Analysis of splice donor and acceptor site function in a transposable gene trap derived from the maize element Activator

Gene trap vectors have been used in insertional mutagenesis in animal systems to clone genes with interesting patterns of expression. These vectors are designed to allow the expression of a reporter gene when the vector inserts into a transcribed region. In this paper we examine alternative splicing events that result in the expression of a GUS reporter gene carried on a Ds element which has been designed as a gene trap vector for plants. We have developed a rapid and reliable method based on PCR to study such events. Many splice donor sites were observed in the 3′ Ac border. The relative frequency of utilisation of certain splice donor and acceptor sites differed between tobacco and Arabidopsis. A higher stringency of splicing was observed in Arabidopsis.     

A New Gene Trap Construct Enriching for Insertion Events Near the 5′ End of Genes

The gene trap approach is based on the integration of a gene trap vector into the genome. This can be done either by electroporation of a plasmid construct or by infection with a viral vector. Commonly used viral gene trap vectors have been shown to select for integrations near the 5 end of genes. To date, no plasmid vector with a similar tendency has been reported. In this paper we describe a new plasmid vector, pKC199geo. This vector contained a short splice acceptor fragment from the Hoxc9 gene, a full length lacZ gene, including an ATG, and a reduced activity, mutant neomycin phosphotransferase gene as a selectable marker. This vector enriched the population of trapped genes in our gene trap screen for insertion events in the 5 end of genes. In the two cases examined the -galactosidase activity pattern accurately reflected the endogenous promotor activity.     

Visualization of site-specific recombination catalyzed by a recombinase from Zygosaccharomyces rouxii in Arabidopsis thaliana

Excision of a DNA segment can occur in Arabidopsis thaliana by reciprocal recombination between two specific recombination sites (RSs) when the recombinase gene (R) from Zygosaccharomyces rouxii is expressed in the plant. To monitor recombination events, we generated several lines of transgenic Arabidopsis plants that carried a cryptic -glucuronidase (GUS) reporter gene which was designed in such a way that expression of the reporter gene could be induced by R gene-mediated recombination. We also made several transgenic lines with an R gene linked to the 35S promoter of cauliflower mosaic virus. Each transgenic line carrying the cryptic reporter gene was crossed with each line carrying the R gene. Activity of GUS in F₁ and F₂ progeny was examined histochemically and recombination between two RSs was analyzed by Southern blotting and the polymerase chain reaction. In seedlings and plantlets of F₁ progeny and most of the F₂ progeny, a variety of patterns of activity of GUS, including sectorial chimerism in leaves, was observed. A small percentage of F₂ individuals exhibited GUS activity in the entire plant. This pattern of expression was ascribed to germinal recombination in the F₁ generation on the basis of an analysis of DNA structure by Southern blotting. These results indicate that R gene-mediated recombination can be induced in both somatic and germ cells of A. thaliana by cross-pollination of parental transgenic lines.     

Accumulation of GC donor splice signals in mammals

Abstract

The GT dinucleotide in the first two intron positions is the most conserved element of the U2 donor splice signals. However, in a small fraction of donor sites, GT is replaced by GC. A substantial enrichment of GC in donor sites of alternatively spliced genes has been observed previously in human, nematode and Arabidopsis, suggesting that GC signals are important for regulation of alternative splicing. We used parsimony analysis to reconstruct evolution of donor splice sites and inferred 298 GT > GC conversion events compared to 40 GC > GT conversion events in primate and rodent genomes. Thus, there was substantive accumulation of GC donor splice sites during the evolution of mammals. Accumulation of GC sites might have been driven by selection for alternative splicing.

Reviewers

This article was reviewed by Jerzy Jurka and Anton Nekrutenko. For the full reviews, please go to the Reviewers' Reports section.     

Splicing of Arabidopsis tRNAMet precursors in tobacco cell and wheat germ extracts

Intron-containing tRNA genes are exceptional within nuclear plant genomes. It appears that merely two tRNA gene families coding for tRNA^Tyr _{G A} and elongator tRNA^Met _CmAU contain intervening sequences. We have previously investigated the features required by wheat germ splicing endonuclease for efficient and accurate intron excision from Arabidopsis pre-tRNA^Tyr. Here we have studied the expression of an Arabidopsis elongator tRNA^Met gene in two plant extracts of different origin. This gene was first transcribed either in HeLa or in tobacco cell nuclear extract and splicing of intron-containing tRNA^Met precursors was then examined in wheat germ S23 extract and in the tobacco system. The results show that conversion of pre-tRNA^Met to mature tRNA proceeds very efficiently in both plant extracts. In order to elucidate the potential role of specific nucleotides at the 3 and 5 splice sites and of a structured intron for pre-tRNA^Met splicing in either extract, we have performed a systematic survey by mutational analyses. The results show that cytidine residues at intron-exon boundaries impair pre-tRNA^Met splicing and that a highly structured intron is indispensable for pre-tRNA^Met splicing. tRNA precursors with an extended anticodon stem of three to four base pairs are readily accepted as substrates by wheat and tobacco splicing endonuclease, whereas pre-tRNA molecules that can form an extended anticodon stem of only two putative base pairs are not spliced at all. An amber suppressor, generated from the intron-containing elongator tRNA^Met gene, is efficiently processed and spliced in both plant extracts.     

分泌蛋白特异性基因陷阱的设计与验证

蛋白质分子向细胞外分泌的过程有赖于信号肽的介导.基因陷阱是功能性基因克隆的一种有效方法,经典的基因陷阱以geo作为报告基因,对所克隆的基因类型没有选择性.将绿脓杆菌外毒素、2A序列、IL-2受体穿膜区以及新霉素磷酸转移酶的基因依次融合构建新型报告基因——peo,该报告基因能够特异性地甄别具有信号肽编码序列的基因.为验证peo对信号肽编码序列的特异性甄别作用,以peo为报告基因,构建了3种质粒载体,分别模拟用基因陷阱载体进行筛选时可能出现的3种情况,通过转染HeLa细胞、G418筛选以及碱性磷酸酶检测,证明peo能够有效地区分信号肽和非信号肽编码序列,以peo为报告基因改造的基因陷阱载体可以用于分泌蛋白基因的筛选.     

Expression analysis of four<Emphasis Type="Italic"> Pinus radiata</Emphasis> male cone promoters in the heterologous host<Emphasis Type="Italic"> Arabidopsis</Emphasis>

Four male cone-specific promoters were isolated from the genome of Pinus radiata D. Don, fused to the -glucuronidase (GUS) reporter gene and analysed in the heterologous host Arabidopsis thaliana (L.) Heynh. The temporal and spatial activities of the promoters PrCHS1, PrLTP2, PrMC2 and PrMALE1 during seven anther developmental stages are described in detail. The two promoters PrMC2 and PrMALE1 confer an identical GUS expression pattern on Arabidopsis anthers. DNA sequence analysis of the PrMC2 and PrMALE1 promoters revealed an 88% sequence identity over 276 bp and divergence further upstream (<40% sequence identity). GUS expression driven by a 276-bp PrMALE1 promoter fragment showed the same pattern in Arabidopsis anthers as observed for the full-length PrMALE1 promoter. Within the 276-bp promoter fragment a region of high homology to a previously described 16-bp anther-box was identified. In gain-of-function experiments the putative PrMALE1 anther-box was fused upstream of a 90-bp CaMV 35S minimal promoter, as a single copy in the sense direction and as an inverted repeat. No GUS expression was conferred to Arabidopsis anthers by either of these two constructs. In a loss-of-function experiment a 226-bp PrMALE1 deletion construct, which did not contain the putative PrMALE1 anther-box, still maintained the originally observed PrMALE1 GUS expression pattern. Hence, gain-of-function as well as loss-of-function experiments consistently showed that the putative anther-box of the PrMALE1 promoter is non-functional in the Arabidopsis genetic background. For the analysis of the four full-length pine promoters PrCHS1, PrLTP2, PrMC2 and PrMALE1, transformation vectors based on pCAMBIA2200 and pCAMBIA1302 were used. It will also be demonstrated in this article that sequences within the T-DNA borders of these vectors caused a characteristic histological background expression in Arabidopsis, with staining observed in vascular tissue of leaves, sepals, roots, filaments of stamens and in stems and pistils.Abbreviation GUS -glucuronidaseGenBank accession numbers for the analysed promoters: AF 337656 (PrCHS1), AF 337655 (PrLTP2), AF 337657 (PrMC2) and AF 337658 (PrMALE1).     

Transgene expression in strawberries driven by a heterologous phloem-specific promoter

Strawberry is susceptible to diseases caused by phytoplasmas, mycoplasma-like prokaryotes restricted to sieve elements in the phloem tissue of infected plants. One strategy to improve strawberry resistance to phytoplasmas involves transgenic expression of anti-microbial peptide genes in phloem. For targeted phloem-specific expression, we constructed a binary vector with an expression cassette bearing the -glucuronidase (GUS) reporter gene (uidA) under control of the Arabidopsis sucrose-H⁺ symporter gene (AtSUC2) promoter. Transgenic strawberry lines were generated with high efficiencies by a modified transformation protocol, which combines the adoption of a 3-day pre-selection period following transformation, and the addition of 10-M thidiazuron to the regeneration medium. Histological GUS activity indicated that the reporter gene was expressed specifically in phloem of leaves, petioles, and roots of transgenic plants. The results suggest that the transformation protocol and the AtSUC2 promoter may be useful for engineering phytoplasma-resistant transgenic strawberries.     

New plant promoter and enhancer testing vectors

We describe here a set of binary vectors suitable forAgrobacterium-mediated gene transfer and specially designed for studying plant promoters. These vectors are based on the use of thegus reporter gene, contain multiple unique restriction sites upstream of thegus gene, and minimal promoters for testing the effect of enhancers or activator elements. In addition, an intron-containinggus (uidA) gene was introduced into one of these vectors in order to examine reporter gene activity in tissues whereAgrobacterium contamination may be a problem or in transient expression assays.     

Activity of the yeast FLP recombinase in Arabidopsis

The coding sequence for FLP recombinase, originally from the 2 plasmid of Saccharomyces cerevisiae, was introduced into Arabidopsis behind the cauliflower mosaic virus 35S promoter. FLP activity was monitored by the glucuronidase activity resulting from inversion of an antisense-oriented GUS reporter gene flanked by a pair of FRT target sites in inverted repeat. FLP-dependent Gus activity was observed in both transient assays and transgenic plants. The FLP system will be useful for a variety of in planta genetic manipulations.     

The chloroplastchIL gene of the green algaChlorella vulgaris C-27 contains a self-splicing group I intron

ThechiL gene product is involved in the light-independent synthesis of chlorophyll in photosynthetic bacteria, green algae and non-flowering plants. The chloroplast genome ofChlorella vulgaris strain C-27 contains the first example of a splitchiL gene, which is interrupted by a 951 bp group I intron in the coding region. In vitro synthesized pre-mRNA containing the entire intron and parts of the flanking exon sequences is able to efficiently self-splice in vitro in the presence of a divalent and a monovalent cation and GTP, to yield the ligated exons and other splicing intermediates characteristic of self-splicing group I introns. The 5 and 3 splice sites were confirmed by cDNA sequencing and the products of the splicing reaction were characterized by primer extension analysis. The absence of a significant ORF in the long P9 region (522 nt), separating the catalytic core from the 3 splice site, makes this intron different from the other known examples of group I introns. Guanosine-mediated attack at the 3 splice site and the presence of G-exchange reaction sites internal to the intron are some other properties demonstrated for the first time by an intron of a protein-coding plastid gene.     

Tissue-dependent enhancement of transgene expression by introns of replacement histone H3 genes of Arabidopsis

Intron-bearing replacement histone H3 genes in Arabidopsis and other plants are highly and constitutively expressed. We demonstrate that the introns located within the 5-untranslated regions (5-UTR) of the two Arabidopsis replacement H3 genes will abolish the cell cycle dependence of an endogenous histone H4 promoter. We demonstrate that these introns, functionally combined with their endogenous promoters, could produce the high and constitutive expression of the replacement H3 genes observed in planta. They strongly increase gene expression whatever the promoter, from the strong 35S CaMV promoter to complete and resected promoters of cell cycle-dependent and replacement histone genes. Quantitative analysis of the extent of reporter gene enhancement in different parts of developing transgenic plantlets, ranging from 2-fold to 70-fold, supports the notion that trans-acting factors are responsible for this effect. Such factors appear most abundant in roots.     

Developmental regulation and tissue-specific differences of heat shock gene expression in transgenic tobacco and Arabidopsis plants

The heat shock (hs) response during plant growth and development was analyzed in tobacco and Arabidopsis using chimaeric -glucuronidase reporter genes (hs-Gus) driven by a soybean hs promoter. Fluorimetric measurements and histochemical staining revealed high Gus activities in leaves, roots, and flowers exclusively after heat stress. The highest levels of heat-inducible expression were found in the vascular tissues. Without heat stress, a developmental induction of hs-Gus was indicated by the accumulation of high levels of Gus in transgenic tobacco seeds. There was no developmental induction of hs-Gus in Arabidopsis seeds. In situ hybridization to the RNA of the small heat shock protein gene Athsp17.6 in tissue sections revealed an expression in heat-shocked leaves but no expression in control leaves of Arabidopsis. However, a high level of constitutive expression of hs gene was detected in meristematic and provascular tissues of the Arabidopsis embryo. The developmental and tissue-specific regulation of the hs response is discussed.Abbreviations hs heat shock - Hsp heat shock protein(s) - hs Gus: heat-inducible Gus gene(s) - HSE heat shock element(s) - HSF heat shock factor - X-gluc 5-bromo-4-chloro-3-indolyl--D-glucuronide - Gus -glucuronidase - DAF days after flowering - SAR scaffold attachment region