Spotting factor (Spf) from the Spotted-dilute system in maize is a member of the En/Spm controlling element family

The Spotted-dilute controlling element system in maize involves an autonomous Spotting factor (Spf), and a receptor at the r1 locus haplotype R1-r(spotted dilute2). Its relationship with other maize transposable element systems is poorly characterized. Through development of a genetic tester that carries receptors for both the Spotted-dilute and the En/Spm controlling element systems, we determined that both receptors respond equally to Spf and En/Spm and that Spf is therefore a member of the En/Spm family of controlling elements.     

Influence of transposable elements on the structure and function of the A1 gene of Zea mays

The structure of the A1 gene of Zea mays was determined by sequencing cDNA and genomic clones. The gene is composed of four exons and three short introns. The 40.1-kd A1 protein is an NADPH-dependent reductase. Germinal derivatives of the mutable a1-m1 allele with either recessive or wild-type phenotype have been isolated. Sequence analysis of these revertant alleles indicates that frame-shift mutations abolish A1 gene function, whereas one additional amino acid within the protein sequence still allows wild-type gene expression. The presence of a second, promoter-like structure, upstream of the functional A1 gene promoter is discussed with respect to its possible involvement in differential expression of the A1 gene. The structure of the a1-m2 8004, 3456 and 4412 alleles, featuring distinguishable phenotypes in the presence of Spm(En), was also determined. In all alleles the 1080-bp-long inhibitor (I) element is located 15 bp upstream of the CAAT box of the A1 gene promoter. The unusual response of a1-m2 alleles to trans-active signals of the Spm(En) element is discussed with respect to the position of the I inserts. Also presented are data on the structure and insertion sites of transposable elements determined by cloning and sequencing of the mutable a1 alleles a1-mpapu, a1-mr 102 and a1-ml.     

Gene Marker Loss Induced by the Transposable Element, En, in Maize

The En/Spm transposable element system in maize includes the functional element, En/Spm and the receptor element I/dSpm. An En receptor has been found that shows En-induced breakage. This En-responsive receptor (designated 1836518) is located on the short arm of chromosome 9, proximal to Wx. In the presence of En, markers distal to the receptor show a loss of gene expression. Kernels heterozygous for aleurone and endosperm marker genes have a variegated appearance. The hypothesis is advanced that this variegation represents a physical loss of the chromosome segments carrying the genes distal to the receptor position. It is the first case of an En-controlled breakage event.     

Diversity of LTR-retrotransposons and <Emphasis Type="Italic">Enhancer/Suppressor</Emphasis><Emphasis Type="Italic">Mutator</Emphasis>-like transposons in cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz)

Cassava (Manihot esculenta Crantz), though a major world crop with enormous potential, is very under studied. Little is known about its genome structure and organisation. Transposable elements have a key role in the evolution of genome structure, and can be used as important tools in applied genetics. This paper sets out to survey the diversity of members of three major classes of transposable element within the cassava genome and in relation to similar elements in other plants. Members of two classes of LTR-retrotransposons, Ty1/copia-like and Ty3/gypsy-like, and of Enhancer/Suppressor Mutator (En/Spm)-like transposons were isolated and characterised. Analyses revealed 59 families of Ty1/copia, 26 families of Ty3/gypsy retrotransposons, and 40 families of En/Spm in the cassava genome. In the comparative analyses, the predicted amino acid sequences for these transposon classes were compared with those of related elements from other plant species. These revealed that there were multiple lineages of Ty1/copia-like retrotransposons in the genome of cassava and suggested that vertical and horizontal transmission as the source of cassava Mecops may not be mutually exclusive. For the Ty3/gypsy elements network, two groups of cassava Megyps were evident including the Arabidopsis Athila lineage. However, cassava En/Spm-like elements (Meens) constituted a single group within a network of plant En/Spm-like elements. Hybridisation analysis supported the presence of transposons in the genome of cassava in medium (Ty3/gypsy and En/Spm) to high (Ty1/copia) copy numbers. Thus the cassava genome was shown to contain diverse members of three major classes of transposable element; however, the different classes exhibited contrasting evolutionary histories.     

Molecular cloning of the a1 locus of Zea mays using the transposable elements En and Mu1

The a1 locus of Zea mays has been cloned using transposable elements as gene tags. The strategy was to make genomic libraries from maize stocks with a1 mutations induced either by En(Spm) or by Robertson's Mutator-system. These libraries were then screened with either Spm-I8 and En1, for the En-containing mutant, or with Mu1 for the Mu-induced mutation. There are many En and Mu1 hybridizing sequences present in the maize genome, however, by a process of cross-screening of the positives from the two libraries and by molecular analysis of the En-positive clones it was possible to identify clones in both libraries carrying all or part of the a1 gene.     

Genetic and molecular analysis of the Spm-dependent a-m2 alleles of the maize a locus

The Suppressor-mutator (Spm) transposable element family of maize consists of the fully functional standard Spm (Spm-s) and many mutant elements. Insertion of an Spm element in or near a gene can markedly alter its expression, in some cases bringing the gene under the control of the mechanisms that regulate expression of the element. To gain insight into such mechanisms, as well as to enlarge our understanding of the Spm element's genetic organization, we have analyzed derivatives of a unique Spm insertion at the maize a locus in which the gene is co-expressed and co-regulated with the element. We describe the genetic properties and the structure of the a locus and Spm element in 9 strains (collectively designated the a-m2 alleles) selected by McClintock from the original a-m2 allele for heritable changes affecting either the Spm element or expression of the a gene. Most of the mutations are intra-element deletions within the 8.3-kb Spm element; many alter both Spm function and expression of the gene. Spm controls a gene expression in alleles with internally deleted, transposition-defective Spm elements and element ends contain the target sequences that mediate Spm's ability to activate expression of the gene. We argue that the properties of the a-m2 alleles reflect the operation of an element-encoded positive regulatory mechanism, as well as a negative regulatory mechanism that affects expression of the element, but appears not to be mediated by an element-encoded gene product.     

Reorientation of the acetyl group of the photoactive bacteriopheophytin in reaction centers of Rhodobacter sphaeroides: an ENDOR/TRIPLE resonance study

The freeze-trapped bacteriopheophytin alpha radical anion phi(*)A- has been investigated by 1H-ENDOR/Special TRIPLE resonance spectroscopy in photosynthetic reaction centers of Rhodobacter sphaeroides, in which the Tyr at position M210 had been replaced by either Phe, Leu, His or Trp. In the wild type reaction center and the mutants YF(M210) and YW(M210) two distinct states of phi(*)A-, denoted I(*)1- and I(*)2-, can be stabilized below 200 K. The state I(*)1 is metastable and relaxes to I(*)2- as the temperature is raised from 135 K to 180 K. The difference in the electronic structure of phi(*)A- between the two states is interpreted in terms of a conformational change of phiA after freeze-trapping, involving a reorientation of the 3-acetyl group with respect to the macrocycle of the bacteriopheophytin. This interpretation is supported by the results of RHF-INDO/SP calculations. In the YH(M210) reaction center only one phiA- state is obtained that is distinct from I(*)1- and I(*)2, and the observed electronic structure indicates an almost in-plane orientation of the 3-acetyl group. This is consistent with the proposal that a hydrogen bond is formed between His M210 and the 3(1)-keto oxygen of phiA that impedes the reorientation of the acetyl group. Only one phi(*)A- state is observed in the YL(M210) reaction center, which is similar to the metastable state I(*)1 in the wild type complex. This result is interpreted in terms of a steric hindrance of the reorientation of the 3-acetyl group that is exerted by the side chain of Leu at position M210. Possible implications of these findings for the mechanism of electron transfer in bacterial reaction centers are discussed.     

Molecular characterization of Vulmar1, a complete mariner transposon of sugar beet and diversity of mariner- and En/Spm-like sequences in the genus Beta.

Transposons of the Tc1-mariner superfamily are widespread in eukaryotic genomes. We have isolated the mariner element Vulmar1 from Beta vulgaris L., which is 3909 bp long and bordered by perfect terminal inverted repeats of 32 bp with homology to terminal inverted repeats of transposons from soybean and rice. According to a characteristic amino acid signature, Vulmar1 can be assigned to the DD39D group of mariner transposons. Vulmar1 is flanked by a 5'-TA-3' target site duplication that is typical for mariner transposons. Southern hybridization revealed that mariner-like copies are highly abundant in Beta species, and sequence analysis of 10 transposase fragments from representative species of the four Beta sections revealed an identity between 34% and 100% after conceptual translation. By fluorescent in situ hybridization, Vulmar1 was detected in distal euchromatin as well as in some intercalary and pericentromeric regions of all B. vulgaris chromosomes. In addition, using PCR, we were able to amplify fragments of the transposase gene of En/Spm-like transposons in the genus Beta. En/Spm-like transposase sequences are highly amplified in four Beta sections and showed a considerable degree of conservation (88.5-100%) at the protein level, while the homology to corresponding regions of En/Spm transposons of other plant species ranges from 49.5% to 62.5%. By fluorescent in situ hybridization, En/Spm-like transposon signals of strong intensity were detected on all chromosomes of B. vulgaris.     

Cytological visualization of DNA transposons and their transposition pattern in somatic cells of maize

Global genomic analysis of transposable element distributions of both natural (En/Spm, Ac-Ds, and MuDR/Mu) and modified (RescueMu) types was performed by fluorescence in situ hybridization (FISH) on somatic chromosomes coupled with karyotyping of each chromosome. In lines without an active transposable element, the locations of silent En/Spm, Ac-Ds, and MuDR/Mu elements were visualized, revealing variation in copy number and position among lines but no apparent locational bias. The ability to detect single elements was validated by using previously mapped active Ac elements. Somatic transpositions were documented in plants containing an engineered Mutator element, RescueMu, via use of the karyotyping system. By analyzing the RescueMu lines, we found that transposition of RescueMu in root-tip cells follows the cut-and-paste type of transposition. This work demonstrates the utility of FISH and karyotyping in the study of transposon activity and its consequences.     

The Spm (En) transposable element controls the excision of a 2-kb DNA insert at the wx allele of Zea mays

The waxy (Wx) locus of Zea mays was cloned from strains carrying the wild-type and wx^m-8 mutant alleles. The receptor component of the Suppressor-Mutator (Spm) controlling element system in the wx^m-8 allele was shown to be a 2 kb long insertion within the transcribed region of the Wx gene. The insertion, termed Spm-I8, is excised during somatic reversion events induced by the autonomous controlling element Enhancer (En), which is an equivalent to Spm. Integration of Spm-I8 into the Wx gene generates a 3-bp target site duplication. Spm-I8 has a 13 bp long inverted repeat at its termini. The ends of the element can be further folded to build a large double-stranded structure consisting of five perfectly matching double-stranded regions of 9–13 bp in length, interrupted by single-stranded loops. A comparison of the wild-type and wx^m-8 alleles revealed two additional insertions 6 (insert-1) and 0.25 (insert-2) kb in length. No En-induced excision of insert-1 and insert-2 could be detected so far. There is remarkable structure and sequence homology between Spm-I8 and the transposable elements Tam1 and Tam2 of Antirrhinum majus at their termini, reflecting a possible evolutionary and/or functional relationship between transposons in different plant species.     

The En/Spm transposable element of Zea mays contains splice sites at the termini generating a novel intron from a dSpm element in the A2 gene

The A2 locus of Zea mays, identified as one of the genes affecting anthocyanin biosynthesis, was cloned using the transposable elements rcy and dSpm as gene tags. The A2 gene encodes a putative protein of 395 amino acids and is devoid of introns. Two a2-m1 alleles, containing dSpm insertions of different sizes, were characterized. The dSpm element from the original state allele has perfect termini and undergoes frequent transposition. The element from the class II state allele is no longer competent to transpose. It has retained the 13 bp terminal inverted repeat but has lost all subterminal sites at the 5' end, which are recognized by tnpA protein, the most abundant product of the En/Spm transposable element system. The relatively high A2 gene expression of one a2-m1 allele is due to removal of almost all dSpm sequences by splicing. The slightly altered A2 enzyme is still functional as shown by complementation of an a2 mutant with the corresponding cDNA. The 5' and 3' splice sites are constituted by the termini of the dSpm element; it therefore represents a novel intron of the A2 gene.     

Efficient insertional mutagenesis in rice using the maize En/Spm elements

We have developed a novel system for insertional mutagenesis in rice (Oryza sativa) based on the maize (Zea mays) enhancer/suppressor mutator (En/Spm) element. In this system, a single T-DNA construct with Spm-transposase and the non-autonomous defective suppressor mutator (dSpm) element is used in conjunction with green fluorescent protein (GFP) and Discosoma sp. Red Fluorescence Protein (DsRed) fluorescent markers to select unlinked stable transpositions of dSpm. Using this system, we could demonstrate high frequencies of unlinked germinal transposition of dSpm in rice. Analysis of dSpm flanking sequences from 353 stable insertion lines revealed that the dSpm insertions appear to be widely distributed on rice chromosomes with a preference for genic regions (70%). The dSpm insertions appear to differ from Activator-Dissociation (Ac-Ds) elements in genomic distribution and exhibit a greater fraction of unlinked transpositions when compared with Ds elements. The results obtained in this study demonstrate that the maize En/Spm element can be used as an effective tool for functional genomics in rice and can complement efforts using other insertional mutagens. Further, the efficacy of the non-invasive fluorescence-based selection system is promising for its application to other crops.     

En/Spm-like transposons in Poaceae species: transposase sequence variability and chromosomal distribution

Belonging to Class II of transposable elements, En/Spm transposons are widespread in a variety of distantly related plant species. Here, we report on the sequence conservation of the transposase region from sequence analyses of En/Spm-like transposons from Poaceae species, namely Zingeria biebersteiniana, Zingeria trichopoda, Triticum monococcum, Triticum urartu, Hordeum spontaneum, and Aegilops speltoides. The transposase region of En/Spm-like transposons was cloned, sequenced, and compared with equivalent regions of Oryza and Arabidopsis from the gene bank database. Southern blot analysis indicated that the En/Spm transposon was present in low (Hordeum spontaneum, Triticum monococcum, Triticum urartu) through medium (Zingeria bieberstiana, Zingeria trichopoda) to relatively high (Aegilops speltoides) copy numbers in Poaceae species. A cytogenetic analysis of the chromosomal distribution of En/Spm transposons revealed the concurence of the chromosomal localization of the En/Spm clusters with mobile clusters of rDNA. An analysis of En/Spm-like transposase amino acid sequences was carried out to investigate sequence divergence between 5 genera--Triticum, Aegilops, Zingeria, Oryza and Arabidopsis. A distance matrix was generated; apparently, En/Spm-like transposase sequences shared the highest sequence homology intra-generically and, as expected, these sequences were significantly diverged from those of O. sativa and A. thaliana. A sequence comparison of En/Spm-like transposase coding regions defined that the intra-genomic complex of En/Spm-like transposons could be viewed as relatively independent, vertically transmitted, and permanently active systems inside higher plant genomes. The sequence data from this article was deposited in the EMBL/GenBank Data Libraries under the accession nos. AY707995-AY707996-AY707997-AY707998-AY707999-AY708000-AY708001-AY708002-AY708003-AY708004-AY708005-AY708005-AY265312.