Activation of the maize transposable element Suppressor-mutator (Spm) in tissue culture

Summary Previous experiments have revealed that the maize transposable element Activator (Ac) may become active during tissue culture. The objective of the present study was to determine whether a second transposable element, Suppressor-mutator (Spm), could also be activated in tissue culture and detected in regenerated maize plants. Approximately 500 R₁ progeny of 143 regenerated plants (derived from 49 embryo cell lines) were crossed as males onto an Spm-responsive tester stock. Spm activity was observed in two R₁ progeny of a single regenerated plant. This plant had been regenerated from Type II (friable embryogenic) callus of an A188 × B73 genetic background after 8 months in culture; the absence of Spm activity in four other plants regenerated from this same callus demonstrates that Spm activity was not present before culturing. Approximately 20 Spm-homologous DNA sequences were detected in each of the inbreds used to initiate the tissue cultures; it is presumed that one of these became active to give rise to Spm activity.     

Transposition of the En/Spm transposable element system in maize (Zea mays L.): reciprocal crosses of a1-m(Au) and a1-m(r) alleles uncover developmental patterns

Transposition studies of the transposon, En/Spm, have dealt with general aspects of the timing of the excision event with regard to DNA replication and plant development, but without describing details of the process. By following the excision events of an En transposon inserted at the a1 locus [a1-m(Au)], several features of this process can be elucidated. In progenies from reciprocal crosses between the a1-m(Au) allele containing an En insert, and a nonautonomous En allele, [a1-m(r) is a deficiency derivative of En], several features of the En at the a1-m(Au) allele can be observed taking place during ear development and during microsporogenesis. First, it has long been known that the distribution of mutant kernel phenotypes on an ear indicates that En transposes late in most of the events during ear development. Second, the phase change of En (presence and absence of activity) is observed during cob development. Third, discordant kernel phenotypes of two ears, reported herein, resulting from a reciprocal cross with the parental phenotype can be deduced to arise from the transposition of En during microsporogenesis and subsequent fertilization, leading to a discordant genotype between endosperm and embryo. The phase change and discordance lead us to conclude that these events can arise from transposition after host DNA replication. It can also be concluded that the activity of the En inserted in this a1-m(Au) allele is not limited to a specific stage or timing during plant development. Further, this study illustrates the power of genetic analysis in the determination of cellular events. Received: 26 May 1999 / Accepted: 11 November 1999     

Transposition of the enhancer controlling element system in maize

Summary Controlling elements have the ability to activate or inactivate standard genes. One of the unique features of controlling elements is their ability to transpose from one position to another on the same chromosome or to another chromosome. In this study, the transposition of the controlling element En was examined in its transposition from its initial site at the al locus to a primary site and then to secondary sites, all within the distal two-thirds of the 3L maizechromosome arm.Stable germinal mutations representing losses of En were selected from three different autonomously mutating al alleles. The insertion of En at a new location was confirmed by crosses of the stable mutants to responsive tester lines. The position of En at the primary transposition site was determined by backcrossing to an al et line using standard three-point tester crosses. Secondary transpositions were detected by Chi-square comparisons of progeny to parental En linkage values.Although En positions were found throughout the segment of chromosome examined, their distribution was not random and some regions of the chromosome were more likely to contain an inserted En. Ens from all three autonomously mutating source alleles showed the same regional preferences. Distributions of transposed Ens on chromosome 3L from the three original unstable al alleles were not significantly different in Chi-square tests. Both primary and secondary sites of insertion were located within these regions. No differences were found between the distribution of primary sites of insertion and the distribution of secondary sites. Statistically significant differences were found between the distributions of transposing and non-transposing Ens.Formerly graduate student, ISU, now with Monsanto Agricultural Products Co., 800 N. Lindbergh Blvd. St. Louis, MO 63166, and Professor of the Department of Agronomy, Iowa State University, Ames, 1A 50011     

Excision of the En/Spm transposable element of Zea mays requires two element-encoded proteins.

An excision assay system for En/Spm was developed in transgenic tobacco. The characteristics of excision and integration are similar to the natural system of Zea mays. In this transgenic model system two En/Spm encoded trans-acting functions, TNPA and TNPD, are required for excision. A biochemical model for transposition is proposed that might also be applicable to other transposable elements.     

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.     

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.     

Sequence comparison of 'states' of a1-m1 suggests a model of Spm (En) action

Two states of the a1-m1 allele featuring different phenotypes in the absence as well as in the presence of Spm or En have been cloned and sequenced.. The insertion site and orientation of the Inhibitor (I) element within the two alleles is identical. The sizes of the I elements differ, being 2.2 kb in state 6078 and 789 bp in state 5719A-1. The internal deletion in state 5719A-1 affects sequences within one side of the terminal inverted repeats of the I element. This alteration can be correlated with the decreased response of this state to the Mutator function of Spm. A model for the interaction between Spm (En)-encoded functions and the receptor element is discussed explaining the phenotypic differences between the states of the locus.     

Transposase activity of the Ac controlling element in maize is regulated by its degree of methylation

Summary The maize controlling element activator, Ac, is capable of self-transposition. We isolated a spontaneously arisen derivative, (wx-m9Ds-cy) of the Ac element present in the wx-m9Ac mutant which does not itself transpose but can be induced to transpose by the presence of an Ac element elsewhere in the genome. The wx-m9Ds-cy derivative reverts to an active Ac form. A comparison of cloned isolates of the three forms of the element shows no differences in restriction enzyme pattern. Southern analysis of the genome organization of the elements shows marked differences in the methylation pattern. The active Ac element is methylated at one end of the element while the inactive derivative wx-m9Ds-cy is completely methylated at all HpaII sites in the element. The revertant Ac is partially demethylated. Reversion of the mutants to the active form appears to be at least a two-step process.     

IRAK-M is a novel member of the Pelle/interleukin-1 receptor-associated kinase (IRAK) family.

The interleukin-1 receptor-associated kinase (IRAK) was first described as a signal transducer for interleukin-1 (IL-1) and has later been implicated in signal transduction of other members of the Toll/IL-1 receptor family. We now report the identification and characterization of a novel IRAK-like molecule. In contrast to the ubiquitously expressed IRAK and IRAK-2, this new IRAK-like molecule is found mainly in cells of monomyeloic origin and is, therefore, designated IRAK-M. Although IRAK-M and IRAK-2 exhibit only a negligible autophosphorylation activity, they can reconstitute the IL-1 response in a 293 mutant cell line lacking IRAK. In addition, we show for the first time that members of the IRAK family are indispensable elements of lipopolysaccharide signal transduction. The discovery of IRAK-M adds another level of complexity to our understanding of signaling by members of the Toll/IL-1 receptor family.     

Eukaryotic initiation factor 4B from wheat and Arabidopsis thaliana is a member of a multigene family

Clones of eukaryotic initiation factor (eIF) 4B from wheat and Arabidopsis thaliana were obtained from cDNA and genomic libraries. The exon/intron organization of the genes from wheat and A. thaliana is very similar. The deduced amino acid sequences for the wheat and Arabidopsis eIF4B proteins showed overall similarity to each other, but very little similarity to eIF4B from other eukaryotes. The recombinant form of eIF4B supports polypeptide synthesis in an in vitro translation system and reacts with antibodies to native wheat eIF4B. In contrast to mammalian eIF4B and eIF4A, the combination of wheat eIF4B and eIF4A does not stimulate RNA-dependent ATP hydrolysis activity; however, wheat eIF4B does stimulate eIF4F and eIF4A RNA-dependent ATP hydrolysis activity. Interestingly, eIF4B does not stimulate eIF(iso)4F and eIF4A hydrolysis activity. Gel filtration experiments indicate wheat eIF4B, like its mammalian counterpart, self-associates to form a homodimer.     

The Egg apparatus 1 gene from maize is a member of a large gene family found in both monocots and dicots

The maize ZmEA1 protein was recently postulated to be involved in short-range pollen tube guidance from the embryo sac. To date, EA1-like sequences had only been identified in monocot species. Using a more conserved C-terminal motif found in the monocot species, numerous ZmEA1-like sequences were retrieved in EST databases from dicot species, as well as from unannotated genomic sequences of Arabidopsis thaliana. RT-PCR analyses were produced for these unannotated genes and showed that these were indeed expressed genes. Further structural and phylogenetic analyses revealed that all members of the EA1-like (EAL) gene family shared a conserved 27–29 amino acid motif, termed the EA box near the C-terminal end, and appear to be secretory proteins. Therefore, the EA box proteins defines a new class of small secretory proteins, some of which being possibly involved in pollen tube guidance. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

In situ detection of transposition of the maize controlling element (Ac) in transgenic soybean tissues

The development of a transposon mutagenesis system in soybean would aid in the isolation of unknown genes. The maize controlling element (Ac) has, therefore, been introduced into the soybean (Glycine max (L.) Merr.) genome byAgrobacterium-mediated transformation.Ac was inserted into the untranslated leader region of the bacterial ß-glucuronidase gene (GUS) such that the excision ofAc resulted in restoration of the GUS gene activity. Excision events of theAc element were monitored by detecting blue cells or sectors in transgenic soybean tissues. Using the GUS gene assay and with hybridization data, we have demonstrated that theAc element transposes in transgenic soybean calli, leaves, stems, and roots.