Characterization of the 5' regulatory region of the mouse angiopoietin-like protein 4.

The 5' regulatory region of the mouse angiopoietin-like protein 4 (mANGPTL4), a remarkably versatile secreted protein responsible for hyperlipidaemia and angiogenesis, was cloned and functionally characterized. Three potential transcriptional start sites were determined by 5'-RACE and found to be at -129, -126 and -118, relative to the translation initiation codon. The activities of the putative promoters were confirmed using a firefly luciferase reporter gene assay system, following transient transfection into COS-1 cells. The PPAR alpha-regulated region and the minimal region required for basal activity of the mANGPTL4 promoter were determined by generating a series of deletion constructs, and were found to be encoded by a sequence between -2761 to -383 and -50 to -30, relative to the transcription start site. Putative recognition sequences for the transcription factor AP2 were identified in the minimal promoter sequences. These results are the first molecular characterization of the regulatory region of this important gene.

Production of functional human selenocysteine-containing KDRF/thioredoxin reductase in E. coli.

In a previous study, we reported the isolation of a cDNA encoding KDRF (KM-102-derived reductase like factor) from the human bone marrow-derived stromal cell line KM-102. Analysis of the sequence of this cDNA revealed it to be the previously reported human thioredoxin reductase cDNA. Human thioredoxin reductase, which was recently isolated from human lung adenocarcinoma NCI-H441 cells as a selenocysteine-containing selenoprotein, and its substrate thioredoxin are thought to be essential for protecting cells from the damage caused by reactive oxygen species. To obtain the selenocysteine-containing recombinant KDRF/thioredoxin reductase, we introduced a secondary structure, which is identical to the selenocysteine insertion signal of Escherichia coli formate dehydrogenase H mRNA, downstream of the TGA in the KDRF/thioredoxin reductase cDNA and expressed it in E. coli. As a result, a significant amount of selenocysteine was incorporated into the C-terminus of the KDRF/thioredoxin reductase protein. The selenocysteine-containing KDRF/thioredoxin reductase showed reducing activities toward human and E. coli thioredoxin, whereas non-selenocysteine-containing KDRF/thioredoxin reductase showed no enzyme activity. Our results suggest that this strategy will be applicable to the production of other mammalian selenocysteine-containing selenoproteins in E. coli.

Molecular cloning and expression of the gene encoding a phospholipase A1 from Aspergillus oryzae.

Phospholipase A1 (PLA1) is a hydrolytic enzyme that catalyzes removal of the acyl group from position 1 of lecithin to form lysolecithin. The genomic DNA and cDNA encoding PLA1 from Aspergillus oryzae were cloned with the mixed deoxyribonucleotide-primed polymerase chain reaction. The PLA1 gene is composed of 1,056 bp and has four exons and three short introns (63, 54, and 51 bp). The deduced amino acid sequence of PLA1 contained the N-terminal sequence of the mature PLA1 analyzed by Edman degradation. PLA1 cDNA has an open reading frame of 885 bp encoding the PLA1 precursor of 295 amino acid residues. The mature PLA1 is composed of 269 amino acid residues, and a prepro-sequence of 26 amino acid residues is at the N-terminal region of the PLA1 precursor. PLA1 has two possible N-glycosylation sites (Asn27 and Asn55). PLA1 has a consensus pentapeptide (-Gly-His-Ser-Xaa-Gly-), which is conserved in lipases. The amino acid sequence of PLA1 showed 47% identity with that of mono- and diacylglycerol lipase from Penicillium camembertii. The PLA1 cDNA was expressed in Saccharomyces cerevisiae KS58-2D, indicating the cloned gene to be functional.

The effect of leustroducsin B on the production of cytokines by human mesenchymal cells.

Leustroducsin B (LSN-B), a novel colony-stimulating factor (CSF) inducer, has been shown to have various biologic activities in vivo. To compare the CSF-inducing activity of LSN-B in vitro with that of the well-known cytokine inducer, interleukin-1beta (IL-1beta), bacterial lipopolysaccharide (LPS), and phorbol 12-myristate 13-acetate (PMA), we measured granulocyte (G)-CSF and granulocyte-macrophage (GM)-CSF levels that were induced with the stimuli in several mesenchymal cells. The results indicated that each stimulant displayed a different profile in the induction of G-CSF and GM-CSF. Next, to investigate if LSN-B induces cytokines other than G-CSF and GM-CSF, we characterized cytokines that were induced with LSN-B from bone marrow stromal cells (BMSCs). The results showed that a variety of cytokines, including G-CSF and GM-CSF, were induced in both clonal and primary BMSCs. From these results, we speculate that LSN-B induces cytokine production via a regulatory pathway distinct from that of IL-1beta, LPS, or PMA and that this signaling of LSN-B might lead to the production of a variety of cytokines in BMSCs. In addition, from our in vitro and in vivo results, we speculate that the biologic activities of LSN-B in vivo might be based on its own cytokine-inducing activity even though the target cell type of LSN-B in vivo remains to be determined.

Cloning and characterization of a novel oxidoreductase KDRF from a human bone marrow-derived stromal cell line KM-102.

A cDNA clone coding for a novel oxidoreductase was cloned from a human bone marrow-derived stromal cell line KM-102. We screened a cDNA library constructed from the mRNA of KM-102 cells stimulated with phorbol 12-myristate 13-acetate and calcium ionophore A23187 using a 32P-labeled 15-mer synthetic oligonucleotide (5'-TAAATAAATAAATAA-3') probe. This probe was designed as a complementary sequence to the three reiterated AUUUA sequences, which are contained in the 3'-untranslated regions of cytokine and some proto-oncogene mRNAs and correlate with rapid mRNA turnover. Then, we obtained one cDNA clone, and further sequence analysis revealed that it coded for a new protein exhibiting 30 to approximately 40% homology with glutathione reductase. By fusion protein analysis, this protein showed reducing activities on 2, 6-dichlorophenol-indophenol and 5,5'-dithio-bis(2-nitrobenzoic acid) but only a weak reducing activity on oxidized glutathione. Although it lacked a stretch of hydrophobic amino acids in its N terminus, it was secreted by monkey kidney-derived COS-1 cells when we introduced the expression plasmid into them and also secreted by a human lung carcinoma cell line A549. Northern blot analysis revealed that the mRNA turnover of this protein was regulated by inflammatory stimuli in KM-102 cells. These results show that this protein may have scavenging enzyme properties and has its mRNA expression regulated in a similar fashion to cytokine genes or proto-oncogenes. Thus, we named it KDRF (KM-102-derived reductase-like factor), and KDRF may play a role in scavenging reactive oxygen intermediates, which are possibly toxic to cells, in response to inflammatory stimuli.

Several short interspersed repetitive elements (SINEs) in distant species may have originated from a common ancestral retrovirus: characterization of a squid SINE and a possible mechanism for generation of tRNA-derived retroposons.

Using labeled transcripts generated in vitro from squid total genomic DNA as a probe, we isolated and characterized a SINE that is present in the squid genome. The squid SINE appears to be derived from a tRNA(Lys). When the consensus sequences of five different SINEs with a tRNA(Lys)-like structure from distantly related species, including squid, were aligned, we found in the tRNA-unrelated region two sequence motifs that were almost identical among these five SINEs. This observation suggests a common evolutionary origin for these SINEs and/or some function(s) for these motifs. Similar sequences were unexpectedly found to be present in sequences complementary to the U5 regions of several mammalian retroviruses whose primer is a tRNA(Lys). On the basis of these findings, we present a model for the generation of SINEs. We propose that they are derived from a "strong-stop DNA" with a primer tRNA(Lys) that is an intermediate in the reverse transcription of certain retroviruses. Our model suggests that a certain group of SINEs may have been generated by horizontal transmission, although it is not clear whether information was transmitted via a similar retrovirus or via an RNA or DNA of a SINE.

Distribution of the salmonid Hpa 1 family in the salmonid species demonstrated by in vitro runoff transcription assay of total genomic DNA: a procedure to estimate repetitive frequency and sequence divergence of a certain repetitive family with a few known sequences.

An in vitro runoff transcription assay of total genomic DNA was developed. As an example of use of this assay, analysis of a highly repetitive sequence in the cherry salmon (Oncorhynchus masou) is described. Total genomic DNA of the cherry salmon was completely digested with Hpa 1, whose site is known to be in the tRNA-unrelated region of the cherry salmon Hpa 1 family. On transcription of the digested DNA in a HeLa cell extract, a discrete-sized RNA of about 100 nucleotides, constituting 70% of the transcripts, was produced, whereas on transcription of the undigested total DNA, only smeared RNA was obtained. In a fingerprint, the oligonucleotides of the discrete transcript from the digested total DNA were very distinct and exactly corresponded to those of a transcript from an Hpa 1 digest of a cloned DNA, but with few extra oligonucleotides. These results showed that the cherry salmon Hpa 1 family constitutes a major repetitive family in the genome of the cherry salmon. For determination of the distribution of the salmonid Hpa 1 family in other salmonid species, the same analysis was applied to DNAs from the chum salmon (Onchorhynchus keta), brown trout (Salmo trutta), Japanese common charr (Salvelinus leucomaenis pluvius), and Japanese huchen (Hucho perryi). The results showed that the salmonid Hpa 1 family is widespread in the genomes of salmonid species. A method and equations are also presented for estimating the relationship between the ratio of a given repetitive family to all the Pol III genes and its average sequence divergence by calculating the molar ratio of the runoff transcript to all the in vitro Pol III transcripts.

Several aspects of total genomic DNA transcription in a HeLa cell extract.

Several aspects of total genomic DNA transcription in a HeLa cell extract were described. (1) By using in vitro transcripts from total genomic DNA as probes, we elucidated several structures of short interspersed elements which are transcribed by RNA polymerase III (designated as Pol III/SINEs. (2) To know whether the repetitive sequence we cloned and sequenced comprises a major or minor family, we developed in vitro run-off transcription assay of total genomic DNA. (3) Unlike in vitro transcription of total genomic DNA from vertebrates, major in vitro transcripts from macronuclear total DNA of Tetrahymena were found to be tRNA themselves.