Removal of the Bloom Syndrome DNA Helicase Extends the Utility of Imprecise Transposon Excision for Making Null Mutations in Drosophila

Transposable elements are frequently used in Drosophila melanogaster for imprecise excision screens to delete genes of interest. However, these screens are highly variable in the number and size of deletions that are recovered. Here, we show that conducting excision screens in mus309 mutant flies that lack DmBlm, the Drosophila ortholog of the Bloom syndrome protein, increases the percentage and overall size of flanking deletions recovered after excision of either P or Minos elements.

Drosophila EcR-B ecdysone receptor isoforms are required for larval molting and for neuron remodeling during metamorphosis

During the metamorphic reorganization of the insect central nervous system, the steroid hormone 20-hydroxyecdysone induces a wide spectrum of cellular responses including neuronal proliferation, maturation, cell death and the remodeling of larval neurons into their adult forms. In Drosophila, expression of specific ecdysone receptor (EcR) isoforms has been correlated with particular responses, suggesting that different EcR isoforms may govern distinct steroid-induced responses in these cells. We have used imprecise excision of a P element to create EcR deletion mutants that remove the EcR-B promoter and therefore should lack EcR-B1 and EcR-B2 expression but retain EcR-A expression. Most of these EcR-B mutant animals show defects in larval molting, arresting at the boundaries between the three larval stages, while a smaller percentage of EcR-B mutants survive into the early stages of metamorphosis. Remodeling of larval neurons at metamorphosis begins with the pruning back of larval-specific dendrites and occurs as these cells are expressing high levels of EcR-B1 and little EcR-A. This pruning response is blocked in the EcR-B mutants despite the fact that adult-specific neurons, which normally express only EcR-A, can progress in their development. These observations support the hypothesis that different EcR isoforms control cell-type-specific responses during remodeling of the nervous system at metamorphosis.

Three new dominant C1 suppressor alleles in Zea mays

Three new dominant suppressor mutations of the C1 transcription regulator gene in maize – C1-IΔ1, C1-IΔ2 and C1-IΔ3 – are described that suppress anthocyanin colouration in kernels similar to the function of the C1-I standard inhibitor. The C1-IΔ mutations were induced by imprecise excision of an En/Spm transposon in the third exon of the C1 gene. These transposon footprints cause a frameshift in the C1 open reading frame that leads to truncated proteins due to an early stop codon 30 amino acids upstream of the wild-type C1 protein. Therefore, the C1-IΔ gene products lack the carboxy-terminal transcriptional activation domain of C1. The C1-I standard allele also lacks this domain and in addition differs in 17 amino acids from the wild-type C1 allele. The new C1-IΔ alleles provide evidence that deletion of the carboxy-terminal activation domain alone is sufficient to generate a dominant suppressive effect on the function of wild-type C1.

Analysis of an enhancer trap expressed in regenerating Drosophila imaginal discs

In Drosophila, imaginal discs are the undifferentiated larval precursors of the pattern of epidermal and sensory neural cells in each adult segment. Although cell fates are already specified by late third instar, disc fragments can either regenerate or duplicate after growth in culture. The outcome depends on signaling between cells across the healed wound and involves a redeployment of the expression patterns of selector genes and other disc pattern genes. We recently used the enhancer-trap method to screen for such genes that are expressed ectopically at the wound-heal site in imaginal discs undergoing regeneration. Here we report the cloning by plasmid rescue of transcribed sequences adjacent to one such enhancer-trap insertion. Using Northern analysis and in situ hybridization we show that one transcript is expressed in the embryo and in imaginal discs in a pattern similar to that of the enhancer trap. We also, by imprecise excision of the enhancer-trap insertion, generated a series of flanking deletions that were mapped using Southern analysis and complementation.Key words: Drosophila, imaginal discs, enhancer traps, regeneration genes.

Imprecise excision of the Caenorhabditis elegans transposon Tc1 creates functional 5' splice sites.

Imprecise excision of the Caenorhabditis elegans transposon Tc1 from a specific site of insertion within the unc-54 myosin heavy chain gene generates either wild-type or partial phenotypic revertants. Wild-type revertants and one class of partial revertants contain insertions of four nucleotides in the unc-54 third exon (Tc1 "footprints"). Such revertants express large amounts of functional unc-54 myosin despite having what would appear to be frameshifting insertions in the unc-54 third exon. We demonstrate that these Tc1 footprints act as efficient 5' splice sites for removal of the unc-54 third intron. Splicing of these new 5' splice sites to the normal third intron splice acceptor removes the Tc1 footprint from the mature mRNA and restores the normal translational reading frame. Partial revertant unc-54(r661), which contains a single nucleotide substitution relative to the wild-type gene, is spliced similarly, except that the use of its new 5' splice site creates a frameshift in the mature mRNA rather than removing one. In all of these revertants, two alternative 5' splice sites are available to remove intron 3. We determined the relative efficiency with which each alternative 5' splice site is used by stabilizing frameshifted mRNAs with smg(-) genetic backgrounds. In all cases, the upstream member of the two alternative sites is used preferentially (> 75% utilization). This may reflect an inherent preference of the splicing machinery for the upstream member of two closely spaced 5' splice sites. Creation of new 5' splice sites may be a general characteristic of Tc1 insertion and excision events.

Inverted DNA repeats: a source of eukaryotic genomic instability.

While inverted DNA repeats are generally acknowledged to be an important source of genetic instability in prokaryotes, relatively little is known about their effects in eukaryotes. Using bacterial transposon Tn5 and its derivatives, we demonstrate that long inverted repeats also cause genetic instability leading to deletion in the yeast Saccharomyces cerevisiae. Furthermore, they induce homologous recombination. Replication plays a major role in the deletion formation. Deletions are stimulated by a mutation in the DNA polymerase delta gene (pol3). The majority of deletions result from imprecise excision between small (4- to 6-bp) repeats in a polar fashion, and they often generate quasipalindrome structures that subsequently may be highly unstable. Breakpoints are clustered near the ends of the long inverted repeats (< 150 bp). The repeats have both intra- and interchromosomal effects in that they also create hot spots for mitotic interchromosomal recombination. Intragenic recombination is 4 to 18 times more frequent for heteroalleles in which one of the two mutations is due to the insertion of a long inverted repeat, compared with other pairs of heteroalleles in which neither mutation has a long repeat. We propose that both deletion and recombination are the result of altered replication at the basal part of the stem formed by the inverted repeats.

Inverted DNA repeats: a source of eukaryotic genomic instability

While inverted DNA repeats are generally acknowledged to be an important source of genetic instability in prokaryotes, relatively little is known about their effects in eukaryotes. Using bacterial transposon Tn5 and its derivatives, we demonstrate that long inverted repeats also cause genetic instability leading to deletion in the yeast Saccharomyces cerevisiae. Furthermore, they induce homologous recombination. Replication plays a major role in the deletion formation. Deletions are stimulated by a mutation in the DNA polymerase delta gene (pol3). The majority of deletions result from imprecise excision between small (4- to 6-bp) repeats in a polar fashion, and they often generate quasipalindrome structures that subsequently may be highly unstable. Breakpoints are clustered near the ends of the long inverted repeats (< 150 bp). The repeats have both intra- and interchromosomal effects in that they also create hot spots for mitotic interchromosomal recombination. Intragenic recombination is 4 to 18 times more frequent for heteroalleles in which one of the two mutations is due to the insertion of a long inverted repeat, compared with other pairs of heteroalleles in which neither mutation has a long repeat. We propose that both deletion and recombination are the result of altered replication at the basal part of the stem formed by the inverted repeats.