Structure of the Bombyx mori fibroin light-chain-encoding gene: upstream sequence elements common to the light and heavy chain.

Two overlapping genomic clones containing the fibroin light-chain (Fib-L)-encoding gene (Fib-L) were obtained from the cosmid library of the silkworm, Bombyx mori J-139, by hybridization with the Fib-L cDNA clone. Sequencing of the 14.6-kb region revealed that Fib-L was 13472 bp long containing seven exons, and that the gene contained a large first intron which occupied about 60% of the gene. Comparison of restriction patterns of the J-139 Fib-L with those of eight other B. mori breeds producing normal-level fibroin demonstrated that considerable restriction-fragment length polymorphisms were present in regions containing the first intron and the 3′-flanking sequence. However, sizes of the Fib-L mRNA and the Fib-L polypeptide were very similar among the nine breeds tested, suggesting that the exon sequences and the splice signals were all well conserved. 5′-Flanking regions of Fib-L and the fibroin heavy-chain (Fib-H)-encoding gene (Fib-H) compared in this study contained three 18-30-bp sequences of high similarity and many 8-10-bp common elements, six of which coincided with the binding sites of homeodomain proteins. Gel retardation assays with the nuclear extracts of the posterior and middle silk glands suggested that protein factors present in the posterior silk-gland nuclei could bind to a set of those common upstream elements.     

Phototropic stimulation does not induce unequal distribution of indole-3-acetic acid in maize coleoptiles

Distribution of endogenous diffusible auxin into agar blocks from phototropically stimulated maize coleoptile tips was studied using a bioassay and a physicochemical assay, to clarify whether phototropism in maize coleoptiles involves a lateral gradient in the amount of auxin. At 50 min after the onset of phototropic stimulation, when the phototropic response was still developing, direct assay of the blocks with the Avena curvature test showed that the auxin activity in the blocks from the shaded half-tips was twice that of the lighted side, at both the first and second positive phototropic curvatures. However, physicochemical determination following purification showed that the amount of indole-3-acetic acid (IAA) was evenly distributed in the blocks from lighted and shaded coleoptile half-tips at both the first and second positive phototropic curvatures. The even distribution of the IAA was also confirmed with the Avena curvature test following purification by HPLC. These results indicate that phototropism in maize coleoptiles is not caused by a lateral gradient of IAA itself and thus cannot be described by the Cholodny-Went theory. Furthermore, the lower auxin activity in the blocks from the lighted half-tips suggests the presence of inhibitor(s) interfering with the action of auxin and their significant diffusion from unilaterally illuminated coleoptile tips.     

Characterization and Intracellular Distribution of Pea Phytochrome I Polypeptides Expressed in E. coli

The pea phytochrome I (PI) cDNA clone, pPP1001, was expressedin E. coli. The plasmid pPP1001 contains pea PI cDNA which coversthe entire coding region with the Shine-Dalgarno consensus sequencejoined upstream of the cDNA in an expression vector pNUT6. ThepPP1001 transformants formed typical inclusion bodies when culturedat 32?C. However, when cultured at 37?C or in the presence ofisopropyl-ß-D-thiogalactopyranoside (IPTG) at 32?C,the bacteria lysed before inclusion body formation. Immuno-stainingwith anti-PI monoclonal antibody, mAP5, of transformants fixedby cold methanol showed that stainable materials were distributedin whole cytoplasmic region. When the inclusion bodies wereobserved clearly, the regions corresponding to the inclusionbodies became difficult to stain. Western blot analysis, however,showed that a ca. 100 kDa PI polypeptide was detected in thefraction from inclusion bodies and a ca. 90 kDa PI polypeptidefrom the soluble fraction. The amino acid sequence analysisof purified 100 kDa PI sample indicated that its amino terminusis blocked. However, minor signals in one experiment yieldeda sequence corresponding to the expected amino terminus of peaPI except for the initiation methionine. One of the anti-peaPI monoclonal antibodies, mAP9, that recognizes the near N-terminusof pea phytochrome was reactive to the 100 kDa polypeptide. (Received June 22, 1990; Accepted November 18, 1990)     

Temporal behavioral variation and division of labor among workers in the primitively eusocial wasp,Polistes jadwigae Dalla Torre

The behavior of workers on 2 nests of the Japanese paper wasp,Polistes jadwigae, was recorded on video tape. Young workers performed pulp collection more frequently than middle-aged and old workers, while foraging of flesh (prey) was done mainly by middle-aged workers. Of the 26 behavioral categories, 7 showed negative, 2 concave, and 1 convex relationship to the workers age. Using behavioral profiles, workers could be classified into 3 age groups: 1) young workers (younger than 10 days old, active intranidal workers), 2) middle-aged workers (11–25 days old, extranidal workers) and 3) old workers (older than 26 days old, ‘idlers’). Idlers engaged in foraging and larval feeding significantly less frequency than the young and middle-aged workers, but they performed alerting toward many alien objects. Task specialization among workers irrespective of age polyethism was also suggested.     

Incorporation of fatty acids by Streptococcus mutans

In a series of investigations into the cariogenicity of Streptococcus mutans, we studied the incorporation of exogenous fatty acids with reference to glucosyltransferase secretion and membrane fatty acid changes. When cells were grown with different fatty acids, both saturated and unsaturated fatty acids were readily incorporated into the membrane lipids and were biotransformed and elongated preferentially to the longer 16- and 18-carbon-chain fatty acids. This incorporation and chain-elongation led to significant changes in fatty acids composition. By adding fatty acids to the medium, it was possible to appropriately modify the degree of unsaturation and the relative ratio between specific fatty acids in the membrane lipids of S. mutans.     

Preparation of colloidal gold for staining proteins electrotransferred onto nitrocellulose membranes

This paper describes a simple method of preparing colloidal gold for staining protein blots. Colloidal gold was prepared from 0.005 or 0.01% HAuCl4 by the addition of formalin as a reductant and potassium hydroxide. Staining of small cell carcinoma tissue extract blotted onto nitrocellulose membranes with this colloidal gold solution resulted in the appearance of a large number of clear wine-red bands. The sensitivity of gold staining was 60 times higher than that of Coomassie brilliant blue staining and almost comparable to that of silver staining of proteins in polyacrylamide gel. The sensitivity of this method was also satisfactory in comparison with that of enzyme immunoblotting. The colloidal gold prepared by this method is usable for routine work.     

New preparation method for 9-anthryldiazomethane (ADAM) as a fluorescent labeling reagent for fatty acids and derivatives

A preparation method for 9-anthryldiazomethane (ADAM) as a fluorescent labeling reagent for carboxylic acids is described. 9-Anthraldehyde hydrazone is oxidized with an organic oxidant, N-chlorosuccinimide, in an organic solvent such as ethyl acetate to give ADAM, and then the reaction mixture is directly used as the reagent solution for the derivatization of carboxylic acids. Both the oxidation and the derivatization reaction are carried out at room temperature, and an aliquot of the derivatization mixture is directly injected into a chromatograph. 9-Anthrylmethyl ester derivatives formed from ADAM and various carboxylic acids are sufficiently separated on a reversed-phase column and are sensitively detected fluorometrically. The present method was applied to the high-performance liquid chromatographic determination of long and short chain fatty acids, keto acids, and hydroxy acids.