Screening insertion libraries for mutations in many genes simultaneously using DNA microarrays

We describe a method to screen pools of DNA from multiple transposon lines for insertions in many genes simultaneously. We use thermal asymmetric interlaced–PCR, a hemispecific PCR amplification protocol that combines nested, insertion-specific primers with degenerate primers, to amplify DNA flanking the transposons. In reconstruction experiments with previously characterized Arabidopsis lines carrying insertions of the maize Dissociation (Ds) transposon, we show that fluorescently labeled, transposon-flanking fragments overlapping ORFs hybridize to cognate expressed sequence tags (ESTs) on a DNA microarray. We further show that insertions can be detected in DNA pools from as many as 100 plants representing different transposon lines and that all of the tested, transposon-disrupted genes whose flanking fragments can be amplified individually also can be detected when amplified from the pool. The ability of a transposon-flanking fragment to hybridize declines rapidly with decreasing homology to the spotted DNA fragment, so that only ESTs with >90% homology to the transposon-disrupted gene exhibit significant cross-hybridization. Because thermal asymmetric interlaced–PCR fragments tend to be short, use of the present method favors recovery of insertions in and near genes. We apply the technique to screening pools of new Ds lines using cDNA microarrays containing ESTs for ≈1,000 stress-induced and -repressed Arabidopsis genes.

Light regulation of type IV pilus-dependent motility by chemosensor-like elements in Synechocystis PCC6803

To optimize photosynthesis, cyanobacteria move toward or away from a light source by a process known as phototaxis. Phototactic movement of the cyanobacterium Synechocystis PCC6803 is a surface-dependent phenomenon that requires type IV pili, cellular appendages implicated in twitching and social motility in a range of bacteria. To elucidate regulation of cyanobacterial motility, we generated transposon-tagged mutants with aberrant phototaxis; mutants were either nonmotile or exhibited an “inverted motility response” (negative phototaxis) relative to wild-type cells. Several mutants contained transposons in genes similar to those involved in bacterial chemotaxis. Synechocystis PCC6803 has three loci with chemotaxis-like genes, of which two, Tax1 and Tax3, are involved in phototaxis. Transposons interrupting the Tax1 locus yielded mutants that exhibited an inverted motility response, suggesting that this locus is involved in controlling positive phototaxis. However, a strain null for taxAY1 was nonmotile and hyperpiliated. Interestingly, whereas the C-terminal region of the TaxD1 polypeptide is similar to the signaling domain of enteric methyl-accepting chemoreceptor proteins, the N terminus has two domains resembling chromophore-binding domains of phytochrome, a photoreceptor in plants. Hence, TaxD1 may play a role in perceiving the light stimulus. Mutants in the Tax3 locus are nonmotile and do not make type IV pili. These findings establish links between chemotaxis-like regulatory elements and type IV pilus-mediated phototaxis.

The Two Genes Encoding Starch-Branching Enzymes IIa and IIb Are Differentially Expressed in Barley1

The sbeIIa and sbeIIb genes, encoding starch-branching enzyme (SBE) IIa and SBEIIb in barley (Hordeum vulgare L.), have been isolated. The 5′ portions of the two genes are strongly divergent, primarily due to the 2064-nucleotide-long intron 2 in sbeIIb. The sequence of this intron shows that it contains a retro-transposon-like element. Expression of sbeIIb but not sbeIIa was found to be endosperm specific. The temporal expression patterns for sbeIIa and sbeIIb were similar and peaked around 12 d after pollination. DNA gel-blot analysis demonstrated that sbeIIa and sbeIIb are both single-copy genes in the barley genome. By fluorescence in situ hybridization, the sbeIIa and sbeIIb genes were mapped to chromosomes 2 and 5, respectively. The cDNA clones for SBEIIa and SBEIIb were isolated and sequenced. The amino acid sequences of SBEIIa and SBEIIb were almost 80% identical. The major structural difference between the two enzymes was the presence of a 94-amino acid N-terminal extension in the SBEIIb precursor. The (β/α)8-barrel topology of the α-amylase superfamily and the catalytic residues implicated in branching enzymes are conserved in both barley enzymes.

Active transposition in zebrafish.

We have identified a new family of Tc1-like transposons in the zebrafish, Danio rerio. The sequence of a candidate active transposon, deduced from sample Tzf elements, shows limited resemblance to the previously described Tdr1 elements of zebrafish. Both the Tzf and the Tdr elements are extremely abundant in zebrafish. We describe here a general strategy for detecting transposition events in a complex genome and demonstrate its utility by selectively monitoring hundreds of potentially active Tzf copies in the zebrafish genome against a background of other related elements. We have followed members of a zebrafish pedigree, using this two-dimensional transposon display strategy, to identify the first examples of active transposition of such elements in vertebrates.

Unique chromosomal regions associated with virulence of an avian pathogenic Escherichia coli strain.

The avian pathogenic Escherichia coli strain (chi)7122 (serotype O78:K80:H9) causes airsacculitis and colisepticemia in chickens. To identify genes associated with avian disease, a genomic subtraction technique was performed between strain (chi)7122 and the E. coli K-12 strain (chi)289. The DNA isolated using this method was found only in strain (chi)7122 and was used to identify cosmid clones carrying unique DNA from a library of (chi)7122 that were then used to map the position of unique DNA on the E. coli chromosome. A total of 12 unique regions were found, 5 of which correspond to previously identified positions for unique DNA sequence in E. coli strains. To assess the role each unique region plays in virulence, mutants of (chi)7122 were constructed in which a segment of unique DNA was replaced with E. coli K-12 DNA by cotransduction of linked transposon insertions in DNA flanking the unique sequence. The resulting replacement mutants were assessed for inability to colonize the air sac and cause septicemia in 2-week-old white Leghorn chickens. Two mutants were found to be avirulent when injected into the right caudal air sac of 2-week-old chickens. One avirulent mutant, designated (chi)7145, carries a replacement of the rfb locus at 44 min, generating a rough phenotype. The second mutant is designated (chi)7146, and carries a replacement at position 0.0 min on the genetic map. Both mutants could be complemented to partial virulence by cosmids carrying sequences unique to (chi)7122.

Formation of chimeric DNA primer extension products by template switching onto an annealed downstream oligonucleotide.

Oligodeoxynucleotide sequences are described that anneal to a template downstream of a priming site. During polymerase-catalyzed extension of the primer, the extending primer shifts from the original template to a segment of the annealed oligonucleotide that acts as an alternative template. The resulting chimeric extended primer has one segment that is complementary to the template and a second segment that is complementary to the oligonucleotide. The influence of the sequence elements of the oligonucleotide and the reaction conditions on template switching have been explored. The sequence requirements for template switching are compared to those for transposon excision.

Identification of a virulence locus encoding a second type III secretion system in Salmonella typhimurium.

Mapping the insertion points of 16 signature-tagged transposon mutants on the Salmonella typhimurium chromosome led to the identification of a 40-kb virulence gene cluster at minute 30.7. This locus is conserved among all other Salmonella species examined but is not present in a variety of other pathogenic bacteria or in Escherichia coli K-12. Nucleotide sequencing of a portion of this locus revealed 11 open reading frames whose predicted proteins encode components of a type III secretion system. To distinguish between this and the type III secretion system encoded by the inv/spa invasion locus known to reside on a pathogenicity island, we refer to the inv/spa locus as Salmonella pathogenicity island (SPI) 1 and the new locus as SPI2. SPI2 has a lower G+C content than that of the remainder of the Salmonella genome and is flanked by genes whose products share greater than 90% identity with those of the E. coli ydhE and pykF genes. Thus SPI2 was probably acquired horizontally by insertion into a region corresponding to that between the ydhE and pykF genes of E. coli. Virulence studies of SPI2 mutants have shown them to be attenuated by at least five orders of magnitude compared with the wild-type strain after oral or intraperitoneal inoculation of mice.

Mg-SINE: a short interspersed nuclear element from the rice blast fungus, Magnaporthe grisea.

A short interspersed nuclear element, Mg-SINE, was isolated and characterized from the genome of the rice blast fungus, Magnaporthe grisea. Mg-SINE was isolated as an insertion element within Pot2, an inverted-repeat transposon from M. grisea and shows typical features of a mammalian SINE. Mg-SINE is present as a 0.47-kb interspersed sequence at approximately 100 copies per haploid genome in both rice and non-rice isolates of M. grisea, indicating a common evolutionary origin. Secondary structure analysis of Mg-SINE revealed a tRNA-related region at the 5' end which folds into a cloverleaf structure. Genomic fusions resulting in chimeric Mg-SINEs (Ch-SINEs) composed of a sequence homologous to Mg-SINE at the 3' end and an unrelated sequence at its 5' end were also isolated, indicating that this and other DNA rearrangements mediated by these elements may have a major effect on the genomic architecture of this fungus.

Genetic rearrangements in the rfb regions of Vibrio cholerae O1 and O139.

The recent emergence of a pathogenic new non-O1 serotype (O139) of Vibrio cholerae has led to numerous studies in an attempt to identify the origins of this new strain. Our studies indicate that O139 strains have clear differences in the surface polysaccharides when compared with O1 strains: the lipopolysaccharide can be described as semi-rough. Southern hybridization with the O1 rfb region demonstrates that O139 strains no longer contain any of the rfb genes required for the synthesis of the O1 O-antigen or its modification and also lack at least 6 kb of additional contiguous DNA. However, O139 strains have retained rfaD and have a single open reading frame closely related to three small open reading frames of the O1 rfb region. This region is closely related to the H-repeat of Escherichia coli and to the transposases of a number of insertion sequence elements and has all the features of an insertion sequence element that has been designated VcIS1. Transposon insertion mutants defective in O139 O-antigen (and capsule) biosynthesis map to the same fragment as VcIS1. Preliminary sequence data of complementing clones indicate that this DNA encodes a galactosyl-transferase and other enzymes for the utilization of galactose in polysaccharide biosynthesis. We propose a mechanism by which both the Ogawa serotype of O1 strains and the O139 serotype strains may have evolved.

Introduction of the transposable element Minos into the germ line of Drosophila melanogaster.

A transposon based on the transposable element Minos from Drosophila hydei was introduced into the genome of Drosophila melanogaster using transformation mediated by the Minos transposase. The transposon carries a wild-type version of the white gene (w) of Drosophila inserted into the second exon of Minos. Transformation was obtained by injecting the transposon into preblastoderm embryos that were expressing transposase either from a Hsp70-Minos fusion inserted into the genome via P-element-mediated transformation or from a coinjected plasmid carrying the Hsp70-Minos fusion. Between 1% and 6% of the fertile injected individuals gave transformed progeny. Four of the insertions were cloned and the DNA sequences flanking the transposon ends were determined. The "empty" sites corresponding to three of the insertions were amplified from the recipient strain by PCR, cloned, and sequenced. In all cases, the transposon has inserted into a TA dinucleotide and has created the characteristic TA target site duplication. In the absence of transposase, the insertions were stable in the soma and the germ line. However, in the presence of the Hsp70-Minos gene the Minos-w transposon excises, resulting in mosaic eyes and germ-line reversion to the white phenotype. Minos could be utilized as an alternative to existing systems for transposon tagging and enhancer trapping in Drosophila; it might also be of use as a germ-line transformation vector for non-Drosophila insects.

Structural domains of IS10 transposase and reconstitution of transposition activity from proteolytic fragments lacking an interdomain linker.

All of the DNA cleavage and strand transfer events required for transposition of insertion sequence IS10 are carried out by a 46-kDa IS10-encoded transposase protein. Limited proteolysis demonstrates that transposase has two principal structural domains, a 28-kDa N-terminal domain (N alpha beta; aa 1-246) and a 17-kDa C-terminal domain (C; aa 256-402). The two domains are connected by a 1-kDa proteolytic-sensitive linker region (aa 247-255). The N-terminal domain N alpha beta can be further subdivided into domains N alpha and N beta by a weaker protease-sensitive site located 6 kDa (53 aa) from the N terminus. The N beta and N alpha beta fragments are capable of nonspecific DNA binding as determined by Southwestern blot analysis. None of the fragments alone is capable of carrying out the first step of transposition, assembly of a synaptic complex containing a pair of transposon ends. Remarkably, complete transposition activity can be reconstituted by mixing fragment N alpha beta and fragment C, with or without the intervening linker region. We infer that the structural integrity of transposase during the transitions involved in the chemical steps of the transposition reaction is maintained independent of the linker, presumably by direct contacts between and among the principal domains. Reconstitution of activity in the absence of the linker region is puzzling, however, because mutations that block strand transfer or affect insertion specificity alter linker region residues. Additional reconstitution experiments demonstrate that the N alpha region is dispensable for formation of a synaptic complex but is required for complexes to undergo cleavage.

The two single-strand cleavages at each end of Tn10 occur in a specific order during transposition.

During Tn10 transposition, the element is excised from the donor site by double-strand cleavages at the two transposon ends. Double-strand cleavage is a central step in the nonreplicative transposition reaction of many transposons in both prokaryotes and eukaryotes. Evidence is presented to show that the Tn10 double-strand cut is made by an ordered, sequential cleavage of the two strands. The transferred strand is cut first, and then the nontransferred strand is cleaved. The single-strand nicked intermediate is seen to accumulate when Mn2+ is substituted for Mg2+ in the reaction or when certain mutant transposases are used. The fact that the transferred strand is cleaved before the non-transferred strand implies that the order of strand cleavages is not the determining factor that precludes a replicative mechanism of transposition.

Structure, inheritance, and transcriptional effects of Pce1, an insertional element within Phanerochaete chrysosporium lignin peroxidase gene lipI.

A 1747-bp insertion within a lignin peroxidase allele of Phanerochaete chrysosporium BKM-F-1767 is described. Pce1, the element, lies immediately adjacent to the fourth intron of lip12. Southern blots reveal the presence of Pce1-homologous sequences in other P. chrysosporium strains. Transposon-like features include inverted terminal repeats and a dinucleotide (TA) target duplication. Atypical of transposons, Pce1 is present at very low copy numbers (one to five copies), and conserved transposase motifs are lacking. The mutation transcriptionally inactivates lip12 and is inherited in a 1:1 Mendelian fashion among haploid progeny. Thus, Pce1 is a transposon-like element that may play a significant role in generating ligninolytic variation in certain P. chrysosporium strains.