The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression.

The complete nucleotide sequence (155 844 bp) of tobacco (Nicotiana tabacum var. Bright Yellow 4) chloroplast DNA has been determined. It contains two copies of an identical 25 339 bp inverted repeat, which are separated by a 86 684 bp and a 18 482 bp single-copy region. The genes for 4 different rRNAs, 30 different tRNAs, 39 different proteins and 11 other predicted protein coding genes have been located. Among them, 15 genes contain introns. Blot hybridization revealed that all rRNA and tRNA genes and 27 protein genes so far analysed are transcribed in the chloroplast and that primary transcripts of the split genes hitherto examined are spliced. Five sequences coding for proteins homologous to components of the respiratory-chain NADH dehydrogenase from human mitochondria have been found. The 30 tRNAs predicted from their genes are sufficient to read all codons if the ;two out of three' and ;U:N wobble' mechanisms operate in the chloroplast. Two sequences which autonomously replicate in yeast have also been mapped. The sequence and expression analyses indicate both prokaryotic and eukaryotic features of the chloroplast genes.

Identification of EGF as an angiogenic factor present in conditioned medium from human salivary gland adenocarcinoma cell clones with varying degrees of metastatic potential.

We have previously shown that conditioned medium (CM) from metastasizing human salivary gland adenocarcinoma cell clones contains factor(s) that stimulate the proliferation and migration of bovine aortic endothelial (BAE) cells, and inhibit the production of collagenases by BAE cells (Azuma M. et al. (1993) Cancer Lett., 73, 85-93). To further characterize this, we evaluated the expression level of epidermal growth factor (EGF) secreted by a non-metastasizing cell clone (HSGc) and its metastasizing cell clones, and analysed the effect of EGF on the biologic behaviors of BAE cells. When the secretion of EGF by cell clones was estimated by enzyme-linked immunosorbent assay, metastasizing cell clones released a large amount of EGF as compared with HSGc. However, the number of EGF receptor was detected consistently at a level that was similar in all cell clones. With regard to the effect of EGF on the malignant potential of cell clones such as proteolytic aggressiveness, EGF did not affect the secretion of both collagenases and their inhibitor from cell clones. Alternatively, exogenous EGF stimulated the proliferation and migration of BAE cells, and inhibited the secretion of collagenases from BAE cells. Neutralization with a neutralizing antibody of EGF released into CM abolished the inhibitory effect of CM on the secretion of collagenases from BAE cells. Thus, the CM-contained factor, which is responsible for the induction of biologic behaviors of BAE cells, can be attributed to EGF.

Effects of media conditioned by a non-metastasizing human salivary gland adenocarcinoma cell clone and metastasizing clones from salivary gland and various other tissues on the proliferation, migration and protease production of bovine aortic endothelial cells in vitro.

We demonstrate the role in tumor-associated angiogenesis of factors released into conditioned medium (CM) from in vitro human salivary gland cell clones with biological phenotypes ranging from non-metastasizing to metastasizing. A non-metastasizing human salivary gland adenocarcinoma cell clone HSGc and its subclone with metastatic potential (Gc2-100 cl-1) were employed. We also used metastasizing cell clones obtained by explant cultures of organs of Gc2-100 cl-1 tumor-bearing nude mouse. The proliferation and migration of bovine aortic endothelial (BAE) cells were significantly stimulated by the addition of CM obtained from Gc2-100 cl-1 and metastasizing cell clones, while CM from HSGc was ineffective. When the effect on protease secretion by BAE cells was examined, CM from Gc2-100 cl-1 and metastasizing cell clones inhibited the secretion of type IV collagenases by BAE cells much more than did CM from HSGc. These findings, therefore, may imply that Gc2-100 cl-1 and metastasizing cell clones secrete angiogenic factors that stimulate not only the proliferation and migration of endothelial cells but also the formation of basement membrane components necessary for the reconstruction of new blood vessels at migrated sites of endothelial cells by preventing the degradation of basement membrane component, type IV collagen.

Site-specific mutagenesis of the human interleukin-1 beta gene: the role of arginine residue at the N-terminal region.

Using the expression system for site-specific mutagenesis in Escherichia coli, we have made deletion mutants at the N-terminal or C-terminal region of human interleukin-1 beta (IL-1 beta) consisting of 153 amino acids. The truncated mutants showed that at least 147 amino acids (numbers 4-150) in IL-1 beta are necessary for the exertion of biological activity. When we changed the arginine at the 4th position (Arg4) in IL-1 beta to other specific amino acids, there was a marked difference in the relative extent of biological and receptor binding activities among the mutants. The order of the mutants was Arg4 = Lys4 greater than Gln4 greater than Gly4 = des-Arg4 greater than Asp4. Our results demonstrate that the arginine residue at the 4th position in IL-1 beta is important, but not essential, for IL-1 beta to exhibit its biological and receptor binding activities, and the positive charge at this site plays a key role for IL-1 beta to exert the activities.

Streptokinase gene variable region classification in streptococci: lack of correlation with post-streptococcal glomerulonephritis.

To investigate a possible causal role of streptokinase (SKase) in acute post-streptococcal glomerulonephritis (APSGN), the major variable region of SKase genes of Streptococcus pyogenes strains isolated from patients with and without APSGN were analyzed using the polymerase chain reaction, restriction enzyme analysis and the direct sequencing of SKase genes. In the APSGN-associated strains, six of nine revealed mutant classes corresponding to the nephritogenic classes I and II proposed by Johnston et al. [1992], the remaining three belonged to non-nephritogenic classes. In twenty strains not associated with APSGN, seventeen belonged to classes I and II, while three were from other classes. The major variable region of the SKase gene shows no apparent relation with induction of APSGN in humans, suggesting that unique classes of streptococcal SKase do not play a role in the pathogenesis of APSGN.

Structure and cotranscription of tobacco chloroplast genes for tRNAGlu(UUC), tRNATyr(GUA) and tRNAAsp(GUC).

The location and nucleotide sequences of tobacco chloroplast genes for tRNAGlu(UUC), tRNATyr(GUA) and tRNAAsp(GUC) have been determined. These genes lie midway between the genes for alpha and beta/epsilon subunits of H+-ATPase on the large single-copy region of the chloroplast DNA. The gene organization is tRNAGlu - 59bp spacer - tRNATyr - 108bp spacer - tRNAAsp on the same DNA strand. Northern blot hybridization studies revealed that these three tRNA genes are cotranscribed. The transcription initiation site was localized at 24 bp upstream from the tRNAGlu coding region and its termination site at 90 bp downstream from the tRNAAsp coding region by S1 mapping. The tricistronic tRNA precursor is thus calculated to be 512 bases long. Its processing was also studied by S1 mapping.

Locations and sequences of tobacco chloroplast genes for tRNAPro(UGG), tRNATrp, tRNAfMet and tRNAGly(GCC): the tRNAGly contains only two base-pairs in the D stem.

The nucleotide sequences of tobacco chloroplast genes for tRNAPro(UGG), tRNATrp, tRNAfMet and tRNAGly(GCC) have been determined. None of these genes contains an intron. One unusual feature is that the tRNAGly contains only two base-pairs (A-U, G-U) in the D stem. These four tRNA genes were located in the known physical map of tobacco chloroplast DNA. Hybridization analysis to chloroplast tRNA revealed that all four tRNA genes are transcribed in vivo.

Detection of Bacteroides fragilis by PCR assay targeting the neuraminidase-encoding gene.

Oligonucleotide primers were designed on the basis of the sequence of the neuraminidase-encoding gene (nanH) of Bacteroides fragilis and used for the specific detection of this anaerobe by the nested PCR assay. Fifty-nine of 60 representative strains of Bact. fragilis were detected, while none of 45 strains of other species generated visible PCR products. The detection limits of Bact. fragilis cells and DNA by the nested PCR were 10 colony-forming units and 10 fg of chromosomal DNA, respectively. The PCR assay targeting the nanH gene has the potential for the detection of Bact. fragilis.

Role of plasminogen activators, metalloproteinases and the tissue inhibitor of metalloproteinase-1 in the metastatic process of human salivary-gland adenocarcinoma cells.

An in vitro system has been established in which conversion from non-metastasizing to metastasizing adenocarcinoma cells can be induced, and subsequently subjected to analysis of the expression of proteases and tissue inhibitor of metalloproteinases-1 (TIMP-1). A human salivary-gland adenocarcinoma cell clone HSGc, with no metastatic ability, was exposed to N-methyl-N-nitrosourea (MNU). Following exposure to MNU, cells with altered morphology were cloned. Upon s.c. inoculation into nude mice, MNU-treated HSGc clones formed metastatic foci in various organs, and then 5 metastasizing clones were isolated. Evaluation of expression of tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), metalloproteinases and TIMP-1 was performed by means of enzyme immunoassay, zymogram, or immunoblot. MNU-treated HSGc and metastasizing clones were found to secrete high levels of tPA, while HSGc produced undetectable levels of this enzyme. Expression of uPA was not observed in any of the cell clones. When the secretion of gelatinolytic enzymes was examined, metastasizing clones produced higher levels of 57- and 32-kDa, but not of 92- or 72-kDa gelatinases, as compared to HSGc cells. Although TIMP-1 was detected in all cell clones, metastasizing clones secreted less TIMP-1 than HSGc cells; in addition, one metastasizing clone produced TIMP-1 with a molecular weight distinct from that of 28-kDa TIMP-1. Our results suggest that the acquisition of metastatic ability by human salivary-gland tumor cells is closely associated with increased secretion of several metalloproteinases as well as decreased or altered TIMP-1 expression.

Amino-terminal region of human macrophage colony-stimulating factor (M-CSF) is sufficient for its in vitro biological activity: molecular cloning and expression of carboxyl-terminal deletion mutants of human M-CSF.

Human T lymphoblastoid cell line CEM-ON belongs to a helper/inducer subclass and secretes M-CSF into medium constitutively. We have isolated a full-length cDNA clone for this factor from a cDNA library of this cell line. The cDNA was 2.5 kb and coded for a 554 amino acid polypeptide precursor including signal sequence. The Northern blot analysis showed that the major transcript of M-CSF is about 4.2 kb. We have studied the expression of not only the wild type plasmid, but also the five C-terminal deletion mutants encoding N-terminal 154, 163, 170, 177, and 185 amino acid residues in monkey COS-1 cells. The results showed that at least C-terminal 377 amino acid residues of human M-CSF are not essential to manifest the in vitro biological activity on murine bone marrow cells.

Stable isotope aided nuclear magnetic resonance study to investigate the receptor-binding site of human interleukin 1 beta.

Resonance assignments for interleukin 1 beta at neutral pH were made by using three-dimensional NMR in combination with specific labeling and double-labeling methods with stable isotopes. On the basis of the present assignments, 15N single-quantum coherence spectra of N-terminal truncated and fusion mutants were compared with that of the wild-type. Although these mutants have reduced biological activity, they showed 15N-SQC spectra similar to that of the wild-type. However, small but significant chemical shift changes were observed for amino acid residues within a loop 86-99, in spite of the modification at the N-terminus, supporting the idea that this loop forms a biologically active part of interleukin 1 beta. Receptor-binding activity was studied for mutants (Asp-93)-, (Leu-93)- and des-(Arg-98)interleukin 1 beta's. The results show significant loss of the receptor-binding activity. The N-terminus, the C-terminus, and the loop 86-99 form a part of the open end of a beta-barrel [Finzel, B. C., Clancy, L. L., Holland, D. R., Muchmore, S. W., Watenpaugh, K. D., & Einspahr, H. M. (1989) J. Mol. Biol. 209, 779-791; Clore, G. M., Wingfield, P. T., & Gronenborn, A. M. (1991) Biochemistry 30, 2315-2323], which forms the receptor-binding site of IL-1 beta.

Site-specific mutagenesis of the human interleukin-1 beta gene: structure-function analysis of the cysteine residues.

Human interleukin-1 beta (IL-1 beta) has two cysteines located at amino acid residues 8 and 71 of the mature protein consisting 153 amino acids. To clarify the role of these characteristic cysteine residues in IL-1 beta, at first, an expression plasmid for site-specific mutagenesis has been constructed by inserting the ori and intergenic region of phage f1 into the IL-1 beta expression vector. The plasmid can be used not only for isolation of the modified IL-1 beta gene but for expression of the mutant protein in Escherichia coli. Using this plasmid, each of the cysteine codons in IL-1 beta gene was changed to serine or alanine codon, or deleted. The modified IL-1 beta showed that the two cysteine residues in IL-1 beta are not essential for biological activity but not to be eliminated for the maintenance of the functional structure of IL-1 beta.