Transposable Element System Research Articles

The defective En-I102 element encodes a product reducing the mutability of the En/Spm transposable element system of Zea mays

Genetic and molecular analysis has revealed a specific En-element of deletion derivative (En-I102) which reduces En/Spm-induced mutability. In the presence of En-I102 the excision frequency of both the autonomous En-1 element and the inhibitor element Spm-I5719A is reduced and excision occurs later in development. The 3697 bp long En-I102 element is derived from En-1 by an internal deletion of 4590 bp removing nucleotides 1862-6451. The promoter at the left end and sequences required for polyadenylation are retained in En-I102. It is transcribed to yield predominantly a 1.8 kb poly(A) RNA. cDNA analysis of this transcript indicated that it contains the coding capacity for a 386 amino acid polypeptide. This polypeptide shares homology with En/Spm encoded functions and we suggest that it interferes with transposition at the protein level.

Spontaneous Activation of Quiescent Uq Transposable Elements during Endosperm Development in Zea Mays.

This study addresses the question of the activation of quiescent transposable elements in maize breeding lines. The a-ruq reporter allele of the Uq transposable element system expresses Uq activity (spots or sectors of spots in otherwise colorless aleurone tissue) when exposed to various genotypes of assorted maize inbred lines lacking any active Uq element. This activation of quiescent Uq elements occurs randomly during the growth of the endosperm. It is concluded that there are components in the genome that enhance the rare activation of quiescent Uq elements. Further, it seems that this activation occurs in the absence of any stress-inducing treatment, but that normal growth conditions provide sufficient stimulus for such activation.

Reactivation of the Mutator transposable element system following gamma irradiation of seed

The bz2-mu1 allele contains a 1.4 kb Mu element insertion in the open reading frame of the bronze-2 locus. This insertion suppresses gene activity. In an active Mutator line, however, the bz2-mu1 allele shows high somatic instability resulting in numerous purple spots of full gene activity against a beige background in the aleurone tissue of the kernel; restoration of gene activity results from excision of the Mu element. In contrast, in plants with an inactive Mutator system, uniformly bronze kernels are found, and the Mu element at bz2-mu1 is stabilized. Accompanying a loss of somatic instability, this Mu element, as well as the Mu elements elsewhere in the genome, have an increased level of DNA modification. Spontaneous reactivation of somatic instability in inactive Mutator lines rarely occurs; however, reactivation can be induced with gamma irradiation. Reactivated plants regain both the spotted kernel phenotype indicative of element excision from the bz2-mu1 reporter allele and diagnostic restriction sites within the Mu elements indicative of a hypomethylated state. The reactivated plants transmit these characters to their progeny. These data support the hypothesis that “genomic shock” can elicit cryptic transposable element activities in maize. Possible mechanisms for inactivation and reactivation of the Mutator transposable element system are also discussed.

Allele-specific and Mutator-associated instability at the Rpl disease-resistance locus of maize

The Rp1 locus of maize determines resistance to the fungal rust pathogen Puccinia sorghi. In an attempt to isolate insertion mutations at Rp1 with the Mutator transposable element system, we found that Rp1 inactivation was common both in lines with Mutator activity and in controls lacking any known transposable element activity. Some alleles of Rp1 are endogenously inactivated as frequently as one in five-hundred, whereas other alleles are over twenty times more stable. In a standard background, no mutations of the Rp1F allele were detected in 7,339 progeny screened. In a Mutator background, 27 Rp1F inactivations were isolated from 35,356 tested seedlings. Although most Rp1F mutant seedlings lost resistance to all of the P. sorghi isolates tested, several retained resistance to a subset of rust biotypes. Some of the resistance specificity profiles obtained in these mutants are unlike any previously identified Rp1 alleles. These data indicate that the Rp1 locus is composed of multiple resistance factors with variable intrinsic stabilities.

Regulation of Mu element copy number in maize lines with an active or inactive Mutator transposable element system.

In the progeny of an active Mutator plant, the number of Mu elements increases on self-pollination and maintains the average parental Mu content on outcrossing to a non-Mutator line; both patterns of transmission require an increase in the absolute number of Mu elements from one generation to the next. The same average copy number of Mu elements is transmitted through the male and female, but there is wide variation in the absolute copy number among the progeny. In inactive Mutator plants-defined both by the loss of somatic instability at a reporter gene (bronze2-mu1) and by modification of the HinfI sites in the terminal inverted repeat sequences of Mu elements - the absolute copy number of Mu elements is fixed in the parent. Thus, in outcrosses Mu element number is halved, and on self-pollination Mu copy number is constant. Reactivation of somatic mutability at cryptic bz2-mu1 alleles in inactive individuals by crossing to an active line seems not to involve an increase in Mu element copy number transmitted by the inactive individual. These and other results suggest that increases in Mu copy number occur late in plant development or in the gametophyte rather than after fertilization.

Concomitant regulation of Mu1 transposition and Mutator activity in maize

The mutagenic activity of the maize transposable element system Mutator can be lost by outcrossing to standard, non-Mutator lines or by repetitive intercrossing of genetically diverse Mutator lines. Lines losing Mutator mutagenic activity in either manner retain high copy numbers (10–15 per diploid genome) of the Mutator-associated Mu transposable elements. Frequent transposition of Mu1-related elements is observed only in active Mutator lines, however. The loss of Mutator activity on intercrossing is correlated with an increase in the copy number of Mu1-like elements to 40–50 per diploid genome, implying a self-encoded or self-activated negative regulator of Mu1 transposition. The outcross loss of Mutator activity is only weakly correlated with a low Mu element copy number and may be due to the loss of a positive regulatory factor encoded by a subset of Mu1-like elements. Transposition of Mu elements in active Mutator lines generates multiple new genomic positions for about half the elements each plant generation. The appearance of Mu1-like elements in these new positions is not accompanied by equally high germinal reversion frequencies, suggesting that Mu1 may commonly transpose via a DNA replicative process.

Aspects of the ac/ds transposable element system in maize

ABSTRACT Studies of the Ac (Activator) transposable element provided the data which led Barbara McClintock to postulate that certain segments of chromosomes could transpose to different locations in the genome. McClintock also recognized the existence of Ds (Dissociation) elements which could transpose, but only in the presence of a trans-acting Ac element elsewhere in the genome. DNA sequences corresponding to Ds and Ac have now been identified, and an understanding of many of the properties of these transposable elements in the maize genome has been acquired in recent years. It is known that cryptic Ac elements and members of at least two families of Ds elements occur in the genome of all maize lines examined. Ds elements also occur in Teosinte and the more distantly related Tripsacum. We discuss the possible origin of these elements and consider the mechanism of activation of cryptic Ac elements. A recent molecular analysis of a transition of an Ac-derived Ds-element back to an active Ac element suggests one molecular mechanism by which changes in the activity state of Ac may occur. Distinctive phenotypes created by controlling elements within a target gene have been shown to be governed by the properties of the insertion element and the position of the insertion within the gene. Genetic effects include modulation of gene expression, alteration of gene products, instability of mutant phenotypes, deletion and duplication of chromosome segments and the production of chromosome rearrangements. We describe an example where a Ds insertion generates an additional intron in the Adhl gene which reduces gene expression through mRNA instability. We also discuss an Ac-dependent modulation of P gene activity in glume and pericarp tissues of maize which may be attributed to an alteration either in patterns of gene expression or the developmental biology of the flower. The molecular consequences of Ac and Ds insertions and excisions are known at the DNA sequence level but little is known of the mechanism of transposition. An initial approach has been to analyse Ac transcription. Preliminary results showing transcription of a limited region of Ac are discussed. The corresponding upstream regions have been linked to the coding region of chloramphenicol acetyltransferase (CAT) and show promoter activity following electroporation into tobacco protoplasts.

Stable non-mutator stocks of maize have sequences homologous to the Mu1 transposable element.

Mutator stocks of maize produce mutants at many loci at rates 20- to 50-fold above spontaneous levels. Current evidence suggests that this high mutation rate is mediated by an active transposable element system, Mu. Members of this transposable element family are found in approximately 10-60 copies in Mutator stocks. We report here an initial characterization of previously undetected sequences homologous to Mu elements in eight non-Mutator inbred lines and varieties of maize that have a normal low mutation rate. All stocks have approximately 40 copies of sequences homologous only to the terminal repeat and show weak homology to an internal probe. In addition, several of the stocks contain an intact Mu element. One intact Mu element and two terminal-specific clones have been isolated from one non-Mutator line, B37. The cloned sequences have been used to demonstrate that in genomic DNA the intact element, termed Mu1.4B37, is modified, such that restriction sites in its termini are not accessible to cleavage by the HinfI restriction enzyme. This modification is similar to that observed in Mutator lines that have lost activity. We hypothesize that the DNA modification of the Mu-like element may contribute to the lack of Mutator activity in B37.

Ds-INDUCED ALLELES AT THE OPAQUE-2 LOCUS OF MAIZE

Transposon mutagenesis has been used to isolate mutable alleles at the Opaque-2 (O2) locus of maize. Plants with the Activator-Dissociation (Ac-Ds) system of transposable elements and O2 were crossed as males to a stable o2 tester line. Among a population of 200,000 kernels, 198 exceptional kernels with somatic instability were recovered. In four cases, designated O2-m1, o2-m2, O2-m3 and O2-m4, variegated phenotypes appeared in F(2) and subsequent generations. Genetic analyses indicated that the presence of Ds near or within the O2 gene was responsible for the observed somatic instability at the O2 locus. The phenotypes of the newly induced alleles were of two types. Alleles O2-m1, O2-m3 and O2-m4, in the absence of Ac, were characterized by kernel phenotypes indistinguishable from the wild type; in the presence of Ac they generated kernels with opaque sectors interspersed within a vitreous background. In contrast, the mutable allele o2-m2, in the absence of Ac, was characterized by kernels with a recessive phenotype similar to o2 recessive mutants. In the presence of Ac, it reverted somatically to wild-type-producing kernels with vitreous spots in an o2 background. The association of the Ds element with the O2 locus may prove a valuable tool directed to the isolation of DNA fragments bearing the O2 gene.

Origin and diversity of mutants controlled by theUqtransposable element system in maize

SUMMARYThree transposable element mutations, displaying an unstable pheno-type at theAlocus, were isolated from lines exhibiting Aberrant Ratio behaviour that originated from maize plants treated with plant RNA viruses. Each of these three unstable mutants is shown to belong to theUq-ruqtransposable element system. The two new mutants, along with the previously describeda-ruqmutant, are nonautonomous in mutability control in that the control of mutability is governed by the segregatingUqregulatory element. By following the distribution of differentUqelements segregating in independently derived genetic lines and utilizing tests of allelism and linkage, certain lineages ofUq-element-containing lines are revealed.

Plant transposable elements generate the DNA sequence diversity needed in evolution

Two germinal and 16 somatic reversion events induced by the Enhancer (En) transposable element system at the wx-8::Spm-I8 allele of Zea mays were cloned and studied by sequence analysis. Excision of the Spm-I8 receptor element from the wx gene results in various mutant DNA sequences. This leads to altered gene products, some of which are still capable of restoring the wild-type phenotype. Possible 'foot-print' sequences that may have arisen by the excision of transposable elements were observed when intron sequences of the wild-type (wx+) and mutant (wx-m8) alleles of the wx gene were compared. The sequence divergence generated by visitation of a locus by plant transposable elements is discussed with respect to the molecular evolution of the new gene functions.

Molecular interactions between the components of the En-I transposable element system of Zea mays

The sequence of the Inhibitor element Spm-I8 isolated from the wx-m8 allele has been determined. The element is 2242 bp in length. Its ends can be folded into long stem and loop structures. In a line containing the autonomous En element (wx-m8+En) we have detected a 2.5-kb transcript hybridizing to Spm-I8. A cDNA copy of this En-specific transcript containing 1.2 kb of the 3' end was cloned and it DNA sequence was determined. The 3' half of the cDNA is homologous to Spm-I8 and the region of homology is interrupted by intervening sequences. In the absence of an autonomous En element two chimeric transcripts are observed in the wx-m8 line which are probably initiated at the wx promoter and terminate in the Spm-I8 insertion. In the presence of the En element, these transcripts are suppressed, possibly by a trans-acting function of En, inhibiting transcription read-through into Spm-I8.

Genetic and molecular analysis of the Enhancer (En) transposable element system of Zea mays

A newly isolated, unstable mutation wx-844::En-1 of Zea mays was proven to be caused by the insertion of the autonomous transposable element En into the Waxy (Wx) gene. Molecular analysis revealed that En-1 is 8.4 kb long, has a 13-bp long perfect inverted repeat at its termini and generates a 3-bp target site duplication. En-1 is integrated into an intron located approximately in the middle of the transcribed region of the Wx gene. Structural evidence is presented indicating that a receptor component (Inhibitor) can arise by internal deletion of an autonomous En element.

Correct temperature induction and developmental regulation of a cloned heat shock gene transformed into the Drosophila germ line.

We have constructed a size variant of the Drosophila hsp28 gene by deleting 207 base pairs of the protein coding region, beginning 33 base pairs downstream of the ATG protein initiation codon. After transformation of Drosophila melanogaster rosy (ry506) flies with this altered gene, using the P transposable element system, it was found that the transformed gene was regulated correctly both after temperature elevation and during the development of the flies. Levels of the variant mRNA were as high as those of the endogenous hsp28 during all patterns of expression, and the variant mRNA appeared in all cases to be processed correctly and to be as stable as the endogenous mRNA. Nevertheless, the chromosomal locus of the transformed gene did not puff after heat shock, suggesting that normal transcription of the gene does not require puffing of the locus. The deleted hsp28 gene retained the reading frame of the endogenous one, and a protein of the expected molecular weight of 18,500 was made after heat shock at levels comparable to those of the endogenous hsp28.

Correct temperature induction and developmental regulation of a cloned heat shock gene transformed into the Drosophila germ line.

We have constructed a size variant of the Drosophila hsp28 gene by deleting 207 base pairs of the protein coding region, beginning 33 base pairs downstream of the ATG protein initiation codon. After transformation of Drosophila melanogaster rosy (ry506) flies with this altered gene, using the P transposable element system, it was found that the transformed gene was regulated correctly both after temperature elevation and during the development of the flies. Levels of the variant mRNA were as high as those of the endogenous hsp28 during all patterns of expression, and the variant mRNA appeared in all cases to be processed correctly and to be as stable as the endogenous mRNA. Nevertheless, the chromosomal locus of the transformed gene did not puff after heat shock, suggesting that normal transcription of the gene does not require puffing of the locus. The deleted hsp28 gene retained the reading frame of the endogenous one, and a protein of the expected molecular weight of 18,500 was made after heat shock at levels comparable to those of the endogenous hsp28.

Transposable Elements in Maize the?Activator-Dissociation (Ac-Ds) System

Although unstable mutants in maize (Zea mays) were described as early as 1914 (Emerson 1914, 1917, 1929; Rhoades 1936, 1938), the first explanation of such mutants in terms of transposable DNA was provided by Barbara McClintock's elegant series of experiments on the activator-dissociation (Ac-Ds) controlling-element system of maize (McClintock 1947,1948, 1951). McClintock demonstrated genetically thatAc and Ds were short regions of DNA which could move (transpose) from one chromosomal location to another. McClintock also established that Ds could transpose only in response to the action of Ac (i.e. both elements were required in the same nucleus for Ds transposition), and that Ac could transpose autonomously (i.e. in the absence of Ds). A total of eight transposable element systems have been recognized in maize, the best characterized of which are Ac(Mp)Ds, Spm and Robertson's mutator (reviewed in Fedoroff 1983; Nevers et al. 1984). All but Robertson's mutator occur as two-element systems, similar to Ac-Ds. Transposable elements have now been shown to be widespread in living organisms-occurring in prokaryotes and lower eukaryotes as well as other higher eukaryotes, including animals.