Spm Element Research Articles

TECHNICAL ADVANCE: Induction of phenotypic variation by activation of genes harbouring a maize Spm element in their promoter regions using a TnpA-VP16 fusion protein

For many crops, a narrowing genetic base is becoming an increasingly significant problem for improvements made through breeding. Commonly used breeding procedures systematically reduce genetic diversity within elite gene pools. Here we describe a new technique for activation of genes in lines carrying Spm or dSpm transposon insertions. Activation of genes in Arabidopsis harbouring Spm or dSpm insertions in their promoters can be induced by over-expression of the TnpA-VP16 fusion protein, which binds Spm ends and activates local transcription. As a result, a variety of phenotypes are recovered from multiple-copy Spm lines in Arabidopsis. Application of this technique to a number of Spm insertion collections in Arabidopsis provides a valuable approach for new insights into plant gene functions. It also provides a proof-of-principle demonstration that the method could be used to generate new variation in elite lines of maize.

The maize LAG1-O mutant suggests that reproductive cell lineages show unique gene expression patterns early in vegetative development.

Patterns of transposable element activity often provide useful information about how and when organisms regulate gene expression. The maize lowered Ac/Ds germinal reversion 1 (LAG1)-O mutation causes unusually low rates of germinal reversion by Ac/Ds-induced alleles even though these same alleles revert frequently and early in somatic development. LAG1-O suppresses Ds transposition at multiple, unlinked loci, and does not affect Spm elements, indicating that the mutation acts in trans and may be specific to Ac/Ds elements. Our data suggest that LAG1-O suppression gradually reduces Ac/Ds activity in the meristem and newly formed leaves until, by the floral transition, transposition is undetectable even with PCR-based assays. This suppression persists during tassel development and does not appear to be released until some point after meiosis. Competitive RT-PCR results show no difference in Ac transposase mRNA levels between LAG1-O and lag1(+) tassels, suggesting that suppression is post-transcriptional. The pattern of LAG1-O expression is consistent with a model in which at least some gene expression specific to those meristem cells that will ultimately give rise to floral tissue and therefore gametes begins very early in plant development, and then persists throughout development.

Multiple independent defective suppressor-mutator transposon insertions in Arabidopsis: a tool for functional genomics.

A new system for insertional mutagenesis based on the maize Enhancer/Suppressor-mutator (En/Spm) element was introduced into Arabidopsis. A single T-DNA construct carried a nonautonomous defective Spm (dSpm) element with a phosphinothricin herbicide resistance (BAR) gene, a transposase expression cassette, and a counterselectable gene. This construct was used to select for stable dSpm transpositions. Treatments for both positive (BAR) and negative selection markers were applicable to soil-grown plants, allowing the recovery of new transpositions on a large scale. To date, a total of 48,000 lines in pools of 50 have been recovered, of which approximately 80% result from independent insertion events. DNA extracted from these pools was used in reverse genetic screens, either by polymerase chain reaction (PCR) using primers from the transposon and the targeted gene or by the display of insertions whereby inverse PCR products of insertions from the DNA pools are spotted on a membrane that is then hybridized with the probe of interest. By sequencing PCR-amplified fragments adjacent to insertion sites, we established a sequenced insertion-site database of 1200 sequences. This database permitted a comparison of the chromosomal distribution of transpositions from various T-DNA locations.

DNA methylation and activity of the maize Spm transposable element.

Maize transposons were the first plant genes known to undergo reversible heritable inactivation. Early in the study of the Suppressor-mutator (Spm) trans-posable element McClintock recognized that certain isolates of the element either cycled between inactive and active phases during development or underwent an inactivation event of longer duration and sufficient stability to be heritable, but which was nonetheless occasionally reversed (McClintock 1957, 1958). In subsequent studies, McClintock developed a deeper understanding of the ways in which the Spm element alternated between active and inactive phases (McClintock 1959, 1961, 1962, 1971). She later reported that theActivator element is also subject to a similar type of reversible inactivation, although the she did not study the Activator element’s inactivation mechanism in as great detail as that of Spm (McClintock 1964, 1965a,b).KeywordsTransposable ElementPromoter MethylationCarnegie InstInactive PhaseMutator ElementThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Maize bronze 1:dSpm insertion mutations that are not fully suppressed by an active Spm.

The Suppressor-mutator (Spm) family of maize transposable elements consists of autonomous Spm elements and nonautonomous defective Spm (dSpm) elements. One characteristic of this family is that the insertion of dSpm elements into a structural gene often permits some level of structural gene expression in the absence of SpM activity, and this structural gene expression is suppressed in trans by Spm activity. The Spm's subterminal repetitive regions (SRRs) contain several iterations of a 12-bp repeat motif. It had been proposed that binding of an Spm-encoded protein to these repeat motifs blocks structural gene transcriptional readthrough, thus suppressing gene expression. The bz-m13 allele of the bronze 1 locus contains a 2.24-kb dSpm insertion in the second exon of a Bz allele. In the absence of Spm activity, bz-m13 displays substantial Bz expression, and this expression is fully suppressed by Spm. Four intra-dSpm deletion derivatives are described in which this Bz expression is only partially suppressed by Spm. Each of these derivatives retains at least 12 SRR repeat motifs. Thus the presence of these repeat motifs is not sufficient to guarantee complete suppression by Spm. Some other property such as secondary structure or element size must play a role.

Maize Spm transposable element has an enhancer-insensitive promoter.

We have used a transient assay system to investigate the promoter region of the maize Suppressor-mutator (Spm) transposable element. All of the sequence required for constitutive promoter activity is confined to the 0.2-kb sequence upstream from the transcription start site of the element at nt 209 and designated the upstream control region. The element's promoter is weak, lacks a conventional TATA box, and depends on the presence of multiple, short repetitive sequence elements. The Spm promoter is quite insensitive to the enhancer sequence of the cauliflower mosaic virus 35S promoter. Enhancer sensitivity can be restored by providing a -30 TATA sequence and removing the G + C-rich sequence encoding the untranslated leader of the element, designated the downstream control region. Although the downstream control region is without effect on Spm promoter activity, it completely inhibits the 35S core promoter and markedly inhibits activity of the complete 35S promoter. The properties of the Spm element's promoter buffer it from both mutational and position-dependent changes in activity. We suggest that the inherent characteristics of the promoter are part of the genetic mechanism that controls the element's transposition frequency, ensuring it remains low and insertion-site independent.

TnpA trans-activates methylated maize Suppressor-mutator transposable elements in transgenic tobacco.

The maize Suppressor-mutator (Spm) transposable element is subject to epigenetic inactivation in transgenic tobacco, as it is in maize. Spm inactivation in tobacco is correlated with increased methylation of sequences near the element's transcription start site. To determine whether element-encoded gene products can promote the reactivation of an inactive element, we investigated the effects of introducing individual CaMV 35S promoter-driven cDNAs for tnpA, tnpB, tnpC and tnpD, the element's four known protein-coding sequences. Introduction of the tnpA cDNA promoted the reactivation of the inactive resident Spm element, as judged by the appearance of regenerants with very early excision events and transposed elements. By contrast, the tnpB, tnpC and tnpD cDNAs had no affect on the activity of the resident Spm element. Similar results were obtained when the element-encoded cDNAs were introduced either by Agrobacterium-mediated retransformation or by a genetic cross. Reactivation of an inactive Spm by the tnpA cDNA is accompanied by reduced methylation of several methylation-sensitive restriction sites near the element's transcription start site. Maintenance of the reactivated Spm element in an active state requires the continued presence of the tnpA cDNA. Elimination of the tnpA cDNA locus by genetic segregation generally results in decreased element activity, as judged by a low frequency of excision events, and is accompanied by increased methylation of the element's 5'-end. Exceptions resembling the phenomenon of "presetting" are also observed in which progeny plants that did not receive the tnpA cDNA locus after meiotic segregation maintain high excision activity and exhibit low methylation levels.

The tnpA and tnpD Gene Products of the Spm Element Are Required for Transposition in Tobacco

The tnpA and tnpD Gene Products of the Spm Element Are Required for Transposition in Tobacco

The tnpA and tnpD gene products of the Spm element are required for transposition in tobacco.

The maize Suppressor-mutator (Spm) element encodes four alternatively spliced transcripts designated tnpA, tnpB, tnpC, and tnpD. tnpA and tnpB are monocistronic, whereas tnpC and tnpD are dicistronic, and the protein-coding sequences of each transcript overlap extensively with those of one or more of the other transcripts. We have analyzed the role of the Spm-encoded gene products in element transposition by using cDNAs with a single open reading frame to (1) complement Spm elements with frameshift mutations and (2) complement each other in a tobacco transposition assay. We report that whereas the tnpA and tnpD gene products are essential for transposition, the tnpB and tnpC gene products are not. We have analyzed the structure of empty donor sites, new insertion sites, and potential transposition intermediates. We discuss the implications of our findings for the mechanism of Spm transposition.

TRIPSACUM ANDERSONII IS A NATURAL HYBRID INVOLVING ZEA AND TRIPSACUM: MOLECULAR EVIDENCE

Cytogenetic evidence suggests that Tripsacum andersonii may be a natural hybrid between Zea and Tripsacum. In this paper we show sequences that hybridize to the transposable elements Mul and Spm are found in T. andersonii and all Zea species examined. However, no hybridizable sequences are observed in the five other Tripsacum species surveyed. These results suggest that Mu and Spm elements became components of the Zea genome after the divergence of Zea and Tripsacum, and they strongly support the cytological evidence that T. andersonii is a Zea‐Tripsacum hybrid. Examination of nuclear ribosomal genes of T. andersonii also supports the hybridization hypothesis and identifies the Zea parent as Zea luxurians. The Tripsacum parent could not be conclusively identified, but the ribosomal gene data suggest that the species of Tripsacum section Fasiculata most closely resemble T. andersonii. Restriction site patterns of two chloroplast DNA sequences indicate that the maternal parent was a species of Tripsacum. These results are complemented by morphological evidence regarding the origin of T. andersonii.

Amplification of genomic sequences flanking transposable elements in host and heterologous plants: a tool for transposon tagging and genome characterization

The isolation of sequences flanking integrated transposable elements is an important step in gene tagging strategies. We have demonstrated that sequences flanking transposons integrated into complex genomes can be simply and rapidly obtained using the polymerase chain reaction. Amplification of such sequences was established in a model system, a transgenic tobacco plant carrying a single Ac element, and successfully applied to the cloning of a specific Spm element from a maize line carrying multiple Spm hybridizing sequences. The described utilization of methylation sensitive restriction enzymes (including those with degenerate recognition sequences) in the generation of templates for amplification will simplify the cloning and mapping of genomic sequences adjacent to transposable elements.

Mutations, epimutations, and the developmental programming of the maize Suppressor‐mutator transposable element

Information about the structure, function and regulation of the maize Suppressor-mutator (Spm) transposable element has emerged from the genetic and molecular characterization of both deletion mutations and an unconventional type of reversible genetic change (epimutation). The element is subject to an epigenetic mechanism that can either stably inactivate it or specify one of a variety of heritable programs of differential element expression in development. The essay explores the relationship between the Spm element's epigenetic developmental programming mechanism and the determinative events central to plant development and differentiation.

Mobility of the maize suppressor-mutator element in transgenic tobacco cells.

Maize Suppressor-mutator (Spm) transposable elements have been introduced into tobacco cells and a visual assay for Spm activity has been developed using a bacterial beta-glucuronidase gene. The Spm element is mobile in tobacco and can trans-activate excision of a transposition-defective Spm (dSpm) element either from a different site on the same transforming Ti plasmid or from a second plasmid. An Spm element expressed from the stronger cauliflower mosaic virus 35S promoter trans-activates transposition of a dSpm element earlier after its introduction into tobacco cells than an element expressed from its own promoter.

The heritable activation of cryptic Suppressor-mutator elements by an active element.

A weakly active maize Suppressor-mutator (Spm-omega) element is able to heritably activate cryptic Spm elements in the maize genome. The spontaneous activation frequency, which is 1-5 x 10(-5) in the present genetic background, increases by about 100-fold in the presence of an Spm-omega and remains an order of magnitude above the background level a generation after removal of the activating Spm-omega. Sectorial somatic reactivation of cryptic elements can be detected phenotypically in kernels. Selection of such kernels constitutes an efficient selection for plants with reactivated Spm elements. Analysis of the reactivation process reveals that it is gradual and proceeds through genetically metastable intermediates that exhibit different patterns of element expression during plant development. Newly reactivated elements tend to return to an inactive form. However, the probability that an element will remain in a heritably active state increases when the element is maintained in the presence of an active Spm element for several generations.

Patterns of developmental and heritable change in methylation of the Suppressor‐mutator transposable element

AbstractGenetic inactivation of the Suppressor‐mutator (Spm) element is correlated with methylation of sequences surrounding the element's transcription initiation site. Several stages in the development of the plant have been identified during which element methylation is reproducibly altered. Loss of element methylation occurs during development of the embryo and early in vegetative growth of the tiller. Element methylation increases during vegetative growth and during development of male and female inflorescences. The susceptibility of element methylation to change during development correlates with the genetic stability of the element's phase of activity. Increases in methylation of sites both upstream and downstream of the Spm element's transcription initiation site parallel increases in the genetic stability of the inactive phase. These results strengthen the likelihood that methylation of C residues within specific regions of the element is important in maintaining the element in an inactive phase and is a component of the molecular mechanism that regulates element expression in plant development.

Molecular mechanisms in the developmental regulation of the maize Suppressor-mutator transposable element.

The maize Suppressor-mutator (Spm) element can exist in one of three heritable forms: (1) a stably active form, (2) a stably inactive form, termed cryptic, and (3) a labile form, here termed programmable, in which the element exhibits one of a variety of heritable developmental programs of expression. Active elements are transcribed and are hypomethylated at sites upstream of the transcription start site, whereas inactive elements are transcriptionally silent and largely methylated at the upstream sites. Active (both stable and programmable), inactive programmable, and cryptic elements are unmethylated, partially methylated, and fully methylated, respectively, at sites within an 0.35-kb 80% G + C region just downstream from the transcription start site. An active Spm element in a genome with a cryptic element promotes its partial demethylation but not its transcriptional activation. In contrast, a trans-acting Spm promotes extensive demethylation and transcriptional activation of an inactive programmable element, as well as its heritable reactivation. These observations define the molecular components of the Spm element's developmental regulatory mechanism. We discuss their general relevance to the developmental regulation of gene expression.

Is the Suppressor-mutator element controlled by a basic developmental regulatory mechanism?

We report the results of genetic studies on derivatives of two different alleles of the maize a locus with an insertion of the Suppressor-mutator (Spm) transposable element in which the element is inactive, but can be reactivated readily. We present evidence that the mechanism that determines whether the element is in an active or inactive phase has two genetically distinguishable components. One determines whether or not the element is genetically active (the phase setting) and the other determines the stability of the setting in development, its heritability, and its phase in the next generation (the phase program). We show that the element's phase can be reset in a reproducible pattern during plant development. We also show that the Spm element can be reprogrammed to undergo a subsequent phase change without a concomitant phase change. The capacity to reset and reprogram the Spm element is differentially expressed within the plant in a pattern that is correlated with the developmental fate of apical and lateral meristems, suggesting the involvement of a basic developmental determination mechanism.

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.

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.

Molecular cloning of the c2 locus of Zea mays, the gene coding for chalcone synthase

The c2 locus of Zea mays, identified as one of the genes affecting anthocyanin biosynthesis, was cloned using the transposable element En (Spm) as a gene tag. The Spm element present at the c2 locus in the autonomously mutating c2-m1 line was isolated using En1 element specific probes. Sequences flanking the element were identified as c2 locus specific and were used to clone the nonautonomous c2-m2 and wild-type alleles. The cloning and analysis of a cDNA complementary to the c2 locus provided evidence that this gene encodes the enzyme chalcone synthase.