Search

Background: Non-alcoholic fatty liver disease (NAFLD) is among the most common causes of chronic liver disease. NAFLD includes a spectrum of clinicopathologic syndromes that includes non-alcoholic steatohepatitis (NASH) that has potential for progression. The pathogenesis of NASH is poorly characterized. Aim: This study was designed to identify differences in hepatic gene expression in patients with NASH and to relate such differences to their clinical characteristics. Design: Consecutive patients undergoing bariatric surgery were prospectively recruited. Extensive clinical data and two liver biopsy specimens were obtained at the time of enrollment. A single hepatopathologist reviewed and classified the liver biopsies. Patients with excessive alcohol use and other causes of liver disease were excluded. A group of 29 NASH patients, 12 with steatosis alone, seven obese controls and six non-obese controls were selected for further investigation. Customized cDNA microarrays containing 5220 relevant genes were designed specifically for this study. Microarray experiments were run in triplicate for each sample and a selected group of genes were confirmed using real-time PCR. Outcome Measure: Differential hepatic gene expressions in patients with NASH as compared with controls. Results: Thirty-four genes with significant differential expression were identified in patients with NASH when compared with non-obese controls. Moreover, 19 of these genes showed no significant expression differences in obese vs. non-obese controls, suggesting a stronger association of these genes to NASH. Conclusions: Several differentially expressed genes in patients with NASH are related to lipid metabolism and extracellular matrix remodeling. Additionally, genes related to liver regeneration, apoptosis, and the detoxification process were differentially expressed. These findings may help clarify the molecular pathogenesis of NASH and identify potential targets for therapeutic intervention.

We have used the promoter, 1st exon and 1st intron of the maize polyubiquitin gene (Ubi-1) for rice transformation experiments and revealed the characteristic expression of Ubi-1 gene: (1) Ubi-1 gene is not regulated systemically but rather individual cells respond independently to the heat or physical stress; (2) Ubi-1 gene changes its tissue-specific expression in response to stress treatment; (3) the expression of Ubi-1 gene is dependent on cell cycle.

Restriction sites for six enzymes were mapped for the plastid DNAs of eight species of oryzoid grasses from temperate North America together with Olyra latifolia using biotin-labeled probes from Oryza sativa. Cladistic analysis of the data indicated the existence of one, most-parsimonious cladogram of 56 steps. Two species of Leersia differed by only one restriction site mutation, and two species of Zizania differed by only two mutations, whereas Luziolafluitans and L. bahiensis differed by 15 mutations. The restriction sites data strongly support two monophyletic clades corresponding to Oryzinae and Zizaniinae, but only weakly support the traditionally hypothesized alliance of Zizaniinae with other monoecious oryzoid grasses. The genera of the temperate North American Oryzeae have been divided into a monoecious group: Zizania, Zizaniopsis, Luziola, and Hydrochloa; and a bisexual group: Oryza and Leersia (Hitchcock, 1935; Hitchcock and Chase, 1951; Hutchinson, 1959). These groups have been variously recognized at tribal or subtribal levels. Stebbins and Crampton (1961) erected two tribes, the Zizanieae and the Oryzeae, following this concept in their tentative scheme of classification. After comparative studies of anatomy, cytology, embryology, and micromorphology, Terrell and Robinson (1974) placed Hydrochloa in Luziola, created a third group, the Luziolinae (Zizaniopsis and Luziola), and considered Zizania to be more closely related to the Oryzinae than to the Luziolinae. Most subsequent treatments (Gould and Shaw, 1983; Tsvelev, 1983; Clayton and Renvoize, 1986; but not

Epithelial ovarian tumors are the most common subtype of ovarian cancer. In this study, we reveal distinct expression signatures of previously uncharacterized ovarian carcinoma subtypes, including endometrioid component of mixed ovarian tumor and Sertoli–Leydig tumor. Both subtypes were compared to the most common and well-characterized ovarian epithelial carcinoma of the serous type. These comparisons were performed by complementaryDNA (cDNA) microarrays allowing high-fidelity measurements of the expression levels of 39,360 human individual cDNA species representing both known and unknown human genes. Functional analysis of differentially expressed genes in Sertoli–Leydig tumor revealed an upregulation in sonic hedgehog pathway, deregulation of several metabolic pathways especially in amino acid metabolism and overexpression of genes associated with protein synthesis, including ribosomal genes.

A set of plasmids has been constructed utilizing the promoter, 5' untranslated exon, and first intron of the maize ubiquitin (Ubi-1) gene to drive expression of protein coding sequences of choice. Plasmids containing chimaeric genes for ubiquitin-luciferase (Ubi-Luc), ubiquitin-beta-glucuronidase (Ubi-GUS), and ubiquitin-phosphinothricin acetyl transferase (Ubi-bar) have been generated, as well as a construct containing chimaeric genes for both Ubi-GUS and Ubi-bar in a single plasmid. Another construct was generated to allow cloning of protein coding sequences of choice on Bam HI and Bam HI-compatible restriction fragments downstream of the Ubi-1 gene fragment. Because the Ubi-1 promoter has been shown to be highly active in monocots, these constructs may be useful for generating high-level gene expression of selectable markers to facilitate efficient transformation of monocots, to drive expression of reference reporter genes in studies of gene expression, and to provide expression of biotechnologically important protein products in transgenic plants.

We have constructed a chimeric gene consisting of the promoter, first exon, and first intron of a maize ubiquitin gene (Ubi-1) and the coding sequence of the bar gene from Streptomyces hygroscopicus. This construct was transferred into rice (Oryza sativa L.) protoplasts via electroporation, and 10 plants were regenerated from calli that had been selected for resistance to exogenously supplied bialaphos. Transgenic plants grown in a greenhouse were resistant to both bialaphos and phosphinothricine at a dosage lethal to untransformed control plants. Evidence of stable integration of the transferred gene into the genome of the regenerated primary transformant plants was obtained from Southern blot analysis. In addition, northern blot analysis indicated expression and proper splicing of the maize ubiquitin gene first intron from the primary chimeric transcript in these transgenic rice plants, and western blot analysis and enzymic assays verified expression of the active bar gene product. Apparent mendelian segregation for bialaphos resistance in T(1) progeny of primary transformants was confirmed.

Two genomic clones (lambda Ubi-1 and lambda Ubi-2) encoding the highly conserved 76 amino acid protein ubiquitin have been isolated from maize. Sequence analysis shows that both genes contain seven contiguous direct repeats of the protein coding region in a polyprotein conformation. The deduced amino acid sequence of all 14 repeats is identical and is the same as for other plant ubiquitins. The use of transcript-specific oligonucleotide probes shows that Ubi-1 and Ubi-2 are expressed constitutively at 25 degrees C but are inducible to higher levels at elevated temperatures in maize seedlings. Both genes contain an intron in the 5' untranslated region which is inefficiently processed following a brief, severe heat shock. The transcription start site of Ubi-1 has been determined and a transcriptional fusion of 0.9 kb of the 5' flanking region and the entire 5' untranslated sequence of Ubi-1 with the coding sequence of the gene encoding the reporter molecule chloramphenicol acetyl transferase (CAT) has been constructed (pUBI-CAT). CAT assays of extracts of protoplasts electroporated with this construct show that the ubiquitin gene fragment confers a high level of CAT expression in maize and other monocot protoplasts but not in protoplasts of the dicot tobacco. Expression from the Ubi-1 promoter of pUBI-CAT yields more than a 10-fold higher level of CAT activity in maize protoplasts than expression from the widely used cauliflower mosaic virus 35S promoter of a 35S-CAT construct. Conversely, in tobacco protoplasts CAT activity from transcription of pUBI-CAT is less than one tenth of the level from p35S-CAT.

Southern blot analysis indicates that the rice genome contains single copies of genes encoding type A (phyA) and type B (phyB) phytochromes. We have isolated overlapping cDNA and genomic clones encoding the entire phyB polypeptide. This monocot sequence is more closely related to phyB from the dicot, Arabidopsis (73% amino acid sequence identity), than it is to the phyA gene in the rice genome (50% identity). These data support the proposal that phyA and phyB subfamilies diverged early in plant evolution and that subsequent divergence accompanied the evolution of monocots and dicots. Moreover, since rice and Arabidopsis phyB polypeptides are more closely related to one another (73% identity) than are monocot and dicot phyA sequences (63–65% identity), it appears that phyB has evolved more slowly than phyA. Sequence conservation between phyA and phyB is greatest in a central core region surrounding the chromophore attachment site, and least toward the amino-terminal and carboxy-terminal ends of the polypeptides, although hydropathy analysis suggests that the overall structure of the two phytochromes has been conserved. Gene-specific Northern blot analysis indicates that, whereas phyA is negatively regulated by phytochrome in rice seedling shoots in the manner typical of monocots, phyB is constitutively expressed irrespective of light treatment. In consequence, phyA and phyB transcripts are equally abundant in fully green tissue. Since Arabidopsis phyB mRNA levels are also unaffected by light, the present results suggest that this mode of regulation is evolutionarily conserved among phyB genes, perhaps reflecting differences in the functional roles of the different phytochrome subfamilies.

The fundamental notion that phytochrome controls plant development through differential regulation of gene expression (Mohr, 1966) is now well supported by direct experimental evidence (Benfey and Chua, 1989; Gilmartin et al., 1990; Kuhlemeier et al., 1987; Nagy et al., 1988; Tobin and Silverthorne, 1985). However, the molecular mechanism by which the photoreceptor transduces its regulatory signal to genes under its control remains unknown. For some time we have approached this question by simultaneously studying the properties of the photoreceptor molecule and the negative autoregulation of phytochrome (phy) genes as a paradigm of phytochrome-regulated gene expression (Colbert, 1988; Lissemore and Quail, 1988; Quail et al., 1987b, 1990). Recent molecular-genetic studies have revealed that phytochrome is encoded by a small family of divergent and differentially regulated genes (Dehesh et al., 1990b; Sharrock and Quail, 1989); have shown that overexpression of the photoreceptor in heterologous, transgenic plants provides a system for directed mutational analysis of functional regions of the polypeptide (Boylan and Quail, 1989; Kay et al., 1989c; Keller et al., 1989); and have begun to provide insight into the cis-regulatory elements and trans-acting factors involved in phy gene transcription (Brace et al., 1989, 1990; Dehesh et al., 1990a).

Bialaphos resistant rice plants expressing a bar gene under the control of the maize ubiquitin promoter which were inoculated with mycelia of the sheath blight disease pathogen, Rhizoctonia solani, and subsequently treated with the herbicide were completely protected from symptomatic infection. Substantial suppression of sheath blight symptoms was also recorded when bialaphos was applied to transgenic plants which had been infected with R. solani two days before herbicide treatment. It may therefore be possible to design new agronomic strategies for the simultaneous control of weeds and fungal pathogens in fields of transgenic plants expressing a bar gene.

Chaboissaea, a New World genus of open, marshy habitats, consists of four species: three taxa endemic to Mexico and one from Argentina and Bolivia. Restriction sites for 14 enzymes were mapped for the chloroplast DNA (cpDNA) plasmids of these four Chaboissaea species. Five hundred five potential restriction sites were detected when using Bealia mexicana, Lycurtus setosus, and Muhlenbergia montania as outgroups. Within Chaboissaea, 56 variable sites were shared among two or more species. Along with unique morphologi- cal characteristics and distinctive allozyme markers, our RFLP data support monophyly of the genus and suggest that Lycurus is its sister group. Cladistic analysis indicates that Chaboissaea subbiflora is the sister to the remaining three species in the genus, followed by C. decumbens; C. atacamensis and C. ligulata are the most derived species in the genus. Chaboissaea atacamensis and C. ligulata are extremely closely related and differ by only four autapomorphies in the latter. All data indicate that C. atacamensis emerged recently and our phylogeny based on cpDNA RFLP analysis suggests that C. ligulata, a species with a large geographical range in Mexico, is its sister taxon. Based on all lines of evidence, the genus appears to have arisen in southwestern North America (Mexico) and recently radiated to South America. RESUMEN. Chaboissaea, es un genero del Nuevo Mundo que habita sitios abiertos y pantanosos; incluye cuatro especies, tres endemicas de Mexico y una localizada en Argentina y Bolivia. Los sitios de restricci6n en catorce enzimas fueron mapeados utilizando el AND del cloropasto de estas cuatro especies del genero Chaboissaea. Ademas fueron detectados 505 potenciales sitios de restricci6n usando Bealia mexicana, Lycurtus setosus, y Muhlenbergia montana como "outgroups." Dentro de Chaboissaea, 56 sitios variables fueron compar- tidos entre dos o mas especies. Junto con caracteristicas morfologicas unicas y marcadores de allozimas distintivos, los datos de RFLP indican la monofilia del genero y sugieren a Lycurus como grupo hermano. El analisis cladistico senlala que Chaboissaea subbiflora es la especie hermana de las tres especies restantes del genero; quienes sequidas por C. decumbens; C. atacamensis y C. ligulata, son las especies mas derivadas. Chaboissaea atacamensis y C. ligulata estan muy cercanamente relacionadas difiriendo esta ultima, solamente en cuatro autapomorfias. Todos los datos senialan que C. atacamensis surgio recientemente y la filogenia basada en el analisis de cpADN RFLP sugiere que C. ligulata, especie con un gran rango geografico en Mexico, es el tax6n hermano. Basados en todas las lineas de evidencia, el genero parece haber surgido en el Sudoeste de Norte America (Mexico) y recientemente se expandi6 a Sur America.

A cDNA library prepared using mRNA isolated from red-light irradiated maize seedlings was screened by a difference procedure for clones that represent red-light regulated mRNA. Two such clones were found to represent mRNA for a chlorophyll a/b binding protein (CAB), and one of these (pAB1084) was used to screen a maize genomic library. One positive genomic clone (lambda AB1084) was isolated and sequenced. The gene represented by lambda AB1084, which we designate maize cab-1, contains extensive nucleotide homology within its protein coding region to CAB genes from other species. The boundaries of the transcribed region of the cab-1 gene were determined by S1 nuclease mapping. The 5' terminus of cab-1 mRNA is located 52-54 nucleotides (nt) upstream of the translation start site and 34 nt downstream of a TATA box. As in the case of petunia CAB genes, several poly(A) addition sites are present in mRNA from the cab-1 gene. The 5' flanking DNA of cab-1 contains sequences related to elements that have been implicated in the light-regulated expression of CAB and rbcS genes in other plant systems. Quantitative Northern blot hybridization analysis using a gene specific probe for cab-1 indicates that the mRNA for this gene is present at 0.4% of the total mRNA and up to 80% of the total CAB mRNA in the leaves of dark-grown seedlings. In consequence, although the degree of up-regulation by white light is only moderate (3- to 6-fold), cab-1 transcripts account for approximately 2% of the mRNA in the leaves of light-grown seedlings.

The regulatory photoreceptor phytochrome controls the transcription of its own phy genes in a negative feedback fashion. We have exploited microprojectile-mediated gene transfer to develop a rapid transient expression assay system for the study of DNA sequences involved in the phytochrome-regulated expression of these genes. The 5'-flanking sequence and part of the structural region of an oat phy gene have been fused to a reporter coding sequence (chloramphenicol acetyltransferase, CAT) and introduced into intact darkgrown seedlings by using high-velocity microprojectiles. Expression is assayable in less than 24 hr from bombardment. The introduced oat phy-CAT fusion gene is expressed and down-regulated by white light in barley, rice, and oat, whereas no expression is detected in three dicots tested, tobacco, cucumber, and Arabidopsis thaliana. In bombarded rice shoots, red/far-red light-reversible repression of expression of the heterologous oat phy-CAT gene shows that it is regulated by phytochrome in a manner parallel to that of the endogenous rice phy genes. These data indicate that the transduction pathway components and promoter sequences involved in autoregulation of phy expression have been evolutionarily conserved between oat and rice. The experiments show the feasibility of using high-velocity microprojectile-mediated gene transfer for the rapid analysis of light-controlled monocot gene promoters in monocot tissues that until now have been recalcitrant to such studies.

We have isolated genomic clones for three loci encoding the phytochrome polypeptide of Zea mays, and have determined the entire sequence of one of them (phyA1) together with approximately 1 kb of 5' flanking DNA. The structure of this gene is highly conserved in comparison with other phytochrome-encoding genes (phy). The deduced amino acid (aa) sequence indicates that the maize phytochrome protein is 1130 aa long (125 kDa). Overall aa sequence identity is 88% with Avena and rice type A phytochromes and 65% with the type A phytochromes of the dicots, pea, zucchini and Arabidopsis. Northern analysis indicates that maize phy transcripts are down-regulated only two- to threefold in etiolated seedlings 3 h after a red light pulse, in contrast to Avena where a ten- to 20-fold decrease is observed. On the other hand, a more than tenfold reduction in maize phy mRNA abundance occurs in seedlings transferred to white light for 24 h. Several conserved sequence elements have been identified by comparison of the maize phyA1 and other monocot phy promoters, suggesting that these common regions may be regulatory elements involved in phy expression.

We have isolated a maize ubiquitin cDNA clone which encodes one partial and three full-length, identical 76 amino acid repeats, in a polyprotein conformation. The deduced amino acid sequence of the mature monomeric polypeptide is identical to that determined for three other plants, barley, oat, and Arabidopsis, and differs from yeast and animal ubiquitin by only two and three amino acids, respectively. Hybridization of the cDNA clone to restriction endonuclease-digested genomic DNA revealed that ubiquitin is encoded by a small multigene family in maize. Northern blot analysis of poly(A)(+) RNA indicated that multiple ubiquitin mRNAs of 2.1, 1.6 and 0.8 kb are produced in maize shoots and roots. The abundance of the largest (2.1 kb) of these transcripts increased transiently 3- to 4-fold over the first 1 to 3 h in seedlings that were subjected to heat shock, and then returned dramatically within 1 h almost to the preshocked level. In contrast, the two smaller transcripts showed little or no change following heat shock. Run-on transcription assays in isolated maize nuclei showed a heat shock-induced increase in ubiquitin run-on transcripts that paralleled the increase in mature 2.1 kb mRNA levels over the first 3 h following the heat shock treatment. This result indicates that heat shock regulates ubiquitin gene expression at least in part at the transcriptional level. The subsequent rapid decline in steady-state mRNA levels, on the other hand, was not preceded by decreased ubiquitin gene transcription, raising the possibility of both transcriptional and posttranscriptional regulation. The run-on transcription assays also revealed a transient 5-fold reduction in rRNA gene transcription following heat shock, indicating that the transcriptional machinery for these genes is selectively sensitive to this stress.