Analysis of Tn5 inversion events inEscherichia coli plasmids

The ability of the bacterial transposon Tn5 to undergo sequence inversion in Rec⁺ Escherichia coli cells as a result of recombination between its duplicated IS50 elements was examined using specially designed plasmid constructs. Surprisingly, recombination events in the IS50 elements that led to crossover and therefore Tn5 inversion could be detected at a frequency of only 10^–5. This was approximately an order of magnitude lower than the frequency of IS50 recombination that led to conversion events (i.e. non-reciprocal recombination) without crossover, and at least two orders of magnitude lower than the frequency of intermolecular recombination between IS50 elements on two different plasmids. These rare conversion and inversion events in Tn5 appeared to be due to intramolecular recombination and not simply to multiple rounds of reciprocal crossing over, since the heterodimeric intermediates that would be generated during the latter process could be readily isolated but were shown to yield a completely different set of plasmid products upon resolution.     

The Evolutionary History of the Transposable Element Penelope in the Drosophila virilis Group of Species

We have used phylogenetic techniques to study the evolutionary history of the Penelope transposable element in the Drosophila virilis species group. Two divergent types of Penelope have been detected, one previously described, clade I, and a new one which we have termed clade III. The phylogeny of some copies of the Penelope clade I element was partially consistent with the species phylogeny of the D. montana subphylad, suggesting cospeciation and allowing the estimation of the evolutionary rate of Penelope. Divergence times of elements found in different species are younger than the age of the species, suggesting horizontal transfer events. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Dmitri Petrov]     

Localization of <Emphasis Type="Italic">pms3</Emphasis>, a gene for photoperiod-sensitive genic male sterility,to a 28.4-kb DNA fragment

Photoperiod-sensitive genic male-sterile (PSGMS) rice, in which pollen fertility is regulated by day-length, originally arose as a natural mutant in the rice cultivar Nongken 58 (Oryza sativa ssp. japonica). Previous studies identified pms3 on chromosome 12 as the locus of the original PSGMS mutation. In this study we have assigned the pms3 locus to a 28.4-kb DNA fragment by genetic and physical mapping. A cross between Nongken 58S (PSGMS line) and DH80 was used to produce an F₂ population of about 7000 plants, from which 892 highly sterile individuals were obtained for recombination analysis. By analyzing recombination events in the sterile individuals using a total of 157 RFLP probes from a BAC contig covering the pms3 region, the pms3 locus was localized to a sub-region of less than 1.7 cM. Further analysis of recombination events using 49 additional probes isolated from this sub-region identified markers flanking the pms3 region on each side; these markers are only 28.4-kb apart. Sequence analysis of this fragment predicted the presence of five ORFs, found high homology with two ESTs in public databases, and detected three SNPs between the mutant and the wild-type parents, which may be helpful for identifying a candidate gene for pms3.     

<Emphasis Type="Italic">Drosophila</Emphasis><Emphasis Type="Italic">P</Emphasis> transposons of the urochordata <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The distribution of polymorphic Alu insertions within the MHC class I HLA-B7 and HLA-B57 haplotypes

There are five polymorphic Alu insertion (POALIN) loci within the major histocompatibility complex (MHC) class I region that have been strongly associated with HLA class I alleles, such as HLA-A1, HLA-A2 and HLA-B57. In order to assess the variability and frequency of POALIN distribution within two common HLA-B haplotypes, we detected the presence of the MHC class I POALIN by PCR in a panel of 15 individuals with HLA-B57 and 47 homozygous individuals with 7.1 AH (HLA-B7, -Cw7, -A3) obtained from the Australian Bone Marrow Donor Registry, and also from four families (25 individuals) containing the HLA-B57 allele. Only two of the 47 HLA-B7 genotypes had a detectable POALIN, whereas all of the HLA-B57 genotypes had at least one or more POALINs present, confirming that certain MHC class I haplotypes are relatively POALIN-free and others are POALIN-enriched. Six distinct HLA-B57 haplotypes, based on differences at the HLA-A locus and three of five POALIN loci, were identified that appear to have evolved by different mechanisms, including either by shuffling different combinations of conserved alpha and beta blocks or by recombination events involving two or more previously generated HLA-B57 haplotypes.     

Male recombination with single and homologous P elements in Drosophila melanogaster

It has previously been shown that, in the presence of a source of P element transposase, male recombination in Drosophila melanogaster is induced at a rate of about 1% in the region of a single P[CaSpeR] element. This paper shows that recombinant chromosomes retain unaltered P[CaSpeR] elements at the original site in a high proportion of cases. This result is incompatible with a simple model in which recombination occurs by resolution of a Holliday junction following P element excision and repair. It has also previously been shown that homozygous regions containing a P element produce male recombination levels of 10–20%, an order of magnitude higher than that given by a single element. This paper shows that reciprocal recombinant chromosomes retaining P[CaSpeR] elements can be combined to produce similarly high levels of recombination. This result potentially allows for recombinant chromosomes from homologous recombination to be analysed at the molecular level in the region of the inserted element.     

Extramacrochaetae,a trans-acting gene of the achaete-scute complex of Drosophila involved in cell communication

Summary Phenotypic analyses of genetic combinations involving the gene extramacrochaetae (emc) reveal its participation in the differentiation of both sensory elements and wing veins. The study of near-amorphic alleles of emc in mitotitc recombination clones indicates that it also affects cell proliferation. These clones show abnormal sizes, shapes and spatial distribution. They differentiate extra sensory elements as well as extra veins. A gain of function mutation in the gene causes opposite phenotypes in both differentiation systems. The effects of the mutant on proliferation and patterning are consistent with the emc gene being involved in the transfer of information between neighbouring cells, which leads to the spatial expression of the achaetescute gene complex and genes involved in vein formation.     

Estimation of gametic frequencies from F2 populations using the EM algorithm and its application in the analysis of crossover interference in rice

The gametes produced in meiosis provide information on the frequency of recombination and also on the interdependence of recombination events, i.e. interference. Using F₂ individuals, it is not possible in all cases to derive the gametes, which have fused, and which provide the information about interference unequivocally when three or more segregating markers are considered simultaneously. Therefore, a method was developed to estimate the gametic frequencies using a maximum likelihood approach together with the expectation maximisation algorithm. This estimation procedure was applied to F₂ mapping data from rice (Oryza sativa L.) to carry out a genome-wide analysis of crossover interference. The distribution of the coefficient of coincidence in dependence on the recombination fraction revealed for all chromosomes increasing positive interference with decreasing interval size. For some chromosomes this mutual inhibition of recombination was not so strong in small intervals. The centromere had a significant effect on interference. The positive interference found in the chromosome arms were reduced significantly when the intervals considered spanned the centromere. Two chromosomes even demonstrated independent recombination and slightly negative interference for small intervals including the centromere. Different marker densities had no effect on the results. In general, interference depended on the frequency of recombination events in relation to the physical length. The strength of the centromere effect on interference seemed to depend on the strength of recombination suppression around the centromere.Electronic Supplementary Material Supplementary material is available for this article at     

Advent of a New Retrotransposon Structure: The Long Form of the Veju Elements

Transposable elements are the main component of plant genomes, especially in grass species. In a previous analysis, we have identified two unusual types of Class I elements, two homologous Veju TRIM elements, but with an unusual long structure. They are formed by the junction of a yet unidentified segment labelled unknown DNA, flanked by the borders of the classical Veju element. Here, we show that the long (Veju_L) and the short forms (Veju_S) coexist within wheat genomes. The associated unknown DNA had always the same origin, and the Veju_L came probably from either illegitimate recombinations or ‘template switching’ between the Veju_S and another unique unknown DNA sequence. This junction then evolved differently within wheat genomes.     

An efficient method for gene disruption in Neurospora crassa

The frequency with which transforming DNA undergoes homologous recombination at a chromosomal site can be quite low in some fungal systems. In such cases, strategies for gene disruption or gene replacement must either select against ectopic integration events or provide easy screening to identify homologous site, double-crossover insertion events. A protocol is presented for efficient isolation of Neurospora crassa strains carrying a definitive null allele in a target gene. The protocol relies on the presence of a selectable marker flanking a disrupted plasmid-borne copy of the gene, and in the case presented led to a seven-fold enrichment for putative homologous site replacement events. In addition, a polymerase chain reaction assay is utilized for rapid identification of homologous recombinants among the remaining candidates. This protocol was used to identify 3 isolates, out of 129 primary transformants, which have a disruption in the Neurospora ccg-1 gene. The method should be applicable to a variety of fungal systems in which two selectable markers can be expressed, including those in which homologous recombination rates are too low to allow easy identification of homologous site insertions by the more traditional molecular method of Southern analysis. In addition to disrupting target genes for the purpose of generating null mutations, this method is useful for the targeting of reporter gene fusions to a native chromosomal site for the purpose of studying gene regulation.     

Evolution of the LINE-like I element in the Drosophila melanogaster species subgroup

LINE-like retrotransposons, the so-called I elements, control the system of I-R (inducer-reactive) hybrid dysgenesis in Drosophila melanogaster. I elements are present in many Drosophila species. It has been suggested that active, complete I elements, located at different sites on the chromosomes, invaded natural populations of D. melanogaster recently (1920–1970). But old strains lacking active I elements have only defective I elements located in the chromocenter. We have cloned I elements from D. melanogaster and the melanogaster subgroup. In D. melanogaster, the nucleotide sequences of chromocentral I elements differed from those on chromosome arms by as much as 7%. All the I elements of D. mauritiana and D. sechellia are more closely related to the chromosomal I elements of D. melanogaster than to the chromocentral I elements in any species. No sequence difference was observed in the surveyed region between two chromosomal I elements isolated from D. melanogaster and one from D. simulans. These findings strongly support the idea that the defective chromocentral I elements of D. melanogaster originated before the species diverged and the chromosomal I elements were eliminated. The chromosomal I elements reinvaded natural populations of D. melanogaster recently, and were possibly introduced from D. simulans by horizontal transmission.     

A two-element Enhancer-Inhibitor transposon system in Arabidopsis thaliana

The Enhancer-Inhibitor (En-I), also known as Suppressor-mutator (Spm-dSpm), transposable element system of maize was modified and introduced into Arabidopsis by Agrobacterium tumefaciens transformation. A stable En/Spm transposase source under control of the CaMV 35S promoter mediated frequent transposition of I/dSpm elements. Transposition occurred continuously throughout plant development over at least seven consecutive plant generations after transformation. New insertions were found at both linked and unlinked positions relative to a transposon donor site. The independent transposition frequency was defined as a transposition parameter, which quantified the rate of unique insertion events and ranged from 7.8% to 29.2% in different populations. An increase as well as a decrease in I/dSpm element copy number was seen at the individual plant level, but not at the population level after several plant generations. The continuous, frequent transposition observed for this transposon system makes it an attractive tool for use in gene tagging in Arabidopsis.     

Mitotic recombination between dispersed but related rRNA genes of Schizosaccharomyces pombe generates a reciprocal translocation

Summary Recombination between dispersed yet related serine tRNA genes of Schizosaccharomyces pombe does occur during mitosis but it is approximately three orders of magnitude less frequent than in meiosis. Two mitotic events have been studied in detail. In the first, a sequence of at least 18 nucleotides has been transferred from the donor sup3 gene on the right arm of chromosome I to the related acceptor gene sup12 on the left arm of the same chromosome, thereby leading to the simultaneous change of 8 bp in the acceptor gene. This event must be explained in terms of recombination rather than mutation. It is assumed that it represents mitotic gene conversion, although it was not possible to demonstrate that the donor gene had emerged unchanged from the event. The second case reflects an interaction between sup9 on chromosome III and sup3 on chromosome I. Genetic and physical analysis allows this event to be described as mitotic gene conversion associated with crossingover. The result of this event is a reciprocal translocation. No further chromosomal aberrations were found among an additional 700 potential intergenic convertants tested. Thus intergenic conversion is much less frequently associated with crossingover than allelic conversion. However, the rare intergenic conversion events associated with crossingover provide a molecular mechanism for chromosomal rearrangements.     

Rearrangements of nitrogen fixation (nif) genes in the heterocystous cyanobacteria

In the vegetative cells of heterocystous cyanobacteria, such asAnabaena, two Operons harbouring the nitrogen fixaton (nif) genes contain two separate intervening DNA elements resulting in the dispersion of genes and impaired gene expression. A 11 kb element disrupts thenifD gene in thenifH, D-K operon. It contains a 11 bp sequence (GGATTACTCCG) directly repeated at its ends and harbours a gene,xisA, which encodes a site-specific recombinase. A large 55 kb element interrupts thefdxN gene in thenifB fdxN-nifS-nifU operon. It contains two 5 bp direct repeats (TATTC) at its ends and accommodates at least one gene,xisF, which encodes another site-specific recombinase. During heterocyst differentiation both the discontinuities are precisely excised by two distinct site-specific recombination events. One of them is brought about by the XisA protein between the 11 bp direct repeats. The second one is caused by the XisF protein and occurs between the 5 bp direct repeats. As a consequence the 11kb and 55 kb elements are removed from the chromosome as circles and functionalnif Operons are created. Nitrogenase proteins are then expressed from the rearranged genes in heterocysts and aerobic nitrogen fixation ensues. How these elements intruded thenif genes and how and why are they maintained in heterocystous cyanobacteria are exciting puzzles engaging considerable research effort currently. The unique developmental regulation of these gene rearrangements in heterocystous cyanobacteria is discussed.     

A comparison of within-plant karyological heterogeneity between agamospermous and sexualTaraxacum (Compositae) as assessed by the nucleolar organiser chromosome

Morphological variation for the NOR chromosome was studied for four half-siblings of a sexual outbreedingTaraxacum, for three siblings of the obligate agamospermT. pseudohamatum, and for two individuals of the agamospermT. brachyglossum. No rearrangement was detected for the 113 chromosomes of sexuals, or for 41 chromosomes of two agamospermous individuals. In the other three agamospermous individuals, 3/16, 5/50, and 5/20 chromosomes showed evidence of chromosomal rearrangement. The majority of rearrangement events (10/13) occurred to the satellite rather than to the body of the NOR-chromosome. It is considered that such high levels of somatic chromosomal rearrangement in agamospermousTaraxacum may be the result of activity by transposable genetic elements. This recombination may be of selective advantage to asexual plants which cannot generate genetic variability through the sexual process.     

Isolation and characterization of a unique protease from sporulating cells of Bacillus subtilis

Two proteases, designated I and II, have been isolated from sporulating cells of Bacillus subtilis. They were partially purified by ammonium sulfate fractionation, Sephadex chromatography and affinity columns. Protease I was found to be similar to an already characterized B. subtilis protease. Protease II is trypsin-like in its substrate specificity and is distinct from protease I in its pH optimum, pH stability, molecular weight, substrate specificity, heat stability and sensitivity to various inhibitors. While both enzymes were produced primarily during sporulation, they attained maximum levels of activity at different times. Distinct functions for these proteases in post exponential B. subtilis are likely.     

Restoring full pollen fertility in transgenic male-sterile tobacco (Nicotiana tabacum L.) by Cre-mediated site-specific recombination

Producing hybrid seed requires an efficient pollination control system to prevent unwanted self-pollination. For further breeding, it would be advantageous to restore pollen fertility in the hybrids. In this work we demonstrate the use of tapetum-specific expression of a stilbene synthase (sts) transgene to induce pollen sterility in tobacco as has been shown previously. The sts-coding region was flanked by loxP recognition sites for Cre-recombinase. From 10 T₀-plants obtained, five proved to be male-sterile. They had smaller flowers with shorter stamina, but the vegetative phenotype was just as in the wild-type. Crossing male-sterile sts-plants with tobacco lines expressing the cre recombinase transgene resulted in site-specific recombination in the hybrids. GUS activity caused by fusion of the tap1-promoter with a promoterless gusA coding region indicated recombination events already in early stages of flower bud development. In all plants which had contained single or double sts-copies before crossing, these were excised, and pollen fertility was fully restored. The phenotype of these restored plants was as in wild-type controls. Contrary, from male sterile plants containing multiple copies of the sts-gene, not all copies were removed, and pollen sterility was maintained.     

Excision of the <Emphasis Type="Italic">nifD</Emphasis> element in the heterocystous cyanobacteria

Heterocyst differentiation in cyanobacteria is accompanied by developmentally regulated DNA rearrangements that occur within the nifD, fdxN, and hupL genes. These genetic elements are excised from the genome by site-specific recombination during the latter stages of differentiation. The nifD element is excised by the recombinase, XisA, located within the element. Our objective was to examine the XisA-mediated excision of the nifD element. To accomplish this, we observed the ability of XisA to excise substrate plasmids that contained the flanking regions of the nifD element in an E. coli host. Using PCR directed mutagenesis, nucleotides in the nifD element flanking regions in substrate plasmids were altered and the effect on recombination was determined. Results indicate that only certain nucleotides within and surrounding the direct repeats are involved in excision. In some nucleotide positions, the presence of a purine versus a pyrimidine greatly affected recombination. Our results also indicated that the site of excision and branch migration occurs in a 6 bp region within the direct repeats. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fine-Scale Crossover Rate Heterogeneity in <Emphasis Type="Italic">Drosophila pseudoobscura</Emphasis>

Broad-scale differences in crossover rate across the genome have been characterized in most genomes studied. Fine-scale differences, however, have only been examined in a few taxa, such as Arabidopsis, yeast, humans, and mice. No prior studies have directly looked for fine-scale recombination rate heterogeneity in Drosophila. We produced 370 Drosophila pseudoobscura containing a crossover event within the 2-megabase (MB) region between the genes yellow and white. We then examined 19 intervals within this region and determined where the crossovers occurred. We found that recombination events occur nonrandomly on a small scale and that mild “hotspots“ of a few kilobases exist in Drosophila. Among the regions studied, recombination rates varied from 1.4 to 52 cM/MB. We also observed a trend toward high codon bias in regions of high recombination. Finally, we identified a significantly positive correlation between recombination rate and simple repeats, as well as the motif CACAC. These sequence features may contribute to broad-scale variation in crossover rate and, thus, shed light on features associated with crossover rate heterogeneity at a genome-wide scale. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Dmitri Petrov]     

Über Plastidenstapel beiBotrydiopsis alpina sowie Anlage und Vermehrung der Stigmen bei dieser undHeterococcus (Xanthophyceae)

Under certain conditions inBotrydiopsis alpina stacks of chloroplasts are formed. They consist of up to 8 elements. In contrast to what is known from other algae in zoosporangia of this species and ofHeterococcus caespitosus, stigmata are formed in early developmental stages. They are reproduced together with the chloroplasts, in which they occupy a position at the edge and near the existing or future incision. At the side of the old stigma a new one is formed, and partitioning of the chloroplast between these two leads to their distribution to the daughter chloroplasts. Young daughter cells in the zoosporangia ofBotrydiopsis alpina contain one chloroplast which undergoes a last unequal division giving rise to one astigmate and usually somewhat smaller and to one stigmate chloroplast. In both species the capacity for locomotion may be suppressed, the presumptive zoospores thereby becoming aplanospores. Autospores in the proper sense were not observed. Their development quite generally is different from that of aplanospores (and zoospores), and both types of spores should be distinguished.

Herrn Professor Dr.Lothar Geitler zum 80. Geburtstag in Verehrung gewidmet.