Molecular characterization of a nonautonomous transposable element (dTph1) of petunia.

An insertion sequence of 283 base pairs has been isolated from the DFR-C gene (dihydroflavonol-4-reductase) of petunia. This insert was found only in a line unstable for the An1 locus (anthocyanin 1, located on chromosome VI) and not in fully pigmented progenitor and revertant lines or in stable white derivative lines. This implies that the An1 locus encodes the DFR-C gene. The unstable An1 system in the line W138 is known to be a two-element system, the autonomous element being located on chromosome I. In the presence of the autonomous element, W138 flowers exhibit a characteristic pattern of red revertant spots and sectors on a white background. In the absence of the autonomous element, the W138 allele gives rise to a stable recessive (white) phenotype. Sequence analysis of progenitor, unstable, and revertant alleles revealed dTph1 to contain perfect terminal inverted repeats of 12 base pairs. In DFR-C, it is flanked by an 8-base pair target site duplication. Sequences homologous to dTph1 are present in at least 50 copies in the line W138. Sequence analysis of An1 revertant alleles indicated that excision, including removal of the target site duplication, is required for reversion to the wild-type phenotype. Derivative stable recessive alleles showed excision of dTph1 and a rearrangement of the target site duplication. dTph1 is the smallest transposable element described to date that is still capable of transposition. The use of dTph1 in tagging experiments and subsequent gene isolation is discussed.     

A transgenic dTph1 insertional mutagenesis system for forward genetics in mycorrhizal phosphate transport of Petunia

The active endogenous dTph1 system of the Petunia hybrida mutator line W138 has been used in several forward-genetic mutant screens that were based on visible phenotypes such as flower morphology and color. In contrast, defective symbiotic phosphate (P_i) transport in mycorrhizal roots of Petunia is a hidden molecular phenotype as the symbiosis between plant roots and fungi takes place below ground, and, while fungal colonization can be visualized histochemically, P_i transport and the activity of P_i transporter proteins cannot be assessed visually. Here, we report on a molecular approach in which expression of a mycorrhiza-inducible bi-functional reporter transgene and insertional mutagenesis in Petunia are combined. Bi-directionalization of a mycorrhizal P_i transporter promoter controlling the expression of two reporter genes encoding firefly luciferase and GUS allows visualization of mycorrhiza-specific P_i transporter expression. A population of selectable transposon insertion mutants was established by crossing the transgenic reporter line with the mutator W138, from which the P _i transporter downregulated ( ptd1 ) mutant was identified, which exhibits strongly reduced expression of mycorrhiza-inducible P_i transporters in mycorrhizal roots.     

Epigenetic interactions among three dTph1 transposons in two homologous chromosomes activate a new excision-repair mechanism in petunia.

Unstable anthocyanin3 (an3) alleles of petunia with insertions of the Activator/Dissociation-like transposon dTph1 fall into two classes that differ in their genetic behavior. Excision of the (single) dTph1 insertion from class 1 an3 alleles results in the formation of a footprint, similar to the "classical" mechanism observed for excisions of maize and snapdragon transposons. By contrast, dTph1 excision and gap repair in class 2 an3 alleles occurs via a newly discovered mechanism that does not generate a footprint at the empty donor site. This novel mechanism depends on the presence of two additional dTph1 elements: one located in cis, 30 bp upstream of the an3 translation start in the same an3 allele, and a homologous copy, which is located in trans in the homologous an3 allele. Absence of the latter dTph1 element causes a heritable suppression of dTph1 excision-repair from the homologous an3 allele by the novel mechanism, which to some extent resembles paramutation. Thus, an epigenetic interaction among three dTph1 copies activates a novel recombination mechanism that eliminates a transposon insertion.     

Insertion mutagenesis and study of transposable elements using a new unstable virescent seedling allele for isolation of haploid petunia lines

The new unstable virescent seedling ( vis* ) allele of a petunia mutant, that has green leaves but white cotyledons with green revertant spots, was used to identify spontaneously occurring haploid petunia lines with active transposable elements. Endogenous transposons were trapped into the single petunia nitrate reductase structural gene ( nia ) using chlorate selection on haploid protoplasts. In two mutant lines, the dTph1 -like transposable element dTph1–3 was inserted at almost the same position but in opposite orientations in the first exon of the nia gene. In a third mutant, a different transposable element was integrated into the fourth exon. This element, called dTph4 , is 787 bp long and has 13 bp terminal inverted repeats of which 12 bp are identical to those of dTph1 . Insertion of dTph1–3 and dTph4 results in an 8 bp duplication of the target site, as already described for dTph1 . In contrast to dTph1 -like elements, dTph4 is present at low copy number in the petunia genome. This can facilitate its use for gene tagging in petunia. The dTph1–3 and dTph4 elements excise frequently, as transposon footprints were found in most of the insertion mutants. The data demonstrate that haploid petunia is an excellent system for gene tagging and for the study of transposable elements.     

A general method to isolate genes tagged by a high copy number transposable element

The Petunia hybrida line W138 contains more than 200 copies of the transposable element dTph1. In W138 progeny these elements give rise to new unstable mutations at high frequency. With the aim of isolating these mutated genes a method was developed to isolate dTph1 flanking sequences unique for mutant plants. This method is based on differential screening of cloned inverse polymerase chain reaction (IPCR) products originating from the mutated plant. It directly yields a probe for the mutated gene which can be used to screen pre-existing cDNA and genomic libraries. This method may be generally applicable to isolate genes tagged by other high copy number transposable elements, like Mutator (Mu) or Dissociation (Ds) in Zea mays.     

Genetic characterisation of Act1, the activator of a non-autonomous transposable element from Petunia hybrida

The line W138 of Petunia hybrida has variegated flowers because it is homozygous for the mutable an1-W138 allele. Excision of the element, causing instability, depends on the presence of the activatorAct1. The previously characterised non-autonomous element dTph1 excises from the dfrC gene in response to Act1. This implies that both non-autonomous elements belong to the same transposable element family. In a range of distantly related cultivars we could detect a single functional Act1 element. Linkage analysis for 11 of these lines showed that Act1 was located on chromosome I in all cases, indicating that the element might be fixed in the genome. A group of cultivars that did not exhibit Act1 activity could be traced back to a recent common origin (Rose of Heaven). Cultivars within this group presumably harbour the same inactivated Act1 element. Among the lines tested were 7 lines representing the two species (P. axillaris and P. integrifolia) from which P. hybrida originated. None of these exhibited Act1 activity. We assume that Act1 is present in an inactive state in these lines and that it was activated upon interspecific crossing. In general, lines representing the two parental species and P. hybrida cultivars contain between 5 and 25 dTph1 elements. The lines R27 and W138, however, contain significantly more dTph1 elements (> 50) than all other lines.     

Analysis by Transposon Display of the behavior of the dTph1 element family during ontogeny and inbreeding of Petunia hybrida

Transposon Display is a high-resolution method that was used here to visualize simultaneously individual members of the dTph1 transposable element family of Petunia hybrida. The method provides a tool for accurate analyses of copy numbers and insertion frequencies, and a means to study the behavior of a family of elements as a whole. Somatic insertion events can be identified and insertion events in a cell of the L2 apical lineage can be distinguished unequivocally from those in a cell outside this lineage. In sublines of the high-copy-number line W137, an average insertion rate equivalent to transposition of 10% of the total number of element copies in each generation was measured, copy number increases of over 20% in four generations were recorded, and element position turnover was analyzed. Insertion events are detected essentially randomly both in time and space. The general applicability of the technique for the analysis of the transpositional behavior of element systems is discussed.     

Genetic and molecular characterization of a-mrh-Mrh, a new mutable system of Zea mays

A new allele of the maize A1 gene, a gene required for anthocyanin pigment biosynthesis, was identified in a genetic stock exhibiting a high frequency of chromosome breakage at the second microspore mitosis. This allele, a-mrh, is unstable in both somatic and germinal tissue when an independent locus, Mrh, is present in the genome. a-mrh was molecularly cloned, and a 246 bp DNA insertion with characteristics of a transposable element was identified within the fourth exon of the gene. Southern blot analysis of germinal derivatives of a-mrh suggests that the DNA insert rMrh is excised from the locus when a wild-type phenotype is restored. Genetic crosses with components of other two-element mutable systems of maize failed to induce mutability. We therefore conclude that rMrh is a member of a new, two-element transposon system of maize. The genetic and molecular characteristics of the elements involved are discussed with respect to stress-activated transposition, response of an element to developmental signals, and a possible new role of plant transposons in gene evolution.     

Transposon Display identifies individual transposable elements in high copy number lines

The dTph1 transposable element family of Petunia hybrida line W138 consists of between 100 and 200 members. A strategy that allows simultaneous detection of individual elements is described. Sequences flanking dTph1 elements are amplified by means of a ligation-mediated PCR. The resulting fragments are locus-specific and can be analysed by polyacrylamide gel electrophoresis. One of the applications of Transposon Display is the isolation of dTph1-tagged genes. Fragments that co-segregate with a mutant phenotype can be extracted from the gel and reamplified, providing access to tagged genes, as demonstrated in a reconstruction experiment. Data on the molecular identification of a phenotypic mutant, isolated in a random tagging experiment is also presented. Upon sequencing, the obtained candidate fragment was found to be identical to part of the previously identified Fbp1 gene.     

Analysis of a Mutator Activity Necessary for Germline Transposition and Excision of Tc1 Transposable Elements in Caenorhabditis Elegans

The Tc1 transposable element family of the nematode Caenorhabditis elegans consists primarily of 1.6-kb size elements. This uniformity of size is in contrast to P in Drosophila and Ac/Ds in maize. Germline transposition and excision of Tc1 are detectable in the Bergerac (BO) strain, but not in the commonly used Bristol (N2) strain. A previous study suggested that multiple genetic components are responsible for the germline Tc1 activity of the BO strain. To analyze further this mutator activity, we derived hybrid strains between the BO strain and the N2 strain. One of the hybrid strains exhibits a single locus of mutator activity, designated mut-4, which maps to LGI. Two additional mutators, mut-5 II and mut-6 IV, arose spontaneously in mut-4 harboring strains. This spontaneous appearance of mutator activity at new sites suggests that the mutator itself transposes. The single mutator-harboring strains with low Tc1 copy number generated in this study should be useful in investigations of the molecular basis of mutator activity. As a first step toward this goal, we examined the Tc1 elements in these low copy number strains for elements consistently co-segregating with mutator activity. Three possible candidates were identified: none was larger than 1.6 kb.     

Chloramphenicol resistance transposable element TnSs1 of Streptococcus suis,a transposon flanked by IS6-family elements

A new transposon, designated TnSs1, which contains a chloramphenicol acetyltransferase gene flanked by direct repeats of an IS6-family element was found in a field isolate of Streptococcus suis. Polymerase chain reaction and hybridization analyses indicated that another field isolate carried the same transposon in a different location on the chromosome. A transposition assay done with a thermosensitive suicide vector showed that, among the seven TnSs1 mutants tested in this study, six formed a cointegrate between the S. suis genome and the vector with the generation of the third copy of the insertion sequence element, and one harbored one copy of TnSs1 on the chromosome as a result of a subsequent resolution step. On transposition, TnSs1 duplicated an 8-bp sequence at the target site.     

The Male Sterility-Associated Pcf Gene and the Normal Atp9-1 Gene in Petunia Are Located on Different Mitochondrial DNA Molecules

A mitochondrial DNA (mtDNA) region termed the S-pcf locus has previously been correlated with cytoplasmic male sterility (CMS) in Petunia. In order to understand the relationship of the S-pcf locus to homologous sequences found elsewhere in mtDNAs of both CMS and fertile lines, the structure of the mitochondrial genome of CMS Petunia line 3688 was determined by cosmid walking. The S-pcf locus, which includes the only copies of genes for NADH dehydrogenase subunit 3 (nad3) and small ribosomal subunit protein 12 (rps12) was found to be located on a circular map of 396 kb, while a second almost identical circular map of 407 kb carries the only copies of the genes for 18S and 5S rRNA (rrn18 and rrn5), the only copy of a conserved unidentified gene (orf25), and the only known functional copy of atp9. Three different copies of a recombination repeat were found in six genomic environments, predicting sub-genomic circles of 277, 266 and 130 kb. The ratio of atp9 to S-pcf mtDNA sequences was approximately 1.5 to 1, indicating that sub-genomic molecules carrying these genes differ in abundance. Comparison of the mtDNA organization of the CMS line with that of the master circle of fertile Petunia line 3704 reveals numerous changes in order and orientation of ten different sectors.     

A Mutant S3 RNase of Petunia inflata Lacking RNase Activity Has an Allele-Specific Dominant Negative Effect on Self-Incompatibility Interactions

Gametophytic self-incompatibility in the Solanaceae is controlled by a multiallelic locus called the S locus. Growth of pollen tubes in the pistil is inhibited when the pollen has one of the two S alleles carried by the pistil. The products of a number of pistil S alleles[mdash]S proteins or S RNases[mdash]have been identified, and their role in controlling the pistil's ability to reject self-pollen has been positively established. In contrast, the existence of pollen S allele products has so far been inferred entirely from genetic evidence. Here, we introduced a modified S3 gene of Petunia inflata encoding an S3 RNase lacking RNase activity into P. inflata plants of the S2S3 genotype to determine whether the production of the mutant protein, designated S3(H93R), would have any effect on the ability of the transgenic plants to reject S2 and S3 pollen. Analysis of the self-incompatibility behavior of 49 primary transgenic plants and the progeny of three plants (H30, H37, and H40) that produced S3(H93R) in addition to producing wild-type levels of endogenous S2 and S3 RNases revealed that S3(H93R) had a dominant negative effect on the function of the S3 RNase in rejecting self-pollen; however, it had no effect on the function of the S2 RNase. One likely explanation of the results is that S3(H93R) competes with the S3 RNase for binding to a common molecule, which is presumably the product of the pollen S3 allele.     

Tcl transposition and mutator activity in a Bristol strain of Caenorhabditis elegans

Summary In most strains of Caenorhabditis elegans with a low copy number of Tc1 transposable elements, germline transposition is rare or undetectable. We have observed low-level Tel transposition in the genome of the C. elegans var. Bristol strain KR579 (unc-13[e51]) resulting in an increase in Tc1 copy number and subsequent mutator activity. Examination of genomic blots from KR579 and KR579derived strains revealed that more Tc1-hybridizing bands were present than in other Bristol strains. A novel Tc1-hybridizing fragment was cloned from a KR579-derived strain. Unique sequence DNA flanking the Tc1 element identified a 1.6 kb restriction fragment length difference between the KR579 and N2 strains consistent with a Tc1 insertion at a new genomic site. The site of insertion of this Tel was sequenced and is similar to the published Tel insertion site consensus sequence. Several isolates of KR579 were established and maintained on plates for a period of 3 years in order to determine if Tc1 copy number would continue to increase. In one isolate, KR1787, a further increase in Tc1 copy number was observed. Examination of the KR1787 strain has shown that it also exhibits mutator activity as assayed by the spontaneous mutation frequency at the unc-22 (twitcher) locus. The KR579 strain differs from most low copy number strains in that it exhibits low-level transposition which has developed into mutator activity.     

Chromosome Walking in the Petunia Inflata Self-Incompatibility (S-) Locus and Gene Identification in an 881-kb Contig Containing S 2 -RNase

Self-incompatibility (SI) in the Solanaceae, Rosaceae and Scrophulariaceae is controlled by the polymorphic S locus, which contains two separate genes encoding pollen and pistil determinants in SI interactions. The S-RNase gene encodes the pistil determinant, whereas the pollen determinant gene, named the pollen S gene, has not yet been identified. Here, we set out to construct an integrated genetic and physical map of the S locus of Petunia inflata and identify any additional genes located at this locus. We first conducted chromosome walking at the S2 locus using BAC clones that contained either S2-RNase or one of the nine markers tightly linked to the S locus. Ten separate contigs were constructed, which collectively spanned 4.4 Mb. To identify additional genes located at the S2 locus, a 328-kb region (part of an 881-kb BAC contig) containing S2-RNase was completely sequenced. Approximately 76% of the region contained repetitive sequences, including transposon-like sequences. Other than S2-RNase, an F-box gene, named PiSLF2 (S2-allele of P. inflata S-locus F-box gene), was the only predicted gene whose deduced amino acid sequence was similar to the sequences of known proteins in the database. Two different cDNA selection methods were used to identify additional genes in the 881-kb contig; 11 groups of cDNA clones were identified in addition to those for S2-RNase and PiSLF2. RT-PCR analysis of expression profiles and PCR analysis of BAC clones and genomic DNA confirmed that seven of these 11 newly identified genes were located in the 881-kb contig.     

Tn554: Isolation and characterization of plasmid insertions

Tn554, a transposon in Staphylococcus aureus that carries determinants of spectinomycin resistance and inducible macrolide-lincosamide resistance, is characterized by a highly efficient transposition, exceptional site specificity for insertion, and inhibition of transposition by a copy of the transposon inserted at its preferred chromosomal site. In this communication we describe the characteristics of a number of rare, secondary-site insertions of Tn554 into several related penicillinase plasmids. These plasmid insertions display considerable variation in the frequencies with which they can act as transposon donors, as well as in the frequencies at which they undergo apparently precise excision. Transposition from the plasmid transposon donors is ordinarily a duplicative process and these subsequent transposition events always return Tn554 to its preferred site in the S. aureus chromosome; such derivatives are indistinguishable from the primary chromosomal insertion from which they were originally derived. We also report an unusual relationship between Tn554 and the transducing phage, φ11, in which Tn554 is frequently transferred independently of its plasmid carrier. We suggest that the bacteriophage may play an important role in the mobility of Tn554, in addition to the usual transduction mechanism, in a process that we have referred to as “hitchhiking.”     

IS1-mediated intramolecular rearrangements: formation of excised transposon circles and replicative deletions.

A system is described which permits visualization and analysis of a number of molecular species associated with transposition activity of the bacterial insertion sequence, IS1, in vivo. The technique involves induction of an IS1 transposase gene carried by a plasmid which also includes an IS1-based transposable element. It is, in principle, applicable to the identification of transposition intermediates as well as unstable transposition products and those which are not detectable by genetic means. Thirteen novel molecular species were detected after 4 h of induction. Five major species were characterized, based on their behaviour as a function of time, on their hybridization patterns and on the nucleotide sequences of the transposon-backbone junctions. All result from intramolecular IS1 transposition events. The two reciprocal partner products of IS1-mediated deletions, the intramolecular equivalent of co-integrates generated by intermolecular transposition, have been identified. Both carry a single copy of the transposable element and present complementary distributions of deletion endpoints. These results establish, by direct physical means, that adjacent IS1-mediated deletions are accompanied by duplication of the element. A second type of molecule identified was an excised circular copy of the transposon, raising the possibility that IS1 is capable of following an intermolecular transposition pathway, via excised transposon circles, leading to direct insertion.     

Partial silencing of the NEC1 gene results in early opening of anthers in Petunia hybrida

The NEC1 gene, previously isolated from Petunia hybrida, is expressed at high levels in nectaries, and in a very localized fashion in stamens, particularly in the anther stomium cells and the upper part of the filament. To elucidate the function of the NEC1 gene, co-suppression was employed for down-regulation of NEC1 expression, and transposon insertion mutagenesis was used to knock out the NEC1 function. Among the transgenic plants and plants carrying dTph1 inserted in the NEC1 gene, an "early open anther" phenotype was observed. In this mutant phenotype, the anthers already open in young flower buds (1.8 cm) that still contain immature pollen, resulting in poor pollen quality and impaired pollen release. The results obtained indicate that NEC1 might be involved in the development of stomium cells, which are ruptured during the normal process of anther dehiscence to release mature pollen. Southern analysis revealed the presence of a highly homologous NEC1-like gene, named NEC2, in the P. hybrida genome. The presence of NEC2 was confirmed by segregation analysis and sequencing of genomic clones. The implications of these results and possible reasons why no visually obvious phenotype in nectaries could be produced by co-suppression or transposon insertion mutagenesis are discussed.