Monophyletic origin of naked barley inferred from molecular analyses of a marker closely linked to the naked caryopsis gene (nud).

To elucidate the origin of naked barley, molecular variation of the marker sKT7 tightly linked to the nud locus was examined. A total of 259 (53 wild, 106 hulled domesticated, and 100 naked domesticated) barley accessions were studied. Restriction analysis of the sKT7 PCR-amplified product revealed the alleles I, II, III, and IV. All four alleles were found in wild barley, but allele IV was found only in a single accession from southwestern Iran. Hulled domesticated accessions showed alleles I, II, or III, but all naked domesticated accessions had allele IV. The distribution of allele IV in wild barley and its pervasive presence in naked domesticated lines support the conclusion that naked barley has a monophyletic origin, probably in southwestern Iran. The available results suggest two scenarios for the origin of naked barley: either directly from a wild barley with allele IV or from a hulled domesticated line with allele IV that later became extinct. Naked domesticated accessions from different regions of the world have extremely homogeneous DNA sequences at the sKT7 locus, supporting the monophyletic origin of naked barley. For allele IV, four haplotypes (IVb to IVe) were found in 30 naked accessions: IVb was predominant (66.7%) and widely distributed, while the other three haplotypes, differing by only one nucleotide at different positions relative to IVb, showed a localized distribution. The geographical distribution of the haplotypes of sKT7 allele IV suggests migration routes of naked domesticated barley in central and eastern Asia.

Efficient fine mapping of the naked caryopsis gene ( nud) by HEGS (High Efficiency Genome Scanning)/AFLP in barley.

The hulled or naked caryopsis character of barley ( Hordeum vulgare L.) is an important trait for edibility and to follow its domestication process. A single recessive gene, nud, controls the naked caryopsis character, and is located on the long arm of chromosome 7H. To develop a fine map around the nud locus efficiently, the HEGS (High Efficiency Genome Scanning) electrophoresis system was combined with amplified fragment length polymorphism (AFLP). From bulked segregant analysis of 1,894 primer combinations, 12 AFLP fragments were selected as linked markers. For mapping, an F(2 )population of 151 individuals derived from a cross between Kobinkatagi (naked type) and Triumph (hulled type) was used. Seven AFLP markers were localized near the nud region. A fine map was developed with one-order higher resolution than before, along with the seven anchor markers. Among the seven linked AFLP markers (KT1-7), KT1, KT2 and KT6 were co-dominant, and the former two were detected for their single-nucleotide polymorphisms (SNPs) in the same length of fragments after electrophoresis with the non-denaturing gels of HEGS. The nud locus has co-segregated with KT3 and KT7, and was flanked by KT2 and KT4, at the 0.3-cM proximal and the 1.2-cM distal side, respectively. Four of these AFLP markers were converted into sequence-characterized amplified region (SCAR) markers, one of which was a dominant marker co-segregating with the nud gene.

A DNA marker closely linked to the vrs1 locus (row-type gene) indicates multiple origins of six-rowed cultivated barley ( Hordeum vulgare L.).

The origin of six-rowed cultivated barley was studied using a DNA marker cMWG699 closely linked to the vrs1 locus. Restriction patterns of the PCR-amplified product of the cMWG699 locus were examined in 280 cultivated ( Hordeum vulgare ssp. vulgare) and 183 wild ( H. vulgare ssp. spontaneum) barleys. Nucleotide sequences of the PCR products were also examined in selected accessions. Six-rowed cultivated barleys were divided into two distinct groups, types I and II. Type I six-rowed cultivated barley was distributed widely while type II six-rowed cultivated barley was found only in the Mediterranean region. The type I sequence was also found in a wild barley accession from Turkmenistan whereas the type II sequence was also found in a two-rowed cultivated barley from North Africa and a wild barley from Morocco. These results suggested that the six-rowed type I and II barleys were derived from two-rowed type I and II barleys, respectively, by independent mutations at the vrs1 locus.

Physical locations of 5S and 18S-25S rDNA in Asian and American diploid Hordeum species with the I genome.

The physical locations of 5S and 18S-25S rDNA sequences in 15 diploid Hordeum species with the I genome were examined by double-target in situ hybridization with pTa71 (18S-25S rDNA) and pTa794 (5S rDNA) clones as probes. All the three Asian species had a species-specific rDNA pattern. In 12 American species studied, eight different rDNA types were found. The type reported previously in H. chilense (the 'chilense' type) was observed in eight American species. The chilense type had double 5S rDNA sites - two sites on one chromosome arm separated by a short distance - and two pairs of major 18S-25S rDNA sites on two pairs of satellite chromosomes. The other seven types found in American species were similar to the chilense type and could be derived from the chilense type through deletion, reduction or addition of a rDNA site. Intraspecific polymorphisms were observed in three American species. The overall similarity in rDNA patterns among American species indicates the close relationships between North and South American species and their derivation from a single ancestral source. The differences in the distribution patterns of 5S and 18S-25S rDNA between Asian and American species suggest differentiation between the I genomes of Asian and American species. The 5S and 18S-25S rDNA sites are useful chromosome markers for delimiting Asian species, but have limited value as a taxonomic character in American species. On the basis of rDNA patterns, karyotype evolution and phylogeny of the I-genome diploid species are discussed.

Barley allele-specific amplicons useful for identifying wheat-barley recombinant chromosomes.

Barley (Hordeum vulgare L.) is potentially a new source of genes for wheat (Triticum aestivum L.) improvement. Wheat-barley chromosome recombinant lines provide a means for introgressing barley genes to wheat genome by chromosome engineering, and since these are expected to occur only rarely in special cytogenetic stocks, an efficient selection skill is necessary to identify them. To convert RFLP markers to barley allele-specific PCR markers useful for effective production of wheat-barley recombinant lines, 91 primer sets derived from RFLP clones which were previously mapped to the barley chromosomes were examined for PCR amplification using 'Chinese Spring' wheat, 'Betzes' barley and the wheat-barley chromosome addition lines. The polymorphisms were detected by an agarose gel electrophoresis of the PCR products without digestion with restriction enzymes. Out of 81 primer sets producing polymorphisms between the wheat and barley genomes, 26 amplified barley chromosome-specific DNAs which were confirmed to be located on the same chromosome as the RFLP markers by using the wheat-barley chromosome addition lines. These amplified DNAs represent barley allele-specific amplicons, which distinguish barley alleles from their wheat homoeologous counterparts. The present investigation revealed a higher probability for obtaining allele-specific amplicons from genomic DNA-derived RFLP markers than from cDNA-derived ones. The barley allele-specific amplicons developed in this study, namely, four for chromosome 2H, two for 3H, seven for 4H, eight for 5H, one for 6H and four for 7H, are suitable for identifying 'Chinese Spring' wheat- 'Betzes' barley recombinant chromosomes. However, one out of eight barley allele-specific amplicons on chromosome 5H did not detect a unique barley band in a 'New Golden' barley chromosome 5H addition line of 'Shinchunaga' wheat, indicating there may be a need to reconstruct allele-specific amplicons with different barley cultivars.

Aluminum tolerance genes on the short arm of chromosome 3R are linked to organic acid release in triticale.

Triticale, a hybrid between wheat and rye, shows a high degree of Al tolerance that is inherited from rye, but the mechanisms of high Al tolerance in both rye and triticale are unknown. We found that the short arm of chromosome 3R carries genes necessary for Al tolerance in triticale (x Triticosecale Wittmark cv Currency). Detailed comparative studies with a 3DS.3RL translocation line (ST22) and a non-substitution line (ST2) were conducted. Root elongation was similarly inhibited by Al in ST2 and ST22 during the first 12 h of Al treatment, but more strongly in ST22 than in ST2 at 18 h and thereafter. The root inhibition induced by other metals (Cu, Cd, and La) was similar between ST2 and ST22, suggesting that the action of the genes for Al tolerance on the short arm of triticale chromosome 3R is highly specific to Al. A 2-fold larger amount of malate and citrate was released from the roots of ST2 than from ST22 at 12 and 18 h after Al treatment, respectively. The marked lag phase in the inhibition of root elongation and the release of organic acids implies that the expression of genes on the short arm of triticale chromosome 3R is induced by Al, and that these genes are necessary for the release of organic acids.

The chromosomal distributions of Ty1-copia group retrotransposable elements in higher plants and their implications for genome evolution.

Retrotransposons make up a major fraction--sometimes more than 40%--of all plant genomes investigated so far. We have isolated the reverse transcriptase domains of the Ty1-copia group elements from several species, ranging in genome size from some 100 Mbp to 23,000 Mbp, and determined the distribution patterns of these retrotransposons on metaphase chromosomes and within interphase nuclei by DNA:DNA in situ hybridization. With some exceptions, the reverse transcriptase domains were distributed over the length of the chromosomes. Exclusion from rDNA sites and some centromeres (e.g., slash pine, 23,000 Mbp, or barley, 5500 Mbp) is frequent, whereas many species exclude retrotransposons from other sites of heterochromatin (e.g., intercalary and centromeric sites in broad bean). In contrast, in the plant Arabidopsis thaliana, widely used for plant molecular genetic studies because of its small genome (c. 100 Mbp), the Ty1-copia group reverse transcriptase gene domains are concentrated in the centromeric regions, colocalizing with the 180 bp satellite sequence pAL1. Unlike the pAL1 sequence, however, the Ty1-copia signal is also detectable as weaker, diffuse hybridization along the lengths of the chromosomes. Possible mechanisms for evolution of the contrasting distributions are discussed. Understanding the physical distribution of retrotransposons and comparisons of the distribution between species is critical to understanding their evolution and the significance for generation of the new patterns of variability and in speciation.

In vitro phosphorylation of bovine cardiac muscle high molecular weight calmodulin binding protein by cyclic AMP-dependent protein kinase and dephosphorylation by calmodulin-dependent phosphatase.

High molecular weight calmodulin binding protein (HMWCaMBP) is one of the major proteins expressed in bovine cardiac muscle. In this study, we report the phosphorylation and dephosphorylation of HMWCaMBP in vitro with a view to understand the function of this protein. The HMWCaMBP was phosphorylated by cAMP-dependent protein kinase with the incorporation of 2.30 mol of phosphate/mol of protein in the presence of EGTA. When phosphorylation was carried out in the presence of Ca2+/calmodulin (CaM), the incorporation of phosphate was reduced to 1.40 mol of phosphate/mol of protein. The decrease in the stoichometry of phosphorylation by Ca2+/CaM appears to be substrate directed i.e. due to the interaction of Ca2+/CaM with HMWCaMBP. The phosphorylated HMWCaMBP was unable to compete for free CaM in a CaM-dependent cyclic nucleotide phosphodiesterase (CaMPDE) assay. These results suggest that the phosphorylation sites may reside in or in proximity to the CaM-binding domain on HMWCaMBP since phosphorylated HMWCaMBP did not inhibit CaMPDE activity. HMWCaMBP was dephosphorylated by CaM-dependent phosphatase, calcineurin.

C-banding analysis on wild Emmer (Triticum dicoccoides Körn) strains with and without spontaneous reciprocal translocations.

C-banding polymorphism was analyzed in eight strains of wild Emmer, Triticum dicoccoides Körn, which included six translocation homozygotes reported previously. Polymorphisms were detected in all of the strains examined, and the breakpoints of five spontaneous translocations were successfully identified by C-bands. Of the eight breakpoints that could be precisely identified, one was located in the centromeric region while the remaining seven were located in proximal to distal euchromatic regions. The two breakpoints of one translocation could only be approximately localized to proximal regions due to the scarcity of C-bands. The present results are in contrast with those observed on T. araraticum, another wild tetraploid wheat belonging to the Timopheevi group, in which most of the breakpoints were located in centromeric regions. In T. dicoccoides, the six translocation chromosome types were derived from the standard karyotype primarily by a mechanism other than centric breakage-fusion.

High molecular weight calmodulin-binding protein is phosphorylated by calmodulin-dependent protein kinase VI from bovine cardiac muscle.

A high molecular weight calmodulin-binding protein (HMW CaMBP) from bovine heart cytosolic fraction was purified to apparent homogeneity. A novel CaM-dependent protein kinase was originally discovered when the total CaM-binding protein fraction from cardiac muscle was loaded on a gel filtration column. The CaM-dependent protein kinase was shown by gel filtration chromatography to have an apparent molecular mass of 36,000 daltons. The CaM-dependent protein kinase has been highly purified by sequential chromatography on DEAE-Sepharose Cl 6B (to remove calmodulin), CaM-Sepharose 4B, phosphocellulose, Sepharose 6B gel filtration and Mono S column chromatographies. The highly purified protein kinase stoichiometrically phosphorylated the HMW CaMBP in a Ca2+/CaM-dependent manner. The phosphorylation resulted in the maximal incorporation of 1 mol of phosphate/mol of the HMW CaMBP. The distinct substrate specificity of this protein kinase indicates that it is not related to the known protein kinases (I, II, III, IV and V) that have been already characterized, therefore we would like to designate this novel kinase as a CaM-dependent protein kinase VI.

High crossability of wild barley (Hordeum spontaneum C. Koch) with bread wheat and the differential elimination of barley chromosomes in the hybrids.

Four bread wheat (Triticum aestivum L.) cultivars, 'Aobakomugi', 'Chinese Spring', 'Norin 61' and 'Shinchunaga', were pollinated with five barley lines/cultivars consisting of three cultivated barley (Hordeum vulgare L.) lines, 'Betzes', 'Kinai 5' and OHL089, and two wild barley (Hordeum spontaneum C. Koch) lines, OUH602 and OUH324. Crossability, expressed as the percentage of embryo formation, varied from 0 to 55.4% among the cross combinations. The two wild barley lines generally had a higher crossability than the previously reported best pollinator, 'Betzes', and some Japanese wheat cultivars were better as the female parent than 'Chinese Spring'. Ninety four hybrid plants were obtained from 250 embryos cultured, and their somatic chromosome numbers ranged from 21 to 36. Eighteen plants were mosaic in chromosome number. Twenty one-chromosome plants appeared most frequently (45.7%) followed by 28-chromosome plants (14.9%). C-banding analysis revealed that elimination of barley chromosomes was mainly responsible for the occurrence of aneuploid plants. In hypoploids derived from 'Betzes'-crosses, chromosome 5 was preferentially eliminated as previously reported, while in hypoploids derived from OUH602-crosses, chromosome 4 was preferentially eliminated. The wild barley line OUH602 may be a useful parent for producing a new wheat-barley addition set because of its high crossability with wheat and a different pattern of chromosome elimination.

The evidence for post-meiotic expression of a testis-specific isoform of a regulatory subunit of calcineurin using a monoclonal antibody.

The expression of a regulatory subunit of calcineurin (CaN beta) during rat spermatogenesis was examined in rat testes using a monoclonal antibody Va1. Results showed that a testis-specific isoform of CaN beta was expressed only 3 weeks after birth, when meiosis begins, and increased in amount depending on the maturation of spermatogenesis. The matured sperm, which consists of only post-meiotic cells, is most likely to have only the testis-specific isoform of CaN beta. The brain type isoform of CaN beta was not detected in rat sperm. Immunoblot analysis of testes from different rodent species by a monoclonal antibody Va1 showed that all rodent species examined had their own homologues corresponding to a testis-specific isoform of CaN beta in rats, although they showed distinctively different molecular weights on SDS-PAGE compared to the testis-specific isoform in rats. Each homologue was shown to be specifically expressed in post-meiotic phase of spermatogenesis, as was seen in rats.

Differences in growth requirements between epithelial and stromal cells derived from rat ventral prostate in serum-free primary culture.

Different procedures of enzymatic digestion of rat prostatic tissue and unique sets of mitogenic factors made it possible to culture practically pure populations of epithelial and stromal cells without previous separation of the two types of cells. Keratin-positive epithelial cells dissociated by trypsin and collagenase from adult rat ventral prostate proliferated in medium WAJC 404 supplemented with epidermal growth factor, insulin, cholera toxin, and bovine pituitary extract. Proliferation of epithelial cells was completely inhibited by dexamethasone as low as 30 nM. On the other hand, fibroblast-like stromal cells released by trypsin digestion required a plastic substratum coated with calf serum or fibronectin, and proliferated in Eagle's minimum essential medium supplemented with cholera toxin, bovine pituitary extract, dexamethasone, and bovine serum albumin. Epidermal growth factor and insulin had negligible effect on proliferation of stromal cells. Physiological concentrations of dihydrotestosterone and estradiol showed no effect on proliferation of both types of cells.

Student accidents in Hawaii's public schools.

A survey of the student accident report forms was conducted in Hawaii public schools. Review of the data gathered during one school year revealed that the intermediate grade level had the highest number of school accidents with males, in a 2.1:1 ratio, more involved in accidents than females. Although no deaths occurred, there were a significant number of school accidents and school days lost to warrant the need for safety education. The accident report forms were reviewed and data summarized by the school nurses working within the schools. Because the information provided in the accident report forms was so varied, it was not possible to make a recommendation as to any specific restriction of activities. However, the survey revealed a need to strengthen the criteria for reporting accidents.