Construction of a fusion gene comprising the Taka-amylase A promoter and the Escherichia coli β-glucuronidase gene and analysis of its expression in Aspergillus oryzae

Summary Northern blot analysis of glucose-grown and starch-grown mycelia of Aspergillus oryzae R11340 was conducted using the cloned Taka-amylase A (TAA) gene as a probe. The amount of mRNA homologous to the TAA gene was increased when this fungus was grown with starch as a sole carbon source. In order to analyze the induction mechanism, we inserted the Escherichia coli uidA gene encoding -glucuronidase (GUS) downstream of the TAA promoter and introduced the resultant fusion gene into the A. oryzae genome. Production of a functional GUS protein was induced by starch, but not by glucose. When the effects of various sugars on expression of the fusion gene were examined, the results suggested that the expression of the fusion gene was under control of the TAA gene promoter.     

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.     

Transformation of Zea mays L. Using Agrobacterium tumefaciens and the Shoot Apex

Agrobacterium tumefaciens is established as a vector for gene transfer in many dicotyledonous plants but is not accepted as a vector in monocotyledonous plants, especially in the important Gramineae. The use of Agrobacterium to transfer genes into monocot species could simplify the transformation and improvement of important crop plants. In this report we describe the use of Agrobacterium to transfer a gene into corn, the regeneration of plants, and detection of the transferred genes in the F₁ progeny. Shoot apices of Zea mays L. variety Funk's G90 were cocultivated with A. tumefaciens EHA 1, which harbored the plasmid pGUS3 containing genes for kanamycin resistance (NPT II) and β-glucuronidase (GUS). Plants developed from these explants within 4 to 6 weeks. Fluorometric GUS assays of leaves and immature seeds from the plants exhibited low GUS activity. Both NOS and GUS gene fragments were amplified by polymerase chain reaction in the DNA isolated from the F₁ generations of one of the original transformed plants. Southern analysis showed both GUS and NPT probes hybridized to DNA in several of the F₁ progeny, demonstrating the incorporation of GUS and NPT II genes into high molecular weight DNA. These data establish successful gene transfer and sexual inheritance of the genes.     

Growth Yields and Maintenance Coefficients of Unadapted and NaCl-Adapted Tobacco Cells Grown in Semicontinuous Culture

Comparison of carbon utilization between unadapted and NaCl (428 millimolar) adapted tobacco (Nicotiana tabacum L.) cells under substrate limited growth conditions was facilitated using semicontinuous culture. Growth yields (Y_g) and maintenance coefficients (m) of unadapted and NaCl adapted cells were similar, indicating that the efficiency of carbon utilization for growth was not altered as a result of salt adaptation and that no additional metabolic costs were associated with growth of adapted cells in the presence of a high concentration (428 millimolar) of NaCl. The Y_g (0.588 grams organic dry weight gain per gram sugar uptake) and m values (0.117 grams sugar uptake per gram organic dry weight per day) were comparable in spite of substantial physiological and biochemical differences that exist between unadapted and NaCl adapted cells. Apparently, a metabolic homeostasis governs biomass production of cells before and after adaptation to salinity.     

13C-n.m.r. analysis of some sulphate derivatives of chitosan.

Positions of substitution with sulphate in three water-soluble sulphated derivatives of chitosan were analysed by 13C n.m.r. The structures of N-acetylchitosan 3,6-O-disulphate, sodium chitosan N-, 6-O-disulphate, and sodium chitosan 6-O-monosulphate were confirmed.     

Comparison of intraspecific nest usurpation between two haplometrotic paper wasp species (Hymenoptera: Vespidae:Polistes)

Intraspecific nest usurpation by foundresses was studied in 2 haplometrotic (solitary founding) species of different subgenera,Polistes (P.) riparius andP. (Polistella) snelleni, in areas where they cohabited. The overall probability for a nest to be usurped by a foreign foundress during the season was about twice as large inP. snelleni as inP. riparius. In both, however, probability of usurpation was largest on late pre-emergence nests, or in late June and early July. InP. riparius, all the usurpers of known origins were those foundresses that had lost their pre-emergence nests to destruction probably by some vertebrates; inP. snelleni, some usurpers had the same history as above, while the others had lost many of all larvae to predation by unknown agents before worker emergence. Usurpers of both species destroyed eggs and younger larvae to much greater extents than older larvae or pupae, and they produced fewer numbers of reproductives in comparison with non-usurping foundresses. We concluded that usurpation behavior has been maintained despite its relatively low productivity because renesting would lead to even lower or no reproductive production.     

Cell type-specific expression of the human renin gene.

We have previously produced transgenic mice carrying the human renin gene, whose expression is regulated in a tissue-specific manner. In the present study, we further characterized expression of the transgene. Northern blot analysis showed that the human renin gene is expressed in the kidney but not in the liver of two lines of transgenic mice with 10 and 50 copies of the transgene, suggesting that the integrated copy number of the human renin gene does not influence the dominant-renal expression pattern. Immunohistochemical study using a monoclonal antibody specific for human renin demonstrated that expression of human renin in the transgenic mouse kidney is confined to the epithelioid juxtaglomerular cells. Transfection experiments indicated that the chloramphenicol acetyltransferase fusion gene containing the 3-kb upstream sequences of the renin gene is activated only in human epithelioid embryonic 293 cells derived from kidney but not in human HepG2 cells from liver. These findings suggest that transfer of the cloned renin gene into mice and in vitro cultured cell lines can give rise to cell type-specific expression.     

Chromosomal genes essential for stable maintenance of the mini-F plasmid in Escherichia coli

We have isolated mutants of Escherichia coli which do not support stable maintenance of mini-F plasmids (delta ccd rep+ sop+). These host mutations, named hop, were classified into five linkage groups on the E. coli chromosome. Genetic analyses of these hop mutations by Hfr mating and P1 transduction showed their loci on the E. coli genetic map to be as follows: hopA in the gyrB-tnaA region, hopB in the bglB-oriC region, hopD between 8 and 15 min, and hopE in the argA-thyA region. Kinetics of stability of the sop+ and delta sop mini-F plasmids in these hop mutants suggest that the hopA mutants are defective in partitioning of mini-F rather than in plasmid replication. The hopB, hopC, and hopD mutants were partially defective in replication of mini-F. The physical structure of the plasmid DNA was normal in hopA, B, C, and D mutants. Large amounts of linear multimers of plasmid DNA accumulated in mutants of the fifth linkage group (hopE). None of the hop mutations in any linkage group affected the normal growth of cells.     

Studies on the development of water maze-learning ability in rats (3). Effect of the learning schedule on learning acquisition

The effect of different learning schedules massed or distributed practice conditions (3 trials a day for 3 days), on water-filled multiple T-maze learning ability of 8-week-old SPF Wistar-Imamichi rats was investigated. Although the mean number of errors decreased day by day in both groups, the number of errors in a given day and the total number of errors in 3 days did not differ significantly between the two groups. A tendency toward a decrease in the number of errors was observed as the trials proceeded in the group with distributed practice but not in the group with massed practice. The result suggests that a certain time period for rest after each trial is necessary to acquire the memory.     

Compressibility-structure relationship of globular proteins

The adiabatic compressibility, -beta s, of 11 globular proteins in water was determined by means of sound velocity measurements at 25 degrees C. All the proteins studied except for subtilisin showed positive -beta s values, indicating the large internal compressibility of the protein molecules. The intrinsic compressibility of proteins free from the hydration effect appeared to be comparable to that of normal ice. The compressibility data for 25 proteins, including 14 reported previously [Gekko, K., & Noguchi, H. (1979) J. Phys. Chem. 83, 2706-2714], were statistically analyzed to examine the correlation of the compressibility with some structural parameters and the amino acid compositions of proteins. It was found that -beta s increases with increasing partial specific volume and hydrophobicity of proteins. The helix element also seemed to be a dynamic domain to increase -beta s. Four amino acid residues (Leu, Glu, Phe, and His) greatly increased -beta s, and another four (Asn, Gly, Ser, and Thr) decreased it. Some empirical equations were derived for the estimation of the -beta s values of unknown proteins on the basis of their amino acid compositions. The volume fluctuations of proteins revealed by the compressibility data were in the range of 30-200 mL/mol, which corresponded to about 0.3% of the total protein volume. The conformational fluctuation seemed to enhance the thermal stability of proteins.