Plasmid pHH1 of Halobacterium salinarium: characterization of the replicon region,the gas vesicle gene cluster and insertion elements

The DNA sequence of the 5.7 kb plasmid pHH9 containing the replicon region of the 150 kb plasmid pHH1 from Halobacterium salinarium was determined. The minimal region necessary for stable plasmid maintenance lies within a 2.9 kb fragment, as defined by transformation experiments. The DNA sequence contained two open reading frames arranged in opposite orientations, separated by an unusually high AT-rich (60–70% A + T) sequence of 350 bp. All H. salinarium strains (H. halobium, H. cutirubrum) investigated harbour endogenous plasmids containing the pHH1 replicon; however, these pHH1-type plasmids differ by insertions and deletions. Adjacent to the replicon, and separated by a copy of each of the insertion elements ISH27 and ISH26, is the 9 kb p-vac region required for gas vesicle synthesis. Analysis of these and other ISH element copies in pHH1 revealed that most of them lack the target DNA duplication usually found with recently transposed ISH elements. These results underline the plasticity of plasmid pHH1.     

Cloning and characterization of a replicon region of the IncHII plasmid pHH1457

A replicative region of the large conjugative plasmid pHH1457 (incompatibility group HII (IncHII)) was cloned. A 1.4-kbp region, in a stable pSBII14 clone, containing a PolI-independent replicon and determinants for the HII incompatibility phenotype, was selected and characterized. High incompatibility with IncHII plasmids was corroborated. Independent replication of the insert was demonstrated by ligation to an antibiotic resistance cassette. pSBII14 was used as a probe to identify IncHII plasmids from other members of the H complex: IncHI (IncHI1, IncHI2 and IncHI3 subgroups). Hybridization experiments revealed a high homology with the replication region of IncHII plasmids, but not with IncHI1 or IncHI3 plasmid prototypes. Homology with IncHI2 plasmids was observed, suggesting the presence of IncHII-like replicons among this subgroup of plasmids. This is the first report of the characterization of an IncHII plasmid maintenance region.     

An interchromosomal gene conversion of the Drosophila dunce locus identified with restriction site polymorphisms: a potential involvement of transposable elements in gene conversion

Summary Females heterozygous for the two alleles dnc ² and dnc ^M14 of the X-linked gene dunce (dnc), and carrying a copy of dnc ⁺ progeny X-chromosomes from recombination experiments. Restriction site polymorphisms have been used as genetic markers to follow the parentage of dnc locus segments in these chromosomes. All six chromosomes are identical with respect to the spectrum of restriction site markers they carry in the dnc ⁺ chromosomal region. In the progeny chromosomes, this region is comprised of sequences like the dnc ^M14 X-chromosome and the translocation copy of dnc ⁺. Sequences flanking the dnc gene in the progeny chromosomes are like the dnc ^M14 chromosome. Internal to the gene but near the 5 end, is a segment from the dnc ⁺ translocation which has apparently originated from an interchromosomal and premeiotic gene conversion event. In addition, two transposable elements have inserted into the progeny chromosomes, one towards the 5 end of dnc and the other near the 3 end. The insertion of these elements occurred premeiotically since all six chromosomes are structurally identical. The data are interpreted with respect to a potential role of transposable element transposition in the process of gene conversion.     

Identification and structural characterization of further DNA elements in the potato and pepper genomes homologous to the transposable element-like insertion Tst1

Summary The molecular cloning and nucleotide sequence of elements from potato and pepper that are related to the recently identified Tst1 element are described. Sequence analysis reveals considerable conservation of sequences internal to both the Tst1 element and two of the related elements identified here. In six potato clones analysed, the II by inverted repeat first identified in the Tst1 element is conserved. Several of the elements are flanked by an 8 by direct repeat. DNA fragments which were amplified from several pepper genomes by polymerase chain reaction (PCR) amplification using the inverted repeat as sequence primers also display considerable conservation of sequences internal to the Tst1 element. These data further support the possibility that Tst1 is a non-autonomous transposable element and that Tst1 might be the first example of a transposable element which occurs in several genera of solanaceous plants.     

Rearrangements at a hobo element inserted into the first intron of the singed gene in the unstable sn 49 system of Drosophila melanogaster

The cytological structure of the X chromosome and the DNA organisation of the singed locus were examined in five singed bristle mutants of Drosophila melanogaster. These mutants are all derived from the unstable mutant singed-49, isolated from a wild population in the Russian Far East in 1975. Rearrangements were found at a site within the first intron of the singed gene, where a hobo element is inserted in these mutants. One rearrangement, which is associated with a strong bristle phenotype, has an inversion between 2D and the location of singed at 7D, which separates the singed promoter from the singed coding region. Two phenotypically wild-type derivatives have smaller rearrangements within the first intron which do not appear to interfere with singed expression. Two derivatives with bristle phenotypes have more complex rearrangements, and one of them shows a dominant or antimorphic phenotype. DNA blotting and in situ hybridisation experiments show that, in addition to these rearrangements at a hobo element inserted at singed, other hobo elements in these strains have been mobilised. This system is therefore similar to others in which functional hobo elements continue to transpose, resulting in elevated rates of mutation and chromosome rearrangement. Received: 19 February 1997 / Accepted: 8 October 1997     

The Sinorhizobium meliloti insertion sequence (IS) elements ISRm102F34-1/ISRm7 and ISRm220-13-5 belong to a new family of insertion sequence elements

The Sinorhizobium meliloti insertion sequence (IS) elements ISRm102F34-1 and ISRm220-13-5 are 1481 and 1550 base pairs (bp) in size, respectively. ISRm102F34-1 is bordered by 15 bp imperfect terminal inverted repeat sequences (two mismatches), whereas the terminal inverted repeat of ISRm220-13-5 has a length of 16 bp (two mismatches). Both insertion sequence elements generate a 6-bp target duplication upon transposition. The putative transposase enzymes of ISRm102F34-1 and ISRm220-13-5 consist of 449 or 448 amino acid residues with predicted molecular weights of 50.7 or 51.3 kDa and theoretical isoelectric points of 10.8 or 11.1, respectively. ISRm102F34-1 is identical in 98.9% of its nucleotide sequence to an apparently inactive copy of an insertion sequence element, designated ISRm7, which flanks the left-end of the nodule formation efficiency (nfe) region of plasmid pRmeGR4b of S. meliloti strain GR4. ISRm102F34-1 and ISRm220-13-5 are closely related since they show an overall identity of 57.0% at the nucleotide sequence level and of 47.3% at the deduced amino acid level of their putative transposases. Both insertion sequence elements displayed significant similarity to the Xanthomonas campestris ISXc6 and its homolog IS1478a. Since none of these insertion sequence elements could be allocated to existing families of insertion sequence elements, a new family is proposed. Analysis of the distribution of ISRm102F34-1/ISRm7 in various local S. meliloti populations sampled from Medicago sativa, Medicago sphaerocarpa and Melilotus alba host plants at different locations in Spain revealed its presence in 35% of the isolates with a copy number ranging from 1 to 5. Furthermore, ISRm102F34-1/ISRm7 homologs were identified in other rhizobial species.     

Ac/Ds transposon mutagenesis in Arabidopsis thaliana: mutant spectrum and frequency of Ds insertion mutants

Using a two-component Ac/Ds system consisting of a stabilized Ac element (Ac_c1) and a non-autonomous element (Ds_A), 650 families of plants carrying independent germinal Ds_A excisions/transpositions were isolated. Progenies of 559 of these Ac_c1/Ds_A families, together with 43 families of plants selected for excision/transposition of wild-type (wt)Ac, were subjected to a broad screening program for mutants exhibiting visible alterations. This resulted in the identification of 48 mutants showing a wide variety of mutant phenotypes, including embryo lethality (24 mutants), chlorophyll defects (5 mutants), defective seedlings (2 mutants), reduced fertility (5 mutants), reduced size (3 mutants), altered leaf morphology (2 mutants), dark green, unexpanded rosette leaves (3 mutants), and aberrant flower or shoot morphology (4 mutants). To test whether these mutants were due to transposon insertions, a series of Southern blot experiments was performed on 28 families, comparing in each case several mutant plants with others showing the wild-type phenotype. A preliminary analysis revealed in 4 of the 28 families analyzed a common, novel Ds_A fragment in all mutant plants, which was present only in heterozygous plants with wt phenotype, as expected for Ds_A insertion mutations. These four mutants included two showing embryo lethality, one with dark green, unexpanded rosette leaves and stunted inflorescences, and one with curly growth of stems, leaves and siliques. Further evidence for Ds_A insertion mutations was obtained for one embryo lethal mutant and for the stunted mutant, while in case of the second embryo lethal mutant, the Ds_A insertion could be separated from the mutant locus by genetic recombination.     

Interaction between the Tam1 and Tam2 transposable elements of Antirrhinum majus

Summary Two stable derivatives of the highly unstable niv-53::Tam1 allele of Antirrhinum majus were analysed. In both derivatives the Tam1 element is integrated at the same site and in the same orientation as in the parental niv-53::Tam1 allele. In both cases the Tam1 element was found to carry a 5 bp deletion (CACTA) in one of its termini. This explains the excision deficiency of these two alleles of Tam1, niv-53::Tam1-46 and niv-53::Tam1-49. Niv-44::Tam2, another stable nivea mutation, carries the 5 kb element Tam2, which is not a derivative of Tam1 but possesses identical terminal inverted repeats. When the stable lines 46 and 49 were corssed with line 44, suprisingly, a high number of the flowers in the F₁ displayed a variegated phenotype. Sequence analysis of two germinal revertants isolated from the heterozygote niv-53::Tam1-46/niv-44::Tam2 shows excision of the Tam2 element. This indicates that Tam2 is a defective element, which can be complemented by an active Tam1 element. However, the variegated F₁ phenotype observed is not inherited monofactorially. Variegation is seen only at particular times of development of the F₁ plants. These phenomena seem to involve both the Tam1 and Tam2 transposable elements.     

Concomitant regulation of Mu1 transposition and Mutator activity in maize

Summary The mutagenic activity of the maize transposable element system Mutator can be lost by outcrossing to standard, non-Mutator lines or by repetitive intercrossing of genetically diverse Mutator lines. Lines losing Mutator mutagenic activity in either manner retain high copy numbers (10–15 per diploid genome) of the Mutator-associated Mu transposable elements. Frequent transposition of Mu1-related elements is observed only in active Mutator lines, however. The loss of Mutator activity on intercrossing is correlated with an increase in the copy number of Mu1-like elements to 40–50 per diploid genome, implying a self-encoded or self-activated negative regulator of Mu1 transposition. The outcross loss of Mutator activity is only weakly correlated with a low Mu element copy number and may be due to the loss of a positive regulatory factor encoded by a subset of Mu1-like elements. Transposition of Mu elements in active Mutator lines generates multiple new genomic positions for about half the elements each plant generation. The appearance of Mu1-like elements in these new positions is not accompanied by equally high germinal reversion frequencies, suggesting that Mu1 may commonly transpose via a DNA replicative process.     

Molecular cloning of the c2 locus of Zea mays, the gene coding for chalcone synthase

Summary The c2 locus of Zea mays, identified as one of the genes affecting anthocyanin biosynthesis, was cloned using the transposable element En (Spm) as a gene tag. The Spm element present at the c2 locus in the autonomously mutating c2-m1 line was isolated using En1 element specific probes. Sequences flanking the element were identified as c2 locus specific and were used to clone the nonautonomous c2-m2 and wild-type alleles. The cloning and analysis of a cDNA complementary to the c2 locus provided evidence that this gene encodes the enzyme chalcone synthase.     

Covalent DNA modification and the regulation of Mutator element transposition in maize

Summary Analyses of the multiple genomic Mu transposable elements in active Mutator lines with several C-methylation sensitive restriction enzymes indicate that Mu elements are undermodified compared with total maize nuclear DNA. Intercrossing of diverse Mutator lines leads to a discrete hypermodification of the Mu elements in a particular plant concurrent with a loss of mutagenic and transpositional potential. The modification events observed appear to be methylation of cytosine at the 5 position in the sequences 5-CG-3 and 5-CNG-3. Some potential C-methylation sites in Mu elements show a higher degree of methylation than others. Once established, the modified Mu state, like the loss of Mutator activity, is stable on outcrossing. Crosses between active Mutator lines with unmodified Mu elements and Mutator-loss lines with modified Mu elements show partial maternal dominance for the modification event. Mutator activity may also be lost thorugh outcrossing in a mechanism not associated with any detected modification events.     

Cloning and characterization of a gene cluster from Bacillus stearothermophilus comprising infC, rpmI and rplT

Summary Using two synthetic deoxyribonucleotide probes encoding segments of the primary structure of initiation factor IF3 from Bacillus stearothermophilus, we identified and cloned a segment of DNA which carries the infC gene. As in Escherichia coli, the infC gene begins with the unusual initiation triplet AUU, and is followed by the structural genes for ribosomal proteins L35 and L20 (rpmI and rplT, respectively).     

Molecular analysis of the loss of somatic instability in the bz2::mu1 allele of maize

Summary Multiple genetic and epigenetic changes were detected within one plant generation at the bz2:: mu1 mutable allele in a population of 118 plants. Loss of somatic instability in bz2::mu1 was usually correlated with methylation of the Mu1 transposable element; in 6 plants, somatic instability was lost as a result of mutations in bz2::mu1. This is a surprisingly high frequency of mutation per allele (2.5%) for the Mutator family, for which germinal revertants occur at a frequency of about 10^–4 per gamete. One germinal excision event was found that contained an 8 by deletion, frameshift mutation in Bronze-2. The three other mutants described occurred as a result of abortive transposition, in which 75–77 by deletions were generated at the junction between Bronze-2 and Mu1. We discuss the possible mechanisms, and the role of host factors in abortive transposition in maize.