Sequence and analysis of chromosome 5 of the plant Arabidopsis thaliana.

The genome of the model plant Arabidopsis thaliana has been sequenced by an international collaboration, The Arabidopsis Genome Initiative. Here we report the complete sequence of chromosome 5. This chromosome is 26 megabases long; it is the second largest Arabidopsis chromosome and represents 21% of the sequenced regions of the genome. The sequence of chromosomes 2 and 4 have been reported previously and that of chromosomes 1 and 3, together with an analysis of the complete genome sequence, are reported in this issue. Analysis of the sequence of chromosome 5 yields further insights into centromere structure and the sequence determinants of heterochromatin condensation. The 5,874 genes encoded on chromosome 5 reveal several new functions in plants, and the patterns of gene organization provide insights into the mechanisms and extent of genome evolution in plants.

[Diagnosis of thyroid nodules by Doppler ultrasonography: a comparison between color Doppler and power Doppler ultrasonography].

The purpose of this study was to assess the usefulness of color and power Doppler imaging in thyroid nodules. The following 4 items were compared between malignant thyroid nodules (34 cases) and benign nodules (51 cases): 1) vascularity; 2) distribution of tumor vessels (none, marginal, peripheral, central); 3) nature of tumor vessels (tortuosity, interruption); and 4) FFT analysis. The distribution of tumor vessels on color Doppler images, nature of tumor vessels on power Doppler images, and the indices of PI, RI, and ATI in FFT analysis were useful in making the differential diagnosis between malignant and benign nodules. In terms of vascularity, including the distribution of tumor vessels on power Doppler images and nature of tumor vessels on color Doppler images, no statistically significant differences were found between malignant and benign nodules. Power Doppler images depicted tumor vessels in more detail than color Doppler images and were considered to extend the application of FFT analysis.

Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions.

The sequence determination of the entire genome of the Synechocystis sp. strain PCC6803 was completed. The total length of the genome finally confirmed was 3,573,470 bp, including the previously reported sequence of 1,003,450 bp from map position 64% to 92% of the genome. The entire sequence was assembled from the sequences of the physical map-based contigs of cosmid clones and of lambda clones and long PCR products which were used for gap-filling. The accuracy of the sequence was guaranteed by analysis of both strands of DNA through the entire genome. The authenticity of the assembled sequence was supported by restriction analysis of long PCR products, which were directly amplified from the genomic DNA using the assembled sequence data. To predict the potential protein-coding regions, analysis of open reading frames (ORFs), analysis by the GeneMark program and similarity search to databases were performed. As a result, a total of 3,168 potential protein genes were assigned on the genome, in which 145 (4.6%) were identical to reported genes and 1,257 (39.6%) and 340 (10.8%) showed similarity to reported and hypothetical genes, respectively. The remaining 1,426 (45.0%) had no apparent similarity to any genes in databases. Among the potential protein genes assigned, 128 were related to the genes participating in photosynthetic reactions. The sum of the sequences coding for potential protein genes occupies 87% of the genome length. By adding rRNA and tRNA genes, therefore, the genome has a very compact arrangement of protein- and RNA-coding regions. A notable feature on the gene organization of the genome was that 99 ORFs, which showed similarity to transposase genes and could be classified into 6 groups, were found spread all over the genome, and at least 26 of them appeared to remain intact. The result implies that rearrangement of the genome occurred frequently during and after establishment of this species.

Stimulation of thrombopoiesis in mice by fibroblast growth factor 9.

Fibroblast growth factor 9 (FGF-9), a novel member of the FGF family, was found to have thrombopoietic activity in vitro and in vivo. In an in vitro megakaryocyte colony-stimulating factor assay, anti-mouse interleukin-6 (IL-6) monoclonal antibody neutralized FGF-9 activity. This suggests that the activity may be exerted via IL-6 induction. BALB/c mice that received subcutaneous FGF-9 injections of 4 to 100 micrograms/day for 2 weeks showed a dose-dependent transient increase in peripheral platelet counts 10 to 12 days after the first treatment. Histologic studies showed a marked increase in megakaryocytes in bone marrow and extramedullary hematopoiesis in the spleen and the liver. Examination of changes in the DNA content of bone marrow megakaryocytes revealed that the ploidy distribution underwent a marked shift 3 days after FGF-9 injection, with a large increase in the 2N megakaryocyte population. The major modal ploidy shifted from the normal 16N to 2N. The number of megakaryocyte progenitor cells in FGF-9-treated mice increased up to 1.5-fold in the bone marrow and 10-fold in the spleen on day 6. These results indicate that FGF-9 acts on the in vivo proliferation of megakaryocytes.

Molecular cloning of a novel cytokine cDNA encoding the ninth member of the fibroblast growth factor family, which has a unique secretion property.

Glia-activating factor (GAF) is a novel heparin-binding growth factor purified from the culture supernatant of a human glioma cell line. It shows a spectrum of activity slightly different from those of other known growth factors. We have isolated the cDNA which encodes human GAF. A homology search revealed that GAF would be the ninth member of the FGF family, and we therefore call it FGF-9. The human FGF-9 cDNA cloned by using oligonucleotide probes encoded a polypeptide consisting of 208 amino acids. Sequence similarity to other members of the FGF family was estimated to be around 30%. Two cysteine residues and other consensus sequences in family members were also well conserved in the FGF-9 sequence. FGF-9 was found to have no typical signal sequence in its N terminus like those in acidic FGF and basic FGF. Acidic FGF and basic FGF are known not to be secreted from cells in a conventional manner. However, FGF-9 was found to be secreted from cells after synthesis despite its lack of a typical signal sequence. It could be detected exclusively in the culture medium of cDNA-transfected COS cells. The amino acid sequence of proteins purified from culture supernatant of the CHO cell line, which was cDNA transfected and selected as a high producer of FGF-9, showed that no peptides were cleaved from the N terminus except the initiation methionine. The rat FGF-9 cDNA was also cloned, and the structural analysis indicated that the PGF-9 gene is highly conserved. Expression of the FGF-9 gene could be detected in the brain and kidney of the adult rat. Restricted gene expression in organs and the unique secretion nature of the protein suggest that FGF-9 plays a physiological role which differs from those of well-characterized acidic FGF and basic FGF.

Novel secretory heparin-binding factors from human glioma cells (glia-activating factors) involved in glial cell growth. Purification and biological properties.

Growth factors for rat primary glial cells were identified in conditioned medium of a human glioma-derived cell line. The factors, designated glia-activating factors (GAFs), were purified to homogeneity by a combination of heparin affinity chromatography, gel filtration, and high performance liquid chromatography on a heparin affinity column and a C4 reversed-phase column. GAFs could be resolved into three peaks by C4 column chromatography. The M(r) values of these three proteins were estimated to be 30,000, 29,000, and 25,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. These M(r) values were in good agreement with the value of 26,000 +/- 3,000 estimated from the elution volume upon gel filtration chromatography under nondenaturing conditions. These data suggested that each of the GAFs consists of a single polypeptide chain and has no subunit structures. These three purified GAFs had almost the same growth-stimulating effect on glial cells in vitro, and the half-maximal dose was around 10(-11) M. Concanavalin A staining and glycopeptide N-glycosidase treatment of GAFs indicated that an asparagine-linked oligosaccharide chain(s) was attached to these three kinds of GAFs. Microsequencing of each GAF revealed a single amino-terminal sequence with no significant homology to any known protein, and the amino-terminal sequence of the 30-kDa GAF included that of the 29-kDa GAF. GAFs also stimulated the cell growth of oligodendrocyte type 2 astrocyte progenitor cells, BALB/c3T3 fibroblasts, and PC-12 cells but not that of human umbilical vein endothelial cells.

Detection and characterization of anti-tumour effector cells in Meth-A-bearing mice treated with recombinant human interleukin 2.

When Meth-A fibrosarcoma-bearing BALB/c mice were injected subcutaneously with 10 micrograms of recombinant human interleukin 2(rIL-2) once a day for 10 days, tumour growth inhibition was in the range of 22-31% of that of the control animals. Anti-tumour effector cells against Meth-A were detected in the spleen cells of the tumour-bearing BALB/c mice injected with rIL-2, using a modified Winn-type neutralization assay with the auxiliary injection of rIL-2. To induce the strongest anti-tumour activity in this assay system, the following were necessary: 1) the effector cells were derived from tumour-bearing BALB/c mice; 2) the donors of the effector cells were injected with rIL-2; 3) the recipient mice in the Winn assay were auxiliarily injected with rIL-2 (a modified Winn assay). The anti-tumour effector activity detected in the modified Winn assay was inhibited by treatment with anti-CD8 or anti-asialo GM1 antibodies plus complement (C), but not completely. We supposed that at least two kinds of anti-Meth-A effector cells with different surface antigens, positive for CD8 and asialo GM1 antigens, were induced in the Meth-A-bearing BALB/c mice injected with rIL-2; these populations seemed to function independently and at least partly as anti-tumour effector cells in this tumour-host system. These spleen cells showed in vitro cytotoxicity against Meth-A cells, which are resistant to NK cells, if the activity was measured in a 24 h 51Cr-release assay in the presence of rIL-2.

Characteristics of mouse lymphoid cells proliferating in vitro by recombinant human interleukin 2 (TGP-3): comparison between normal and nude mice.

Various lymphoid cells obtained from BALB/c and BALB/c nu/nu mice were cultured in vitro with recombinant human interleukin 2 (rIL 2), and the characteristics of responder cells to rIL 2 were analyzed. Spleen cells, lymph node cells, and thymocytes except for bone marrow cells obtained from BALB/c mice remarkably proliferated in response to rIL 2. On the other hand, among lymphoid cells obtained from BALB/c nu/nu mice, only lymph node cells showed significant proliferation by rIL 2. Flow cytometric analysis revealed that mainly two types of lymphoid cells were proliferating in response to rIL 2 in BALB/c mice, i.e., Thy 1+, Lyt 1-, Lyt 2- and Thy 1+, Lyt 1-, Lyt 2+ cells. On the other hand, most of the proliferating cells were Thy 1+, Lyt 1-, Lyt 2- cells in BALB/c nu/nu mice. Treatment with various antibodies plus complement revealed that the majority of IL 2-responsive cells in BALB/c mice were Thy 1+, Lyt 1+, and Lyt 2+, although a minor part of them were Thy 1-, Lyt 1-, and Lyt 2-. On the other hand, a predominant type of the IL 2-responsive cells in BALB/c nu/nu mice were Thy 1-, Lyt 1-, and Lyt 2-, though some were Thy 1+. Nonspecific killer activity against tumor cells increased to variable extents in all of the lymphoid cells of both strains after culture with rIL 2. Our results indicate that mouse responder cells to rIL 2 have the following characteristics. First, the responder cells exist abundantly among spleen, lymph nodes, and thymus in normal mice, though their cell lineages are heterogeneous; one is of T cell lineage and the other of natural killer (NK) cell lineage. Second, nude mice are defective in the responder cells of T cell lineage but not of NK cell lineage. Moreover, the responder cells in nude mice predominantly accumulate in the lymph nodes but not other lymphoid organs.

Suppression of pulmonary tumour metastasis in mice by recombinant human interleukin-2: role of asialo GM1-positive cells.

Recombinant human interleukin-2 (rIL-2) suppressed metastatic tumour colony formation in the lungs of C57BL/6 mice bearing Lewis lung carcinoma (3LL). In tumour-bearing mice given rIL-2, non-specific killer cells that were cytotoxic not only against natural killer-sensitive YAC-1 cells but also against 3LL cells in an in vitro 51Cr-release assay were concomitantly induced as tumour metastasis was suppressed. These non-specific killer cells were mostly removed by treatment with anti-Thy 1.2 or anti-asialo GM1 antibody plus complement (C) in vitro but not with anti-Lyt 1.2 or anti-Lyt 2.2 plus C, indicating that they were positive for Thy 1 and asialo GM1 but not for Lyt 1 and Lyt 2. In order to explore the mechanism by which rIL-2 suppressed tumour metastasis, we examined the clearance of intravenously injected 51Cr-labelled 3LL cells in the lungs of mice given rIL-2. The rate of tumour cell clearance was increased. This enhanced clearance was almost completely removed by injecting anti-asialo GM1 antibody. In addition, the injection of anti-asialo GM1 antibody also depleted most of the non-specific killer cells induced by administering rIL-2. These results indicate that asialo GM1-positive cells are not only cytotoxic in vitro but also play a critical role in the clearance of 3LL cells in the lungs in vivo. Our results indicate that asialo GM1-positive cells play an important role as anti-metastatic effector cells in suppressing the metastasis of 3LL cells in mice given rIL-2.

Characteristics of murine non-specific killer cells induced in vivo by recombinant human interleukin-2.

We have examined the induction of murine non-specific killer cells in vivo and in vitro by purified recombinant human interleukin-2 (rIL-2), and compared their characteristics with respect to killing ability, cell surface phenotypes, and antibody-dependent cell-mediated cytotoxicity (ADCC). C57BL/6 spleen cells cultured with rIL-2 were remarkably cytotoxic against a variety of tumour cells in a 4-hr 51Cr-release assay. Treatment with various antibodies (anti-Thy 1, anti-Lyt 1, anti-Lyt 2, and anti-asialo GM1) plus complement (C) showed that anti-Thy 1 or anti-asialo GM1 antibody plus C removed a majority of killer activity (80% and 66%, respectively). In addition, an increase in ADCC was detected in the spleen cells cultured with rIL-2. These ADCC effector cells were indistinguishable from non-specific killer cells by the cell surface phenotypes. A single administration of rIL-2 in vivo induced only transient and marginal enhancement of non-specific killer activity of spleen cells in C57BL/6 mice. On the other hand, when 10 micrograms of rIL-2 were administered daily by bolus to C57BL/6 mice, the activity increased gradually for about 10 days and reached a plateau. This enhanced non-specific killer activity rapidly decreased and returned to normal by 72 hr after the administration was stopped. The non-specific killer cells induced in vivo in this manner were not only greatly cytotoxic against natural killer (NK)-sensitive tumour cells but were also significantly cytotoxic against NK-resistant tumour cells. Most of the killer activity (more than 90%) was specifically removed by treatment with anti-Thy 1 or anti-asialo GM1 antibody plus C. An increase in ADCC was detected concurrently with an increase in non-specific killer activity in vivo, and both effector cells were indistinguishable by their cell surface phenotypes. These results indicate that a majority of non-specific killer cells induced both in vivo and in vitro by rIL-2 have some common features. Our results also suggest that these cells belong to the same lineage as NK cells, although they are thought to be at different stages from resident NK cells.

Production of cytotoxic factor(s) in human T cell lines transformed by a human retrovirus.

Human T cell lines, MT-2, TCL-Ter, TCL-Haz, and TCL-Kan which were transformed by a human retrovirus, constitutively produced cytotoxic factor(s) (CF) in the culture supernatants. In these cell lines, MT-2 produced the largest amount of CF. The amount of CF produced by MT-2 was 9-10 or 3-4 times larger than that produced by a human B cell line, RPMI 1788, or normal peripheral blood leukocytes stimulated with mitogens and phorbol ester. The kinetics of the production by MT-2 was similar in media with and without serum. The activity was stable at 56 degrees C for 30 min but was lost at 80 degrees C for 30 min and at pH 2 for 20 hr. On gel filtration, the molecular weight of the factor produced by MT-2 was approximately 90,000. On isoelectric focusing, the activity was recovered in the fraction at pH 6.5-7.0.

Comparison of the biological properties of purified natural and recombinant human interleukin-2.

We compared the biological properties of the purified recombinant human IL-2 derived from E. coli with those of purified natural IL-2. Both had almost the same specific in vitro activities on a weight basis to support long-term proliferation of IL-2 dependent human peripheral blood lymphocytes, a mouse killer T cell line, and a mouse natural killer cell line; induce killer cells in normal mouse spleen cells; and induce antibody forming cells in nude mouse spleen cells. No differences in these biological activities were found between two forms of natural IL-2 that were separable by reverse phase high performance liquid chromatography.

Purification and partial sequence analysis of human interleukin-2 derived from peripheral blood leukocytes.

Human peripheral blood leukocyte-derived interleukin-2 (IL-2) was resolved by DEAE-cellulose column chromatography into three peaks of activity, IL-2A, B, and C, with isoelectric points of 7.2, 6.6, and 7.9, respectively. IL-2 A, B, and C were further purified by reverse phase high performance liquid chromatography and resolved into two apparently homogeneous peaks each with identical molecular weight: A-1 and A-2 (Mr17000); B-1 and B-2 (Mr17500); and C-1 and C-2 (Mr14400). The amino acid compositions and partial NH2-terminal amino acid sequences of these molecular species were consistent with those predicted from IL-2 cDNA sequences derived from Jurkat and peripheral blood leukocytes.

Correlation of interferon gamma inducing ability to sugar binding specificities of various lectins.

Various lectins were examined to determine possible induction of gamma interferon (IFN-gamma) in human leukocytes. Among the seven positive lectins (Concanavalin A, pea lectin, lentil lectin, rice bran agglutinin, pokeweed mitogen, wheat germ agglutinin, phytohemagglutinin-P), six except rice bran agglutinin belonged to those which recognize carbohydrate chains connected to polypeptide through a glycosylamine linkage between N-acetylglucosamine (GlcNAc) and asparagine residues. The specificity of carbohydrate chain recognition of rice bran agglutinin, residual one positive lectin, has not been reported. Induction of IFN-gamma by wheat germ agglutinin, one of the positive lectins, was inhibited by the addition of GlcNAc during the induction, but not by the addition of glucose, galactose, alpha-methylmannose, N-acetylgalactosamine, N-acetylmannosamine, and lactose.