Multiplex polymerase chain reaction (PCR) technique has recently been applied to gain many advantages in molecular genetics. The present study focused on the development of 15 new microsatellite markers with multiplex PCR systems in ayu, Plecoglossus altivelis, an important freshwater fish in Japan. All loci were followed Mendelian inheritance in 27 F1 progeny except for the one locus. The number of alleles per locus ranged from nine to 44 and the observed heterozygosities ranged from 0.680 to 0.980 in 50 unrelated individuals. The results indicate that these new microsatellite markers are useful for studies of linkage mapping and population genetics for the species.

The forkhead box genes (Fox genes) are expressed in many tissues during embryogenesis and their products play a key role in organogenesis. We cloned two Fox genes from an embryonic cDNA library of medaka (Oryzias latipes). One was MeHNF3 beta, a homologue of HNF3 beta (FoxA2). The other was Mefkh1 (medaka-forkhead1), related to HNF3 gamma (FoxA3). We found that the expression pattern of FoxA3 differs in some aspects between medaka and zebrafish. Mefkh1 is expressed in the dorsal marginal zone, embryonic shield, polster, gut and slightly axial mesoderm, whereas zebrafish FoxA3 is expressed in the yolk syncytial layer (YSL), axial mesoderm, polster, gut and posterior neural crest cells (Odenthal and Nusslein-Volhard, 1998, Dev Genes Evol 208, 245-258). In zebrafish, the level of expression of FoxA3 in the axial mesoderm is similar to that of FoxA2 (axial); in medaka, FoxA3 (Mefkh1) expression is mainly detected in the polster, and FoxA2 (MeHNF3 beta) is expressed in the axial mesoderm. This indicates that the combined functioning of FoxA2 and FoxA3 in the organogenesis of the polster and axial mesoderm differs between medaka and zebrafish.

The abnormalities of haploid medaka embryos were characterized by comparative analysis of histologic sections and expression patterns of some developmental marker genes between haploids and diploids to clarify whether medaka haploids are useful for identifying mutants. During gastrulation, an obvious defect was first observed as a delay of epiboly and involution. This delay was shown to be caused not by the perturbation of mesoderm induction, but by widespread cell death and disorganization of cell arrangement in the blastoderm. This disorganization of cell arrangement was also detected in various organs, such as the brain, somite and notochord, at a late developmental stage. Ten days after fertilization, a small head and a short body axis were formed; these changes were also observed in haploid embryos in other species, but their cause is unknown. Based on the expression patterns of HNF3β and goosecoid , it was demonstrated that a short and impotent prechordal plate induced near the marginal zone in haploid embryos was responsible for this defect. However, in these experiments it was also demonstrated that many major organs in haploids, such as the somite and notochord, differentiated incompletely but were present. Therefore, it was concluded that haploid screening is suitable for identifying mutations revealed by an obvious phenotype, such as dorsoventral polarity.

Genetic analyses of sex determination have identified sex chromosomes in many teleost fish species. However, there are several cases for which sex ratios do not fit perfectly with the expectations of heterogametic systems, suggesting the influence of either minor sex determining genes or environmental influences on the process of sex differentiation. The frequent absence of sex chromosome markers makes the identification of minor sex-determining genes very difficult. It is easier to test first the hypothesis of environmental sex determination (ESD) by studying the temperature effect, since temperature-dependent sex determination has been demonstrated to occur in several vertebrate groups including 1 fish species. To contribute to a better understanding of fish sex determination, we have tested the effects of high temperatures on sex ratios of Oreochromis niloticus, and have attempted to isolate sex chromosome molecular markers in Leporinus elongatus. Treatments of O. niloticus fry at 36 degrees C applied for 10 days and more, and starting 1 week after fertilization markedly increased the proportion of males, and progeny-testing these males confirmed that some of them are sex-reversed genetic females. Two non-coding sequences of L. elongatus Z and W chromosomes were cloned by genomic subtraction. They cross-hybridized with the genome of a close species without providing sex-specific patterns. A collection of L. elongates individuals was subjected to gonadal and chromosomal sexing, and DNA hybridization with both sequences. These analyses revealed 3 individuals having atypical W chromosomes. Interestingly, 2 of these were males having a ZW karyotype. We assume that these atypical sex chromosome arise by exchanges between Z and W chromosomes, and that a transition between female and male heterogamety is underway in this species.

In order to study the divergence of teleost sex chromosomes, subtractive cloning was carried out between genomic DNA of males and females of the rainbow trout (XX/XY) and of Leporinus elongatus (ZW/ZZ). Inserts cloned in a plasmid vector were individually tested on Southern blots of DNA of males and females for sex specificity. No sex-specific insert was obtained from trout, but two out of ten inserts cloned from L. elongatus showed sex-specific patterns in this species: one corresponds to a sequence present on both Z and W chromosomes, while the other is W specific. Sequences of these two inserts show neither clear homology with other known sequences, nor an open reading frame. They cross-hybridize with the genomic DNA of Leporinus friderici, but without sex-specific patterns. Twenty-four L. elongatus adults were sexed by gonadal observation, chromosomed examination and Southern hybridization with one or the other insert. Ten males and 11 females had chromosomes and hybridization patterns typical of their sex. One ZW female was recognized as a male with the W-specific probe. This was also the case for two unusual ZW males, one having a male hybridization pattern with the other probe. These three atypical individuals may result from single genetic exchanges between four regions of the Z and the W, giving rise to three atypical W chromosomes. Finding males with such atypical heterochromosomes in a female heterogametic species may indicate that a gradual transition occurs between the heterogametic systems.

A transparent glass plate was roughened at the submicron level, and then a chemically adsorbed monolayer of a fluorocarbon compound was formed along the glass surface. The monolayer was prepared using the chemical coupling reaction accompanying dehydrochlorination between a chlorosilyl group of the heptadeca-fluorodecyltrichlorosilane (CF 3 (CF 2 ) 7 (CH 2 ) 2 SiCl 3 : HFTS) surface-active agent and a hydroxyl group on the glass surface. The contact angle of the glass surface relative to water was approximately 155 degrees with transmittance kept at 92%. This glass plate may be useful for various products, such as motor vehicles and building window glass.

A new concept for plasma generation which enhances plasma density at low pressures through the use of a high-frequency rotating electric field is introduced. A 50-MHz electric power was applied to three electrodes which are positioned with a triangular symmetry. A phase shift of 120° between the electrodes brought about uniform and high plasma density at a pressure of 1 Pa. The nonuniformity of Ar gas plasma generated was within 10% over a 6” wafer using a tuned Langmuir probe electron density measurement. Polysilicon etchings were made by both SF6/O2 and Cl2/O2 plasmas. The achieved polysilicon etch-rate was over 200 nm/min for both kinds of plasmas at an electric power of 90 W along with a 13.56 MHz RF bias power of 30 W applied to the substrate. High etch-rate selectivity between polysilicon and oxide of 45 for SF6/O2 and 152 for Cl2/O2 was realized. A very low etch-rate nonuniformity of 1.5% was attained for Cl2/O2.

A new doping method for the vertical sidewall of a trench by electron cyclotron resonance plasma is described. The plasma was produced under a pressure of 5×10−4 Torr. A doped layer was formed uniformly along the sidewall of a trench with subhalf micron width and an aspect ratio of 6.2. By using a de-ionized water cooling system, the wafer temperature was maintained below 120 °C and the boron dopant was introduced without damage to the photoresist.

Nine induced tetraploid females were artificially inseminated by UV-irradiated sperm collected from diploid males, in order to induce the gynogenetic development of their ova. Most of the resulting embryos were diploid (or minor aneuploids). Several gynogenetic tetraploids, likely to issue from unreduced ova, were also detected in these progenies. The same females fertilized by normal sperm of diploid males gave a majority of triploids and several pentaploids, while the fertilization by normal sperm of tetraploid males gave rise to a majority of tetraploids and one hexaploid. The same crosses, after the eggs had been heat-shocked to double the maternal genetic contribution, yielded about three-quarters pentaploids and one quarter haploids (normal sperm of diploids), or three-quarters hexaploids and one quarter diploids (normal sperm of tetraploids). These haploids and diploids are likely to result from androgenesis.