The molecular analysis ofbrown eye color mutations isolated from geographically discrete populations ofDrosophila melanogaster

A large proportion of spontaneous mutations inDrosophila melanogaster strains of laboratory origin are associated with insertions of mobile DNA elements. As a first step toward determining whether spontaneous laboratory mutations are predictive for mutational events occurring in the wild, recessivebrown (bw) eye color mutants were isolated. By inbreeding the progeny of wild-caughtDrosophila melanogaster females,bw mutations were isolated from seven separate geographic sites distributed among Japan, California, Siberia and Hungary. Among a total of 14 mutations studied, no case of transposon mutagenesis was found. At least 4 mutations are associated with small deletions in thebw gene. The remainder are inseparable from wild-typebw by Southern analysis and are presumed to be basepair changes or very small indels. Although only two spontaneousbw mutants of laboratory origin have been analyzed molecularly, one is a mobile element insertion.     

Nucleotide Sequence Surrounding the Locus Marker D21S246 on Human Chromosome 21

Most ofthe human Not I linking clones identified to date areconsidered to be derived from CpG islands because ofthe recognitionsequence of this enzyme, and CpG islands have been reportedto be located around the 5' regions of genes. As a pilot study,we determined the complete nucleotide sequence (41,924 bp) ofa human cosmid clone (LL21NC02Q7A10) containing the marker D21S246originating from a Not I linking clone. As a result of sequenceanalysis, we successfully mapped and revealed the genomic genestructure for KIAA0002 previously reported as a cDNA clone.This gene consists of 15 exons and was shown to exist at theD21S246 locus on human chromosome 21q21.3–q22.1. Theseresults demonstrated that genomic marker-anchored DNA sequencingis a useful approach for the human genome project.     

Purification and characterization of a novel enzyme, alpha-neoagarooligosaccharide hydrolase (alpha-NAOS hydrolase), from a marine bacterium, Vibrio sp. strain JT0107.

A novel enzyme, alpha-neoagarooligosaccharide hydrolase (EC 3.2.1.-), which hydrolyzes the alpha-1,3 linkage of neoagarooligosaccharides to yield agaropentaose (O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro-alpha-L-galactopyranosyl (1-->3)-D-galactose], agarotriose [O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro- alpha-L-galactopyranosyl (1-->3)-D-galactose], agarobiose [O-beta-D-galactopyranosyl(1-->4)-3,6-anhydro-L-galactose], 3,6-anhydro-L-galactose, and D-galactose was isolated from the marine bacterium Vibrio sp. strain JT0107 and characterized. This enzyme was purified 383-fold from cultured cells by using a combination of ammonium sulfate precipitation, successive anion-exchange column chromatography, gel filtration, and hydroxyapatite chromatography, gel filtration, and hydroxyapatite chromatography. The purified protein gave a single band (M(r), 42,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Estimation of the M(r) by the gel filtration method gave a value of 84,000, indicating that the enzyme is dimeric. Amino acid sequence analysis revealed it to have a single N-terminal sequence that has no sequence homology to any other known agarases. The optimum temperature and pH were 30 degrees C and 7.7, respectively. The Km and maximum rate of metabolism for neoagarobiose were 5.37 mM and 92 U/mg of protein, respectively.     

Identification of a basic glycoprotein induced by ethylene in primary leaves of azuki bean as a cationic peroxidase.

Ethylene causes the accumulation of seven different proteins (each designated AZxx according to its molecular mass, xx in kD) in excised primary leaves of azuki bean (Vigna angularis) (F. Ishige, H. Mori, K. Yamazaki, H. Imaseki [1991] Plant Cell Physiol 32: 681-690). A complementary DNA encoding an ethylene-induced basic glycoprotein, AZ42, from azuki bean was cloned and its complete nucleotide sequence was determined. Characterization of the cDNA was accomplished by monitoring expression of an immunoreactive protein in Escherichia coli that harbored the cDNA and by the identification of a partial amino acid sequence that was the same as that determined from the purified protein. An open reading frame (1071 base pairs) in the cDNA encoded a protein of 357 amino acids with a molecular mass of 39.3 kD. The amino acid sequence contained three regions that are highly conserved among peroxidases from eight different plants. Purified AZ42 exhibited peroxidase activity. The basic glycoprotein induced by ethylene was identified as a cationic isozyme of peroxidase. The corresponding mRNA was not present in leaves that had not been treated with ethylene, but it appeared after 1 h of treatment with ethylene and its level increased for the next 15 h. Accumulation of the mRNA was also induced after wounding or treatment with salicylate. The wound-induced increase in the level of the mRNA was suppressed by 2,5-norbornadiene, but the salicylate-induced increase was not.     

A comparison of sweating responses during exercise and recovery in terms of sweating rate and body temperature

Based on the hypothesis that the relation between sweating rate and body temperature should be different during exercise and rest after exercise, we compared the sweating response during exercise and recovery at a similar body temperature. Healthy male subjects performed submaximal exercise (Experiment 1) and maximal exercise (Experiment 2) in a room at 27° C and 35% relative humidity. During exercise and recovery of 20 min after exercise, esophageal temperature (Tes), mean skin temperature, mean body temperature ( ), chest sweating rate ( ), and the frequency of sweat expulsion (F _SW) were measured. In both experiments, andF _SW were clearly higher during exercise than recovery at a similar body temperature (Tes, ). was similar during exercise and recovery, or a little less during the former, at a similarF _SW. It is concluded that the sweating rate during exercise is greater than that during recovery at the same body temperature, due to greater central sudomotor activity during exercise. The difference between the two values is thought to be related to non-thermal factors and the rate of change in mean skin temperature.     

Embryonic Development and Postnatal Changes in Free d-Aspartate and d-Serine in the Human Prefrontal Cortex

Abstract: We have analyzed free chiral amino acids (aspartate and serine) in the human frontal cortex at different ontogenic stages (from 14 weeks of gestation to 101 years of age) by HPLC with fluorometric detection after derivatization with N-tert -butyl-oxycarbonyl- l -cysteine and o -phthaldialdehyde. Exceptionally high levels of free d -aspartate and d -serine were demonstrated in the fetal cortex at gestational week 14. The ratios of d -aspartate and of d -serine to the total corresponding amino acids were also high, at 0.63 and 0.27, respectively. The concentration of d -aspartate dramatically decreased to a trace level by gestational week 41 and then remained very low during all postnatal stages. In contrast, the frontal tip contained persistently high levels of d -serine throughout embryonic and postnatal life, whereas the d -amino acid content in adolescents and aged individuals was about half of that in the fetuses. Because d -aspartate and d -serine are known to have selective actions at the NMDA-type excitatory amino acid receptor, the present data suggest that these d -amino acids might play a pivotal role in cerebral development and functions that are related to the NMDA receptor.     

Rapid determination of tetracycline antibiotics in serum by reversed-phase high-performance liquid chromatography with fluorescence detection

A rapid and accurate determination of tetracycline antibiotics in human serum by reversed-phase high-performance liquid chromatography with fluorescence detection has been developed, based on protein precipitation in serum. Various reagents for precipitation were investigated, and 24% trichloroacetic acid in methanolic solution gave the maximum recovery (at least 94.3%) and interference-free chromatograms of different three tetracyclines. At a concentration of 0.5 μg/ml, the precision (relative standard deviation) ranged from 1.12 to 1.94%. In the range 0.04–10.0 μg/ml for oxytetracycline and chlorotetracycline and 0.01–10.0 μg/ml for tetracycline, linear responses were observed. The detection limits of this method were 10–35 ng/ml for all three antibiotics. The proposed method was applied to the determination of serum concentrations in subjects receiving tetracycline antibiotics.     

Efficient and gentle elution of antibodies from an immobilized polypeptide antigen BT saturated magnesium chloride

Summary A model antigen, rabbit immunoglobulin G, was immobilized onto polyester cloth by adsorption. The antigen cloth was reacted with sheep anti-rabbit IgG antibody. Antibody bound to the antigen cloth was nearly quantitatively eluted by saturated MgCl₂, whereas a commercial antibody eluent slowly eluted only about 70 % of the antibody. Exposure of antibody to saturated MgCl₂ for 30 min resulted in no loss of immunoactivity. Saturated MgCl₂, therefore, is an ideal eluent in immunoaffinity purification of antibodies.     

Excess carotenoids disturb prospective cell-to-cell recognition system in mating responses ofPhycomyces blakesleeanus

Carotenogenic mutants ofPhycomyces, which accumulate excess β-carotene or its intermediates, always failed in zygospore development. No improvement occurred when such mutants were mated together with a helper wild type of the same mating type against the wild type of the opposite mating type. Addition of excess synthesized pheromone, trisporin B, also failed to improve the zygospore development, though the mating response was significantly activated in the early stages and abundant zygophores were formed. Exceptional acceleration of the zygospore development under these experimental conditions occurred in a regulatory albino mutant (carA), which does not accumulate excess intermediate carotenoids. Chemically- or genetically-induced ovarproduction of β-carotene or lycopene also inhibited the zygospore development. These results imply that the zygospore development ofPhycomyces is maximal when the intracellular amount of β-carotene is optimal (=wild type), and that pheromones act mainly in the early stages of mating, while other factors such as the cell-to-cell recognition system may also be involved in the later stages. Intracellular accumulation of excess β-carotene or its intermediates probably disturb such later-stage factors.     

Molecular Evolution of the Duplicated Amy Locus in the Drosophila Melanogaster Species Subgroup: Concerted Evolution Only in the Coding Region and an Excess of Nonsynonymous Substitutions in Speciation

From the analysis of restriction maps of the Amy region in eight sibling species belonging to the Drosophila melanogaster species subgroup, we herein show that the patterns of duplication of the Amy gene are almost the same in all species. This indicates that duplication occurred before speciation within this species subgroup. From the nucleotide sequence data, we show a strong within-species similarity between the duplicated loci in the Amy coding region. This is in contrast to a strong similarity in the 5' and 3' flanking regions within each locus (proximal or distal) throughout the species subgroup. This means that concerted evolution occurred only in the Amy coding region and that differentiated evolution between the duplication occurred in the flanking regions. Moreover, when comparing the species, we also found a significant excess of nonsynonymous substitutions. In particular, all the fixed substitutions specific to D. erecta were found to be nonsynonymous. We thus conclude that adaptive protein evolution occurred in the lineage of D. erecta that is a ``specialist' species for host plants and probably also occurs in the process of speciation in general.