Cassettes for seed-specific expression tested in transformed embryogenic cultures of soybean

Soybean (Glycine max [L.] Merrill) lectin is a seed protein that accumulates in protein bodies of cotyledons during seed development. We have constructed two expression cassettes containing the 5′ and 3′ regions of the soybean lectin gene connected by aNot I restriction site. One vector also contains the 32 amino acid signal sequence. Using polymerase chain reaction (PCR), the coding region of the β-glucuronidase (uidA) gene was inserted into theNot I site of each vector. We tested the function of the expression cassettes in transformed embryogenic cultures of soybean. Development-specific GUS expression was observed in developing somatic embryos transformed with the chimeric lectin promoter-GUS constructs as determined by histochemical assays. Our data indicate that these cassettes could be used to drive expression of foreign genes to modify embryo-specific traits of soybean as protein quality or quantity in the seed.     

A highly conserved nuclear gene for low-level phylogenetics: elongation factor-1 alpha recovers morphology-based tree for heliothine moths

Molecular systematists need increased access to nuclear genes. Highly conserved, low copy number protein-encoding nuclear genes have attractive features for phylogenetic inference but have heretofore been applied mostly to very ancient divergences. By virtue of their synonymous substitutions, such genes should contain a wealth of information about lower-level taxonomic relationships as well, with the advantage that amino acid conservatism makes both alignment and primer definition straightforward. We tested this postulate for the elongation factor-1 alpha (EF-1 alpha) gene in the noctuid moth subfamily Heliothinae, which has probably diversified since the middle Tertiary. We sequenced 1,240 bp in 18 taxa representing heliothine groupings strongly supported by previous morphological and allozyme studies. The single most parsimonious gene tree and the neighbor-joining tree for all nucleotides show almost complete concordance with the morphological tree. Homoplasy and pairwise divergence levels are low, transition/transversion ratios are high, and phylogenetic information is spread evenly across gene regions. The EF-1 alpha gene and presumably other highly conserved genes hold much promise for phylogenetics of Tertiary age eukaryote groups.      

Structure-function analysis of the DNA binding domain of Saccharomyces cerevisiae ABF1.

To localize the DNA binding domain of the Saccharomyces cerevisiae Ars binding factor 1 (ABF1), a multifunctional DNA binding protein, plasmid constructs carrying point mutations and internal deletions in the ABF1 gene were generated and expressed in Escherichia coli. Normal and mutant ABF1 proteins were purified by affinity chromatography and their DNA binding activities were analyzed. The substitution of His61, Cys66 and His67 respectively, located in the zinc finger motif in the N-terminal region (amino acids 40-91), eliminated the DNA binding activity of ABF1 protein. Point mutations in the middle region of ABF1, specifically at Leu353, Leu399, Tyr403, Gly404, Phe410 and Lys434, also eliminated or reduced DNA binding activity. However, the DNA binding activity of point mutants of Ser307, Ser496 and Glu649 was the same as that of wild-type ABF1 protein and deletion mutants of amino acids 200-265, between the zinc finger region and the middle region (residues 323-496) retained DNA binding activity. As a result, we confirmed that the DNA binding domain of ABF1 appears to be bipartite and another DNA binding motif, other than the zinc finger motif, is situated between amino acid residues 323 and 496.     

Properties of acetylcholinesterase reconstituted in liposomes of a different charge

Acetylcholinesterase (AChE) purified from mouse brain was reconstituted in liposomes of a different charge, and the properties of liposome-associated AChE were investigated. Relative to the K_m value (38.5 M) of AChE bound to a neutral liposome, the value of AChE reconstituted in a negatively-charged liposome decreased to 23.3 M, whereas that of AChE in a positively-charged liposome increased to 90.9 M. Additionally, AChE bound to a positively-charged liposome expressed a wider range of optimum pH than the enzyme in a negatively-charged liposome. In a stability study, it was found that soluble AChE was unstable at pH 5.5 and 7.4, while it was relatively stable at pH 10. Noteworthy, the immobilization of AChE to liposome enhanced the stability of soluble enzyme at acidic and neutral pH. Moreover, in the stabilization of the enzyme, a neutral liposome was more effective than charged liposomes, of which a positively-charged liposome was more effective than a negatively-charged liposome at acidic pH. Based on these results, it is proposed that while the K_m value and the pH dependence of AChE activity are affected by the charge of liposome, the stability of AChE is determined mainly by a hydrophobic binding to a phospholipid membrane.This work was supported in part by Agency for Defense Development.     

Surface ultrastructure of the tegument of Clonorchis sinensis newly excysted juveniles and adult worms.

The tegumental structures of newly excysted juveniles and adult worms of Clonorchis sinensis were studied using scanning and transmission electron microscopy. After excystation the juvenile's tegumental surface is characterized by knoblike protuberances and is armed almost entirely with numerous rows of small spines encircling the body. These spines are double- or triple-pointed on the anterior portion of the body and become single-pointed posteriorly. Four types of presumed sensory structures were observed as follow: A) ciliated knoblike papillae and B) nonciliated platelike papillae, both of which are arranged in rougly a bilaterally symmetrical pattern dorsally, ventrally, and laterally; C) rounded swellings of nonciliated papillae on the lips of the ventral and oral suckers, which are characterized in the transmission electron microscope by a rounded dense body in the apical bulb; and D) a sensory receptor with a bulbous projection having the appearance of a modified cilium, which was not found with SEM likely owing to its being enclosed by an extension of the tegument. In full-grown adult worms, the tegumental surface is knobbed or lobulated in various forms without surface spines. The tegumental structures in the adults appear to be clearly differentiated from those in the juveniles. Upraised, buttonlike papillae, each topped by a short cilium, which are similar to the Type A papillae in the juveniles, are distributed thickly around the oral and ventral suckers, and are rather randomly scattered over the remainder of the body. Some nonciliated swollen papillae were found on the lip of the ventral sucker.     

Delayed hypersensitivity in murine salmonellosis: specificity of footpad reaction in mice infected with rough mutants of Salmonella typhimurium

Delayed type (footpad) hypersensitivity (DTH) in BALB/c mice immunized with rough mutant strains of Salmonella typhimurium LT2 was examined. Injection of live organisms of an Rb mutant TV148 strain induced DTH in mice, while injection of the heat-killed organisms did not. The mice immunized with live organisms of the Ra, Rb, Rc, Rd, and Re mutant strains showed positive footpad reactions to the heat-killed cell antigen of LT2 (wild type) strain. The mice immunized with the Rb mutant strain also showed positive footpad swellings in response to heat-killed cell antigens of S. paratyphi A, S. paratyphi B, S. typhi, S. enteritidis, and S. cholerae-suis. Furthermore, positive reactions to antigens of Escherichia coli and Shigella flexneri were seen in the TV148-immunized mice, but the mice did not respond to heat-killed organisms of Pseudomonas aeruginosa or Staphylococcus aureus. The cross-reactive footpad reaction to E. coli could be transferred adoptively with T cells prepared from the spleens of TV148-immunized mice into syngeneic recipients. These results suggest that the cross-reactive DTH antigen(s) is widely distributed among related organisms such as Shigella and Escherichia.     

Genetic recombination in Micromonospora rosaria by protoplast fusion

Auxotrophic strains of Micromonospora rosaria were isolated by N-methyl-N'-nitro-N'-nitrosoguanidine mutagenesis and used in intraspecific recombination by protoplast fusion. High-frequency fusion of protoplasts of M. rosaria strains was induced by polyethylene glycol (molecular weight, 1,000) (PEG 1,000). The optimum concentration of PEG 1,000 for fusion of M. rosaria was 50% (wt/vol). PEG 4,000 was slightly better than PEG 1,000 at concentrations lower than 50% (wt/vol). The recombinant frequency did not increase after treatment with PEG 1,000 (50% [wt/vol]) for longer than 20 min. Under these conditions, fusion with many auxotrophic strains of M. rosaria resulted in a high frequency of formation of true recombinants (sometimes more than 10%). Additionally, when ros (rosamicin nonproducing) strains were crossed by protoplast fusion; about 5% of the resultant prototrophic recombinants were shown to have the ros+ (rosamicin producing) characteristic restored. Rosamicin production by M. rosaria colonies was clearly distinguished by the broth overlay method. The results of fusion experiments between ros and ros+ strains indicated that either the chromosomal mutation or pleiotrophic effect of some auxotrophic markers is involved.     

Monomer diffusion and polymer alignment in domains of sickle hemoglobin.

We have used polarized absorbance to observe the process of monomer accretion and polymer alignment which occurs in domains of sickle hemoglobin that are formed and maintained by laser photolysis. These diffusion and alignment processes have been studied as a function of initial concentration and temperature (initial and final), as well as beam size and domain number. Monomers are found to diffuse into growing polymer domains with a rate that is essentially temperature and concentration independent, but which depends on the size of the final domain boundaries, and the number of domains within a boundary. The final concentrations achieved are very close to those found in packed centrifugation experiments (50-55 g/dl) and are approximately independent of starting temperature and concentration. The influx of monomers is accompanied by polymer alignment, and the amount aligned is proportional to the amount diffused throughout the process. We propose that polymer alignment controls the influx of added monomers into the growing domain.     

Cloning of cDNA for mosquito lysosomal aspartic protease. Sequence analysis of an insect lysosomal enzyme similar to cathepsins D and E.

A cDNA coding for the lysosomal aspartic protease from the mosquito (mLAP) was cloned and sequenced. The mLAP cDNA is 1420 base pairs long with an open reading frame of 387 amino acids. The deduced amino acid sequence contains a signal pre-propeptide sequence of 18 amino acids followed by 369 amino acids with a 35-amino acid putative pro-enzyme domain in the NH2-terminal. The amino acid sequence of mLAP is 92 and 81% similar to human cathepsin D and cathepsin E, respectively. Typical cleavage sites for cathepsin D processing into light and heavy chains are lacking in mLAP. A single glycosylation site occurs in the mLAP sequence at a position corresponding to the first glycosylation site of cathepsins D. The mLAP sequence shares putative phosphorylation determinants, which in cathepsins D are linked to the formation of mannose 6-phosphate. In the mosquito fat body, lysosomal enzymes specifically degrade organelles involved in the biosynthesis and secretion of vitellogenin. The mLAP mRNA accumulates to its highest level 24 h after initiation of vitellogenin synthesis and 12 h before the peak of mLAP protein accumulation and its enzymatic activity. Translational regulation of mLAP mRNA may occur. The 5'-untranslated region of mLAP mRNA is similar to elements conferring negative translational control by steroids.