Molecular characterization and expression profiles of IscA1 gene in a long-distance migrant, Agrotis segetum

Cryptochrome (CRYs) proteins have been elucidated as the molecular basis for magnetoreception in Drosophila, and a putative magnetic receptor (named IscA1) protein may aslo be involved in sensing magnetic fields in Drosophila. However, whether IscA1 has a conserved role in diverse animals and functions in orientation during animal migration is unknown. Here we report on the cloning and sequencing of the IscA1 gene from Agrotis segetum, which encodes a predicted protein IscA1 that has 131 amino acids and two conserved iron-sulphur cluster binding domains. Multiple sequence alignment and phylogenetic analysis were used to show that IscA1 had a relatively high homology from species of Noctuoidea. Quantitative polymerase chain reaction showed that IscA1 was ubiquitously expressed in adult organs and, among all developmental stages, expression was higher in adults. When Agrotis segetum was exposed to 14?h light/10?h dark, IscA1 expression also showed daily oscillations, and constant light or dark disturbed these oscillations. IscA1 expression levels in a migratory population were higher than in a reared population and higher in a southward migratory population than in a northward. These findings suggest that the IscA1 gene in A. segetum might be associated with migration and provide a molecular basis for further study on the functions of IscA1 gene in magnetoreception and potential control of the turnip moth.     

Locus-specific primers for LMW glutenin genes on each of the group 1 chromosomes of hexaploid wheat

To reveal the chromosomal location of three known low-molecular-weight (LMW) glutenin genes in wheat, we designed and used three sets of sequence-specific primers in polymerase chain reactions (PCR) on Chinese Spring and its derived group 1 aneuploid nullisomic-tetrasomic stocks. Two sets proved to be chromosome specific and amplified sequences from the Glu-A3 and Glu-D3 loci, respectively. The third set was apparently composed of conserved sequences as it produced PCR products in each of the aneuploids. Two of these products were cloned, and their sequences differed from the known LMW glutenin genes at several positions. Again, primer sets specific for these sequences were designed. One set was directed to the Glu-A3 locus, the second set resulted in two PCR products differing in length, one of which was located on chromosome 1B and the other on 1D. Primer sets constructed for the latter two sequences were specific for the Glu-B3 and Glu-D3 loci, respectively. Hence, primer sets specific for each of the three homoeologous chromosomes of the group 1 (1A, 1B, 1D) are available. In addition, these locus-specific primers were assayed for their ability to distinguish among wheat cultivars. PCR products amplified with one of the Glu-A3-specific primer sets showed length polymorphisms in various wheat varieties. Varieties carrying the 1RS.1BL translocated chromosomes could be recognized by the absence of a PCR product when the Glu-B3 primer set was used. These results suggest that PCR with locus-specific primers can be useful in the molecular genetic analysis of hexaploid wheat.     

Genetic and physical characterization of theLR1 leaf rust resistance locus in wheat (Triticum aestivum L.)

The objective of this study was to characterize the leaf rust resistance locusLr1 in wheat. Restriction fragment length polymorphism (RELP) analysis was performed on the resistant lineLr1/6^*Thatcher and the susceptible varieties Thatcher and Frisal, as well as on the segregating F₂ populations. Seventeen out of 37 RFLP probes mapping to group 5 chromosomes showed polymorphism betweenLr1/6^*Thatcher and Frisal, whereas 11 probes were polymorphic between the near-isogenic lines (NILs)Lr1/6^*Thatcher and Thatcher. Three of these probes were linked to the resistance gene in the segregating F₂ populations. One probe (pTAG621) showed very tight linkage toLr1 and mapped to a single-copy region on chromosome 5D. The map location of pTAG621 at the end of the long arm of chromosome 5D was confirmed by the absence of the band in the nulli-tetrasomic line N5DT5B of Chinese Spring and a set of deletion lines of Chinese Spring lacking the distal part of 5DL. Twenty-seven breeding lines containing theLr1 resistance gene in different genetic backgrounds showed the same band asLr1/6^*Thatcher when hybridized with pTAG621. The RFLP marker was converted to a sequence-tagged-site marker using polymerase chain reaction (PCR) amplification. Sequencing of the specific fragment amplified from both NILs revealed point mutations as well as small insertion/deletion events. These were used to design primers that allowed amplification of a specific product only from the resistant lineLr1/6^*Thatcher. This STS, specific for theLr1 resistance gene, will allow efficient selection for the disease resistance gene in wheat breeding programmes. In addition, the identification of a D-genome-specific probe tightly linked toLr1 should ultimately provide the basis for positional cloning of the gene.     

Intestinal tube formation in Caenorhabditis elegans requires vang-1 and egl-15 signaling

Understanding how epithelial organs form during morphogenesis is a major problem in developmental biology. In the present paper, we provide a detailed analysis of vang-1, the only homolog of the planar cell polarity protein Strabismus/Van Gogh in Caenorhabditis elegans. We demonstrate that during organogenesis of the intestine, (i) VANG-1 specifically interacts with PDZ 2 domain of DLG-1 (Discs large) and becomes phosphorylated by the kinase domain of the FGF-like receptor tyrosine kinase EGL-15; (ii) VANG-1 is predominantly restrained to the cell cortex but relocates to the apical junction; and (iii) in vang-1 embryos epithelial cells of the intestine are not correctly arranged along the anterior-posterior axis. To investigate what determines the disposition of the VANG-1 protein, either truncated protein forms were expressed in the intestine or RNAi was used to remove the functions of gene products previously shown to be involved in apical junction formation. Removal of the VANG-1 PDZ binding motif “− ESAV” and depletion of dlg-1 or let-413 gene functions interferes with the localization of VANG-1. In addition, egl-15 embryos show a premature relocation of VANG-1 to the apical junction, causing defects that resemble those observed in mutant vang-1 embryos and after intestine-specific overexpression of full-length vang-1. Finally, the localization of VANG-1 depends on DSH-2, a homolog of the planar cell polarity protein Dishevelled and depletion phenocopies vang-1 and egl-15 phenotypes in the embryonic intestine.     

Molecular characterization of two y-type high molecular weight glutenin subunit alleles 1Ay12 and 1Ay8 from cultivated einkorn wheat (Triticum monococcum ssp. monococcum)

Two y-type high molecular weight glutenin subunits (HMW-GSs) 1Ay12^? and 1Ay8^? from the two accessions PI560720 and PI345186 of cultivated einkorn wheat (Triticum monococcum ssp. monococcum, AA, 2n = 2x = 14), were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The mobility of 1Ay12^? and 1Ay8^? was similar to that of 1Dy12 and 1By8 from common wheat Chinese Spring, respectively. Their ORFs respectively consisted of 1812 bp and 1935 bp, encoding 602 and 643 amino acid residues with the four typical structural domains of HMW-GS including signal peptide, conserved N-, and C-terminal and central repetitive domains. Compared with the most similar active 1Ay alleles previous published, there were a total of 15 SNPs and 2 InDels in them. Their encoding functions were confirmed by successful heterogeneous expression. The two novel 1Ay alleles were named as 1Ay12^? and 1Ay8^? with the accession No. JQ318694 and JQ318695 in GenBank, respectively. The two alleles were classed into the two distinct groups, Phe-type and Cys-type, which might be relevant to the differentiation of Glu-A1-2 alleles. Of which, 1Ay8^? belonged to Cys-type group, and its protein possessed an additional conserved cysteine residue in central repetitive region besides the six common ones in N- and C-terminal regions of Phe-type group, and was the second longest in all the known active 1Ay alleles. These results suggested that the subunit 1Ay8^? of cultivated einkorn wheat accession PI345186 might have a potential ability to strengthen the gluten polymer interactions and be a valuable genetic resource for wheat quality improvement.     

Purification and characterization of Put1p from Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the PUT1 and PUT2 genes are required for the conversion of proline to glutamate. The PUT1 gene encodes Put1p, a proline dehydrogenase (PRODH) enzyme localized in the mitochondrion. Put1p was expressed and purified from Escherichia coli and shown to have a UV-visible absorption spectrum that is typical of a bound flavin cofactor. A K_m value of 36 mM proline and a k_cat = 27 s⁻¹ were determined for Put1p using an artificial electron acceptor. Put1p also exhibited high activity using ubiquinone-1 (CoQ₁) as an electron acceptor with a k_cat = 9.6 s⁻¹ and a K_m of 33 μM for CoQ₁. In addition, knockout strains of the electron transfer flavoprotein (ETF) homolog in S. cerevisiae were able to grow on proline as the sole nitrogen source demonstrating that ETF is not required for proline utilization in yeast.     

Chronological expression of Wnt target genes Ccnd1, Myc, Cdkn1a, Tfrc, Plf1 and Ramp3

The canonical Wnt pathway regulates several biological processes including development, cell growth and proliferation via consecutive gene regulation. A high number of target genes of the Wnt pathway has been identified, but the chronological order of target gene expression is still elusive. This order is supposed to be crucial for the controlled course of events downstream of the activated Wnt pathway. Here we present the expression chronologies of the target genes Ccnd1 (encoding for cyclin D1), Myc (c-Myc), Cdkn1a (p21^CIP1/WAF1), Tfrc (Transferrin receptor 1), Plf1 (Proliferin-1) and Ramp3 (Receptor activity-modifying protein 3) in C57MG cells after stimulation with Wnt-3a. We discriminated between immediate (below 1 h), early (between 1 and 6 h), intermediate (between 6 and 12 h) and late (after 12 h) targets. According to this classification Myc and Tfrc belong to the immediate target genes; Ccnd1, Plf1 and Ramp3 are early target genes and Cdkn1a is an intermediate target gene.     

Detection and characterization of a glutenin subunit with unusual high Mr at the Glu-A1 locus in hexaploid wheat

A hexaploid wheat landrace collected from the Baluchistan province of Pakistan was found to possess a novel high-molecular-weight glutenin subunit (HMW-GS). The subunit has a very slow electrophoretic mobility as revealed by SDS-PAGE, and its molecular weight is comparable to that of the highest molecular weight glutenin subunit (2.2 encoded in the D-genome) reported so far in hexaploid wheat varieties and landraces of Japanese origin. Evidence obtained from (PCR) gene amplification studies using the primers specific for Glu-1 loci proved that the gene coding for this novel subunit belongs to the Glu-A1 locus located on the long arm of chromosome 1A. Digestion of the amplified gene (PCR product) with restriction enzymes indicated that the novel gene differs from prevailing Glu-A1 alleles (null, 1 and 2^*) by an extra DNA fragment of approximately 600 base pairs. The results also indicated that the novel subunit is most probably a derivative of subunit 2^* that has very likely incorporated the 600-bp fragment following a process of unequal crossing over. The present findings were further substantiated by reserved phase high performance liquid chromatography (RP-HPLC) analysis.     

Molecular cloning,characterization and expression analysis of ATG1 in the silkworm, Bombyx mori

Atg1 is a Serine/Threonine protein kinase that plays a pivotal role in autophagy. A complete coding sequence of ATG1 is not available for the silkworm, Bombyx mori which is a good model for studying the autophagic process.     

Thermodynamic characterization of OsGID1-gibberellin binding using calorimetry and docking simulations

Gibberellins (GAs) are phytohormones regulating various developmental processes in plants. In rice, the initial GA-signaling events involve the binding of a GA to the soluble GA receptor protein, GID1. Although X-ray structures for certain GID1/GA complexes have recently been determined, an examination of the complexes does not fully clarify how GID1s discriminate among different GAs. Herein, we present a study aimed at defining the types of forces important to binding via a combination of isothermal titration calorimetry (ITC) and computational docking studies that employed rice GID1 (OsGID1), OsGID1 mutants, which were designed to have a decreased possible number of hydrogen bonds with bound GA, and GA variants. We find that, in general, GA binding is enthalpically driven and that a hydrogen bond between the phenolic hydroxyl of OsGID1 Tyr134 and the C-3 hydroxyl of a GA is a defining structural element. A hydrogen-bond network that involves the C-6 carboxyl of a GA that directly hydrogen bonds the hydroxyl of Ser198 and indirectly, via a two-water-molecule network, the phenolic hydroxyl of Tyr329 and the NH of the amide side-chain of Asn255 is also important for GA binding. The binding of OsGID1 by GA(1) is the most enthalpically driven association found for the biologically active GAs evaluated in this study. This observation might be a consequence of a hydrogen bond formed between the hydroxyl at the C-13 position of GA(1) and the main chain carbonyl of OsGID1 Phe245. Our results demonstrate that by combining ITC experiments and computational methods much can be learned about the thermodynamics of ligand/protein binding.     

Molecular characterization and genomic organization of low molecular weight glutenin subunit genes at the Glu-3 loci in hexaploid wheat (Triticum aestivum L.)

In this study, we report on the molecular characterization and genomic organization of the low molecular weight glutenin subunit (LMW-GS) gene family in hexaploid wheat (Triticum aestivum L.). Eighty-two positive BAC clones were identified to contain LMW-GS genes from the hexaploid wheat ‘Glenlea’ BAC library via filter hybridization and PCR validation. Twelve unique LMW glutenin genes and seven pseudogenes were isolated from these positive BAC clones by primer-template mismatch PCR and subsequent primer walking using hemi-nested touchdown PCR. These genes were sequenced and each consisted of a single-open reading frame (ORF) and untranslated 5′ and 3′ flanking regions. All 12 LMW glutenin subunits contained eight cysteine residues. The LMW-m-type subunits are the most abundant in hexaploid wheat. Of the 12 LMW-GS, 1, 2 and 9 are i-type, s-type and m-type, respectively. The phylogenetic analysis suggested that the LMW-i type gene showed greater differences to LMW-s and LMW-m-type genes, which, in turn, were more closely related to one another. On the basis of their N-terminal sequences, they were classified into nine groups. Fingerprinting of the 82 BAC clones indicated 30 BAC clones assembled into eight contigs, while the remaining clones were singletons. BAC end sequencing of the 82 clones revealed that long terminal repeat (LTR) retrotransposons were abundant in the Glu-3 regions. The average physical distance between two adjacent LMW-GS genes was estimated to be 81 kb. Most of LMW-GS genes are located in the d-genome, suggesting that the Glu-D3 locus is much larger than the Glu-B3 locus and Glu-A3 locus. Alignments of sequences indicated that the same type (starting with the same N-terminal sequence) LMW-GS genes were highly conserved in the homologous genomes between hexaploid wheat and its donors such as durum wheat and T. tauschii. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular characterization and expression analysis of ubiquitin-activating enzyme E1 gene in Citrus reticulata

Ubiquitin-activating enzyme E1 (UBE1) catalyzes the first step in the ubiquitination reaction, which targets a protein for degradation via a proteasome pathway. UBE1 plays an important role in metabolic processes. In this study, full-length cDNA and DNA sequences of UBE1 gene, designated CrUBE1, were obtained from ‘Wuzishatangju’ (self-incompatible, SI) and ‘Shatangju’ (self-compatible, SC) mandarins. 5 amino acids and 8 bases were different in cDNA and DNA sequences of CrUBE1 between ‘Wuzishatangju’ and ‘Shatangju’, respectively. Southern blot analysis showed that there existed only one copy of the CrUBE1 gene in genome of ‘Wuzishatangju’ and ‘Shatangju’. The temporal and spatial expression characteristics of the CrUBE1 gene were investigated using semi-quantitative RT-PCR (SqPCR) and quantitative real-time PCR (qPCR). The expression level of the CrUBE1 gene in anthers of ‘Shatangju’ was approximately 10-fold higher than in anthers of ‘Wuzishatangju’. The highest expression level of CrUBE1 was detected in pistils at 7 days after self-pollination of ‘Wuzishatangju’, which was approximately 5-fold higher than at 0 h. To obtain CrUBE1 protein, the full-length cDNA of CrUBE1 genes from ‘Wuzishatangju’ and ‘Shatangju’ were successfully expressed in Pichia pastoris. Pollen germination frequency of ‘Wuzishatangju’ was significantly inhibited with increasing of CrUBE1 protein concentrations from ‘Wuzishatangju’.     

Cloning,characterization and heterelogous expression of the INU1 gene from Cryptococcus aureus HYA

The INU1 gene (Accession number: JX073660) encoding exo-inulinase from Cryptococcus aureus HYA was cloned and characterized. The gene had an open reading frame (ORF) of 1653 bp long encoding an inulinase. The coding region of the gene was not interrupted by any intron. It encoded 551 amino acid residues of a protein with a putative signal peptide of 23 amino acids and the calculated molecular mass of 59.5 kDa. The protein sequence deduced from the inulinase structural gene contained the inulinase consensus sequences (WMNDPNGL), (RDP), ECP, FS and Q. It also had two conserved putative N-glycosylation sites. The inulinase from C. aureus HYA was found to be closely related to that from Kluyveromyces marxianus and Pichia guilliermondii. The inulinase gene without the signal sequence was subcloned into pPICZaA expression vector and expressed in Pichia pastoris X-33. The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 60 kDa was found. Enzyme activity assay verified the recombinant protein as an inulinase. A maximum inulinase activity of 16.3 ± 0.24 U/ml was obtained from the culture supernatant of P. pastoris X-33 harboring the inulinase gene. The optimal temperature and pH for action of the enzyme were 50 °C and 5.0, respectively. A large amount of monosaccharides were detected after the hydrolysis of inulin with the purified recombinant inulinase.