A comprehensive characterization of single-copy T-DNA insertions in the Arabidopsis thaliana genome

T-DNA flanking sequences were isolated from 112 Arabidopsis thaliana single-copy T-DNA lines and sequence mapped to the chromosomes. Even though two T-DNA insertions mapped to a heterochromatic domain located in the pericentromeric region of chromosome I, expression of reporter genes was detected in these transgenic lines. T-DNA insertion did not seem to be biased toward any of Arabidopsis' five chromosomes. The observed distribution of T-DNA copies in intergenic sequence versus gene sequence (i.e. 5-upstream regions, coding sequences and 3-downstream regions) appeared randomly. An evaluation of T-DNA insertion frequencies within gene sequence revealed that integration into 5-upstream regions occurred more frequently than expected, whereas insertions in coding sequences (exons and introns) were found less frequently than expected based on random distribution predictions. In the majority of cases, single-copy T-DNA insertions were associated with small or large rearrangements such as deletions and/or duplications of target site sequences, deletions and/or duplications of T-DNA sequences, and gross chromosomal rearrangements such as translocations. The accuracy of integration was similarly high for both left- and right-border sequences. These results may be called upon when making detailed molecular analyses of transgenic plants or T-DNA induced mutants.     

Transgene structures in T-DNA-inserted rice plants

T-DNA is commonly used for delivery of foreign genes and as an insertional mutagen. Although ample information exists regarding T-DNA organization in dicotyledonous plants, little is known about the monocot rice. Here, we investigated the structure of T-DNA in a large number of transgenic rice plants. Analysis of the T-DNA borders revealed that more than half of the right ends were at the cleavage site, whereas the left ends were not conserved and were deleted up to 180 bp from the left border (LB) cleavage site. Three types of junctions were found between T-DNA and genomic DNA. In the first, up to seven nucleotide overlaps were present. The frequency of this type was much higher in the LB region than at the right border (RB). In the second type, which was more frequent in RB, the link was direct, without any overlaps or filler DNA. Finally, the third type showed filler DNA between T-DNA and the plant sequences. Out of 171 samples examined, 77 carried the vector backbone sequence, with the majority caused by the failure of T-strand termination at LB. However, a significant portion also resulted from co-integration of T-DNA and the vector backbone to a single locus. Most linkages between T-DNA and the vector backbone were formed between two 3 ends or two 5 ends of the transferred DNAs. The 3 ends were mostly linked through 3–6 bp of the complementing sequence, whereas the 5 ends were linked through either precise junctions or imprecise junctions with filler DNA.     

Repeated sequences from theArabidopsis thaliana genome function as enhancers in transgenic tobacco

Sixteen segments ofArabidopsis thaliana DNA that function as enhancers in transgenic tobacco plants were isolated using the pROA97 enhancer cloning vehicle and library transformation ofNicotiana tabacum. The sequences were compared for AT content, homology, repeated motifs, and expression pattern in transgenicN. tabacum. The sequences were average with respect to the AT content ofA. thaliana DNA. They could be placed into seven homology groups. Five of the sequences are single-copy sequences. The remaining eleven sequences represent two homology groups. Homology Group I contains seven sequences with minor differences. Homology Group II contains four sequences with minor differences. Two repeated motifs were identified (5-CCTCT-3 and 5-AAGGAT-3). Both repeated motifs are found in other plant enhancers, and in the promoter region of the cauliflower mosaic virus 35S gene. In the 35S gene TATA region, the motifs can form two alternative stem-loop structures. The TATATAA sequence is located in the loop region of both stem-loop structures.     

A model for the mechanism of precise integration of a microinjected transgene

A unique transgenic mouse line has undergone transgene integration in a very precise fashion. The phenotype displayed by mice of the line followed the predicted inheritance patterns for X-linked transgene insertion which has been confirmed. In order to investigate the mechanism of integration the DNA sequence of the transgene and cellular junctions have been determined. A comparison between wild type and transgenic mutant sequences at the site of insertion revealed that there was no loss or rearrangement of cellular DNA upon integration of the transgene. The cellular sequences at the transgene 5 and 3 joins are contiguous in the wild type. The integrant exists as a head to tail tandem dimer with minimal loss of sequence compared with the injected monomer. Analysis of the site of insertion has revealed a 5 bp homology between the 5 end of the transgene and the cellular sequences. In addition, adjacent to the site of insertion within the cellular sequences, there are several sequence motifs implicated in recombination events including a clustering of strong consensus sites for DNA topoisomerase type I and a region of homology to the human minisatellite consensus core sequence, theEscherichia coli Chi site and the meiotic recombination hotspot within the E gene of the murine major histocompatibility complex. This clustering of features is likely to have been factorial in the integrity of the insertion event. A model depicting the mechanism of this precise integration is proposed.     

Structural variability in the genome of the Thermoproteus tenax virus TTV1

Summary Six variants of the TTV1 genome, including the primary isolate, have been characterized. DNA sequence comparison of wild-type virus (WT) and one of the variants (VT3) showed that differences are due to insertions and deletions that were confined to contiguous portions of two distinctClaI fragments. Seven similar short DNA sequences (30–102 bp) were involved in the variation. The deletions and insertions of these short DNA sequences occurred in every case adjacent to the 8 by consensus sequence 5-ACXCCTAC-3 which formed the 5 flank of the segments involved.     

Exon shuffling in anther-specific genes from sunflower

We describe here the nucleotide sequence of an anther-specific gene (sf18) from sunflower, encoding a proline- and glycine-rich polypeptide with a hydrophobic amino-terminus (signal peptide). The gene is split by a 211 by intron and is partially related to another anther-specific gene (sf2) from sunflower with which it shares important sequence stretches in the 5 coding and upstream regions. We propose that the two genes have originated via exon shuffling, during which a copy of a DNA segment including the promoter region as well as a signal peptide coding sequence has been transferred into the upstream region of two different potential coding sequences, generating two novel genes which display the same specificity of expression and which both encode an extracellular protein. While the 5 region of the intron is highly conserved as part of the transferred region and may play a role in the selection of the 5 splice site, a common octanucleotide at the 3 end of the intron of the two genes might be involved in 3 splice site selection.     

Chloroplast DNA sequences integrated into an intron of a tomato nuclear gene

Summary DNA sequences capable of hybridizing with chloroplast DNA have previously been reported to exist in the nuclear genome of higher plants. Here we show that the third intron of the cultivated tomato (Lycopersicon esculentum) nuclear gene Cab-7, which resides on chromosome 10 and which we recently cloned and sequenced, contains two DNA fragments derived from the coding region of the chloroplast gene psbG. The first fragment, 133 bp long, is located at a site 63 bp from the 3 end of the 833 bp intron. The exact sequence of the 11 nucleotides at the 3 end of the inserting chloroplast sequence is also found at the 5 border of the insertion. A small (107 bp) chloroplast DNA fragment is inserted near the middle of the intron, again with the 3 end of the inserting element (6 bp) duplicated at the 5 border of the insertion. The second insert is a subfragment of the first insert, and is most likely directly derived from it. The psbG insertion sequence was found to be present in the Cab-7 gene of all tomato species examined but not in species from related genera (e.g. Solanum, Petunia, Nicotiana), suggesting that the original transposition event (chloroplast to nucleus) occurred relatively recently-since the divergence of the genus Lycopersicon from other genera in the family Solanaceae, but before radiation of species in that genus.     

Sequence analysis of linked maize 22 kDa α-zein genes

We have determined the nucleotide sequence of a 7343 bp zein genomic clone (gZ22.8H3) from the maize inbred W64A. Computer-aided analysis of the DNA sequence revealed two contiguous 22 kDa -zein genes. The 5 gene (gZ22.8) encodes a complete polypeptide and contains putative regulatory sequences in both the 5 and 3 flanking regions that are typical of zein genes. In contrast, the 3 gene (gZ22.8) appears to be a pseudogene, because it contains numerous insertions and deletions that would prevent translation of the mRNA. Alignment of the 5 and 3 flanking sequences of both genes indicated that they resulted from a 3.3 kb DNA duplication event.     

Molecular cloning,characterization and expression of Mn-superoxide dismutase from the rubber tree (Hevea brasiliensis)

The gene and the RNA from Arabidopsis thaliana for the plastid-located glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) and their encoded product have been studied. The gene (designated ATS1) was isolated by screening a DASH genomic library for cross-hybridization with a radiolabeled probe prepared from cDNA for GPAT from squash. cDNA clones representing the mRNA were isolated by screening a ZAPII cDNA library for hybridization with a radiolabeled probe prepared from a DNA fragment of ATS1. The nucleotide sequences of the gene and the cDNA were determined, and the 5 end of the RNA was mapped by primer extension. Sequences similar to the TATA box, polyadenylation sequences and intron-splicing sequences were found at the expected locations. The pre-mRNA was 3288 nucleotides long and contained 5 and 3-untranslated sequences of 57 and 442 nucleotides, respectively. The coding sequence of 1377 nucleotides was interrupted by 11 introns of 1412 nucleotides in total and the 3-untranslated sequence contained another intron of 94 nucleotides. The open-reading frame encoded a polypeptide of 459 amino acid residues, the amino acid sequence of which was highly homologous to those of precursors to plastid-located GPATs from squash and pea. The enzymatic activity of a gene product that was over-produced in Escherichia coli confirmed the indentity of the gene.Abbreviations ACP acyl carrier protein - GPAT glycerol-3-phosphate acyltransferase - IPTG isopropyl--thiogalactopyranoside.     

Integrons: Novel DNA elements which capture genes by site-specific recombination

Integrons are unusual DNA elements which include a gene encoding a site-specific DNA recombinase, a DNA integrase, and an adjacent site at which a wide variety of antibiotic resistance and other genes are found as inserts. One or more genes can be found in the insert region, but each gene is part of an independent gene cassette. The inserted genes are expressed from a promoter in the conserved sequences located 5 to the genes, and integrons are thus natural expression vectors. A model for gene insertion in which circular gene cassettes are inserted individually via a single site-specific recombination event has been proposed and verified experimentally. The gene cassettes include a gene coding region and, at the 3 end of the gene an imperfect inverted repeat, a 59-base element. The 59-base elements are a diverse family of elements which function as sites recognized by the DNA integrase. Site-specific insertion of individual genes thus represents a further mechanism which contributes to the evolution of the genomes of Gram-negative bacteria and their plasmids and transposons.Members of the most studied class of integrons, which include thesulI gene in the conserved sequences, are believed to be mobile DNA elements on the basis that they are found in many independent locations, and a discrete boundary is found at the outer end of the 5-conserved segment. However, the length of the 3-conserved segment is variable in the integrons examined to date, and it is likely that this variability has arisen as the result of insertion and deletion events. Though the true extent of the 3-conserved segment remains to be determined, it seems likely that these integrons are mobile DNA elements. The second known class of integrons comprises members of the Tn7 transposon family.     

Sequencing and analysis of the cos region of the lactococcal bacteriophage c2

The cohesive termini of the DNA genome of the lactococcal bacteriophage c2 were directly sequenced and appeared to be complementary, non-symmetrical, 9-nucleotide single-stranded, 3 extended DNAs, with the following sequence: 5-GTTAGGCTT-3 3-CAATCCGAA-5. DNA located on either side of the cohesive ends was sequenced and several repeats and a region with the potential for a DNA bend were found. Previously sequenced cos regions of 13 other bacteriophages were also examined for similar sequence features. All of the bacteriophages from gram-positive hosts had 3 extended DNA termini, in contrast to the bacteriophages from gram-negative hosts, which had 5 extended DNA termini. All bacteriophages had a region of dyad symmetry close to the cohesive termini. A 7.3 kb DNA fragment of the c2 genome containing the cos sequences was cloned; transduction experiments demonstrated that these cloned sequences could act as a substrate for packaging enzymes of phage c2.     

Purification and characterization of the 5′ → 3′ exonuclease domain-deleted Thermus filiformis DNA polymerase expressed in Escherichia coli

The gene encoding 5 3 exonuclease domain-deleted Tfi DNA polymerase, named 5 3 Exo^– Tfi fragment, from Thermus filiformis was expressed in Escherichia coli under the control of the tac promoter on a high-copy plasmid, pJR. The expressed enzyme was purified 27-fold with a 19% yield and a specific activity of 2621 U mg^–1 protein. The 5 3 exonuclease domain of Tfi DNA polymerase was removed without significant effect on enzyme activity and stability. PCR conditions for the 5 3 Exo^– Tfi fragment were more tolerant to the buffer composition as compared to the full-length enzyme.     

Cloning of the fibroin gene from the oak silkworm, Antheraea yamamai and its complete sequence

The nucleotide sequences containing an entire genomic region and 5 upstream region of Antheraea yamamai fibroin gene have been determined. The gene consists of an initial exon encoding 14 amino acids, an intron (150 bp), and a long second exon coding for 2641 amino acids. The fibroin coding sequence shows a specialized organization with a highly repetitive region flanked by non repetitive 5 and 3 ends. Northern blot analyses confirmed that fibroin gene is actively expressed in the posterior silk gland of the final instar larvae of Antheraea yamamai.     

Action of nereistoxin on recombinant neuronal nicotinic acetylcholine receptors expressed in<Emphasis Type="Italic"> Xenopus laevis</Emphasis> oocytes

Nereistoxin (NTX), a natural neurotoxin from the salivary glands of the marine annelid worm Lumbriconereis heteropoda, is highly toxic to insects. Its synthetic analogue, Cartap, was the first commercial insecticide based on a natural product. We have used voltage-clamp electrophysiology to compare the actions of NTX on recombinant nicotinic acetylcholine receptors (nicotinic AChRs) expressed in Xenopus laevis oocytes following nuclear injection of cDNAs. The recombinant nicotinic AChRs investigated were chicken 7, chicken 42 and the Drosophila melanogaster/chicken hybrid receptors SAD/2 and ALS/2. No agonist action of NTX (0.1–100 M) was observed on chicken 7, chicken 42 and the Drosophila/chicken hybrid nicotinic AChRs. Currents elicited by ACh were reduced in amplitude by NTX in a dose-dependent manner. The toxin was slightly more potent on recombinant Drosophila/vertebrate hybrid receptors than on vertebrate homomeric (7) or heteromeric (42) nicotinic AChRs. Block by NTX of the chicken 7, chicken 42 and the SAD/2 and ALS/2 Drosophila/chicken hybrid receptors is in all cases non-competitive. Thus, the site of action on nicotinic AChRs of NTX, to which the insecticide Cartap is metabolised in insects, differs from that of the major nicotinic AChR-active insecticide, imidacloprid.     

Developmental regulatory elements in the 5′ flanking DNA of the Drosophila choline acetyltransferase gene

Summary Choline acetyltransferase (ChAT, EC 2.3.1.6) catalyzes the production of the neurotransmitter acetylcholine, and is an essential factor for neurons to be cholinergic. We have analyzed regulation of the Drosophila ChAT gene during development by examining the -galactosidase expression pattern in transformed lines carrying different lengths of 5 flanking DNA fused to a lacZ reporter gene. The largest fragment tested, 7.4 kb, resulted in the most extensive expression pattern in embryonic and larval nervous system and likely reflects all the cis-regulatory elements necessary for ChAT expression. We also found that 5 flanking DNA located between 3.3 kb and 1.2 kb is essential for the reporter gene expression in most of the segmentally arranged embryonic sensory neurons as well as other distinct cells in the CNS. The existence of negative regulatory elements was suggested by the observation that differentiating photoreceptor cells in eye imaginal discs showed the reporter gene expression in several 1.2 kb and 3.3 kb transformants but not in 7.4 kb transformants. Furthermore, we have fused the 5 flanking DNA fragments to a wild type ChAT cDNA and used these constructs to transform Drosophila with a Cha mutant background. Surprisingly, even though different amounts of 5 flanking DNA resulted in different spatial expression patterns, all of the positively expressing cDNA transformed lines were rescued from lethality. Our results suggest that developmental expression of the ChAT gene is regulated both positively and negatively by the combined action of several elements located in the 7.4 kb upstream region, and that the more distal 5 flanking DNA is not necessary for embryonic survival and development to adult flies. Correspondence to: P.M. Salvaterra     

Conservation and change of the developmentally crucial fushi tarazu gene in Drosophila

Summary We have studied the evolutionary changes occurring in the noncoding regions around the developmentally important fushi tarazu (ftz) gene in a total of 11 species in the genus Drosophila. Previous molecular developmental studies have identified DNA elements both 3 and 5 to the coding region which are important in proper regulation of expression of the Drosophila melanogaster ftz gene. We show here that these same elements are the most evolutionarily conserved regions in the vicinity of the gene homologs. Parts of some control elements are more conserved than exonic sequences. Not only is there sequence conservation, but the relative position, orientation, and distances among the control elements remain conserved. One quite significant difference does exist between the two major subgenera studied, Sophophora and Drosophila: namely, an inversion of the ftz unit with respect to other genes in the Antennapedia complex, ANT-C. As a comparison, we applied similar analysis to a housekeeping gene-rosy (ry), or Xdh. In contrast, DNA sequences 5 to the ry coding region revealed little evolutionary conservation. These studies bear out the proposition that functionally important DNA sequences remain more conserved through evolutionary time than do less functionally important sequences. This proposition could be tested in the present case because we could predict a priori from the developmental studies which DNA regions should be most conserved.     

Mass-spectrometric quantitation of cytokinins in tobacco crown-gall tumours induced by mutated octopine Ti plasmids of Agrobacterium tumefaciens

The levels of the major cytokinins, zeatin, zeatin riboside, zeatin riboside-5-monophosphate and zeatin-7-glucoside were measured in tobacco (Nicotiana tabacum L.) crown-gall tissues carrying insertion and deletion mutations in the T-DNA. Measurements were made by combined gas chromatography-mass spectrometry using selected ion monitoring with ¹⁵N- and ²H-labelled internal standards. The results demonstrate that, relative to wild-type tumour tissue, cytokinin levels are considerably elevated in tissues lacking functional T-DNA auxin-biosynthetic genes. From a detailed analysis of the major cytokinin metabolites it is concluded that a reduction in the extent of cytokinin degradation via N⁶-side-chain cleavage is an important factor leading to increased cytokinin levels in these tissues.Abbreviations IAA indole-3-acetic acid - SIM selected ion monitoring - Z zeatin - [7G]Z zeatin-7-glucoside - [9R]Z zeatin-9-riboside - [9R-5P]Z zeatin riboside-5-monophosphate     

Characterization of the supervirulent virG gene of the Agrobacterium tumefaciens plasmid pTiBo542.

Summary The virG gene of the Agrobacterium tumefaciens Ti plasmid pTiBo542 has previously been reported to elicit stronger vir gene expression than its counterpart in the pTiA6 plasmid, a property we call the superactivator phenotype. The DNA sequence of the pTiBo542 virG gene was determined and compared to that of the pTiA6 gene. The DNA sequences of these genes differ at 16 positions: two differences are in the promoter regions, 12 are in the coding regions, and two are in the 3 untranslated regions. The 3 end of the pTiA6 virG gene also contains a probable insertion sequence that is not found downstream of the pTiBo542 gene. The base pair differences in the two coding regions result in only two amino acid differences, both in the amino-terminal halves of the proteins. Five hybrid virG genes were constructed and used to activate the expression of a virB::lacZ gene fusion. Differences in the coding regions of these genes accounted for most of the superactivator phenotype, while differences at the promoter and 3 untranslated regions also contributed. These findings suggest that the properties of these VirG proteins and their quantities are important for vir gene induction, and also suggest a long-term selective pressure for mutations contributing to differences between these two genes.     

Targeted DNA integration within different functional gene domains in yeast reveals ORF sequences as recombinational cold-spots

The efficiency of gene targeting within different segments of genes in yeast was estimated by transforming yeast cells with double-stranded integrative plasmids, bearing functional gene domains [promoter (P), ORF (O) and terminator (T)] derived from the common genetic markers HIS3, LEU2 , TRP1 and URA3. Transformation experiments with circular plasmids carrying a single gene domain demonstrated that the 5 and 3 flanking DNA regions (P and T) of the HIS3 and URA3 genes are preferred as sites for plasmid integration by several fold over the corresponding ORFs. Moreover, when plasmids bearing combinations of two or three regions were linearized to target them to a specific site of integration, three of the ORFs were found to be less preferred as sites for plasmid integration than their corresponding flanking regions. Surprisingly, in up to 50% of the transformants obtained with plasmids that had been linearized within coding sequences, the DNA actually integrated into neighbouring regions. Almost the same frequencies of ORF mis-targeting were obtained with plasmid vectors containing only two functional domains (PO or OT) of the gene URA3, demonstrating that this event is not the consequence of competition between homologous DNA regions distal to the ORF. Therefore, we suggest that coding sequences could be considered to be cold spots for plasmid integration in yeast.Communicated by A. Aguilera