Identification of transposon-like elements in non-coding regions of tomato ACC oxidase genes

1-aminocyclopropane-1-carboxylate (ACC) oxidase, which catalyses the terminal step in ethylene biosynthesis, is encoded by a small multigene family in tomato that is differentially expressed in response to developmental and environmental cues. In this study we report the isolation and sequencing of approximately 2 kb of 5′-flanking sequence of three tomato ACC oxidase genes (LEACO1, LEACO2, LEACO3) and the occurrence of class I and class II mobile element-like insertions in promoter and intron regions of two of them. The LEACO1 upstream region contains a 420-bp direct repeat which is present in multiple copies in the tomato genome and is very similar to sequences in the promoters of the tomato E4 and 2A11 genes. The region covering the repeats resembles the remnant of a retrotransposon. Two copies of a small transposable element, belonging to the Stowaway inverted repeat element family, have been found in the 5′-flanking sequence and the third intron of LEACO3. Received: 8 August 1996 / Accepted: 4 November 1996     

Sequence and characterization of twoHSP70 genes in the colonial protochordateBotryllus schlosseri

Two genes belonging to the heat shock protein 70 gene family have been cloned from the colonial protochordateBotryllus schlosseri. The two intronless genes(HSP70.1 andHSP70.2) exhibit 93.6% sequence identity within the predicted coding region, and 83.3% and 81.7% sequence identity in the 5′ and 3′ flanking regions, respectively. The predicted amino acid sequences are 95% identical and contain several signatures characteristic of cytoplasmic eukaryoticHSP70 genes (Gupta et al. 1994; Rensing and Maier 1994). Northern blotting and sequence analysis suggest that both genes are heat-inducible merebees of theHSP70 gene family. Given these characteristics,HSP70.1 andHSP70.2 appear to be good candidates for protochordate homologues of the major histocompatibility complex-linkedHSP70 genes of human, mouse, and rat (Milner and Campbell 1990; Walter et al. 1994). Further experiments to determine whether there is functional evidence for such similarity are in progress. The nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank nucleotide sequence databases and have been assigned the accession numbers US 1901 (HSP70.1) and US 1902 (HSP70.2)     

Structure and expression of the three MHC-linked HSP70 genes

A duplicated locus encoding the major heat shock-induced protein HSP70 is located in the major histocompatibility complex (MHC) class III region 92 kilobases (kb) telomeric to the C2 gene. Nucleotide sequence analysis of the two intronless genes, HSP70-1 and HSP70-2, has shown that they encode an identical protein product of 641 amino acids. A third intronless gene, HSP70-Hom, has also been identified 4 kb telomeric to the HSP70-1 gene. This encodes a more basic protein of 641 amino acids which has 90% sequence similarity with HSP70-1. In order to investigate the expression of the three (MHC)-linked HSP70 genes individually by northern blot analysis, we have isolated locus-specific probes from the 3 untranslated regions of the genes. The HSP70-1 and HSP70-2 genes have been shown to be expressed at high levels as a 2.4 kb mRNA in cells heat-shocked at 42°C. HSP70-1 is also expressed constitutively at very low levels. The HSP70-Hom gene, which has no heat shock consensus sequence in its 5 flanking sequence, is expressed as a 3 kb mRNA at low levels both constitutively and following heat shock.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M34267-9. Address correspondence and offprint requests to: R. D. Campbell.     

The Tall Fescue Turf Grass Class I Chitinase Gene <Emphasis Type="Italic">FaChit1</Emphasis> Is Activated by Fungal Elicitors,Dehydration, Ethylene,and Mechanical Wounding

The cDNA, genomic DNA, and promoter sequence of FaChit1, a class I chitinase gene from Festuca arundinacea, were isolated and characterized in the present work. The deduced amino acid sequence of FaChit1 contains the chitin binding, catalytic, and proline and glycine-rich domains characteristic for most class I chitinases, but no C-terminal extension region. FaChit1 is induced effectively by fungal elicitors, dehydration, and ethylene, but only slightly by mechanical wounding. To identify potential stress-related cis-acting elements, 5′ sequences 935, 651, and 233 bp upstream of the FaChit1 start codon were fused to the GUS reporter gene and analyzed in transgenic tobacco. The results indicated that the 935 bp fragment closely mirrored endogenous gene expression and that the 651 bp fragment was sufficient to direct reporter the gene expression in response to fungal elicitors, ethylene, dehydration, or mechanical wounding due to both known and presently uncharacterized cis-acting elements. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Examination of the Intron in the meiosis-specific recombination gene REC114 in Saccharomyces

REC114 is one of 10 genes known to be required for the initiation of meiotic recombination in Saccharomyces cerevisiae. It is transcribed only in meiosis, and our previous sequence analysis suggested the presence of an intron in the 3′ end of the gene. Hypotheses in the literature have suggested, because of its unusual location, either that the putative intron in REC114 is likely to be necessary for expression, or that there may actually be no intron present. This work demonstrates that REC114 does have an intron and is one of only three genes in yeast with introns located in the 3′ end. Furthermore, the 3′ splice site utilized in REC114 is a very rare AAG sequence; only three other genes in yeast use this nonconsensus sequence. The splicing of REC114 does not require MER1, a gene known to be involved in meiosis-specific RNA processing. In fact, an intronless copy of REC114 can complement a null rec114 mutation. Thus, it does not appear that the intron is essential for expression of REC114. Although the intron is not absolutely required for meiotic function, it is conserved in evolution; two other species of yeast contain an intron at the same location in their REC114 genes. Received: 16 October 1996 / Accepted: 10 February 1997     

The gene cluster inlC2DE of Listeria monocytogenes contains additional new internalin genes and is important for virulence in mice

In this work we identified and characterized a gene cluster containing three internalin genes of Listeria monocytogenes EGD. These genes, termed inlG, inlH and inlE, encode proteins of 490, 548 and 499 amino acids, respectively, which belong to the family of large, cell wall-bound internalins. The inlGHE gene cluster is flanked by two listerial house-keeping genes encoding proteins homologous to the 6-phospho-β-glucosidase and the succinyl-diaminopimelate desuccinylase of E. coli. A similar internalin gene cluster, inlC2DE, localised to the same position on the L. monocytogenes EGD chromosome was recently described in a different isolate (Dramsi S, Dehoux P, Lebrun M, Goossens PL, Cossart P (1997) Infect Immun 65: 1615–1625). Sequence comparison of the two inl gene clusters indicates that inlG is a new internalin gene, while inlH was generated by a site-specific recombination, leading to an in-frame deletion which removed the 3′-terminal end of inlC2 and the 5′-terminal part of inlD. The third gene of the inlGHE cluster, inlE, is almost identical to the previously reported inlE gene. Our data show that the inlGHE gene cluster is probably transcribed from a major PrfA- independent promoter located upstream of inlG. PCR analysis revealed the presence of the newly identified inl genes inlG and inlH in most L. monocytogenes isolates tested. A mutant which has lost inlG, inlH and inlE by an in-frame deletion exhibited, after oral infection of mice, a significant loss in virulence and shows drastically reduced numbers of viable bacteria in both liver and spleen when compared to the wild-type strain. Received: 21 April 1998 / Accepted: 5 June 1998     

Molecular analysis of the Aureobasidium pullulans ura3 gene encoding orotidine-5′-phosphate decarboxylase and isolation of mutants defective in this gene

Orotidine-5′-phosphate decarboxylase (OMP decarboxylase) catalyses the final step in the pyrimidine biosynthesis, the conversion of orotidine-5′-phosphate (OMP) to uridine-5′-phosphate. The ura3 gene of Aureobasidium pullulans, encoding OMP decarboxylase, was isolated from an Aureobasidium genomic library constructed in the plasmid pBlueskriptSK⁻. The ura3 gene of A. pullulans has an open reading frame of 271 amino acid residues. Analysis of the sequence revealed the presence of two introns. In the predicted amino acid sequence there are regions of strong homology to the equivalent genes of Aspergillus niger, Neurospora crassa, Phycomyces blakesleeanus and Homo sapiens. The ura3 gene is the third Aureobasidium gene that has been cloned and analysed. We have also isolated ura3 mutants by selection of ethyl methanesulphonate mutagenised cells on 5-fluoroorotic acid. Transformation of these A. pullulans mutant strains to prototrophy showed the functionality of the cloned gene. Received: 16 July 1999 / Revision received: 20 September 1999 / Accepted: 24 September 1999     

Identification and characterization of the duplicate rice sucrose synthase genes <Emphasis Type="Italic">OsSUS5</Emphasis> and <Emphasis Type="Italic">OsSUS7</Emphasis> which are associated with the plasma membrane

Systematic searches using the complete genome sequence of rice (Oryza sativa) identified OsSUS7, a new member of the rice sucrose synthase (OsSUS) gene family, which shows only nine single nucleotide substitutions in the OsSUS5 coding sequence. Comparative genomic analysis revealed that the synteny between OsSUS5 and OsSUS7 is conserved, and that significant numbers of transposable elements are scattered at both loci. In particular, a 17.6-kb genomic region containing transposable elements was identified in the 5′ upstream sequence of the OsSUS7 gene. GFP fusion experiments indicated that OsSUS5 and OsSUS7 are largely associated with the plasma membrane and partly with the cytosol in maize mesophyll protoplasts. RT-PCR analysis and transient expression assays revealed that OsSUS5 and OsSUS7 exhibit similar expression patterns in rice tissues, with the highest expression evident in roots. These results suggest that two redundant genes, OsSUS5 and OsSUS7, evolved via duplication of a chromosome region and through the transposition of transposable elements.     

Isolation of the maize <Emphasis Type="Italic">Zpu1</Emphasis> gene promoter and its functional analysis in transgenic tobacco plants

By screening a genomic library of maize, a 2.2 kb 5′ flanking fragment of Zpu1 gene, encoding the pullulanase-type starch debranching enzyme, was isolated. Promoter fragments of various lengths, including the full 5′ flanking sequence (−2267 to −1) (Z1), a 3′ deletion (−2267 to −513) (Z5) and three 5′ deletions extending to −1943 (Z2), −1143 (Z3) and −516 (Z4) upstream of the translational initiation codon (ATG), were fused to the GUS reporter gene and introduced into tobacco. When these constructs were tested in transgenic tobacco plants, seed-preferred GUS activity was observed in pZ1-transgenic lines. In pZ2-transgenic lines, the GUS activity was not only restricted to seeds, but was also detected in calyxes, petals, stamens and mature leaves. At the same time, negligible GUS activity was detected in roots, stems, young leaves, stigmas and ovaries from the transgenic tobacco plants, which had integrated the full isolated sequence of Zpu1 promoter or its deletions. Deletion analysis indicated that the promoter contained a putative positive cis-regulatory element and the proximal region (−516 to −1) was essential for directing the expression of gus reporter gene. Analysis of GUS activity during the fruit development and seed germination suggested that Zpu1 promoter is active both in starch anabolism and in starch catabolism, which is consistent with the function of the endogenous gene in maize. GUS activity in leaves under light and darkness confirmed that Zpu1 promoter functions in the starch degradation of photosynthetic tissues in the dark phase of the diurnal cycle.     

Male Gametic Cell-specific Histone <Emphasis Type="Italic">gH2A</Emphasis> Gene of <Emphasis Type="Italic">Lilium longiflorum</Emphasis>: Genomic Structure and Promoter Activity in the Generative Cell

A genomic clone containing the gH2A gene, a histone variant specifically expressed in male gametic cells within the pollen of Lilium longiflorum, was isolated. Sequence analysis revealed that the coding region of the gene is interrupted by one intron, as is the case with the somatic type of plant histone H2A genes, suggesting derivation from the same ancestral gene containing one intron. In addition, a 2.8-kbp fragment of the 5′ upstream region of gH2A contained TATA and CAAT boxes, but neither a plant histone-specific regulatory DNA element nor vegetative cell-specific cis-elements were found. A histochemical study of stable transformants demonstrated that the 5′ upstream region of the gene can drive gene expression specifically in the generative cell of pollen; no activity was detectable in the vegetative cell or in other reproductive and vegetative tissues of transgenic Nicotiana tabacum. These results strongly suggest that the generative cell can direct specific gene expression, that this expression may be regulated by a putative male gametic factor, and that the gH2A promoter may therefore serve as a useful male gametic cell fate marker in angiosperms.     

Differential excision patterns of the <Emphasis Type="Italic">En</Emphasis>-transposable element at the <Emphasis Type="Italic">A2</Emphasis> locus in maize relate to the insertion site

Defined mutant alleles with resident transposons display characteristic patterns of germinal and somatic reversion, and heritable changes in the timing and frequency of reversions, which have been termed “change of state” by McClintock, constantly arise. Several mechanisms were proposed to account for these changes. They may be ascribed to the structure and composition of the elements themselves (composition hypothesis) or to their location (position hypothesis). In the current study, insertion positions were determined for three autonomous En-controlled mutable alleles of the A2 locus in maize that show different somatic reversion patterns. A relationship was observed between En insertion positions in the single coding region of the intronless A2 gene and anthocyanin variegation patterns in the aleurone. An insertion in the 5′ region of the coding sequence produced a very late somatic variegation pattern, whereas two early variegation patterns were caused by En insertions in the 3′ region of the coding sequence.     

Characterization of a pollen-preferential gene, BAN102, from Chinese cabbage

We isolated and characterized a pollen-preferential gene, BAN102, from Chinese cabbage and analyzed the activity of its promoter. There were three or four copies of the BAN102 gene in the Chinese cabbage genome that specifically expressed in pollen and pollen tube. There were 2137 bp of BAN102 genomic clone comprising 186 bp of protein coding region, and 1178 bp of 5′ and 773 bp of 3′ non-coding regions. TATA box were located at 1071 nt of the promoter region while the polyadenylation signal and polyadenylation site were at 1470 and 1486 nt of the 3′ non-coding region. BLAST search of BAN102 sequence showed that coding region of BAN102 gene was the greatest percent similarity with arabinogalactan protein (AGP23) gene from Arabidopsis thaliana. Promoter analysis using GUS gene as a reporter showed that the pollen-specificity of BAN102 resided within the −112 to −44 bp of proximal promoter from the transient expression in tobacco and Chinese cabbage plants.     

A series of repetitive DNA sequences are associated with human core and H1 histone genes

Summary Repetitive DNA sequences, derived from the human β-globin gene cluster, were mapped within a series of human genomic DNA segments containing core (H2A, H2B, H3 and H4) and H1 histone genes. Cloned recombinant λCH4A phage with human histone gene inserts were analyzed by Southern blot analysis using the following³²P-labeled (nick translated) repetitive sequences as probes:Alu I,Kpn I and LTR-like. A cloned DNA designated RS002-5′C6 containing (i)a (TG)₁₆ simple repeat, (ii) an (ATTTT)n repeat and (iii)a 52 base pair alternating purine and pyrimidine sequence was also used as a radiolabelled hybridization probe. Analysis of 12 recombinant phage, containing 6 arrangements of core histone genes, indicated the presence ofAlu I,Kpn and RS002-5′C6 repetitive sequences. In contrast, analysis of 4 human genomic DNA segments, containing both core and H1 histone genes, indicated the presence of onlyAlu I family sequences. LTR-like sequences were not detected in association with any of the core or H1 histone genes examined. These results suggest that human histone and β-globin genes share certain aspects of sequence organization in flanking regions despite marked differences in their overall structure and pattern of expression.     

Partial Sequence of a Sponge Mitochondrial Genome Reveals Sequence Similarity to Cnidaria in Cytochrome Oxidase Subunit II and the Large Ribosomal RNA Subunit

A 2550-bp portion of the mitochondrial genome of a Demosponge, genus Tetilla, was amplified from whole genomic DNA extract and sequenced. The sequence was found to code for the 3′ end of the 16S rRNA gene, cytochrome c oxidase subunit II, a lysine tRNA, ATPase subunit 8, and a 5′ portion of ATPase subunit 6. The Porifera cluster distinctly within the eumetazoan radiation, as a sister group to the Cnidaria. Also, the mitochondrial genetic code of this sponge is likely identical to that found in the Cnidaria. Both the full COII DNA and protein sequences and a portion of the 16S rRNA gene were found to possess a striking similarity to published Cnidarian mtDNA sequences, allying the Porifera more closely to the Cnidaria than to any other metazoan phylum. The gene arrangement, COII—tRNA^Lys—ATP8—ATP6, is observed in many Eumetazoan phyla and is apparently ancestral in the metazoa. Received: 24 November 1997 / Accepted: 14 September 1998