,AAT-1, ADA, GPI, andPEP-A, was observed.     

Genetic variability predicts common frog (Rana temporaria) size at metamorphosis in the wild

We investigated associations between genetic variability and two fitness-related traits--size and age at metamorphosis--in two subartic populations of the common frog, Rana temporaria. We found that metamorphic size was positively correlated with individual heterozygosity (as estimated using eight microsatellite loci) and that maternal heterozygosity also explained a significant amount of variation in this trait. In contrast, age at metamorphosis was only explained by environmental factors. Since size at metamorphosis is positively correlated with fitness in amphibians, these results suggest that genetic variability may be an important component of individual fitness in common frogs. The environmental variation underlying timing of metamorphosis may indicate that strong selection pressure on this trait in the Nordic environment is likely to override genetic effects.     

Inheritance of enzymes and blood proteins in the leopard frog,Rana pipiens: Three linkage groups established

Individuals from natural populations of the leopard frog, Rana pipiens, were analyzed for electrophoretic differences in blood proteins and enzymes from an amputated digit. The proteins examined represent products of 72 loci. Presumptive heterozygotes at multiple loci were selected for experimental crosses. Mendelian inheritance of 18 protein variations were demonstrated in the offspring. Tests for linkage or independent assortment were performed for 75 locus pairs. Three linkage groups were established. Linkage group 1 contains two loci, aconitase-1 (Acon1) and serum albumin (Alb), with a 19% recombination frequency between them. Linkage group 2 contains four loci, glyoxalase (Gly), acid phosphatase-1 (Ap1), acid phosphatase-2 (AP2), and esterase-5 (Est5). The data show the relationships Gly-21.1%-AP1-0%-AP2-6.3%-Est5, and Gly-25.6%-Est5. Linkage group 3 consists of four closely linked esterase loci. The data, Est1-5.1%-Est6, Est6-1.8%-Est10-1.9%-Est4 and Est6-3.0%-Est4, do not establish a complete order but suggest that Est10 is between Est4 and Est6. These results, with data demonstrating apparent independent assortment of 67 other locus pairs, provide a foundation for establishing the frog genetic map.The project was supported by Grant No. RR-00572 from the Division of Research Resources, National Institutes of Health. This paper is contribution No. C-87 from the Amphibian Facility, George W. Nace, Director.     

Distinct specific status of the Korean brown frog, Rana amurensis coreana (Amphibia: Ranidae)

We used morphological and genetic data to assess the taxonomic status of Rana amurensis coreana and R. a. amurensis. Morphological comparisons revealed these two subspecies to be different from each other in size of body, nature of tubercles on dorsal stripe, degree of development in toe webbing, and condition of lateral spots of trunk. They were also different in sequence of mitochondrial 16S rRNA gene, with genetic distance as large as those observed among different species of brown frogs. Therefore, Korean populations previously considered a subspecies of R. amurensis should be regarded as a distinct species (=R. coreana).     

Plasma immunoreactive calcitonin in the frog (Rana pipiens)

1. Plasma immunoreactive calcitonin (iCT) and ionic calcium [( Ca]i) were measured in intact frogs (Rana pipiens) within complete 24 hr light-dark cycles over an 18 month period. 2. Plasma iCT exhibits an annual periodicity about the annual mean of 10.0 ng/ml, with an amplitude of 5.4 ng/ml that peaks in October. 3. Within an annual cycle, a significant inverse association exists between the basal monthly levels of plasma iCT and [Ca]i for animals maintained in freshwater control conditions. 4. When subjected to a high calcium environment during the latter half of the year, plasma [Ca]i and iCT were elevated above control levels but exhibited independent cyclic patterns. 5. A distinct seasonal response of increased iCT in a high calcium environment may be related to the secretory activity of the ultimobranchial glands and physiological responsiveness to other calcemic hormones; e.g. parathyroid hormone and vitamin D.     

AFLP linkage map of the Japanese quail Coturnix japonica

The quail is a valuable farm and laboratory animal. Yet molecular information about this species remains scarce. We present here the first genetic linkage map of the Japanese quail. This comprehensive map is based solely on amplified fragment length polymorphism (AFLP) markers. These markers were developed and genotyped in an F2 progeny from a cross between two lines of quail differing in stress reactivity. A total of 432 polymorphic AFLP markers were detected with 24 TaqI/EcoRI primer combinations. On average, 18 markers were produced per primer combination. Two hundred and fifty eight of the polymorphic markers were assigned to 39 autosomal linkage groups plus the ZW sex chromosome linkage groups. The linkage groups range from 2 to 28 markers and from 0.0 to 195.5 cM. The AFLP map covers a total length of 1516 cM, with an average genetic distance between two consecutive markers of 7.6 cM. This AFLP map can be enriched with other marker types, especially mapped chicken genes that will enable to link the maps of both species and make use of the powerful comparative mapping approach. This AFLP map of the Japanese quail already provides an efficient tool for quantitative trait loci (QTL) mapping.     

Genetic variation in relation to demography of peripheral pool frog populations (Rana lessonae)

Summary Genetic variation in an isolated northern metapopulation of the pool frog (Rana lessonae) in Sweden was compared to that of Central European populations using enzyme electrophoresis and literature data. Of the 31 loci scored, two (EST-2 andIDH-2) were polymorphic while no variation occurred in seven of the eight loci which are polymorphic in Central European populations.The heterozygosity level of the Swedish pool frogs is very low compared to that of other anuran populations, but their mean proportion of fertilized eggs within egg masses (97.5%) was not lower than in more heterozygous species, and their body size-specific fecundity did not differ from that of Polish conspecifics. The low genetic variability of the Swedish pool frogs is discussed in relation to features of the local populations such as size (N), calculated effective size (N _e ) reproductive success and probable history. It is concluded that long-term strong fluctuations in population size caused by reporductive failure in cold years have contributed more to the low genetic variability than could a single founder event due to a recent introduction by man.     

Genetic evaluation of the HSP70 protein in the Japanese quail (Coturnix japonica)

Heat stress is one of the main problems in modern aviculture, since it affects birds especially in the final phase of rearing, causing bird mortality and economic losses to the aviculturist. The quail, as most birds, has difficulties in dissipating heat. However, little is known about the mechanism that controls the responses of the organism to stressor agents. Therefore, the study of heat shock proteins (HSPs) in these birds is important. A 960-bp portion of HSP70 was amplified using oligonucleotide primers specific for chickens. The fragment was sequenced, since it was the same protein, although some modifications have been observed. It showed 98% homology with HSP70 stress protein in Gallus gallus and 99% homology with Numida meleageris.