In vivo analysis of intron processing using splicing-dependent reporter gene assays

The mechanisms of intron recognition and processing have been well-studied in mammals and yeast, but in plants the biochemistry of splicing is not known and the rules for intron recognition are not clearly defined. To increase understanding of intron processing in plants, we have constructed new pairs of vectors, pSuccess and pFail, to assess the efficiency of splicing in maize cultured cells. In the pFail series we use translation of pre-mRNA to monitor the amount of unspliced RNA. We inserted an ATG codon in the Bz2 (Bronze-2) intron in frame with luciferase: this construct will express luciferase activity only when splicing fails. In the pSuccess series the spliced message is monitored by inserting an ATG upstream of the Bz2 intron in frame with luciferase: this construct will express luciferase activity only when splicing succeeds. We show here, using both the wild-type Bz2 intron and the same intron with splice site mutations, that the efficiency of splicing can be estimated by the ratio between the luciferase activities of the vector pairs. We also show that mutations in the unique U-rich motif inside the intron can modulate splicing. In addition, a GC-rich insertion in the first exon increases the efficiency of splicing, suggesting that exons also play an important role in intron recognition and/or processing.     

Introduction of a plant intron into the luciferase gene ofPhotinus pyralis

We describe a new luciferase reporter gene,luc ^INT, for early detection of luciferase activity inAgrobacterium transformation studies, and present improved techniques for the extraction of luciferase that decrease the time needed to quantitate luciferase activity. Theluc ^INT reporter gene combines the PIV2 intron fromGUS ^INT withluc ^*, the modified luciferase gene.luc ^INTis expressed in plant cells but not inAgrobacterium, allowing earlier detection of gene expression in the presence ofAgrobacterium during transformations in tobacco leaf discs. Stable expression levels ofluc ^INT andluc ^* in tobacco suspension cultures are compared for two different promoters. The nucleotide sequence data for the gene will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number U84006.     

Evolution of the recA gene and the molecular phylogeny of bacteria

The DNA sequences of the recA gene from 25 strains of bacteria are known. The evolution of these recA gene sequences, and of the derived RecA protein sequences, is examined, with special reference to the effect of variations in genomic G + C content. From the aligned RecA protein sequences, phylogenetic trees have been drawn using both distance matrix and maximum parsimony methods. There is a broad concordance between these trees and those derived from other data (largely 16S ribosomal RNA sequences). There is a fair degree of certainty in the relationships among the Purple or Proteobacteria, but the branching pattern between higher taxa within the eubacteria cannot be reliably resolved with these data. Correspondence to: A.T. Lloyd     

Sequence-specific interactions of a maize factor with a GC-rich repeat in the phosphoenolpyruvate carboxylase gene

Summary A plant nuclear protein PEP-I, which binds specifically to the promoter region of the phosphoenolpyruvate carboxylase (PEPC) gene, was identified. Methylation interference analysis and DNA binding assays using synthetic oligonucleotides revealed that PEP-I binds to GC-rich elements. These elements are directly repeated sequences in the promoter region of the PEPC gene and we have suggested that they may be cis-regulatory element of this gene. The consensus sequence of the element is CCCTCTCCACATCC and the CTCC is essential for binding of PEP-I. PEP-I is present in the nuclear extracts of green leaves, where the PEPC gene is expressed. However, no binding was detected in tissues where the PEPC gene is not expressed in vivo, such as roots or etiolated leaves. Thus, PEP-1 is the first factor identified in plants which has different binding activity in light-grown compared with dark-grown tissue. PEP-I binding is also tissue-specific, suggesting that PEP-1 may function to coordinate PEPC gene expression with respect to light and tissue specificity. This report describes the identification and characterization of the sequences required for PEP-1 binding.     

FramePlot: a new implementation of the Frame analysis for predicting protein-coding regions in bacterial DNA with a high G+C content

FramePlot is a web-based tool for predicting protein-coding regions in bacterial DNA with a high G + C content, such as Streptomyces. The graphical output provides for easy distinction of protein-coding regions from non-coding regions. The plot is a clickable map. Clicking on an ORF provides not only the nucleotide sequence but also its deduced amino acid sequence. These sequences can then be compared to the NCBI sequence database over the Internet. The program is freely available for academic purposes at http://www.nih.go.jp/jun/cgi-bin/frameplot.pl.     

Expression in transgenic tobacco of the bacterial neomycin phosphotransferase gene modified by intron insertions of various sizes

A plant selectable marker gene consisting of cauliflower mosaic virus expression signals and the proteincoding sequence of bacterial neomycin phosphotransferase was modified by insertion of an intron sequence from a storage protein gene, phaseolin. Correct and efficient splicing of the resulting mosaic RNA was observed in transgenic tobacco plants. The insertion of various linkers or gradual increase of intron size by addition in both orientations of internal intron sequences from another plant gene (parsley, 4-coumarate ligase) had little or no effect on the precision of slicing. The gene activity measured by selectability assay in the protoplast transformation showed that only introns enlarged to 1161 bases and longer caused decreased selectability. The suitability of such mosaic marker genes for studies of RNA splicing, DNA recombination and early events after infection of plants with Agrobacterium is discussed.     

Nuclear suppression of a mitochondrial RNA splice defect: nucleotide sequence and disruption of the MRS3 gene

Summary A mitochondrial RNA splice defect in the first intron of the COB gene (bI1) can be suppressed by a dominant nuclear mutation SUP-101. Starting with a gene bank of yeast nuclear DNA from a SUP-101 suppressor strain cloned in the YEp13 plasmid, we have isolated a recombinant plasmid which exerts a suppressor activity similar to the SUP-101 allele. The N3(2) insert of this plasmid contains an open reading frame (ORF) of 1014 bp which is transcribed to a 12 S RNA. Deletion of the 5 end of this ORF and its upstream sequences abolishes the suppressor activity. The N3(2) insert thus carries a functional gene (called MRS3) which can suppress a mitochondrial splice defect. The chromosomal equivalent of the cloned gene has been mapped to chromosome 10. Disruption of this chromosomal gene has no phenotypic effect on wild-type cells.     

Transgenic maize plants expressing a fungal phytase gene

Maize seeds are the major ingredient of commercial pig and poultry feed. Phosphorus in maize seeds exists predominantly in the form of phytate. Phytate phosphorus is not available to monogastric animals and phosphate supplementation is required for optimal animal growth. Undigested phytate in animal manure is considered a major source of phosphorus pollution to the environment from agricultural production. Microbial phytase produced by fermentation as a feed additive is widely used to manage the nutritional and environmental problems caused by phytate, but the approach is associated with production costs for the enzyme and requirement of special cares in feed processing and diet formulation. An alternative approach would be to produce plant seeds that contain high phytase activities. We have over-expressed Aspergillus niger phyA2 gene in maize seeds using a construct driven by the maize embryo-specific globulin-1 promoter. Low-copy-number transgenic lines with simple integration patterns were identified. Western-blot analysis showed that the maize-expressed phytase protein was smaller than that expressed in yeast, apparently due to different glycosylation. Phytase activity in transgenic maize seeds reached approximately 2,200 units per kg seed, about a 50-fold increase compared to non-transgenic maize seeds. The phytase expression was stable across four generations. The transgenic seeds germinated normally. Our results show that the phytase expression lines can be used for development of new maize hybrids to improve phosphorus availability and reduce the impact of animal production on the environment.     

DNA methylation in the Alcohol dehydrogenase-1 gene of maize

Using a battery of methylation-sensitive restriction enzymes, cytosine methylation at 23 sites in a 7.6 kb region surrounding the Alcohol dehydrogenase-1 (Adh1) gene was measured in DNA prepared from immature maize cobs. Both the 5 upstream region and the entire coding region were hypomethylated in the two alleles examined. Methylation in Adh1 is independent of changes in Mutator transposable element methylation. The role of DNA methylation in Adh1 gene regulation is discussed.     

Phylogeny of Drosophila and related genera inferred from the nucleotide sequence of the Cu,Zn Sod gene

The phylogeny and taxonomy of the drosophilids have been the subject of extensive investigations. Recently, Grimaldi (1990) has challenged some common conceptions, and several sets of molecular data have provided information not always compatible with other taxonomic knowledge or consistent with each other. We present the coding nucleotide sequence of the Cu,Zn superoxide dismutase gene (Sod) for 15 species, which include the medfly Ceratitis capitata (family Tephritidae), the genera Chymomyza and Zaprionus, and representatives of the subgenera Dorsilopha, Drosophila, Hirtodrosophila, Scaptodrosophila, and Sophophora. Phylogenetic analysis of the Sod sequences indicates that Scaptodrosophila and Chymomyza branched off the main lineage before the major Drosophila radiations. The presence of a second intron in Chymomyza and Scaptodrosophila (as well as in the medfly) confirms the early divergence of these two taxa. This second intron became deleted from the main lineage before the major Drosophila radiations. According to the Sod sequences, Sophophora (including the melanogaster, obscura, saltans, and willistoni species groups) is older than the subgenus Drosophila; a deep branch splits the willistoni and saltans groups from the melanogaster and obscura groups. The genus Zaprionus and the subgenera Dorsilopha and Hirtodrosophila appear as branches of a prolific “bush” that also embraces the numerous species of the subgenus Drosophila. The Sod results corroborate in many, but not all, respects Throckmorton's (King, R.C. (ed) Handbook of Genetics. Plenum Press, New York, pp. 421–469, 1975) phylogeny; are inconsistent in some important ways with Grimaldi's (Bull. Am. Museum Nat. Hist. 197:1–139, 1990) cladistic analysis; and also are inconsistent with some inferences based on mitochondrial DNA data. The Sod results manifest how, in addition to the information derived from nucleotide sequences, structural features (i.e., the deletion of an intron) can help resolve phylogenetic issues. Correspondence requests to: F. J. Ayala     

The calmodulin-stimulated ATPase of maize coleoptiles is a 140000-Mr polypeptide

The calmodulin-stimulated ATPase of maize (Zea mays L.) coleoptiles has been purified by calcium-dependent binding to a calmodulin affinity column. In the presence of protease inhibitors (phenylmethylsulfonylfluoride and chymostatin) a polypeptide of relative molecular mass (M_r) 140000 (±10000) is obtained on sodium-dodecylsulphate polyacrylamide gels. This polypeptide is recognised specifically by an affinity-purified polyclonal antibody to mammalian calmodulin-stimulated calcium-pumping ATPases and is of similar M_r to the erythrocyte-membrane calcium pump (138000 M_r).Abbreviations EGTA ethylene glycol-bis(-aminoethylether)-N,N,N,N-tetraacetic acid - M_r apparent molecular mass - SDS sodium dodecyl sulphate     

细菌ncRNA基因预测工具的评估

<正>细菌非编码RNA(ncRNA)在RNA的转录调节、染色体复制、RNA加工与修饰、m RNA翻译与稳定性、蛋白质降解与转运以及细菌感染等许多过程中都发挥着重要作用[1]。目前,基于实验鉴定的细菌ncRNA,已开发出不同的生物信息学预测方法[3-6],其中比较基因组学和转录单元预测方法是目前最常用的方法。比较基因组学和转录单元预测方法的结合使用,具有较高的特异性,但大多数方法的普适性较低,不     

Tagging of a maize gene involved in kernel development by an activated Uq transposable element

Summary A quiescent Uq transposable element has been activated in a maize plant treated with 5-aza-2-deoxycyti-dine. This activated Uq cosegregates with a heritable dominant miniature (Mn) kernel phenotype, indicating its physical association with a maize miniature locus (Mn:: Uq). The Mn:: Uq mutant is dominant in producing a miniature seed phenotype of variable size and in reducing seedling vigor in the early growth stage. Genetic experiments indicate that the Mn:: Uq mutant also affects the activity of the male gametophyte, whereby pollen germination is inhibited, thus lacking pollen tube growth resulting in the male nontransmissibility of this mutant. Proof for the Uq element in this mutant is derived by its ability to transactivate the standard a-ruq reporter allele to yield spotted aleurone tissue. However, the Mn:: Uq mutant does not transactivate a normally Uq-responsive c-ruq allele, suggesting a structural difference between the two ruq receptors at the A1 and C1 loci. It is anticipated that cloning of the Uq transposable element would facilitate the molecular cloning and characterization of the maize miniature gene.Journal Paper No. J-13425 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa 50011, USA, Project No. 2850