Comparative genomic analysis of CIPK gene family in <Emphasis Type="Italic">Arabidopsis</Emphasis> and <Emphasis Type="Italic">Populus</Emphasis>

Calcium serves as a second messenger in various signal transduction pathways in plants. CBL-interacting protein kinases (CIPKs), which have a variety of functions, are involved in calcium signal transduction. Previous, the studies on CIPK family members focused on Arabidopsis and rice. Here, we present a comparative genomic analysis of the CIPK gene family in Arabidopsis and poplar, a model tree species. Twenty-seven potential CIPKs were identified from poplar using genome-wide analysis. Like the CIPK gene family from Arabidopsis, CIPK genes from poplar were also divided into intron-free and intron-harboring groups. In the intron-harboring group, the intron distribution of CIPKs is rather conserved during the genome evolutionary process. Many homologous gene pairs were found in the CIPK gene family, indicating duplication events might contribute to the amplification of this gene family. The phylogenetic comparison of CIPKs in combination with intron distribution analysis revealed that CIPK genes from both Arabidopsis and poplar might have an ancient origin, which formed earlier than the separation of these two eudicot species. Our genomic and bioinformatic analysis will provide an important foundation for further functional dissection of the CBL-CIPK signaling network in poplars. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Heterologous co-production of <Emphasis Type="Italic">Thermobifida fusca</Emphasis> Cel9A with other cellulases in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The processive endoglucanase Cel9A of the moderately thermophilic actinomycete Thermobifida fusca was functionally produced in Saccharomyces cerevisiae. Recombinant Cel9A displayed activity on both soluble (carboxymethylcellulose) and insoluble (Avicel) cellulose substrates confirming its processive endoglucanase activity. High-performance anionic exchange chromatography analyses of soluble sugars released from Avicel revealed a cellobiose/glucose ratio of 2.5 ± 0.1. Growth by the recombinant strain on amorphous cellulose was possible due to the sufficient amount of glucose cleaved from the cellulose chain. This is the first confirmed report of S. cerevisiae growing on a cellulosic substrate as sole carbohydrate source while only expressing one recombinant gene. To improve the cellulolytic capability of S. cerevisiae and to investigate the level of synergy among cellulases produced by a recombinant host, the cel9A gene was co-expressed with four cellulase-coding genes of Trichoderma reesei: two endoglucanases cel5A (egII) and cel7B (egI), and two cellobiohydrolases cel6A (cbhII) and cel7A (cbhI). Synergy, especially between the Cel9A and the two cellobiohydrolases, resulted in a higher cellulolytic capability of the recombinant host.     

Study of tauopathies by comparing <Emphasis Type="Italic">Drosophila</Emphasis> and human <Emphasis Type="Italic">tau</Emphasis> in <Emphasis Type="Italic">Drosophila</Emphasis>

The microtubule-binding protein tau has been investigated for its contribution to various neurodegenerative disorders. However, the findings from transgenic studies, using the same tau transgene, vary widely among different laboratories. Here, we have investigated the potential mechanisms underlying tauopathies by comparing Drosophila (d-tau) and human (h-tau) tau in a Drosophila model. Overexpression of a single copy of either tau isoform in the retina results in a similar rough eye phenotype. However, co-expression of Par-1 with d-tau leads to lethality, whereas co-expression of Par-1 with h-tau has little effect on the rough eye phenotype. We have found analogous results by comparing larval proteomes. Through genetic screening and proteomic analysis, we have identified some important potential modifiers and tau-associated proteins. These results suggest that the two tau genes differ significantly. This comparison between species-specific isoforms may help to clarify whether the homologous tau genes are conserved. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This study was supported by the National Science Foundation of China (30270341; 30630028), the Multidisciplinary Program (Brain and Mind) of the Chinese Academy of Sciences, the Major State Basic Research Program (“973 program”; G2000077800; G2006CB806600; 2006CB911003), the Precedent Project of Important Intersectional Disciplines in the Knowledge Innovation Engineering of the Chinese Academy of Sciences (KJCX1-09-03).     

<Emphasis Type="Italic">S</Emphasis> locus-linked F-box genes expressed in anthers of <Emphasis Type="Italic">Hordeum bulbosum</Emphasis>

Diploid Hordeum bulbosum (a wild relative of cultivated barley) exhibits a two-locus self-incompatibility (SI) system gametophytically controlled by the unlinked multiallelic loci S and Z. This unique SI system is observed in the grasses (Poaceae) including the tribe Triticeae. This paper describes the identification and characterization of two F-box genes cosegregating with the S locus in H. bulbosum, named Hordeum S locus-linked F-box 1 (HSLF1) and HSLF2, which were derived from an S ₃ haplotype-specific clone (HAS175) obtained by previous AMF (AFLP-based mRNA fingerprinting) analysis. Sequence analysis showed that both genes encode similar F-box proteins with a C-terminal leucine-rich repeat (LRR) domain, which are distinct from S locus (or S haplotype-specific) F-box protein (SLF/SFB), a class of F-box proteins identified as the pollen S determinant in S-RNase-based gametophytic SI systems. A number of homologous F-box genes with an LRR domain were found in the rice genome, although the functions of the gene family are unknown. One allele of the HSLF1 gene (HSLF1-S ₃) was expressed specifically in mature anthers, whereas no expression was detected from the other two alleles examined. Although the degree of sequence polymorphism among the three HSLF1 alleles was low, a frameshift mutation was found in one of the unexpressed alleles. The HSLF2 gene showed a low level of expression with no tissue specificity as well as little sequence polymorphism among the three alleles. The multiplicity of S locus-linked F-box genes is discussed in comparison with those found in the S-RNase-based SI system. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB511822–AB511825 and AB511859–AB511862.     

Identification and expression analysis of rainbow trout <Emphasis Type="Italic">pumilio</Emphasis>-1 and <Emphasis Type="Italic">pumilio</Emphasis>-2

Pumilio is a sequence-specific RNA-binding protein that regulates translation from the relevant mRNA. The PUF-domain, the RNA-binding motif of Pumilio, is highly conserved across species. In the present study, we have identified two pumilio genes (pumilio-1 and pumilio-2) in rainbow trout and analyzed their expression patterns in its tissues. Pumilio-1 mRNA and pumilio-2A mRNA code for typical full length Pumilio proteins that contain a PUF-domain, whereas pumilio-2B mRNA is a splice variant of pumilio-2 and encodes a protein that lacks the PUF-domain. We have also identified a novel 72-bp exon that has not been reported in other animal species but is conserved in fish species. The insertion of this novel exon leads to the expression of an isoform of the Pumilio-2 protein with a slightly altered conformation of the PUF-domain. Pumilio-1 mRNA and pumilio-2A mRNA (irrespective of the presence of the 72-bp exon) are expressed in both the brain and ovaries at high levels, whereas pumilio-2B mRNA is expressed at low levels in all the rainbow trout tissues examined. Western blot analysis also indicates that the full length Pumilio proteins are expressed predominantly in the brain and ovaries. These data suggest that the Pumilio proteins have physiological roles and are involved in regulatory mechanisms in rainbow trout.This work was in part supported by a grant from the Akiyama Foundation to E.I. Nucleotide sequence data for rainbow trout pumilio-1 and pumilio-2 have been deposited in the DDBJ/EMBL/GenBank databases.     

Common genetic variation in eight genes of the <Emphasis Type="Italic">GH</Emphasis>/<Emphasis Type="Italic">IGF1</Emphasis> axis does not contribute to adult height variation

Stature (adult height) is one of the most heritable human traits, yet few genes, if any, have been convincingly associated with adult height variation in the general population. Here, we selected 150 tag SNPs from eight candidate genes in the growth hormone (GH)/insulin-like growth factor-1 (IGF1) axis (GHR, GHRH, GHRHR, IGF1, IGFALS, IGFBP3, JAK2, STAT5B), and genotyped them in ∼2,200 individuals ascertained for short or tall stature. Nominally significant tag SNPs were then tested in three additional replication cohorts, including a family-based panel to rule out spurious associations owing to population stratification. Across the four height cohorts (N = 6,075 individuals), we did not observe any consistent associations between stature and common variants (≥5% minor allele frequency) in these eight genes, including a common deletion of the growth hormone receptor gene exon 3. Tests of epistatic interactions between these genes did not yield any results beyond those expected by chance. Although we have not tested all genes in the GH/IGF1 axis, our results indicate that common variation in these GH/IGF1 axis genes is not a major determinant of stature, and suggest that if common variation contributes to adult height variation in the general population, the variants are in other, possibly unanticipated genes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Mitochondrial <Emphasis Type="Italic">atpA</Emphasis> gene is altered in a new <Emphasis Type="Italic">orf220</Emphasis>-type cytoplasmic male-sterile line of stem mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>)

The purpose of this research is to identify the probable mitochondrial factor associated with cytoplasmic male sterility (cms) by comparative analysis of cms and its isogenic maintainer lines in stem mustards. Dramatic variations in the morphology of floral organs were observed in cms stem mustard. Mitochondrial atpA gene was shown to be altered in cms compared with that in its maintainer line, of which mitochondrial atpA gene from its maintainer line was sequenced to encode 507 amino acids. It was indicative of high homology with mitochondrial atpA genes from other species, even as high as 94% in similarity with Oryza sativa in terms of amino acid constituents. However, only 429 amino acids were deduced in cms showing 83% similarity with atpA gene from its maintainer line. Two copies were observed in its maintainer line, but only one was found in cms. Such numerous differences of mitochondrial atpA gene between cms and its maintainer lines may not be the results of evolutionary divergence but the rearrangements of mitochondria. Expression of mitochondrial atpA gene was shown to be down-regulated in cms by using Northern blot. Consequently, mitochondrial ATP synthesis was severely decreased more than one fold in cms stem mustard indicating deficiency in mitochondrial ATP synthesis in this type of cms. Therefore, we deduced that mitochondrial atpA gene altered in cms could be associated with male-sterility in this type of cms. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Jing-Hua Yang and Yan Huai contributed equally to this work.     

Characterisation and expression of monosaccharide transporters in lupins, <Emphasis Type="Italic">Lupinus polyphyllus</Emphasis> and <Emphasis Type="Italic">L. albus</Emphasis>

Monosaccharide transporter (MST) genes of Lupinus polyphyllus and L. albus were cloned, expressed and characterised. The isolation and functional characterisation of a cDNA clone and its corresponding genomic clone of a sugar transporter from L. polyphyllus (LpSTP1) is reported. Phylogenetic comparison of the nucleic and amino acid sequences showed the highest similarity to the AtSTP1 gene from Arabidopsis thaliana, which encodes a high affinity sugar transporter. The similar topology as well as the substrate specificity and expression pattern of LpSTP1 encoded protein additionally support the high similarity to the AtSTP1 gene product. The 1,590 bp LpSTP1 cDNA clone was heterologously expressed in yeast resulting in a fully functional specific sugar transporter. This transformation restored the viability of a yeast deletion mutant, which is devoid of all intrinsic MSTs and thus unable to take up and grow on hexose-containing media. The LpSTP1 protein is postulated to be a high-affinity MST since it supported growth best on media containing 0.2% hexose. Tissue-specific expression of LaSTP1 in L. albus was assayed by real-time PCR, which revealed that the lupin STP1 is mainly expressed in flower buds, flowers and young leaves. The results suggest that the main role of LaSTP1 is to catalyse monosaccharide import in sink tissues to meet increased carbohydrate demand during plant development. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Alterations of <Emphasis Type="Italic">ROBO1/DUTT1</Emphasis> and <Emphasis Type="Italic">ROBO2</Emphasis> loci in early dysplastic lesions of head and neck: clinical and prognostic implications

Deletion of chromosomal 3p12.3 was suggested to be associated with dysplastic lesions of head and neck. This region harbors two candidate tumor suppressors ROBO1/DUTT1, ROBO2 and two non-coding RNAs (ncRNAs) located at intron 2 of ROBO1/DUTT1. Aim of this study is to understand the role of these genes in development of head and neck squamous cell carcinoma. A collection of 72 dysplastic lesions and 116 HNSCC samples and two oral cancer cell lines were analyzed for ROBO1/DUTT1 and ROBO2 deletion and promoter methylation. ROBO1/DUTT1, ROBO2 and two ncRNAs mRNA expression were analyzed by Q-PCR. Immunohistochemical analysis of ROBO1/DUTT1 and ROBO2 was performed. Alterations of these genes were correlated with different clinicopathological parameters. High frequency of molecular alterations (deletion/methylation) was seen in ROBO1/DUTT1 than ROBO2. In mild dysplastic lesions both of these genes showed high molecular alterations and remained more or less constant in subsequent stages. Q-PCR analysis showed reduced expression of these genes and the two ncRNAs. In vitro demethylation experiment by 5-aza-dC showed upregulation of ROBO1/DUTT1 and ROBO2 while the expression of the ncRNAs remained unchanged. Immunohistochemical analysis of ROBO1/DUTT1 and ROBO2 showed concordance with their mRNA expression and molecular alterations. Poor patients’ outcome was predicted in the cases with alteration of ROBO1/DUTT1 along with tobacco addiction and nodal involvement. Our data suggests (a) ROBO1/DUTT1 and the ncRNAs are transcribed from different promoters, and (b) inactivation of ROBO1/DUTT1 could be used as molecular signature for early detection and prognosis of the head and neck cancer. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular characterization of the <Emphasis Type="Italic">Arginine decarboxylase</Emphasis> gene family in rice

Arginine decarboxylase (ADC) is a key enzyme in plants that converts arginine into putrescine, an important mediator of abiotic stress tolerance. Adc genes have been isolated from a number of dicotyledonous plants but the oat and rice Adc genes are the only representatives of monocotyledonous species described thus far. Rice has a small family of Adc genes, and OsAdc1 expression has been shown to fluctuate under drought and chilling stress. We identified and characterized a second rice Adc gene (OsAdc2) which encodes a 629-amino-acid protein with a predicted molecular mass of 67 kDa. An unusual feature of the OsAdc2 gene is the presence of an intron and a short upstream open reading frame in the 5′-UTR. Sequence comparisons showed that OsAdc2 is more closely related to the oat Adc gene than to OsAdc1 or to its dicot homologs, and mRNA analysis showed that the two rice genes are also differently regulated. Whereas OsAdc1 is expressed in leaf, root and stem, OsAdc2 expression is restricted to stem tissue. Protein expression was investigated with specific antibodies against ADC1 and ADC2, corroborating the mRNA data. We discuss the expression profiles of OsAdc1 and OsAdc2 and potential functions for the two corresponding proteins.     

Isolation of the <Emphasis Type="Italic">B</Emphasis> incompatibility factor mutants in <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

 B incompatibility factor mutants (Bmut) in Pleurotus ostreatus were recovered from common-B mating heterokaryons resulted from matings between wild-type monokaryons with different A but the same B factors (A1B2 and A2B2) after NTG mutagenesis. The mutant monokaryons such as A1B2mut and A2B2mut were observed to have regularly uninucleated hyphal cells and to be compatible with each other. Matings between A1B2mut and A2B2mut monokaryons produced stable heterokaryons (A1B2mut + A2B2mut) that had binucleated hyphal cells with true clamp connections and formed normal fruit-bodies. Mating tests using basidiospore progeny from each of these heterokaryons revealed the bipolar mating pattern. Genetic analysis suggested that the mutation of B factor in P. ostreatus might occur in the B incompatibility factor genes. Received: August 3, 2001 / Accepted: January 18, 2002     

Time Course of <Emphasis Type="Italic">c-fos</Emphasis>, <Emphasis Type="Italic">vasopressin</Emphasis> and <Emphasis Type="Italic">oxytocin</Emphasis> mRNA Expression in the Hypothalamus Following Long-Term Dehydration

Summary 1. This study presents a time course analysis of the messenger RNA (mRNA) levels of c-fos, vasopressin (VP), and oxytocin (OT) in the paraventricular (PVN) and supraoptic nucleus (SON), following acute and chronic dehydration by water deprivation. 2. Male Wistar rats were separated into five groups: nondehydrated (control group) and dehydrated for 6, 24, 48 and 72 h. Following water deprivation, animals were decapitated, their blood was collected for hematocrit, osmolality, and plasma sodium measurements, and brains were removed for dissection of both PVN and SON. 3. As expected, the hematocrit, osmolality, plasma sodium, and weight loss were increased after water deprivation. In SON, a significant increase in both VP and OT mRNA expression was observed 6 h after dehydration reaching a peak at 24 h and returning to basal levels of expression at 72 h. In the PVN, an increase in both VP and OT mRNA expression occurred 24 h after dehydration. At 72 h the VP and OT mRNA expression levels had decreased but they were still at higher levels than those detected in control animals. 4. These results suggest that SON is the first nucleus to respond to the dehydration stimulus. Additionally, we also observed an increase in c-fos mRNA expression in both PVN and SON 6 h after water deprivation, which progressively decreased 24, 48, and 72 h after the onset of water deprivation. Therefore, it is possible that c-fos may be involved in the modulation of VP and OT genes, regulating the mRNA expression levels on a temporally distinct basis within the PVN and SON.