Differential expression of major histocompatibility complex class II genes on murine macrophages associated with T cell cytokine profile and protective/suppressive effects

Protective/suppressive major histocompatibility complex (MHC) class II alleles have been identified in humans and mice where they exert a disease-protective and immunosuppressive effect. Various modes of action have been proposed, among them differential expression of MHC class II genes in different types of antigen-presenting cells impacting on the T helper type 1 (Th1)–Th2 balance. To test this possibility, the expression of H-2 molecules from the four haplotypes H-2b, H-2d, H-2k, and H-2q was determined on bone marrow-derived macrophages (BMDMs) and splenic B cells. The I-Ab and I-Ek molecules, both well characterized as protective/suppressive, are expressed at a high level on almost all CD11b+ BMDMs for 5–8 days, after which expression slowly declines. In contrast, I-Ad, I-Ak, and I-Aq expression is lower, peaks over a shorter period, and declines more rapidly. No differential expression could be detected on B cells. In addition, the differential MHC class II expression found on macrophages skews the cytokine response of T cells as shown by an in vitro restimulation assay with BMDMs as antigen-presenting cells. The results indicate that macrophages of the protective/suppressive haplotypes express MHC class II molecules at a high level and exert Th1 bias, whereas low-level expression favors a Th2 response. We suggest that the extent of expression of the class II gene gates the back signal from T cells and in this way controls the activity of macrophages. This effect mediated by polymorphic nonexon segments of MHC class II genes may play a role in determining disease susceptibility in humans and mice.

Differential Expression and Functions of Cortical Myosin IIA and IIB Isotypes during Meiotic Maturation, Fertilization, and Mitosis in Mouse Oocytes and Embryos

To explore the role of nonmuscle myosin II isoforms during mouse gametogenesis, fertilization, and early development, localization and microinjection studies were performed using monospecific antibodies to myosin IIA and IIB isotypes. Each myosin II antibody recognizes a 205-kDa protein in oocytes, but not mature sperm. Myosin IIA and IIB demonstrate differential expression during meiotic maturation and following fertilization: only the IIA isoform detects metaphase spindles or accumulates in the mitotic cleavage furrow. In the unfertilized oocyte, both myosin isoforms are polarized in the cortex directly overlying the metaphase-arrested second meiotic spindle. Cortical polarization is altered after spindle disassembly with Colcemid: the scattered meiotic chromosomes initiate myosin IIA and microfilament assemble in the vicinity of each chromosome mass. During sperm incorporation, both myosin II isotypes concentrate in the second polar body cleavage furrow and the sperm incorporation cone. In functional experiments, the microinjection of myosin IIA antibody disrupts meiotic maturation to metaphase II arrest, probably through depletion of spindle-associated myosin IIA protein and antibody binding to chromosome surfaces. Conversely, the microinjection of myosin IIB antibody blocks microfilament-directed chromosome scattering in Colcemid-treated mature oocytes, suggesting a role in mediating chromosome–cortical actomyosin interactions. Neither myosin II antibody, alone or coinjected, blocks second polar body formation, in vitro fertilization, or cytokinesis. Finally, microinjection of a nonphosphorylatable 20-kDa regulatory myosin light chain specifically blocks sperm incorporation cone disassembly and impedes cell cycle progression, suggesting that interference with myosin II phosphorylation influences fertilization. Thus, conventional myosins break cortical symmetry in oocytes by participating in eccentric meiotic spindle positioning, sperm incorporation cone dynamics, and cytokinesis. Although murine sperm do not express myosin II, different myosin II isotypes may have distinct roles during early embryonic development.

Differential expression of dystrophin isoforms and utrophin during dibutyryl-cAMP-induced morphological differentiation of rat brain astrocytes

We have identified isoforms of dystrophin and utrophin, a dystrophin homologue, expressed in astrocytes and examined their expression patterns during dibutyryl-cAMP (dBcAMP)-induced morphological differentiation of astrocytes. Immunoblot and immunocytochemical analyses showed that full-length-type dystrophin (427 kDa), utrophin (395 kDa), and Dp71 (75 kDa), a small-type dystrophin isoform, were coexpressed in cultured nondifferentiated rat brain astrocytes and were found to be located in the cell membrane. During morphological differentiation of the astrocytes induced by 1 mM dBcAMP, the amount of Dp71 markedly increased, whereas that of dystrophin and utrophin decreased. Northern blot analyses revealed that dBcAMP regulates the mRNA levels of Dp71 and dystrophin but not that of utrophin. dBcAMP slightly increased the amount of the β-dystroglycan responsible for anchoring dystrophin isoforms and utrophin to the cell membrane. Immunocytochemical analyses showed that most utrophin was observed in the cytoplasmic area during astrocyte differentiation, whereas Dp71 was found along the cell membrane of the differentiated astrocytes. These findings suggest that most of the dystrophin/utrophin-dystroglycan complex on cell membrane in cultured astrocytes was replaced by the Dp71-dystroglycan complex during morphological differentiation. The cell biological roles of Dp71 are discussed.

Hypoxia-induced gene expression profiling in the euryoxic fish Gillichthys mirabilis

Hypoxia is important in both biomedical and environmental contexts and necessitates rapid adaptive changes in metabolic organization. Mammals, as air breathers, have a limited capacity to withstand sustained exposure to hypoxia. By contrast, some aquatic animals, such as certain fishes, are routinely exposed and resistant to severe environmental hypoxia. Understanding the changes in gene expression in fishes exposed to hypoxic stress could reveal novel mechanisms of tolerance that may shed new light on hypoxia and ischemia in higher vertebrates. Using cDNA microarrays, we have studied gene expression in a hypoxia-tolerant burrow-dwelling goby fish, Gillichthys mirabilis. We show that a coherent picture of a complex transcriptional response can be generated for a nonmodel organism for which sequence data were unavailable. We demonstrate that: (i) although certain shifts in gene expression mirror changes in mammals, novel genes are differentially expressed in fish; and (ii) tissue-specific patterns of expression reflect the different metabolic roles of tissues during hypoxia.

BodyMap incorporated PCR-based expression profiling data and a gene ranking system

BodyMap is a human and mouse gene expression database that is based on site-directed 3′-expressed sequence tags generated at Osaka University. To date, it contains more than 300 000 tag sequences from 64 human and 39 mouse tissues. For the recent release, the precise anatomical expression patterns for more than half of the human gene entries were generated by introduced amplified fragment length polymorphism (iAFLP), which is a PCR-based high-throughput expression profiling method. The iAFLP data incorporated into BodyMap describe the relative contents of more than 12 000 transcripts across 30 tissue RNAs. In addition, a newly developed gene ranking system helps users obtain lists of genes that have desired expression patterns according to their significance. BodyMap supports complete transfer of unique data sets and provides analysis that is accessible through the WWW at http://bodymap.ims.u-tokyo.ac.jp.

Delineating developmental and metabolic pathways in vivo by expression profiling using the RIKEN set of 18,816 full-length enriched mouse cDNA arrays

We have systematically characterized gene expression patterns in 49 adult and embryonic mouse tissues by using cDNA microarrays with 18,816 mouse cDNAs. Cluster analysis defined sets of genes that were expressed ubiquitously or in similar groups of tissues such as digestive organs and muscle. Clustering of expression profiles was observed in embryonic brain, postnatal cerebellum, and adult olfactory bulb, reflecting similarities in neurogenesis and remodeling. Finally, clustering genes coding for known enzymes into 78 metabolic pathways revealed a surprising coordination of expression within each pathway among different tissues. On the other hand, a more detailed examination of glycolysis revealed tissue-specific differences in profiles of key regulatory enzymes. Thus, by surveying global gene expression by using microarrays with a large number of elements, we provide insights into the commonality and diversity of pathways responsible for the development and maintenance of the mammalian body plan.

Differential Expression of Three Members of the 1-Aminocyclopropane-1-Carboxylate Synthase Gene Family in Carnation1

We investigated the expression patterns of three 1-aminocyclopropane-1-carboxylate (ACC) synthase genes in carnation (Dianthus caryophyllus cv White Sim) under conditions previously shown to induce ethylene biosynthesis. These included treatment of flowers with 2,4-dichlorophenoxyacetic acid, ethylene, LiCl, cycloheximide, and natural and pollination-induced flower senescence. Accumulation of ACC synthase transcripts in leaves following mechanical wounding and treatment with 2,4-dichlorophenoxyacetic acid or LiCl was also determined by RNA gel-blot analysis. As in other species, the carnation ACC synthase genes were found to be differentially regulated in a tissue-specific manner. DCACS2 and DCACS3 were preferentially expressed in styles, whereas DCACS1 mRNA was most abundant in petals. Cycloheximide did not induce increased accumulation of ACC synthase transcripts in carnation flowers, whereas the expression of ACC synthase was up-regulated by auxin, ethylene, LiCl, pollination, and senescence in a floral-organ-specific manner. Expression of the three ACC synthases identified in carnation did not correspond to elevated ethylene biosynthesis from wounded or auxin-treated leaves, and there are likely additional members of the carnation ACC synthase gene family responsible for ACC synthase expression in vegetative tissues.

Differential Expression of Genes for Cyclin-Dependent Protein Kinases in Rice Plants1

Cyclin-dependent protein kinases (CDKs) play key roles in regulating the eukaryotic cell cycle. We have analyzed the expression of four rice (Oryza sativa) CDK genes, cdc2Os1, cdc2Os2, cdc2Os3, and R2, by in situ hybridization of sections of root apices. Transcripts of cdc2Os1, cdc2Os2, and R2 were detected uniformly in the dividing region of the root apex. cdc2Os1 and cdc2Os2 were also expressed in differentiated cells such as those in the sclerenchyma, pericycle, and parenchyma of the central cylinder. By contrast, signals corresponding to transcripts of cdc2Os3 were distributed only in patches in the dividing region. Counterstaining of sections with 4′,6-diamidino-2-phenylindole and double-target in situ hybridization with a probe for histone H4 transcripts revealed that cdc2Os3 transcripts were abundant from the G2 to the M phase, but were less abundant or absent during the S phase. The levels of the Cdc2Os3 protein and its associated histone H1-kinase activity were reduced by treatment of cultured cells with hydroxyurea, which blocks cycling cells at the onset of the S phase. Our results suggest that domains other than the conserved amino acid sequence (the PSTAIRE motif) have important roles in the function of non-PSTAIRE CDKs in distinct cell-cycle phases.

Comprehensive copy number and gene expression profiling of the 17q23 amplicon in human breast cancer

The biological significance of DNA amplification in cancer is thought to be due to the selection of increased expression of a single or few important genes. However, systematic surveys of the copy number and expression of all genes within an amplified region of the genome have not been performed. Here we have used a combination of molecular, genomic, and microarray technologies to identify target genes for 17q23, a common region of amplification in breast cancers with poor prognosis. Construction of a 4-Mb genomic contig made it possible to define two common regions of amplification in breast cancer cell lines. Analysis of 184 primary breast tumors by fluorescence in situ hybridization on tissue microarrays validated these results with the highest amplification frequency (12.5%) observed for the distal region. Based on GeneMap'99 information, 17 known genes and 26 expressed sequence tags were localized to the contig. Analysis of genomic sequence identified 77 additional transcripts. A comprehensive analysis of expression levels of these transcripts in six breast cancer cell lines was carried out by using complementary DNA microarrays. The expression patterns varied from one cell line to another, and several overexpressed genes were identified. Of these, RPS6KB1, MUL, APPBP2, and TRAP240 as well as one uncharacterized expressed sequence tag were located in the two common amplified regions. In summary, comprehensive analysis of the 17q23 amplicon revealed a limited number of highly expressed genes that may contribute to the more aggressive clinical course observed in breast cancer patients with 17q23-amplified tumors.

Differential Expression and Internal Feedback Regulation of 1-Aminocyclopropane-1-Carboxylate Synthase, 1-Aminocyclopropane-1-Carboxylate Oxidase, and Ethylene Receptor Genes in Tomato Fruit during Development and Ripening1

We investigated the feedback regulation of ethylene biosynthesis in tomato (Lycopersicon esculentum) fruit with respect to the transition from system 1 to system 2 ethylene production. The abundance of LE-ACS2, LE-ACS4, and NR mRNAs increased in the ripening fruit concomitant with a burst in ethylene production. These increases in mRNAs with ripening were prevented to a large extent by treatment with 1-methylcyclopropene (MCP), an ethylene action inhibitor. Transcripts for the LE-ACS6 gene, which accumulated in preclimacteric fruit but not in untreated ripening fruit, did accumulate in ripening fruit treated with MCP. Treatment of young fruit with propylene prevented the accumulation of transcripts for this gene. LE-ACS1A, LE-ACS3, and TAE1 genes were expressed constitutively in the fruit throughout development and ripening irrespective of whether the fruit was treated with MCP or propylene. The transcripts for LE-ACO1 and LE-ACO4 genes already existed in preclimacteric fruit and increased greatly when ripening commenced. These increases in LE-ACO mRNA with ripening were also prevented by treatment with MCP. The results suggest that in tomato fruit the preclimacteric system 1 ethylene is possibly mediated via constitutively expressed LE-ACS1A and LE-ACS3 and negatively feedback-regulated LE-ACS6 genes with preexisting LE-ACO1 and LE-ACO4 mRNAs. At the onset of the climacteric stage, it shifts to system 2 ethylene, with a large accumulation of LE-ACS2, LE-ACS4, LE-ACO1, and LE-ACO4 mRNAs as a result of a positive feedback regulation. This transition from system 1 to system 2 ethylene production might be related to the accumulated level of NR mRNA.

Differential Expression of Alternative Oxidase Genes in Soybean Cotyledons during Postgerminative Development1

The expression of the alternative oxidase (AOX) was investigated during cotyledon development in soybean (Glycine max [L.] Merr.) seedlings. The total amount of AOX protein increased throughout development, not just in earlier stages as previously thought, and was correlated with the increase in capacity of the alternative pathway. Each AOX isoform (AOX1, AOX2, and AOX3) showed a different developmental trend in mRNA abundance, such that the increase in AOX protein and capacity appears to involve a shift in gene expression from AOX2 to AOX3. As the cotyledons aged, the size of the mitochondrial ubiquinone pool decreased. We discuss how this and other factors may affect the alternative pathway activity that results from the developmental regulation of AOX expression.

Differential Expression of the S-Adenosyl-l-Methionine Synthase Genes during Pea Development1

Two genes coding for S-adenosyl-l-methionine synthase (SAMS, EC 2.5.1.6) were previously isolated from pea (Pisum sativum) ovaries. Both SAMS genes were highly homologous throughout their coding regions but showed a certain degree of sequence divergence within the 5′ and the 3′ untranslated regions. These regions have been used as gene-specific probes to analyze the differential expression of SAMS1 and SAMS2 genes in pea plants. The ribonuclease protection assay revealed different expression patterns for each individual gene. SAMS1 was strongly expressed in nearly all tissues, especially in roots. SAMS2 expression was weaker, reaching its highest level at the apex. Following pollination, SAMS1 was specifically up-regulated, whereas SAMS2 was expressed constitutively. The up-regulation of SAMS1 during ovary development was also observed in unpollinated ovaries treated with auxins. In unpollinated ovaries an increase in SAMS1 expression was observed as a consequence of ethylene production associated with the emasculation process. In senescing ovaries both SAMS1 and SAMS2 genes showed increased expression. Ethylene treatment of unpollinated ovaries led to an increase in the SAMS1 mRNA level. However, SAMS2 expression remained unchangeable after ethylene treatment, indicating that SAMS2 induction during ovary senescence was not ethylene dependent. SAMS mRNAs were localized by in situ hybridization at the endocarp of developing fruits and in the ovules of senescing ovaries. Our results indicate that the transcriptional regulation of SAMS genes is developmentally controlled in a specific way for each gene.

Tomato Fructokinases Exhibit Differential Expression and Substrate Regulation1

Two divergent genes encoding fructokinase, Frk1 and Frk2, have been previously shown to be expressed in tomato (Lycopersicon esculentum L.) and have now been further characterized with regard to their spatial expression and the enzymic properties of the encoded proteins. Frk1 and Frk2 mRNA levels were coordinately induced by exogenous sugar, indicating that both belong to the growing class of sugar-regulated genes. However, in situ hybridization indicated that Frk1 and Frk2 were expressed in a spatially distinct manner, with Frk2 mRNA primarily localized in cells of the fruit pericarp, which store starch, and Frk1 mRNA distributed ubiquitously in pericarp tissue. To evaluate the biochemical characteristics of the products of the Frk1 and Frk2 genes, each cDNA was expressed in a mutant yeast (Saccharomyces cerevisiae) line defective in hexose phosphorylation and unable to grow on glucose or fructose (Fru). Both Frk1 and Frk2 proteins expressed in yeast conferred the ability to grow on Fru and exhibited fructokinase activity in vitro. Although both Frk1 and Frk2 both utilized Fru as a substrate, only Frk2 activity was inhibited at high Fru concentrations. These results indicate that Frk2 can be distinguished from Frk1 by its sensitivity to substrate inhibition and by its temporal and spatial pattern of expression, which suggests that it plays a primary role in plant cells specialized for starch storage.

Differential Expression of a Novel Gene in Response to Coronatine, Methyl Jasmonate, and Wounding in the Coi1 Mutant of Arabidopsis1

Coronatine is a phytotoxin produced by some plant-pathogenic bacteria. It has been shown that coronatine mimics the action of methyl jasmonate (MeJA) in plants. MeJA is a plant-signaling molecule involved in stress responses such as wounding and pathogen attack. In Arabidopsis thaliana, MeJA is essential for pollen grain development. The coi1 (for coronatine-insensitive) mutant of Arabidopsis, which is insensitive to coronatine and MeJA, produces sterile male flowers and shows an altered response to wounding. When the differential display technique was used, a message that was rapidly induced by coronatine in wild-type plants but not in coi1 was identified and the corresponding cDNA was cloned. The coronatine-induced gene ATHCOR1 (for A. thaliana coronatine-induced) is expressed in seedlings, mature leaves, flowers, and siliques but was not detected in roots. The expression of this gene was dramatically reduced in coi1 plants, indicating that COI1 affects its expression. ATHCOR1 was rapidly induced by MeJA and wounding in wild-type plants. The sequence of ATHCOR1 shows no strong homology to known proteins. However, the predicted polypeptide contains a conserved amino acid sequence present in several bacterial, animal, and plant hydrolases and includes a potential ATP/GTP-binding-site motif (P-loop).