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Cucumisin is a subtilisin-like serine protease (subtilase) that is found in the juice of melon fruits (Cucumis melo L.). It is synthesized as a preproprotein consisting of a signal peptide, NH(2)-terminal propeptide, and 67-kDa protease domain. We investigated the role of this propeptide (88 residues) in the cucumisin precursor. Complementary DNAs encoding the propeptides of cucumisin, two other plant subtilases (Arabidopsis ARA12 and rice RSP1), and bacterial subtilisin E were expressed in Escherichia coli independently of their mature enzymes. The cucumisin propeptide strongly inhibited cucumisin in a competitive manner with a K(i) value of 6.2 ± 0.55 nm. Interestingly, cucumisin was also strongly inhibited by ARA12 and RSP1 propeptides but not by the subtilisin E propeptide. In contrast, the propeptides of cucumisin, ARA12, and RSP1 did not inhibit subtilisin. Deletion analysis clearly showed that two hydrophobic regions, Asn(32)-Met(38) and Gly(97)-Leu(103), in the cucumisin propeptide were important for its inhibitory activity. Site-directed mutagenesis also confirmed the role of a Val(36)-centerd hydrophobic cluster within the Asn(32)-Met(38) region in cucumisin inhibition. Circular dichroism spectroscopy revealed that the cucumisin propeptide had a secondary structure without a cognate protease domain and that the thermal unfolding of the propeptide at 90 °C was only partial and reversible. A tripeptide, Ile(35)-Val(36)-Tyr(37), in the Asn(32)-Met(38) region was thought to contribute toward the formation of a proper secondary structure necessary for cucumisin inhibition. This is the first report on the function and structural information of the propeptide of a plant serine protease.

Of the four known globin genes that exist in the fresh-water crustacean Daphnia magna, several are individually induced by hypoxia, lending pale normoxic animals a visible red color when challenged by oxygen deprivation. The promoter regions of the Daphnia globin genes each contain numerous hypoxia response elements (HREs) as potential binding sites for hypoxia-inducible factors (HIFs). Daphnia HIF, bound to human HRE sequences, was detected in extracts from hypoxic (red), but not normoxic (pale), animals. Taking advantage of the phylogenetically conserved HIF/HRE recognition, we employed heterologous transfections of HIF-expressing human and Drosophila cells to model HIF signaling in Daphnia. These experiments revealed that three functional HREs within the promoter of the D. magna globin-2 gene cooperate for maximal hypoxic induction of a downstream luciferase reporter gene. Two of these three cis-elements, at promoter positions -258 and -107, are able to specifically bind human, Drosophila, or Daphnia HIF complexes in vitro. The same two sites are also necessary for maximal induction of reporter transcription under low oxygen tension in the presence of either endogenous human or overexpressed Drosophila HIF proteins. The third motif of the globin-2 gene promoter, a CACGTG palindrome at position -146, functions as a docking site for an unknown constitutive transcription factor. In human cells, this -146 complex interferes with HIF occupancy at the adjacent -107 HRE and thus controls the extent of HIF-mediated hypoxic activation of the downstream target.

Cucumisin, a subtilisin-like serine protease, is expressed at high levels in the fruit of melon (Cucumis melo L.) and accumulates in the juice. We investigated roles of the promoter regions and DNA-protein interactions in fruit-specific expression of the cucumisin gene. In transient expression analysis, a chimeric gene construct containing a 1.2-kb cucumisin promoter fused to a beta-glucuronidase (GUS) reporter gene was expressed in fruit tissues at high levels, but the promoter activities in leaves and stems were very low. Deletion analysis indicated that a positive regulatory region is located between nucleotides -234 and -214 relative to the transcriptional initiation site. Gain-of-function experiments revealed that this 20-bp sequence conferred fruit specificity and contained a regulatory enhancer. Gel mobility shift experiments demonstrated the presence of fruit nuclear factors that interact with the cucumisin promoter. A typical G-box (GACACGTGTC) present in the 20-bp sequence did not bind fruit protein, but two possible cis-elements, an I-box-like sequence (AGATATGATAAAA) and an odd base palindromic TGTCACA motif, were identified in the promoter region between positions -254 and -215. The I-box-like sequence bound more tightly to fruit nuclear protein than the TGTCACA motif. The I-box-like sequence functions as a negative regulatory element, and the TGTCACA motif is a novel enhancer element necessary for fruit-specific expression of the cucumisin gene. Specific nucleotides responsible for the binding of fruit nuclear protein in these two elements were also determined.

The FGF-3 gene is constitutively expressed in tumorigenic clones from the SW613-S human colon carcinoma cell line but is silent in non-tumorigenic clones. We have investigated the transcriptional mechanisms responsible for this differential expression. Mapping of DNase I-hypersensitive sites throughout the FGF-3 gene and the region extending 15 kilobases upstream disclosed differences in the patterns obtained between tumorigenic and non-tumorigenic cells. Transient expression assays carried out with a reporter gene driven by FGF-3 promoter fragments of various lengths (0.143 to 11 kilobases) did not reproduce the differential regulation of the resident gene between the two cell types. The same constructs did exhibit a differential activity in stable transfectants, suggesting the involvement of a chromatin-based mechanism in this regulation. Under these conditions, even the 143-base pair minimal promoter fragment was able to drive the differential expression of the reporter gene. During the course of these analyses, several transcriptional modulatory elements (mainly activators) were identified in the FGF-3 upstream region and were found to colocalize with DNase I-hypersensitive sites. Moreover, a putative new promoter was discovered 6 kilobases upstream of FGF-3. Altogether, these data provide a basis for the elucidation of the complex regulation of the human FGF-3 gene.

Hemoglobin (Hb) purified from the water flea, Daphnia magna, reared under hypoxia was analyzed by two-dimensional gel electrophoresis. The Hb was shown to be composed of six major subunit chain species (designated as DHbA to DHbF). The NH2-terminal amino acid sequences of DHbA, DHbB, DHbC, and DHbF are different from one another, indicating that at least four Hb genes are present in D. magna. The NH2-terminal amino acid sequences of DHbD and DHbE are the same as those of DHbA and DHbB, respectively. The six Hb chains were also found in the animal reared under normoxia in small amounts and with altered composition; the extent of decrease under normoxia was higher in the amounts of DHbC, DHbD, and DHbF than those of others. These results indicate that the Hb genes are differentially regulated by the ambient oxygen concentration. Four Hb genes constituting a cluster in the order, dhb4, dhb3, dhb1, and dhb2, were found on the chromosome of D. magna. The complete nucleotide sequences of the dhb1, dhb2, and dhb3 genes and their cDNAs showed that the genes have a seven-exon, six-intron structure. The structure consists of an intron separating an exon encoding a secretory signal sequence, two large repeated regions of a three-exon, two-intron structure that encode each a domain containing a heme-binding site, and an intron bridging the two repeated regions. The deduced amino acid sequences of the gene products showed higher than 79% identity to one another and showed unique features conserved in D. magna Hb chains. The analysis also suggested that DHbB (or DHbE), DHbF, and DHbC are encoded by the dhb1, dhb2, and dhb3 genes, respectively.

Cucumisin is a thermostable alkaline serine protease that is found in the juice of melon fruits (Cucumis melo L.). We have determined the complete nucleotide sequence of a cucumisin cDNA (2,552 nucleotides) and deduced the corresponding amino acid sequence. The open reading frame of the cDNA consists of 731 codons and encodes a large precursor (molecular weight, 78,815) relative to the observed size of mature native cucumisin (67 kDa). Sequence comparisons reveal that cucumisin has several features in common with the microbial proteases of the subtilisin family. The highly conserved sequences to the proximal regions of the catalytic triad amino acids Asp, His, and Ser, together with the substrate binding site in subtilisin, can be found within the deduced amino acid sequence of the protease domain of the cucumisin precursor. Cucumisin is the first known plant protease with such characteristics. Examination of the primary structure of cucumisin revealed that it is synthesized as a precursor, consisting of four functional domains: a possible signal peptide (22 amino acid residues), an NH2-terminal pro-sequence (88 residues), a 54-kDa protease domain (505 residues), which is the active enzyme domain of the 67-kDa native cucumisin, and a 14-kDa COOH-terminal polypeptide (116 residues), which arises by limited autolysis of the 67-kDa native cucumisin. This structure of cucumisin suggests that it is probably synthesized as an inactive precursor.

A DNA sequence of 816 base pairs encompassing the entire Erwinia amylovora lipoprotein gene was determined. Sequence comparison between E. amylovora, Escherichia coli, and Serratia marcescens suggests that the structure of the lipoprotein has been highly conserved under the constraint of efficient gene expression selecting promoter structure, mRNA secondary structure, and codon usage in addition to the polypeptide function. The sequence also suggests that the lpp gene of the three bacteria diverged sequentially in the course of evolution.

The alpha-amylase of Bacillus licheniformis (BLA) is stable and active at high temperature. More than 80% of its activity is retained after heat treatment at 90 degrees C for 30 min, and the optimum temperature for its activity is 80-85 degrees C. In contrast, the alpha-amylase of Bacillus amyloliquefaciens (BAA), the amino acid sequence of which shows 80% homology with that of BLA, is rapidly inactivated at 90 degrees C. Various chimeric genes were constructed from the structural genes for the two enzymes, and their products were analyzed for stability as to irreversible thermoinactivation. Two regions in the amino acid sequence of BLA comprising Gln178 (region I) and the 255th-270th residues (region II), respectively, were shown to determine the thermostability of BLA. Region I plays a major role in determining the thermostability. By means of site-directed mutagenesis of the BAA gene, deletion of Arg176 and Gly177 in region I and substitutions of alanine for Lys269 and aspartic acid for Asn266 in region II were shown to be responsible for the enhancement of the thermostability. Mutant BAAs containing the above deletion and substitutions showed almost the same thermostability as BLA as to irreversible thermoinactivation. Nevertheless, the mutant BAAs showed a temperature optimum as low as that of BAA (65 degrees C), indicating that they are still susceptible to reversible inactivation at temperatures higher than 65 degrees C.