Transposable elements generate novel spatial patterns of gene expression in Antirrhinum majus

The pallida gene of A. majus encodes a product required for the synthesis of red flower pigment. We have shown that the unstable pallida(recurrens) mutation is due to the insertion of the Tam3 transposable element near the promoter of the gene. Imprecise excision of Tam3 alters pallida gene expression and generates new spatial patterns or different intensities of flower pigmentation. Distinct spatial patterns may also result from rearrangements induced by Tam3 that alter the relative position of the pallida gene. Changes in Tam3 structure or position result in new unstable phenotypes. These findings suggest that genes may be rendered genetically hypervariable as a consequence of transposable element insertion and excision.     

Pigmentation mutants produced by transposon mutagenesis in Antirrhinum majus

New pigmentation mutants were generated by transposon mutagenesis in Antirrhinum majus, in three previously described loci, nivea, delila and incolorata, and two new loci, daphne and olive. The wild-type olive gene is required for the production of dark-green leaves, and the daphne gene for the synthesis of flavones. Five out of the six mutants were both germinally and somatically unstable, indicating that they resulted from transposon insertions. Molecular analysis of the mutant at nivea (niv-600) showed that it was caused by insertion of a new transposon, Tam4. The sequence of Tam4 suggests that it is unable to transpose autonomously and that it is related to Tam1 and Tam2. All three of these transposons have identical inverted repeats, produce 3 bp target duplications, leave similar excision footprints and share at one end a 600-700 bp region containing many palindromic copies of a motif sequence, possibly required in cis for transposition. The somatic excision of Tam4 in niv-600 is at a very low rate compared to germinal excision but it can be activated by crossing to lines carrying derivative alleles of a Tam1 insertion at niv. Molecular analysis of four different pigmentation mutants has shown that insertions of Tam1, Tam2, Tam3 and Tam4 have been obtained, illustrating the potential of general transposon mutagenesis for trapping and isolating new transposons as well as for tagging genes.     

Transposition of IS2 into the hemB gene of Escherichia coli K-12.

Genetic studies of the hemB gene in Escherichia coli have resulted in the recovery of both stable and unstable mutant strains. The stable strains have been shown to result from large deletions. This study demonstrates that unstable strains result from the insertion of transposable element IS2 primarily into the 5' region of the structural gene; the instability results from precise excision of the element, producing strains with both high and low frequencies of reversion. This first report of IS2 insertion into hemB suggests that this gene may be a preferred target for insertion of this transposable element.     

Enhanced Tn10 and mini-Tn10 precise excision in DNA replication mutants of Escherichia coli K12

The precise excision of transposon Tn10 and a mini-Tn10 derivative, inserted in the gal or lac operons, was studied in dnaB252 and dnaE486 temperature-sensitive mutants of Escherichia coli. dnaB codes for a DNA replication helicase and dnaE for the alpha subunit of DNA polymerase III. Mutations in these genes were found to enhance, at the permissive temperature, the precise excision of both genetic elements. The increase factor was much more pronounced for the dnaB252 mutant with the transposons inserted in gal. The stimulated excision was only partially affected by a recA null mutation but was significantly reduced by introduction of recF null or ruvA mutations. A model involving template switching of the polymerase between the direct repeats flanking the transposons, on the same strand or between sister strands, could account for the observed results.     

Tagging and Cloning of a Petunia Flower Color Gene with the Maize Transposable Element Activator

We report here the use of the maize transposable element Activator (Ac) to isolate a dicot gene. Ac was introduced into petunia, where it transposed into Ph6, one of several genes that modify anthocyanin pigmentation in flowers by affecting the pH of the corolla. Like other Ac-mutable alleles, the new mutation is unstable and reverts to a functional form in somatic and germinal tissues. The mutant gene was cloned using Ac as a probe, demonstrating the feasibility of heterologous transposon tagging in higher plants. Confirmation that the cloned DNA fragment corresponded to the mutated gene was obtained from an analysis of revertants. In every case examined, reversion to the wild-type phenotype was correlated with restoration of a wild-type-sized DNA fragment. New transposed Acs were detected in many of the revertants. As in maize, the frequency of somatic and germinal excision of Ac from the mutable allele appears to be dependent on genetic background.     

ZBED Evolution: Repeated Utilization of DNA Transposons as Regulators of Diverse Host Functions

ZBED genes originate from domesticated hAT DNA transposons and encode regulatory proteins of diverse function in vertebrates. Here we reveal the evolutionary relationship between ZBED genes and demonstrate that they are derived from at least two independent domestication events in jawed vertebrate ancestors. We show that ZBEDs form two monophyletic clades, one of which has expanded through several independent duplications in host lineages. Subsequent diversification of ZBED genes has facilitated regulation of multiple diverse fundamental functions. In contrast to known examples of transposable element exaptation, our results demonstrate a novel unprecedented capacity for the repeated utilization of a family of transposable element-derived protein domains sequestered as regulators during the evolution of diverse host gene functions in vertebrates. Specifically, ZBEDs have contributed to vertebrate regulatory innovation through the donation of modular DNA and protein interacting domains. We identify that C7ORF29, ZBED2, 3, 4, and ZBEDX form a monophyletic group together with ZBED6, that is distinct from ZBED1 genes. Furthermore, we show that ZBED5 is related to Buster DNA transposons and is phylogenetically separate from other ZBEDs. Our results offer new insights into the evolution of regulatory pathways, and suggest that DNA transposons have contributed to regulatory complexity during genome evolution in vertebrates.     

A low copy number, copia-like transposon in maize.

Bs1, a transposable element that moved into the maize Adh1 gene following barley stripe mosaic virus infection, is shown to be present in 1-5 copies in all maize and teosinte lines tested. Bs1 sequences do not hybridize with the genome of barley stripe mosaic virus. The insertion of Bs1 is bounded by 304-bp perfect direct repeats, similar in structure to Ty1 in yeast, copia and related elements in Drosophila, and vertebrate pro-retroviruses, but different from all other known plant transposons. No free copies of the terminal sequences or large internal deletions of Bs elements could be detected. Bs1 is apparently not related to several transposons which moved into the Shrunken gene in lines made genetically unstable by barley stripe mosaic virus infection, suggesting that this virus may cause genome shock, resulting in a generalized liberation of transposons in response to environmental stress.     

Epistatic analysis of the roles of the RAD27 and POL4 gene products in DNA base excision repair in S. cerevisiae

The cellular role of the DNA polymerase encoded by the Saccharomyces cerevisiae POL4 gene is unclear. We have used an epistasis analysis to investigate whether the proteins encoded by the POL4 and RAD27 genes participate in alternative, non-redundant subpathways of DNA base excision repair (BER). We constructed strains in which the genes were deleted singly or in combination and have examined their sensitivity to DNA damaging agents as well as spontaneous mutation frequency. The double deletion strain is no more sensitive to damaging agents and has no higher spontaneous mutation frequency than the most sensitive single mutant. These data indicate that the protein encoded by the POL4 gene does not participate in a non-redundant subpathway of base excision repair under these conditions. We discuss the implications of these results in light of the recent classification of the POL4 gene product as a member of the DNA polymerase lambda family.     

Distinct mechanisms determine transposon inheritance and methylation via small interfering RNA and histone modification

Heritable, but reversible, changes in transposable element activity were first observed in maize by Barbara McClintock in the 1950s. More recently, transposon silencing has been associated with DNA methylation, histone H3 lysine-9 methylation (H3mK9), and RNA interference (RNAi). Using a genetic approach, we have investigated the role of these modifications in the epigenetic regulation and inheritance of six Arabidopsis transposons. Silencing of most of the transposons is relieved in DNA methyltransferase (met1), chromatin remodeling ATPase (ddm1), and histone modification (sil1) mutants. In contrast, only a small subset of the transposons require the H3mK9 methyltransferase KRYPTONITE, the RNAi gene ARGONAUTE1, and the CXG methyltransferase CHROMOMETHYLASE3. In crosses to wild-type plants, epigenetic inheritance of active transposons varied from mutant to mutant, indicating these genes differ in their ability to silence transposons. According to their pattern of transposon regulation, the mutants can be divided into two groups, which suggests that there are distinct, but interacting, complexes or pathways involved in transposon silencing. Furthermore, different transposons tend to be susceptible to different forms of epigenetic regulation.     

Identification of genes required for excision of CTnDOT, a Bacteroides conjugative transposon

Integrated self-transmissible elements called conjugative transposons have been found in many different bacteria, but little is known about how they excise from the chromosome to form the circular intermediate, which is then transferred by conjugation. We have now identified a gene, exc, which is required for the excision of the Bacteroides conjugative transposon, CTnDOT. The int gene of CTnDOT is a member of the lambda integrase family of recombinases, a family that also contains the integrase of the Gram-positive conjugative transposon Tn916. The exc gene was located 15 kbp from the int gene, which is located at one end of the 65 kbp element. The exc gene, together with the regulatory genes, rteA, rteB and rteC, were necessary to excise a miniature form of CTnDOT that contained only the ends of the element and the int gene. Another open reading frame (ORF) in the same operon and upstream of exc, orf3, was not essential for excision and had no significant amino acid sequence similarity to any proteins in the databases. The deduced amino acid sequence of the CTnDOT Exc protein has significant similarity to topoisomerases. A small ORF (orf2) that could encode a small, basic protein comparable with lambda and Tn916 excision proteins (Xis) was located immediately downstream of the CTnDOT int gene. Although Xis proteins are required for excision of lambda and Tn916, orf2 had no effect on excision of the element. Excision of the CTnDOT mini-element was not affected by the site in which it was integrated, another difference from Tn916. Our results demonstrate that the Bacteroides CTnDOT excision system is tightly regulated and appears to be different from that of any other known integrated transmissible element, including those of some Bacteroides mobilizable transposons that are mobilized by CTnDOT.     

Effort and Contribution of T-DNA Insertion Mutant Library for Rice Functional Genomics Research in China: Review and Perspective

With the completion of the rice (Oryza sativa L.) genome‐sequencing project, the rice research community proposed to characterize the function of every predicted gene in rice by 2020. One of the most effective and high‐throughput strategies for studying gene function is to employ genetic mutations induced by insertion elements such as T‐DNA or transposons. Since 1999, with support from the Ministry of Science and Technology of China for Rice Functional Genomics Programs, large‐scale T‐DNA insertion mutant populations have been generated in Huazhong Agricultural University, the Chinese Academy of Sciences and the Chinese Academy of Agricultural Sciences. Currently, a total of 372,346 mutant lines have been generated, and 58,226 T‐DNA or Tos17 flanking sequence tags have been isolated. Using these mutant resources, more than 40 genes with potential applications in rice breeding have already been identified. These include genes involved in biotic or abiotic stress responses, nutrient metabolism, pollen development, and plant architecture. The functional analysis of these genes will not only deepen our understanding of the fundamental biological questions in rice, but will also offer valuable gene resources for developing Green Super Rice that is high‐yielding with few inputs even under the poor growth conditions of many regions of Africa and Asia.      

Latheo, a New Gene Involved in Associative Learning and Memory in Drosophila Melanogaster, Identified from P Element Mutagenesis

Genetic dissection of learning and memory in Drosophila has been limited by the existence of ethyl methanesulfonate (EMS)-induced mutations in only a small number of X-linked genes. To remedy this shortcoming, we have begun a P element mutagenesis to screen for autosomal mutations that disrupt associative learning and/or memory. The generation of "P-tagged" mutant alleles will expedite molecular cloning of these new genes. Here, we describe a behavior-genetic characterization of latheoP1, a recessive, hypomorphic mutation of an essential gene. latheoP1 flies perform poorly in olfactory avoidance conditioning experiments. This performance deficit could not be attributed to abnormal olfactory acuity or shock reactivity-two task-relevant "peripheral" behaviors which are used during classical conditioning. Thus, the latheoP1 mutation appears to affect learning/memory specifically. Consistent with chromosomal in situ localization of the P element insertion, deficiencies of the 49F region of the second chromosome failed to complement the behavioral effect of the latheoP1 mutation. Further complementation analyses between latheoP1 and lethal alleles, produced by excision of the latheoP1 insert or by EMS or gamma-rays, in the 49F region mapped the latheo mutation to one vital complementation group. Flies heterozygous for latheoP1 and one of two EMS lethal alleles or one lethal excision allele also show the behavioral deficits, thereby demonstrating that the behavioral and lethal phenotypes co-map to the same locus.     

Non-clonability correlates with genomic instability: a case study of a unique DNA region

Instability of eukaryotic DNA in constructs propagated in prokaryotic hosts is a frequently observed phenomenon. With the exception of a very high A+T-content and the presence of multiple repetitions, no general rule at the basis of this phenomenon is actually known. The intergenic spacer located between the pi and alpha(D) chicken alpha-type globin genes is frequently deleted from recombinant phages and plasmids. Here we have cloned this DNA fragment using a specially designed bacterial strain (SURE competent cells, Stratogene). Comparative analysis of DNA of recombinant clones bearing deletions and clones containing the intact genomic DNA fragment has revealed two important DNA sequence motifs that contribute to the unclonability of eukaryotic DNA in prokaryotic cells. First, the similarity to bacterial transposons (i.e. the presence of repeats flanking a several kilobase DNA fragment) may cause the loss of the fragment during propagation of the recombinant DNA in E. coli. Second, a high content of rotationally correlated kinkable elements (TG*CA steps) may result in non-clonability of the DNA sequence. Interestingly, the latter type of "unclonable" DNA sequence motifs identified in the globin gene domain is unstable (frequently rearranged) also in the eukaryotic chromosome resulting in a local polymorphism. In the chicken domain of alpha globin genes this unstable DNA sequence seems to be partially protected by interaction with nuclear matrix proteins.     

Insertions of a Novel Class of Transposable Elements with a Strong Target Site Preference at the R Locus of Maize

The r locus of maize regulates anthocyanin synthesis in various tissues of maize through the production of helix-loop-helix DNA binding proteins capable of inducing expression of structural genes in the anthocyanin biosynthetic pathway. The complex r variant, R-r:standard (R-r), undergoes frequent mutation through a variety of mechanisms including displaced synapsis and crossing over, and intrachromosomal recombination. Here we report a new mechanism for mutation at the R-r complex: insertion of a novel family of transposable elements. Because the elements were first identified in the R-p gene of the R-r complex, they have been named P Instability Factor (PIF). Two different PIF elements were cloned and found to have identical sequences at their termini but divergent internal sequences. In addition, the PIF elements showed a marked specificity of insertion sites. Six out of seven PIF-containing derivatives examined had an element inserted at an identical location. Two different members of the PIF element family were identified at this position. The seventh PIF-containing derivative examined had the element inserted at a distinct position within r. Even at this location, however, the element inserted into a conserved target sequence. The timing of PIF excision is unusual. Germinal excision rates can range up to several percent of progeny. Yet somatic sectors are rare, even in lines exhibiting high germinal reversion rates.     

Suppression of Recj Exonuclease Mutants of Escherichia Coli by Alterations in DNA Helicases II (Uvrd) and IV (Held)

The recJ gene encodes a single-strand DNA-specific exonuclease involved in homologous recombination. We have isolated a pseudorevertant strain in which recJ mutant phenotypes were alleviated. Suppression of recJ was due to at least three mutations, two of which we have identified as alterations in DNA helicase genes. A recessive amber mutation, ``uvrD517(am),' at codon 503 of the gene encoding helicase II was sufficient to suppress recJ partially. The uvrD517(am) mutation does not eliminate uvrD function because it affects UV survival only weakly; moreover, a uvrD insertion mutation could not replace uvrD517(am) as a suppressor. However, suppression may result from differential loss of uvrD function: mutation rate in a uvrD517(am) derivative was greatly elevated, equal to that in a uvrD insertion mutant. The second cosuppressor mutation is an allele of the helD gene, encoding DNA helicase IV, and could be replaced by insertion mutations in helD. The identity of the third cosuppressor ``srjD' is not known. Strains carrying the three cosuppressor mutations exhibited hyperrecombinational phenotypes including elevated excision of repeated sequences. To explain recJ suppression, we propose that loss of antirecombinational helicase activity by the suppressor mutations stabilizes recombinational intermediates formed in the absence of recJ.