Evolution of Ac and Dsl elements in select grasses (Poaceae)

We present data on evolution of the Ac/Ds family of transposable elements in select grasses (Poaceae). An Ac-like element was cloned from a DNA library of the grass Pennisetum glaucum (pearl millet) and 2387 bp of it have been sequenced. When the pearl millet Ac-like sequence is aligned with the corresponding region of the maize Ac sequence, it is found that all sequences corresponding to intron II in maize Ac are absent in pearl millet Ac. Kimura's evolutionary distance between maize and pearl millet Ac sequences is estimated to be 0.429±0.020 nucleotide substitutions per site. This value is not significantly different from the average number of synonymous substitutions for coding regions of the Adh1 gene between maize and pearl millet, which is 0.395±0.051 nucleotide substitutions per site. If we can assume Ac and Adh1 divergence times are equivalent between maize and pearl millet, then the above calculations suggest Ac-like sequences have probably not been strongly constrained by natural selection. The level of DNA sequence divergence between maize and pearl millet Ac sequences, the estimated date when maize and pearl millet diverged (25–40 million years ago), coupled with their reproductive isolation/lack of current genetic exchange, all support the theory that Ac-like sequences have not been recently introduced into pearl millet from maize. Instead, Ac-like sequences were probably present in the progenitor of maize and pearl millet, and have thus existed in the grasses for at least 25 million years. Ac-like sequences may be widely distributed among the grasses. We also present the first 2 Dsl controlling element sequences from teosinte species: Zea luxurians and Zea perennis. A total of 10 Dsl elements had previously been sequenced from maize and a distant maize relative, Tripsacum. When a maximum likelihood network of genetic relationships is constructed for all 12 sequenced Dsl elements, the 2 teosinte Dsl elements are as distant from most maize Dsl elements and from each other, as the maize Dsl elements are from one another. Our new teosinte sequence data support the previous conclusion that Dsl elements have been accumulating mutations independently since maize and Tripsacum diverged. We present a scenario for the origin of Dsl elements.     

Molecular evolutionary characterization of an Activator (Ac)-like transposable element sequence from pearl millet (Pennisetum glaucum) (Poaceae)

We present data on the evolution of the Ac/Ds family of transposable elements in select grasses (Poaceae). A defective Ac-like element was cloned from a DNA library of the grass Pennisetum glaucum (pearl millet) and its entire 4531 bp sequence has been determined. When the pearl millet Ac-like sequence is aligned with the maize Ac sequence, it is found that there is approximately 70% DNA similarity in the central region spanning most of maize Ac exon II and all of exon III. In addition, there are two smaller regions of similarity at the Ac terminii. Besides these three major structural similarities, Pennisetum Ac has two large regions, one 5 and one 3, that show little similarity to Zea Ac. Furthermore, most of the sequences corresponding to intron II in maize Ac are absent in pearl millet Ac. Kimura's evolutionary distance between the central region of maize and pearl millet Ac sequences is estimated to be 0.429±0.020 nucleotide substitutions per site. This value is not significantly different from the average number of synonymous substitutions for coding regions of the Adh1 gene between maize and pearl millet, which is 0.395±0.051 nucleotide substitutions per site. If we assume Ac and Adh1 divergence times are equivalent between maize and pearl millet, then the above calculations suggest Ac-like sequences have probably not been strongly constrained by natural selection. Conserved DNA and amino acid sequence motifs are also examined. The level of DNA sequence divergence between maize and pearl millet Ac sequences, the estimated date when maize and pearl millet diverged (25–40 million years ago), coupled with their reproductive isolation/lack of current genetic exchange, all support the theory that Ac-like sequences have not been recently introduced into pearl millet from maize. Instead, Ac-like sequences were probably present in the progenitor of maize and pearl millet and have thus existed in the grasses for at least 25 million years.     

Characterization of an unusual <Emphasis Type="Italic">Ds</Emphasis> transposable element in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>: Insertion of an abortive circular transposition intermediate

The maize Ac/Dstransposable elements, which belong to the hAT transposon superfamily, are widely used as insertional mutagens in numerous plant species. Molecular studies suggest that Ac/Ds elements transpose in a conservative non-replicative fashion; however the molecular mechanism of transposition remains unclear. We describe here the identification of an unusual Ds element, Ds-mmd1, in a transgenic Arabidopsis line. Ds-mmd1 is rearranged relative to the original Ds element, such that the original 5 and 3 ends are internal and previously internal sequences are the new 5 and 3 termini of Ds-mmd1. Short duplications of plant genomic DNA and Ds sequences are present at the Ds-mmd1 junctions, suggesting that a circular Dsmolecule was part of the events that created the Ds-mmd1 element. In addition, a revertant analysis on mmd1 plants demonstrated that Ds-mmd1 can be eliminated from the genome in an Ac-dependent process.     

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.     

Complex structure of the ribosomal DNA spacer of Cucumis sativus (cucumber)

Summary The nuclear 18 S, 5.8 S and 25 S ribosomal RNA genes (rDNA) of Cucumis sativus (cucumber) occur in at least four different repeat types of 10.2, 10.5, 11.5, and 12.5 kb in length. The intergenic spacer of these repeats has been cloned and characterized with respect to sequence organization. The spacer structure is very unusual compared to those of other eukaryotes. Duplicated regions of 197 bp and 311 bp containing part of the 3 end of the 25 S rRNA coding region and approximately 470 bp of 25 S rRNA flanking sequences occur in the intergenic spacer. The data from sequence analysis suggest that these duplications originate from recombination events in which DNA sequences of the original rDNA spacer were paired with sequences of the 25 S rRNA coding region. The duplicated 3ends of the 25 S rRNA are separated from each other mostly by a tandemly repeated 30 bp element showing a high GC-content of 87.5%. In addition, another tandemly repeated sequence of 90 bp was found downstream of the 3flanking sequences of the 25 S rRNA coding region. These results suggest that rRNA coding sequences can be involved in the generation of rDNA spacer sequences by unequal crossing over.     

Molecular evolution of human T-cell leukemia virus

Summary In light of previous data, which suggested thatAc-like sequences might have undergone a significant radiation in the recent past, I examined the copy number ofAc-like sequences in representatives of all theZea taxa, both maize and teosinte. The maize and teosinte samples contained approximately equal numbers ofAc-like sequences. FewAc-like sequences were in unmethylated regions of DNA. Unmethylated elements were distributed randomly among both maize and teosinte lines. The appearance in a line of a discrete band resulting from digestion with one methylation-sensitive restriction enzyme was correlated with the appearance of discrete bands with other methylation-sensitive bands. This suggests that individualAc-like elements are occasionally demethylated in many sites. No unmethylated element having restriction fragments of the lengths predicted from the publishedAc sequence was seen in the approximately 326 elements examined.     

Phylogenetic analysis of organellar DNA sequences in the Andropogoneae: Saccharinae

To study the phylogenetics of sugarcane (Saccharum officinarum L.) and its relatives we sequenced four loci on cytoplasmic genomes (two chloroplast and two mitochondrial) and analyzed mitochondrial RFLPs generated using probes for COXI, COXII, COXIII, Cob, 18S+5S, 26S, ATPase 6, ATPase 9, and ATPase (D'Hont et al. 1993). Approximately 650 bp of DNA in the intergenic spacer region between rbcL and atpB and approximately 150 bp from the chloroplast 16S rDNA through the intergenic spacer region tRNA^val gene were sequenced. In the mitochondrial genome, part of the 18S rRNA gene and approximately 150 bp from the 18S gene 3 end, through an intergenic spacer region, to the 5S rRNA gene were sequenced. No polymorphisms were observed between maize, sorghum, and Saccharum complex members for the mitochondrial 18S internal region or for the intergenic tRNA^val chloroplast locus. Two polymorphisms (insertion-deletion events, indels) were observed within the 18S-5S mitochondrial locus, which separated the accessions into three groups: one containing all of the Erianthus, Eccoilopus, Imperata, Sorghum, and 1 Miscanthus species; a second containing Saccharum species, Narenga porphyrocoma, Sclerostachya fusca, and 1 presumably hybrid Miscanthus sp. from New Guinea; and a third containing maize. Eighteen accessions were sequenced for the intergenic region between rbcL and atpB, which was the most polymorphic of the regions studied and contained 52 site mutations and 52 indels, across all taxa. Within the Saccharum complex, at most 7 site mutations and 16 indels were informative. The maternal lineage of Erianthus/Eccoilopus was nearly as divergent from the remaining Saccharum complex members as it was from sorghum, in agreement with a previous study. Sequences from the rbcL-atpB spacer were aligned with GENBANK sequences for wheat, rice, barley, and maize, which were used as outgroups in phylogenetic analyses. To determine whether limited intra-complex variability was caused by under sampling of taxa, we used seven restriction enzymes to digest the PCR-amplified rbcL-atpB spacer of an additional 36 accessions within the Saccharum complex. This analysis revealed ten restriction sites (none informative) and eight length variants (four informative). The small amount of variation present in the organellar DNAs of this polyploid complex suggests that either the complex is very young or that rates of evolution between the Saccharum complex and outgroup taxa are different. Other phylogenetic information will be required to resolve systematic relationships within the complex. Finally, no variation was observed in commercial sugarcane varieties, implying a world-wide cytoplasmic monoculture for this crop.     

Characterization of the horse (<Emphasis Type="Italic">Equus caballus</Emphasis>)<Emphasis Type="Italic"> IGHA</Emphasis> gene

Nucleotide sequences of the immunoglobulin constant heavy chain genes of the horse have been described for IGHM, IGHG and IGHE genes, but not for IGHA. Here, we provide the nucleotide sequence of the genomic IGHA gene of the horse (Equus caballus), including its secretion region and the transmembrane exon. The equine IGHA gene shows the typical structure of a mammalian IGHA gene, with only three exons, separated by two introns of similar size. The hinge exon is located at the 5 end of the CH2 exon and encodes a hinge region of 11 amino acids, which contains five proline residues. The coding nucleotide sequence of the secreted form of the equine IGHA gene shares around 72% identity with the human IGHA1 and IGHA2 genes, as well as the bovine, ovine, porcine and canine IGHA genes, without distinct preference for any of these species. The same species also cluster together in a phylogenetic tree of the IGHA coding regions of various mammals, whereas rodent, rabbit, marsupial and monotreme IGHA genes each build a separate cluster.The nucleotide sequences reported in this paper have been assigned the EMBL/GenBank accession numbers AY247966 and AY351982     

Development of transgenic sorghum for insect resistance against the spotted stem borer (Chilo partellus)

Transgenic sorghum plants expressing a synthetic cry1Ac gene from Bacillus thuringiensis (Bt) under the control of a wound-inducible promoter from the maize protease inhibitor gene (mpiC1) were produced via particle bombardment of shoot apices. Plants were regenerated from the transformed shoot apices via direct somatic embryogenesis with an intermittent three-step selection strategy using the herbicide Basta. Molecular characterisation based on polymerase chain reaction and Southern blot analysis revealed multiple insertions of the cry1Ac gene in five plants from three independent transformation events. Inheritance and expression of the Bt gene was confirmed in T₁ plants. Enzyme-linked immunosorbant assay indicated that Cry1Ac protein accumulated at levels of 1–8 ng per gram of fresh tissue in leaves that were mechanically wounded. Transgenic sorghum plants were evaluated for resistance against the spotted stem borer (Chilo partellus Swinhoe) in insect bioassays, which indicated partial resistance to damage by the neonate larvae of the spotted stem borer. Reduction in leaf damage 5 days after infestation was up to 60%; larval mortality was 40%, with the surviving larvae showing a 36% reduction in weight over those fed on control plants. Despite the low levels of expression of Bt -endotoxin under the control of the wound-inducible promoter, the transgenic plants showed partial tolerance against first instar larvae of the spotted stem borer.     

Isolation and sequence analysis of the genomic DNA fragment encoding an aspartic proteinase inhibitor homologue from potato (Solanum tuberosum L.)

A genomic DNA clone encoding an aspartic proteinase inhibitor of potato was isolated from a lambda EMBL3 phage library using the aspartic proteinase inhibitor cDNA as a hybridization probe. The gene has all characteristic sequences normally found in eucaryotic genes. Typical CAAT and TATA box sequences were found in the 5-upstream region. In this part are also two putative regulatory AGGA box sequences located. In the genomic sequence there are no intron sequences interrupting the coding region. An open reading frame of the gene encodes a precursor protein of 217 amino acids which shows high percent identity with the aspartic proteinase inhibitor cDNA.     

Nucleotide sequence of the maize chloroplast rpo B/C1/C2 operon: Comparison between the derived protein primary structures from various organisms with respect to functional domains

Summary The genes (rpo B/C₁/C₂) coding for the , , subnits of maize (Zea mays) chloroplast RNA polymerase have been located on the plastome and their nucleotide sequences established. The operon is part of a large inversion with respect to the tobacco and spinach chloroplast genomes and is flanked by the genes trnC and rps2. Notable features of the nucleotide sequence are the loss of an intron in rpoC₁, and an insertion of approximately 450 by in rpOC₂ compared to the dicotyledons tobacco, spinach and liver-wort. The derived amino acid sequence of this additional monocotyledon specific sequence is characterized by acidic heptameric repeat units containing stretches of glutamic acid, tyrosines and leucines with regular spacing. Other structural motifs, such as a nucleotide binding domain in the subunit and a zinc finger in the subunit, are compared at the amino acid level throughout the RNA polymerase subunits with the enzymes from other organisms in order to identify functionally important conserved regions.The sequence data presented in this paper will appear in the EMBL/Gen Bank/DDBJ Nucleotide Databases under the accession number X17318     

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.     

Comparative Structural and Functional Characterization of Sorghum and Maize Duplications Containing Orthologous Myb Transcription Regulators of 3-Deoxyflavonoid Biosynthesis

Sequence characterization of the genomic region of sorghum yellow seed 1 shows the presence of two genes that are arranged in a head to tail orientation. The two duplicated gene copies, y1 and y2 are separated by a 9.084 kbp intergenic region, which is largely composed of highly repetitive sequences. The y1 is the functional copy, while the y2 may represent a pseudogene; there are several sequence indels and rearrangements within the putative coding region of y2. The y1 gene encodes a R2R3 type of Myb domain protein that regulates the expression of chalcone synthase, chalcone isomerase and dihydroflavonol reductase genes required for the biosynthesis of 3-deoxyflavonoids. Expression of y1 can be observed throughout the plant and it represents a combination of expression patterns produced by different alleles of the maize p1. Comparative sequence analysis within the coding regions and flanking sequences of y1, y2 and their maize and teosinte orthologs show local rearrangements and insertions that may have created modified regulatory regions. These micro-colinearity modifications possibly are responsible for differential patterns of expression in maize and sorghum floral and vegetative tissues. Phylogenetic analysis indicates that sorghum y1 and y2 sequences may have arisen by gene duplication mechanisms and represent an evolutionarily parallel event to the duplication of maize p2 and p1 genes.     

PREM-1, a putative maize retroelement has LTR (long terminal repeat) sequences that are preferentially transcribed in pollen

A family of highly dispersed repetitive elements, designated PREM-1, which are transcribed primarily during pollen development, has been identified in maize. Sequence data from six PREM-1-containing genomic clones suggest that the PREM-1 sequences are the LTRs of a family of putative retroelements. PREM-1 LTRs are estimated to be present in about 10 000 to 40 000 copies in the maize genome. Although related sequences have been detected in sorghum and crab grass, highly homologous sequences appear to be specific to the genus Zea (maize and teosinte). A diverse group of RNAs that contain portions of the PREM-1 sequence at their 3 ends are transcribed in pollen; highest levels appear in early uninucleate microspores. The PREM-1-containing cDNAs do not appear to code for protein products since stop codons are present in all three reading frames. The possible significance of expression of retroelements in the male gametophyte, in terms of transposition of DNA, is discussed.     

A candidate autonomous version of the wheat MITE <Emphasis Type="Italic">Hikkoshi</Emphasis> is present in the rice genome

A miniature inverted-repeat transposable element (MITE), designated as Hikkoshi, was previously identified in the null Wx-A1 allele of Turkish bread wheat lines. This MITE is 165 bp in size and has 12-bp terminal inverted repeats (TIRs) flanked by 8-bp target site duplications (TSDs). Southern and PCR analyses demonstrated the presence of multiple copies of Hikkoshi in the wheat genome. Database searches indicated that Hikkoshi MITEs are also present in barley, rice and maize. A 3.4-kb element that has Hikkoshi-like TIRs flanked by 8-bp TSDs has now been identified in the rice genome. This element shows high similarity to the 5 subterminal region of the wheat Hikkoshi MITE and contains a transposase (TPase) coding region. The TPase has two conserved domains, ZnF_TTF and hATC, and its amino acid sequence shows a high degree of homology to TPases encoded by Tip100 transposable elements belonging to the hAT superfamily. We designated the 3.4-kb element as OsHikkoshi. Several wheat clones deposited in EST databases showed sequence similarity to the TPase ORF of OsHikkoshi. The sequence information from the TPase of OsHikkoshi will thus be useful in isolating the autonomous element of the Hikkoshi system from wheat.     

The molecular cloning of a phospholipase A2 from Bothrops jararacussu snake venom: Evolution of venom group II phospholipase A2's may imply gene duplications

The sequence coding for a snake venom phospholipase A₂ (PLA₂), BJUPLA₂, has been cloned from a Bothrops jararacussu venom gland cDNA library. The cDNA sequence predicts a precursor containing a 16-residue signal peptide followed by a molecule of 122 amino acid residues with a strong sequence similarity to group II snake venom PLA₂'s. A striking feature of the cDNA is the high sequence conservation of the 5 and 3 untranslated regions in cDNAs coding for PLA₂'s from a number of viper species. The greatest sequence variation was observed between the regions coding for the mature proteins, with most substitutions occurring in nonsynonymous sites. The phylogenetic tree constructed by alignment of the amino acid sequence of BJUPLA₂ with group II PLA₂'s in general groups them according to current taxonomical divisions and/or functional activity. It also suggests that gene duplications may have occurred at a number of different points during the evolution of snake venom group II PLA₂'s.The nucleotide sequence reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number X76289.Correspondence to: A.M. Moura-da-Silva     

Mechanism of Ds1 excision from the genome of maize streak virus

Summary We have previously shown that the maize transposable element Ds1 introduced into maize plants by agroinfection can be excised from the genome of geminivirus maize streak virus (MSV). Excision depended strictly on the presence of an active Ac element in the plants. In this study, the excision products or footprints left in the MSV genome after Ds1 excision were extensively characterized and the effects of flanking sequences on Ds1 excision were analysed. Most types of footprints obtained were comparable to those described for Ds1 excision in the maize genome, and could be explained by the models proposed for excision of plant transposable elements. In two revertants, however, some terminal sequences of the Ds1 element were found to have been left behind at the excision site. The finding of this novel type of Ds1 footprint indicated that gene conversion events occurred during and/or after Ds1 excision from the MSV genome. A partial deletion of one copy of the 8 by duplications flanking the Ds1 element had no effect on the frequency or on the types of footprints of Ds1 excision from the MSV genome. Thus, the duplicated 8 by sequences flanking the transposable element are not involved in Ds1 excision. These results, as well as a statistical analysis of the modifications of the bases flanking the Ds1 element after excision, are discussed in terms of excision models.     

Sequence of the 18S-5S ribosomal gene region and the cytochrome oxidase II gene from mtDNA of Zea diploperennis

Summary The coding and flanking sequences of the 18S-5S ribosomal RNA genes and the cytochrome oxidase subunit II gene of Zea diploperennis mitochondrial DNA have been determined and compared to the corresponding sequences of normal maize (Zea mays L.) Both length and substitution mutations are found in the coding region of the 18S rRNA gene, whereas only one substitution mutation is found in the coding region of cytochrome oxidase II. Sequence divergence between maize and Zea diploperennis is about one-tenth of that between wheat and maize. The rate of nucleotide divergence by base substitution is less for plant mitochrondrial genes than for comparable genes in animal mitochondria.