The<Emphasis Type="Italic"> GAOLAOZHUANGREN2</Emphasis> gene is required for normal glucose response and development of<Emphasis Type="Italic"> Arabidopsis</Emphasis>

Recent studies of glucose (Glc) sensing and signaling have revealed that Glc acts as a critical signaling molecule in higher plants. Several Glc sensing-defective Arabidopsis mutants have been characterized in detail, and the corresponding genes encoding Glc-signaling proteins have been isolated. However, the full complexity of Glc signaling in higher plants is not yet fully understood. Here, we report the identification and characterization of a new Glc-insensitive mutant, gaolaozhuangren2 (glz2), which was isolated from transposon mutagenesis experiments in Arabidopsis. In addition to its insensitivity to Glc, the glz2 plant exhibits several developmental defects such as short stature with reduced apical dominance, short roots, small and dark-green leaves, late flowering and female sterility. Treatment with 4% Glc blocked expression of the OE33 gene in wild-type plants, whereas expression of this gene was unchanged in the glz2 mutant plants. Taken together, our results suggest that the GLZ2 gene is required for normal glucose response and development of Arabidopsis.Mingjie Chen and Xiaoxiang Xia contributed equally to this work.     

An insertional mutation in the rice<Emphasis Type="Italic"> PAIR2</Emphasis> gene,the ortholog of<Emphasis Type="Italic"> Arabidopsis ASY1</Emphasis>, results in a defect in homologous chromosome pairing during meiosis

To elucidate the genetic system that establishes homologous chromosome pairing in monocot plants, we have isolated an asynaptic mutant of rice, designated pair2 (homologous pairing aberration in rice meiosis 2), in which 24 completely unpaired univalents are observed at pachytene and diakinesis. The mutation was caused by an insertion of the retrotransposon Tos17, as demonstrated by complementation of the mutation by transformation with the corresponding wild-type gene. The gene in which the element was inserted is orthologous to the ASY1 gene of Arabidopsis thaliana and the HOP1 gene of Saccharomyces cerevisiae. Mature PAIR2 mRNA and several splicing variants were found to be highly expressed in wild-type reproductive tissues, and lower expression was also detected in vegetative tissues. In situ hybridization and BrdU incorporation experiments revealed that PAIR2 expression is specifically enhanced in male and female meiocytes, but not in those at pre-meiotic S phase or in the pollen maturation stages. The results obtained in this study suggest that the PAIR2 gene is essential for homologous chromosome pairing in meiosis, as in the case of the genes ASY1 and HOP1. The study also suggested the possibility that a highly homologous copy of the PAIR2 gene located on a different chromosome is in fact a pseudogene.Communicated by G. Jürgens     

Identification of plant-regulated genes in<Emphasis Type="Italic"> Ustilago maydis</Emphasis> by enhancer-trapping mutagenesis

To identify plant-induced genes in the maize pathogenic fungus Ustilago maydis we have developed a genetic screen that combines REMI (restriction enzyme mediated integration) mutagenesis with enhancer trapping using the gene for Green Fluorescent Protein (GFP) as vital reporter. Of 2350 insertion mutants isolated, three were shown to express GFP only after the fungus had come into contact with the host maize plant. One of the genes tagged was mfa1, which encodes the pheromone precursor, while the second gene, pig2 , codes for a product that showed similarity to protein disulfide isomerase. The third integration event had occurred in a locus which we designated the p -locus. This locus contains 11 genes in a 24-kb stretch. Of these, pig3 , 4 , 5 , 6 and 7 show a plant-regulated expression pattern, while the other genes found at the locus (designated npi) do not. Of the plant-regulated genes only two were found to be similar to database entries: the pig4 product is related to membrane transporters of the major facilitator family, while the pig6 protein shows similarity to multidrug transporters. Detailed expression studies revealed that the five plant-regulated genes at the p -locus differ in their expression profiles. Mutants deleted for each of them showed no apparent phenotype, while the npi1 gene appeared to be essential. A viable deletion encompassing the entire p -locus could be generated when npi1 function was provided ectopically. This deletion mutant also showed no obvious alteration in virulence.Communicated by C. P. HollenbergThe first three authors contributed equally to this work     

Development of PCR-based SNP markers for rice blast resistance genes at the<Emphasis Type="Italic"> Piz</Emphasis> locus

We assessed the utility of single-nucleotide polymorphisms (SNPs) and small insertion/deletion polymorphisms (InDels) as DNA markers in genetic analysis and breeding of rice. Toward this end, we surveyed SNPs and InDels in the chromosomal region containing the Piz and Piz-t rice blast resistance genes and developed PCR-based markers for typing the SNPs. Analysis of sequences from a blast-susceptible Japanese cultivar and two cultivars each containing one of these genes revealed that SNPs are abundant in the Piz and Piz-t regions (on average, one SNP every 248 bp), but the number of InDels was much lower. The dense distribution of SNPs facilitated the generation of SNP markers in the vicinity of the genes. For typing these SNPs, we used a modified allele-specific PCR method. Of the 49 candidate allele-specific markers, 33 unambiguously and reproducibly discriminated between the two alleles. We used the markers for mapping the Piz and Piz-t genes and evaluating the size of DNA segments introgressed from the Piz donor cultivar in Japanese near-isogenic lines containing Piz. Our findings suggest that, because of its ability to generate numerous markers within a target region and its simplicity in assaying genotypes, SNP genotyping with allele-specific PCR is a valuable tool for gene mapping, map-based cloning, and marker-assisted selection in crops, especially rice.Communicated by D.J. Mackill     

<Emphasis Type="Italic">AVAG2</Emphasis> is a putative D-class gene from an ornamental asparagus

Members of the AGAMOUS (AG) family of MADS-box genes play important roles in regulating the development of reproductive organs in flowering plants. To elucidate the molecular mechanisms of floral development in Asparagus virgatus, we isolated and characterized an Asparagus AG-homologue, AVAG2. AVAG2 contains an open reading frame that encodes a deduced protein with 234 amino acid residues. Phylogenetic analysis indicated that AVAG2 belongs to the D-lineage of the AG gene family. AVAG2 mRNA was detected in the flower, but not in vegetative organs. Moreover, in in situ hybridization experiments, AVAG2 signals were observed in the stamens and carpels during early flower development, and appeared in the ovule only at later developmental stages. This suggests that the AVAG2 gene is involved in ovule formation. Thus, our expression data support the phylogenetic analysis indicating that AVAG2 belongs to the D-class gene family.     

ADrosophila homologue of theSchizosaccharomyces pombe act2 gene

Diverse proteins that are 35% to 55% identical to actins have been discovered recently in yeasts, nematodes, and vertebrates. In order to study these proteins systematically and relate their functions to those of conventional actins, we are isolating the corresponding genes from the genetically tractable eukaryote,Drosophila melanogaster. Here we report the isolation and partial characterization of aDrosophila homologue of theSchizosaccharomyces pombe act2 gene. Degenerate oligonucleotide primers specifying peptides that are highly conserved within the actin protein superfamily were used in conjunction with polymerase chain reaction (PCR) to amplify a portion of theDrosophila gene that we have namedactr66B. The corresponding full-length cDNA sequence encodes a protein of 418 residues that is 65% identical to the product of theS. pombe act2 gene, 80% identical to the bovineact2 homologue, but only 48% identical to the principalDrosophila cytoplasmic actin encoded by theAct5C actin gene. Alignment of the yeast, bovine, andDrosophila actin-related proteins shows that they have four peptide insertions, relative to conventional actins, three of which are well placed to modify actin polymerization and one that is likely to perturb the binding of myosin. Locations of two of the fiveactr66B introns are conserved betweenDrosophila and yeast genes, further attesting that they evolved from a common ancestor and are likely to encode proteins having similar functions. We demonstrate that theDrosophila gene is located on the left arm of chromosome 3, within subdivision 66B. Finally, we show by RNA blot-hybridization that the gene is expressed at low levels, relative to conventional nonmuscle actin, in all developmental stages. From these and other observations we infer that the actr66B protein is a minor component of all cells, perhaps serving to modify the polymerization, structure, and dynamic behavior of actin filaments. Our work was supported by grants from the NIH and the Muscular Dystrophy Association to E.A.F. Sequences described herein have been filed in the GenBank Database under Accession Number X71789.     

<Emphasis Type="Italic">odd-skipped</Emphasis> homologs function during gut development in<Emphasis Type="Italic"> C. elegans</Emphasis>

Genes in the odd-skipped (odd) family encode a discrete subset of C2H2 zinc finger proteins that are widely distributed among metazoan phyla. Although the initial member (odd) was identified as a Drosophila pair-rule gene, various homologs are expressed within each of the three germ layers in complex patterns that suggest roles in many pathways beyond segmentation. To further investigate the evolutionary history and extant functions of genes in this family, we have initiated a characterization of two homologs, odd-1 and odd-2, identified in the genome of the nematode, Caenorhabditis elegans. Sequence comparisons with homologs from insects (Drosophila and Anopheles) and mammals suggest that two paralogs were present within an ancestral metazoan; additional insect paralogs and both extant mammalian genes likely resulted from gene duplications that occurred after the split between the arthropods and chordates. Analyses of gene function using RNAi indicate that odd-1 and odd-2 play essential and distinct roles during gut development. Specific expression of both genes in the developing intestine and other cells in the vicinity of the gut was shown using GFP-reporters. These results indicate primary functions for both genes that are most like those of the Drosophila paralogs bowel and drumstick, and support a model in which gut specification represents the ancestral role for genes in this family.Edited by C. Desplan     

The dominant MHC class I gene is adjacent to the polymorphic<Emphasis Type="Italic"> TAP2</Emphasis> gene in the duck,<Emphasis Type="Italic"> Anas platyrhynchos</Emphasis>

We are investigating the expression and linkage of major histocompatibility complex (MHC) class I genes in the duck (Anas platyrhynchos) with a view toward understanding the susceptibility of ducks to two medically important viruses: influenza A and hepatitis B. In mammals, there are multiple MHC class I loci, and alleles at a locus are polymorphic and co-dominantly expressed. In contrast, in lower vertebrates the expression of one locus predominates. Southern-blot analysis and amplification of genomic sequences suggested that ducks have at least four loci encoding MHC class I. To identify expressed MHC genes, we constructed an unamplified cDNA library from the spleen of a single duck and screened for MHC class I. We sequenced 44 positive clones and identified four MHC class I sequences, each sharing approximately 85% nucleotide identity. Allele-specific oligonucleotide hybridization to a Northern blot indicated that only two of these sequences were abundantly expressed. In chickens, the dominantly expressed MHC class I gene lies adjacent to the transporter of antigen processing (TAP2) gene. To investigate whether this organization is also found in ducks, we cloned the gene encoding TAP2 from the cDNA library. PCR amplification from genomic DNA allowed us to determine that the dominantly expressed MHC class I gene was adjacent to TAP2. Furthermore, we amplified two alleles of the TAP2 gene from this duck that have significant and clustered amino acid differences that may influence the peptides transported. This organization has implications for the ability of ducks to eliminate viral pathogens.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers AY294416–22     

Members of two gene families encoding ubiquitin-conjugating enzymes, AtUBC1-3 and AtUBC4-6, fromArabidopsis thaliana are differentially expressed

Covalent attachment of ubiquitin to other intracellular proteins is essential for many physiological processes in eukaryotes, including selective protein degradation. Selection of proteins for ubiquitin conjugation is accomplished, in part, by a group of enzymes designated E2s or ubiquitin-conjugating enzymes (UBCs). At least six types of E2s have been identified in the plantArabidopsis thaliana; each type is encoded by a small gene family. Previously, we described the isolation and characterization of two three-member gene families, designatedAtUBC1-3 andAtUBC4-6, encoding two of these E2 types. Here, we investigated the expression patterns, of theAtUBC1-3 andAtUBC4-6 genes by the histochemical analysis of transgenicArabidopsis containing the corresponding promoters fused to the -glucuronidase-coding region. Staining patterns showed that these genes are active in many stages of development and some aspects of cell death, but are not induced by heat stress. Within the two gene families, individual members exhibited both overlapping and complementary expression patterns, indicating that at least one member of each gene family is expressed in most cell types and at most developmental stages. Different composite patterns of expression were observed between theAtUBC1-3 andAtUBC4-6 families, suggesting distinct biochemical and/or physiological functions for the encoded E2s inArabidopsis.     

Gene expression of chloroplast translation elongation factor Tu during maize chloroplast biogenesis

We have examined the expression of a maize nucleartuf gene(tufA) coding for the chloroplast translation elongation factor EF-Tu. Southern analysis revealed that the maize chloroplast EF-Tu was encoded by at least two distinct genes in the nuclear genome. In order to know the effect of light on the expression of thetufA gene during maize chloroplast biogenesis, we have analyzed the steady-state level of thetufA mRNAs by Northern analysis. The steady-state level of thetufA mRNAs was similar in both continuous light- and dark-grown seedlings. The level of thetufA mRNAs also maintained at relatively same level during light-induced greening of etiolated seedlings and all examined developmental stages. These results indicate that the gene expression of the maize chloroplast EF-Tu is rarely light-regulated at it’s mRNA level during chloroplast biogenesis.     

Cloning of a novel gene,<Emphasis Type="Italic">Cymg1</Emphasis>, related to family 2 cystatins and expressed at specific stages of mouse testis development

We have cloned a novel gene,Cymg1 (GenBank accession number AY600990), from a mouse testis cDNA library.Cymg1 is located in 2G3 of mouse chromosome 2. The cDNA includes an open reading frame that encodes 141 amino acid residues. The encoded polypeptide has a cysteine protease inhibitor domain found in the family 2 cystatins but lacks critical consensus sites important for cysteine protease inhibition. These characteristics are seen in the proteins of the CRES subfamily of the family 2 cystatins which are expressed specifically in the reproductive tract. CYMG1 protein shows 44% identity with mouse CRES and 30% identity with mouse cystatin C. Northern blot analysis showed that theCymg1 gene was specifically expressed in adult mouse testis. RT-PCR also showed thatCymg1 was expressed in testis and spermatogonial cells.Cymg1 expression level varied in the different developmental stages of mouse testis, and were coincidental with spermatogenesis and sex maturation. These results indicate thatCymg1 may play important roles in mouse spermatogenesis and sex maturation     

The pea<Emphasis Type="Italic"> END1</Emphasis> promoter drives anther-specific gene expression in different plant species

END1 was isolated by an immunosubtractive approach intended to identify specific proteins present in the different pea (Pisum sativum L.) floral organs and the genes encoding them. Following this strategy we obtained a monoclonal antibody (mAbA1) that specifically recognized a 26-kDa protein (END1) only detected in anther tissues. Northern blot assays showed that END1 is expressed specifically in the anther. In situ hybridization and immunolocalization assays corroborated the specific expression of END1 in the epidermis, connective, endothecium and middle layer cells during the different stages of anther development. END1 is the first anther-specific gene isolated from pea. The absence of a practicable pea transformation method together with the fact that no END1 homologue gene exists in Arabidopsis prevented us from carrying out END1 functional studies. However, we designed functional studies with the END1 promoter in different dicot species, as the specific spatial and temporal expression pattern of END1 suggested, among other things, the possibility of using its promoter region for biotechnological applications. Using different constructs to drive the uidA (-glucuronidase) gene controlled by the 2.7-kb isolated promoter sequence we have proven that the END1 promoter is fully functional in the anthers of transgenic Arabidopsis thaliana (L.) Heynh., Nicotiana tabacum L. (tobacco) and Lycopersicon esculentum Mill. (tomato) plants. The presence in the –330-bp region of the promoter sequence of three putative CArG boxes also suggests that END1 could be a target gene of MADS-box proteins and that, subsequently, it would be activated by genes controlling floral organ identity.Abbreviations GUS -Glucuronidase - uidA -Glucuronidase gene - Nos Nopaline synthase gene - nptII Neomycin phosphotransferase II gene - SEM Scanning electron microscopy GenBank accession numbers for the END1 cDNA and the END1 promoter: AY 091466 and AY 324651, respectively     

Characterization of <Emphasis Type="Italic">twist</Emphasis> and <Emphasis Type="Italic">snail</Emphasis> gene expression during mesoderm and nervous system development in the polychaete annelid <Emphasis Type="Italic">Capitella</Emphasis> sp. I

To investigate the evolutionary history of mesoderm in the bilaterian lineage, we are studying mesoderm development in the polychaete annelid, Capitella sp. I, a representative lophotrochozoan. In this study, we focus on the Twist and Snail families as candidate mesodermal patterning genes and report the isolation and in situ expression patterns of two twist homologs (CapI-twt1 and CapI-twt2) and two snail homologs (CapI-sna1 and CapI-sna2) in Capitella sp. I. CapI-twt1 is expressed in a subset of mesoderm derivatives during larval development, while CapI-twt2 shows more general mesoderm expression at the same stages. Neither twist gene is detected before the completion of gastrulation. The two snail genes have very distinct expression patterns. At cleavage and early gastrula stages, CapI-sna1 is broadly expressed in precursors of all three germ layers and becomes restricted to cells around the closing blastopore during late gastrulation; CapI-sna2 expression is not detected at these stages. After gastrulation, both snail genes are expressed in the developing central nervous system (CNS) at stages when neural precursor cells are internalized, and CapI-sna1 is also expressed laterally within the segmental mesoderm. Based on the expression patterns in this study, we suggest a putative function for Capitella sp. I twist genes in mesoderm differentiation and for snail genes in regulating CNS development and general cell migration during gastrulation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular phylogeny of theChironomus genus deduced from nucleotide sequences of two nuclear genes,ssp 160 and the globin 2b gene

Two genes were employed to study phylogenetic relatedness of theChironomus species: the protein-coding, salivary gland-specificssp160 gene, and the globin 2b (gb2b) gene. By using PCR, it was demonstrated that all the 38Chironomus species analyzed possess thegb2b gene, while only 13 have thessp160 gene. Partial nucleotide sequences of the genes of 22 species were determined. The data obtained were employed to construct phylogenetic trees which appeared to be topologically similar and revealed five groups of phylogenetically closely related species. Combining the data obtained in the studies of nuclear and mitochondrial genes, a molecular-data-based scenario could be suggested for theChironomus genus evolution.