Effects of an antisense napin gene on seed storage compounds in transgenic Brassica napus seeds

To manipulate the quantity and quality of storage components in Brassica napus seeds, we have constructed an antisense gene for the storage protein napin. The antisense gene was driven by the 5-flanking region of the B. napus napin gene to express antisense RNA in a seed-specific manner. Seeds of transgenic plants with antisense genes often contained reduced amounts of napin. In some transgenic plants, no accumulation of napin was observed. However, the total protein content of transgenic and wild-type seeds did not differ significantly. Seeds lacking napin accumulated 1.4 to 1.5 times more cruciferin than untransformed seeds, although the oleosin content was not affected. Fatty acid content and composition in the seeds of transgenic plants were also analyzed by gas chromatography. Though the total fatty acid content of the transformants was the same as that of non-transformants, there was a reduction in 18:1 contents and a concomitant increase of 18:2 in seeds with reduced napin levels. This observed change in fatty acid composition was inherited in the next generation.     

The promoters of two carboxylases in a C4 plant (maize) direct cell-specific, light-regulated expression in a C3 plant (rice)

C₄ plants have two carboxylases which function in photosynthesis. One, phosphoenolpyruvate carboxylase (PEPC) is localized in mesophyll cells, and the other, ribulose bisphosphate carboxylase (RuBPC) is found in bundle sheath cells. In contrast, C₃ plants have only one photosynthetic carboxylase, RuBPC, which is localized in mesophyll cells. The expression of PEPC in C₃ mesophyll cells is quite low relative to PEPC expression in C₄ mesophyll cells. Two chimeric genes have been constructed consisting of the structural gene encoding β-glucuronidase (GUS) controlled by two promoters from C₄ (maize) photosynthetic genes: (i) the PEPC gene (pepc) and (ii) the small subunit of RuBPC (rbcS). These constructs were introduced into a C₃ cereal, rice. Both chimeric genes were expressed almost exclusively in mesophyll cells in the leaf blades and leaf sheaths at high levels, and no or very little activity was observed in other cells. The expression of both genes was also regulated by light. These observations indicate that the regulation systems which direct cell-specific and light-inducible expression of pepc and rbcS in C₄ plants are also present in C₃ plants. Nevertheless, expression of endogenous pepc in C₃ plants is very low in C₃ mesophyll cells, and the cell specificity of rbcS expression in C₃ plants differs from that in C₄ plants. Rice nuclear extracts were assayed for DNA-binding protein(s) which interact with a cis-regulatory element in the pepc promoter. Gel-retardation assays indicate that a nuclear protein with similar DNA-binding specificity to a maize nuclear protein is present in rice. The possibility that differences in pepc expression in a C₃ plant (rice) and C₄ plant (maize) may be the result of changes in cis-acting elements between pepc in rice and maize is discussed. It also appears that differences in the cellular localization of rbcS expression are probably due to changes in a trans-acting factor(s) required for rbcS expression.     

The Presequence of a Precursor to the {delta}-Subunit of Sweet Potato Mitochondrial F1ATPase is not Sufficient for the Transport of {beta}-Glucuronidase (GUS) into Mitochondria of Tobacco, Rice and Yeast Cells

A precursor to the     

Functions of milk protein gene 5′ flanking regions on human growth hormone gene

Fragments containing 5′ flanking regions of four bovine milk protein genes—alpha lactalbumin (bαLA), alpha S₁ casein (bαS₁CN), beta casein (bβCN), kappa casein (b_kCN)—and mouse whey acidic protein (mWAP) gene were prepared by PCR and ligated to human growth hormone (hGH) gene. These recombinant DNAs were microinjected into rat embryos to produce transgenic rats, and the functions of the 5′ regions to direct secretion of hGH in the milk were tested. Although milk was obtained only in 5 of 19 mWAP/hGH rat lines, more than two-thirds of the rats carrying the other four DNAs produced milk. More than 80% of the lactated rats carrying bαLA/, bβCN/, and mWAP/hGH, and 33% of the laclated bαS₁CN/hGH rats secreted detectable amounts of hGH (> 0.05 μg/ml) in the milk. In some rats, the hGH concentrations in the milk were comparable to or more than that of the corresponding milk protein in bovine milk. The ranges of hGH concentrations in the milk of bαLA/, bβCN/, bαS₁CN/, and mWAP/hGH rats were 1.13–4,360 μg/ml, 0.11–10,900 μg/ml, 86.8–6,480 μg/ml, and 6.87–151 μg/ml, respectively. HGH was also detected in the sera of these rats, and some abnormalities of growth and reproduction were observed. All but one virgin mWAP/hGH rat secreted up to 0.0722 μg/ml of hGH in the serum, and more than half of them showed abnormal fat accumulations at their abdomen. None of the bαCN/hGH rats secreted detectable amount of hGH into their milk, whereas 8 of the 11 lines secreted hGH into their sera. For the production of hGH in transgenic rat milk, the 5′ region of bαS₁CN was shown most suitable, because the bαS₁CN/hGH rat secreted > 6,000 μg/ml of hGH into the milk and could be reproduced. © 1994 Wiley-Liss, Inc.     

Regulation of L-alanine-initiated germination ofBacillus subtilis spores by alanine racemase

Summary Germination ofBacillus subtilis spores was initiated by L-Ala and competitively inhibited by D-Ala, suggesting the presence of an alanine receptor. The spores showed alanine racemase activity in the spore coat. To investigate the role of alanine racemase (L D) on germination, net racemase activity was determined using diphenylamine as a germination inhibitor and germination was measured using D-penicillamine as a racemase inhibitor. Apparent affinity of L-Ala to the germinant receptor was more than 1000 times higher than that to the racemase. Germination increased in the presence of D-penicillamine, when the concentration of L-Ala was low and that of spores was high. Racemase activity was optimal at 65°C at pH 9.0 and germination at 43°C at pH 7.2. Under unfavorable growth conditions such as high population of spores in limited nutrients, high temperature and high pH, spore alanine racemase converted the germinant actively to the inhibitor and this conversion may regulate germination for survival of the population.     

Site-directed mutagenesis of histidine residues in Clostridium perfringens alpha-toxin.

Mutagenesis of H-68 or -148 in Clostridium perfringens alpha-toxin resulted in complete loss of hemolytic, phospholipase C, sphingomyelinase, and lethal activities of the toxin. These activities of the variant toxin at H-126 or -136 decreased by approximately 100-fold of the activities of the wild-type toxin. Mutation at H-46, -207, -212, or -241 showed no effect on the biological activities, indicating that these residues are not essential for these activities. The variant toxin at H-11 was not detected in culture supernatant and in cells of the transformant carrying the variant toxin gene. Wild-type toxin and the variant toxin at H-148 bound to erythrocytes in the presence of Ca2+; however, the variant toxins at H-68, -126, and -136 did not. Co2+ and Mn2+ ions stimulated binding of the variant toxin at H-68, -126, and -136 to membranes in the presence of Ca2+ and caused an increase in hemolytic activity. Wild-type toxin and the variant toxins at H-68, -126, and -136 contained two zinc atoms in the molecule. Wild-type toxin inactivated by EDTA contained two zinc atoms. These results suggest that wild-type toxin contains two tightly bound zinc atoms which are not coordinated to H-68, -126, and -136. The variant toxin at H-148 possessed only one zinc atom. Wild-type toxin and the variant toxin at H-148 showed [65Zn]2+ binding, but the variant toxins at H-68, -126, and -136 did not. Furthermore, [65Zn]2+ binding to wild-type toxin was competitively inhibited by unlabeled Zn2+, Co2+, and Mn2+. These results suggest that H-68, -126, and -136 residues bind an exchangeable and labile metal which is important for binding to membranes and that H-148 tightly binds one zinc atom which is essential for the active site of alpha-toxin.     

Light-Induced CREB Phosphorylation and Gene Expression in Rat Retinal Cells

Abstract: The signal pathway for light-induced expression of c- fos and the neuropeptide somatostatin (SS) in rat retinal cells was investigated. Flashing light induced c- fos and SS mRNA in the inner nuclear layer and the ganglion cell layer. As both c- fos and SS genes have a cyclic AMP response element (CRE) in their promoters, CRE-binding protein (CREB) phosphorylation in retinal cells was examined with a phospho-CREB-specific antibody. Both flashing light and administration of the L-type Ca²⁺ channel activator Bay K 8644 induced phosphorylation of CREB in the nuclei of the amacrine cells and the ganglion cells where c- fos /SS mRNAs were expressed. These cells could be double-stained with anti-calmodulin kinase II (anti-CaM kinase II) monoclonal antibody and phospho-CREB-specific polyclonal antiserum after Bay K 8644 administration, indicating the colocalization of phosphorylated CREB at Ser¹³³ and CaM kinase II in the neural retina.     

Genetic Variation in VP7 Gene of Human Rotavirus Serotype 2 (G2 Type) Isolated in Japan,China, and Pakistan

Sequence analysis of the gene encoding the major neutralization glycoprotein (VP7) was performed on sixteen human isolates of serotype 2 of rotavirus in Japan, China, and Pakistan and their genetic variations were examined. Comparative studies of their nucleotide and deduced amino acid sequences between the sixteen isolates and the HU5 strain revealed an overall homology of more than 94%. A higher degree of homology in nucleotides was observed among the sixteen isolates than between HU5 and the isolates. A total of thirteen amino acid residues frequently converted to another amino acid. Out of the thirteen, five amino acid residues belonging to the major neutralizing epitope regions (C, E, and F in this communication) converted frequently. From the amino acid sequences three subtypes, subtype 1, subtype 2, and intermediate, were suggested to be classified as previously reported for serotype 1 (Xin et al, Virology, 1993, 197: 813-816).