Molecular and histological characterizations of the Om(2D) mutants in Drosophila ananassae.

A series of transposon-induced optic morphology (Om) mutants found in a hypermutable marker stock of Drosophila ananassae provides a useful system for analyzing the molecular mechanism of eye morphogenesis. In the present study, one of the 25 Om loci so far reported, Om(2D), has been subjected to histological and molecular analyses as a first step toward understanding the role of Om genes in eye morphogenesis. Histological abnormalities observed during eye morphogenesis of the mutant, i.e. cell death within the eye-antennal discs of third instar larvae, and loss of the lamina, disorganized ommatidia and atrophied optic lobes in adults, were all comparable to those reported with various eye morphology mutants of D. melanogaster. Approximately 25 kb of genomic DNA including the Om (2D) locus was cloned by tom tagging. Southern blot and cloning analyses of two alleles of the Om (2D) locus revealed that insertions of the tom element occurred at three sites within 359 bp; two tandemly arrayed toms sharing one long terminal repeat at the junction and an internally deleted tom were present 359 bp apart from each other in Om (2D) 63, while a single tom in reverse orientation was present within the 359 bp in Om (2D) 10a. Host DNA sequences at the three insertion sites were TATAT or AATAT, and ATAT was duplicated upon the tom insertion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification ofTnr3, aSuppressor-Mutator/Enhancer-like transposable element from rice

We isolated members of the retroposon family p-SINE1 in rice and found that one member contained an insertion. A 3-bp sequence at the insertion site within p-SINE1 appeared duplicated. The insertion sequence, 1536 bp in length, carried imperfect inverted repeats of about 13 bp at its termini which begin with 5-CACTA--- -3; these repeats are similar to those found in members of theEn/Spm transposable element family. These results indicate that the insertion sequence is a transposable element belonging to theEn/Spm family and is thus namedTnr3 (transposable element inrice no.3). In fact,Tnr3 carried long subterminal regions containing direct and inverted repeats of short DNA sequences of 15 bp, another characteristic of theEN/Spm family. The subterminal repeat sequences inTnr3 are, however, of two kinds, although they share homology with each other.Tnr3 and its relatives were present in multiple copies in rice. Considering the length ofTnr3, it cannot represent an autonomous type element, but is a non-autonomous element probably derived by deletion from an autonomous transposon.     

The isolation of Ant1, a transposable element from Aspergillus niger

A transposable element has been isolated from the industrially important fungus Aspergillus niger (strain N402). The element was identified as an insertion sequence within the coding region of the nitrate reductase gene. It had inserted at a TA site and appeared to have duplicated the target site upon insertion. The isolated element was found to be 4798 by in length and contained 37-bp inverted, imperfect, terminal repeats (ITRs). The sequence of the central region of the element revealed an open reading frame (designated ORF1) which showed similarity, at the amino acid level, to the transposase of the Tc1/mariner class of DNA transposons. Another sequence within the central region of the element showed similarity to the 3 coding and downstream untranslated region of the amyA gene of A. niger. Sequence homology and structural features indicate that this element, which has been named Ant1 (A. niger transposon 1), is related to the Tc1/mariner group of DNA transposons. Ant1 is apparently present as a single copy in strain N402 of A. niger.     

Complete nucleotide sequence and genome organization of a Drosophila transposable genetic element, 297

The complete nucleotide sequence of 297, a Drosophila copia-like transposable element, was determined and compared with those of other similar Drosophila elements and mammalian retrovirus proviruses. It was found that 297 contains three long open reading frames, comparable in sizes and locations with gag, pol, and env genes in the proviruses of replication-competent retroviruses in vertebrates. The first and second open reading frames of 297 exhibit sequence homologies to gag and pol, respectively, of Moloney murine leukaemia virus. In particular, as with 17.6, another Drosophila copia-like element, the second open reading frame of 297 was shown to be very similar in its entire organization to the retroviral pol gene and to consist of three enzymatic domains. By contrast, no appreciable homology was found between the third open reading frame of 297 and the retroviral env gene. It is also suggested that 297 and 17.6 are a peculiar pair of copia-like elements recently diverged from a common progenitor.     

The new class 11 transposon Tn163 is plasmid-borne in two unrelated Rhizobium leguminosarum biovar viciae strains

Tn163 is a transposable element identified in Rhizobium leguminosarum bv. viciae by its high insertion rate into positive selection vectors. The 4.6 kb element was found in only one further R. leguminosarum bv. viciae strain out of 70 strains investigated. Both unrelated R. leguminosarum bv. viciae strains contained one copy of the transposable element, which was localized in plasmids native to these strains. DNA sequence analysis revealed three large open reading frames (ORFs) and 38 bp terminal inverted repeats. ORF1 encodes a putative protein of 990 amino acids displaying strong homologies to transposases of class 11 transposons. ORF2, transcribed in the opposite direction, codes for a protein of 213 amino acids which is highly homologous to DNA invertases and resolvases of class II transposons. Homology of ORF1 and ORF2 and the genetic structure of the element indicate that Tn163 can be classified as a class II transposon. It is the first example of a native transposon in the genus Rhizobium. ORF3, which was found not to be involved in the transposition process, encodes a putative protein (256 amino acids) of unknown function. During transposition Tn163 produced direct repeats of 5 bp, which is typical for transposons of the Tn3 family. However, one out of the ten insertion sites sequenced showed a 6 by duplication of the target DNA; all duplicated sequences were A/T rich. Insertion of Tn163 into the sacB gene revealed two hot spots. Chromosomes of different R. leguminosarum bv. viciae strains were found to be highly refractory to the insertion of Tn163.     

A new insertion sequence element,ISLdl1, in Lactobacillus delbrueckii subsp. lactis ATCC 15808

A new insertion sequence element designated ISLdl1 has been isolated and characterized from Lactobacillus delbrueckii subsp. lactis ATCC 15808. It is the first IS element of L. delbrueckii subsp. lactis described. ISLdl1 is a 1508 bp element flanked by 26 bp imperfect inverted repeats, and generates an 8 bp AT-rich target duplication upon insertion. It contains one ORF encoding a protein of 455 amino acids. This protein shows significant homology to the transposases of the ISL3 family and to other bacterial transposases and putative transposases, and no homology to other proteins. Based on these structural features, ISLdl1 belongs to the ISL3 family. ISLdl1 is present in about 10-12 copies in the genome of ATCC 15808 based on Southern hybridization analysis. Location sites of eight ISLdl1 copies have been determined in more detail by cloning and sequencing one or both of the flanking regions of each ISLdl1 copy. ISLdl1 or ISLdl1-like IS elements were found exclusively in Lactobacillus delbrueckii species and in all strains of subsp. lactis tested. The nucleotide sequence of ISLdl1 is deposited under the accession number AJ302652.     

MAGGY, a retrotransposon in the genome of the rice blast fungusMagnaporthe grisea

Full-length copies of a previously described repetitive DNA sequence (CH2-8) were isolated from the genome of theMagnaporthe grisea strain 2539. One copy of the complete element was sequenced and found to resemble agypsy-like LTR retrotransposon. We named this element MAGGY (MAGnaporthe GYpsy-like element). MAGGY contains two internal ORFs putatively encoding Gag, Pol and Env-like proteins which are similar to peptides encoded by retroelements identified in other filamentous fungi. MAGGY was found to be widely distributed amongM. grisea isolates from geographically dispersed locations and different hosts. It was present in high copy number in the genomes of all nine rice-pathogenic isolates examined. By contrast,M. grisea strains isolated from other Gramineae were found to possess varying copy numbers of MAGGY and in some cases the element was completely absent. The wide distribution of MAGGY suggests that this element invaded the genome ofM. grisea prior to the evolution of rice-specific form(s). It may since have been horizontally transmitted to other sub-specific groups. One copy of MAGGY, corresponding to the element we sequenced, was located at identical locations in the genomes of geographically dispersed strains, suggesting that this copy of the element is a relatively ancient insertion.     

Insertion of the Drosophila transposable element copia generates a 5 base pair duplication

To examine the details of insertion for the D. malanogaster transposable element copia, we have isolated three independent pairs of genomic fragments which correspond to occupied and unoccupied target sites for insertion. Restriction endonuclease analysis suggests that sites with and without an element differ by a simple 5000 bp insertion. Direct DNA sequence analysis demonstrates that a 5 bp sequence, present once in the target DNA at the site of insertion, is found on both sides of the element after insertion. The 5 bp sequences which are duplicated are different in each case. Moreover, there does not appear to be any sequence homology among these three independent insertion sites     

Bi-directional Duplex Promoters with Duplicated Enhancers Significantly Increase Transgene Expression in Grape and Tobacco

Novel bi-directional duplex promoters (BDDP) were constructed by placing two identical core promoters divergently on both upstream and downstream sides of their duplicated enhancer elements. Estimates of promoter function were obtained by creating versions of CaMV 35S and CsVMV BDDPs that contained reporter marker genes encoding beta-glucuronidase (GUS) and enhanced green fluorescent protein (EGFP) interchangeably linked either to the upstream or downstream core promoters. GUS was used for quantitative analysis of promoter function, whereas, EGFP allowed visual qualitative evaluation. In addition, the GUS and EGFP genes placed in downstream positions were modified by translational fusion with neomycin phosphotransferase (NPTII) to allow simultaneous monitoring of promoter activity and selection of stable transformants. These versions of BDDP were compared with each other and with equivalent unidirectional constructs by evaluating their expression in grape and tobacco. For 35S promoter constructs tested in grape somatic embryos (SE), BDDP exhibited transient GUS expression 206- and 300-fold greater in downstream and upstream configurations, respectively, compared to a unidirectional 35S core promoter. Compared with a unidirectional double enhanced 35S promoter, BDDPs exhibited 0.5- and 3-fold increased GUS expression from downstream and upstream core promoters, respectively. The same differences in expression levels determined quantitatively with GUS were distinguished qualitatively with EGFP. Constructs using CsVMV core promoters yielded results relative to those obtained with 35S promoter. For example, the upstream BDDP CsVMV core promoter provided a 200-fold increase in GUS expression compared to a unidirectional core promoter. However, CsVMV promoter was found to have higher promoter activity than 35S promoter in both BDDP and unidirectional constructs. Incorporation of an additional duplicated enhancer element to BDDPs resulted in increased expression. For example, a 35S BDDP with two divergently arranged duplicated enhancer elements resulted in over a 6-fold increase in GUS expression in stably transformed tobacco plants compared to a BDDP with one duplicated enhancer element. Data demonstrate that BDDP composed of divergently-arranged core promoters separated by duplicated enhancers, all derived from a single promoter sequence, can be used to significantly enhance transgene expression and to direct synchronized expression of multiple transgenes.     

Identification of a new insertion element, similar to gram-negative IS26, on the lactose plasmid of Streptococcus lactis ML3.

In Streptococcus lactis ML3, the lactose plasmid (pSK08) forms cointegrates with a conjugal plasmid (pRS01). It has been proposed that cointegration is mediated by insertion sequences (IS) present on pSK08 (D. G. Anderson and L.L. McKay, J. Bacteriol. 158:954-962, 1984). We examined the junction regions of the cointegrate pPW2 and the corresponding regions of pSK08 (donor) and pRS01 (target) and identified a new IS element on pSK08 (ISS1S) which was involved in and duplicated during formation of pPW2. ISS1S was 808 base pairs (bp) in size, had 18-bp inverted repeats (GGTTCTGTTGCAAAGTTT) at its ends, contained a single long open reading frame encoding a putative protein of 226 amino acids, and generated 8-bp direct repeats of target DNA during cointegrate formation. An iso-IS element, ISS1T, which is duplicated in some other cointegrate plasmids, was also found on pSK08. ISS1T was also 808 bp in size and was identical to ISS1S in sequence except for 4 bp, none of which altered the inverted repeats or amino acid sequence of the open reading frame. Comparison of ISS1 with gram-negative IS26 revealed strong homologies in size (820 bp), sequence of inverted repeats (GGCACTGTTGCAAA), size of direct repeats generated after cointegration (8 bp), and number, size, and amino acid sequence (44.5% identical) of the open reading of frame.     

Host-handling behavior of the parasitoid,Diglyphus minoeus [Hym.: Eulophidae], parasitizing the honeysuckle leaf-miner,Phytomyza lonicerae [Dipt.: Agromyzidae]

The host-handling behavior of individual female waspsDiglyphys minoeus which attack the larvae of the honeysuckle leaf-miner,Phytomyza lonicerae, include 4 types of behavior: probing, ovipositor insertion, host-feeding, and resting. Ovipositor insertion may either be injection of venom, probing of the host by the sensilla on the ovipositor's tip or egg laying. Three types of attacks were distinguished: oviposition attack for unparasitized larvae, host-feeding attack for unparasitized host larvae and host rejection for previously parasitized host larvae. Oviposition attack was characterized by frequent alternation between probing and ovipositor insertion, long duration of ovipositor insertion and resting, and the long duration of host-handling. Resting behavior is thought to protect the progeny against superparasitism, host-feeding by other wasps or hosts' recovery from paralysis. Host-feeding attack was characterized by frequent alternation between host-feeding and ovipositor insertion and long duration of host-feeding. Host rejection was composed mainly of probing and ovipositor insertion and short handling time.      

Identification of Tn4430, a transposon of Bacillus thuringiensis functional in Escherichia coli

Summary The mobile genetic element Tn4430, originating from the gram-positive bacterium, Bacillus thuringiensis, and previously described as the Th-sequence, is the first transposon isolated from the genus Bacillus. In the present work a gene (APH-III) conferring resistance to kanamycin was inserted into this 4.2 kb transposon. Transposition experiments showed that Tn4430APH-III could transpose in the gram-negative host Escherichia coli when its insertion functions were supplied by an intact copy of Tn4430. By transposing Tn4430APH-III directly onto pBR322, it was possible to determine the nucleotide sequence of the terminal inverted repeats of Tn4430 and of the target DNA site. Identical 38 bp in inverted orientation are situated at each end of the transposon and there is a direct duplication of 5 bp at the insertion site. Thus, it is clear that Tn4430 is closely related to the transposons belonging to the Tn3 family (class II elements).     

Diversification of the barley and wheat <Emphasis Type="Italic">blt101</Emphasis>/<Emphasis Type="Italic">wpi6</Emphasis> promoters by the <Emphasis Type="Italic">Xumet</Emphasis> element without affecting stress responsiveness

BLT101-family plasma membrane proteins are found in a wide range of organisms from bacteria to nematodes and are involved in the regulation of cellular cation concentration under stress conditions. A comparison of the promoter regions of barley blt101 and its wheat ortholog, wpi6, revealed highly conserved nucleotide sequences between both genes and a unique insertion of a Xumet element in the blt101 promoter. The Xumet insertion occurred between a putative abscisic acid-responsive element (ABRE) and the dehydration-responsive element/c-repeat (DRE/CRT) within the blt101 promoter. However, blt101 and wpi6 were induced similarly in response to ABA, drought and low temperature, suggesting that the insertion does not affect promoter functions. The Xumet insertion in the blt101/wpi6 promoter region was detected in five barley cultivars, but absent in two wheat cultivars tested, suggesting that the insertion is barley-specific. Genomic Southern blot analysis revealed a large number of Xumet sequences interspersed in the barley genome, whereas only one or very few copies are present in the wheat genome. The data suggested that an expansion in copy number of Xumet elements occurred in the barley genome through evolution.     

Molecular cloning of an insertion sequence-like element from <Emphasis Type="Italic">Vibrio anguillarum</Emphasis> and its functional identification in <Emphasis Type="Italic">E. coli</Emphasis>

The tdh (thermostable direct hemolysin) gene occurs in some strains of Vibrio species. All tdh genes are flanked by insertion sequence-like elements (ISV). All previous attempts have failed to detect transposition of these ISVs. In this work, we have built a transposition detection system in E. coli and succeeded in detecting the transposition of an insertion sequence-like element at a frequency of Km^r mutants of 7.2 × 10⁻⁶. A specific flanking sequence (5′-Py-Pu-3′) was found on either side of the target duplication.     

Insertion mutations at the maizeOpaque2 locus induced by transposable element familiesAc, En/Spm andBg

Eight independently isolated unstable alleles of theOpaque2 (O2) locus were analysed genetically and at the DNA level. The whole series of mutations was isolated from a maize strain carrying a wild-typeO2 allele and the transposable elementActivator (Ac) at thewx-m7 allele. Previous work with another unstable allele of the same series has shown that it was indeed caused by the insertion of anAc element. Unexpectedly, the remaining eight mutations were not caused by the designatedAc element, but by other insertions that are structurally similar or identical to one of two different autonomous transposable elements. Six mutations were caused by the insertion of a transposable element of theEnhancer/Suppressor-Mutator (En/Spm) family. Two mutations were the result of the insertion of a transposable element of theBergamo (Bg) family. Genetic tests carried out with plants carrying the unstable mutations demonstrated that all were caused by the insertion of an autonomous transposable element.