Isolation and characterization of IS 31831, a transposable element from Corynebacterium glutamicum

A transposable element from a coryneform bacterium, Corynebacterium glutamicum ATCC 31831 was isolated and characterized. The element IS 31831 is a 1453 bp insertion sequence with 24 bp imperfect terminal inverted repeats. It contains one open reading frame highly homologous at the amino acid level to the transposase of IS 1096 from Mycobacterium smeg-matis. Both IS 31831 and IS 1096 exhibit several common characteristics suggesting that they constitute a new family of insertion sequences. IS 31831 was isolated by taking advantage of the sucrose sensitivity of coryneform bacteria conferred by expression of the Bacillus subtilis sacB gene. An Escherichia coli/ Corynebacterium shuttle vector useful for the isolation of transposable elements from the coryneform group of bacteria was constructed.     

Thehermit transposable element of the Australian sheep blowfly,Lucilia cuprina, belongs to thehAT family of transposable elements

We report the cloning ofhermit, a member of thehAT family of transposable elements from the genome of the Australian sheep blowfly,Lucilia cuprina. Hermit is 2716 bp long and is 49% homologous to the autonomoushobo element,HFL1, at the nucleic acid level.Hermit has 15 bp terminal inverted repeats that share 10 bp with the terminal inverted repeats ofHFL1. Conceptual translation reveals a 583 residue open reading frame (ORF) that is 64% similar and 42% identical to theHFL1 ORF. However, the sequence of thehermit element contains two frameshifts within the putative ORF, indication thathermit is an inactive element. Analysis ofL. cuprina strains from within and outside Australia suggested thathermit is present as a single copy in all the genomes analysed.     

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.     

DNA sequence of the transposable element IS1

Summary The nucleotide sequence of an IS1 element recently transposed into the lacI gene is reported. This sequence is nearly identical to one previously reported for another IS1 element (Ohtsubo and Ohtsubo, 1978). The implications of this similarity are discussed. The sizes of potential polypeptides encoded in the IS1 DNA have been determined and possible roles for these peptides in the illegitimate recombination events mediated by the element are considered.     

Description and characterization of IS994, a putative IS3 family insertion sequence from the salmon pathogen, Renibacterium salmoninarum

The motor properties of myosin reside in the globular S1 region of the myosin heavy chain (MHC) subunit. All vertebrates express a family of MHC isoforms in skeletal muscle that have a major influence on the mechanical properties of the various fiber types. Differences in molecular composition of S1 among MHC isoforms within a species have not been studied to any great detail. Presently, we have isolated, cloned and sequenced the S1 subunit of four MHC isoforms from skeletal muscle in Rana pipiens that are specifically expressed in four mechanically divergent fiber types. Paired analysis showed that the overall amino acid identity was higher between the three S1 isoforms expressed in twitch fibers than between the twitch and tonic isoforms. Relatedness in amino acid composition was evaluated in regions reported to govern cross-bridge kinetics. Surface loops 1 and 2, thought to influence motor velocity and ATPase, respectively, were both highly divergent between isoforms. However, the divergence in the loops was roughly equal to that of the amino-terminal region, a domain considered less important for motor function. We tested the hypothesis that the loops are more conserved in pairs of isoforms with more similar kinetics. Comparisons including other vertebrate species showed no tendency for loops from pairs with similar kinetics to be more conserved. These data suggest that the overall structure of loops 1 and 2 is not critical in regulating the kinetic properties of R. pipiens S1 isoforms. Cloning of this family of frog S1 isoforms will facilitate future structure/function studies of the molecular basis of variability in myosin cross-bridge kinetics.     

Molecular cloning and characterization of a beta-L-Arabinobiosidase in Bifidobacterium longum that belongs to a novel glycoside hydrolase family

Extensin is a glycoprotein that is rich in hydroxyprolines linked to β-L-arabinofuranosides. In this study, we cloned a hypBA2 gene that encodes a novel β-L-arabinobiosidase from Bifidobacterium longum JCM 1217. This enzyme does not have any sequence similarity with other glycoside hydrolase families but has 38-98% identity to hypothetical proteins in Bifidobacterium and Xanthomonas strains. The recombinant enzyme liberated L-arabinofuranose (Araf)-β1,2-Araf disaccharide from carrot extensin, potato lectin, and Araf-β1,2-Araf-β1,2-Araf-β-Hyp (Ara(3)-Hyp) but not Araf-α1,3-Araf-β1,2-Araf-β1,2-Araf-β-Hyp (Ara(4)-Hyp) or Araf-β1,2-Araf-β-Hyp (Ara(2)-Hyp), which indicated that it was specific for unmodified Ara(3)-Hyp substrate. The enzyme also transglycosylated 1-alkanols with retention of the anomeric configuration. This is the first report of an enzyme that hydrolyzes Hyp-linked β-L-arabinofuranosides, which defines a new family of glycoside hydrolases, glycoside hydrolase family 121.     

Molecular cloning and characterization of CYP719, a methylenedioxy bridge-forming enzyme that belongs to a novel P450 family,from cultured Coptis japonica cells

Two cytochrome P450 (P450) cDNAs involved in the biosynthesis of berberine, an antimicrobial benzylisoquinoline alkaloid, were isolated from cultured Coptis japonica cells and characterized. A sequence analysis showed that one C. japonica P450 (designated CYP719) belonged to a novel P450 family. Further, heterologous expression in yeast confirmed that it had the same activity as a methylenedioxy bridge-forming enzyme (canadine synthase), which catalyzes the conversion of (S)-tetrahydrocolumbamine ((S)-THC) to (S)-tetrahydroberberine ((S)-THB, (S)-canadine). The other P450 (designated CYP80B2) showed high homology to California poppy (S)-N-methylcoclaurine-3'-hydroxylase (CYP80B1), which converts (S)-N-methylcoclaurine to (S)-3'-hydroxy-N-methylcoclaurine. Recombinant CYP719 showed typical P450 properties as well as high substrate affinity and specificity for (S)-THC. (S)Scoulerine was not a substrate of CYP719, indicating that some other P450, e.g. (S)-cheilanthifoline synthase, is needed in (S)-stylopine biosynthesis. All of the berberine biosynthetic genes, including CYP719 and CYP80B2, were highly expressed in selected cultured C. japonica cells and moderately expressed in root, which suggests coordinated regulation of the expression of biosynthetic genes.     

On the divergence of members of a transposable element family

Statistical properties of the amount of divergence of members of a transposable element family are studied. The analysis is based on the model proposed by Langley et al. [5], describing the evolution of a family of selectively neutral transposable elements in a finite haploid population of size 2N. By considering the time back to the most recent common ancestor of two copies, both the probability of identity and the moments of the number of sites that differ between two sampled copies are obtained. Our analytic results are consistent with the numerical results of Ohta [8] for a similar model. The effects of gene conversion are also examined. In agreement with Slatkin [9], we find that gene conversion has a minimal effect on the probability of identity providing that the rate of deletion is sufficiently large.     

Physical and genetic analysis of IS110, a transposable element of Streptomyces coelicolor A3(2)

Summary On at least three independent occasions a 1.6 kb segment of Streptomyces coelicolor DNA was detected in apparently the same location in an attP-deleted derivative of the temperature phage C31 that carried a selectable viomycin resistance gene. This sequence (termed IS110) allowed integration of the phage (giving viomycin-resistant transductants) at homologous sequences (detected by Southern hybridisation) at several locations in the S. coelicolor genome. The inserted prophages facilitated genetic mapping of two IS110 copies in the chromosomal linkage map. A third copy did not exhibit simple segregation with chromosomal markers, and there appeared to be a frequent DNA rearrangement close to this copy. Some variation in the number of copies of IS110 and their location has taken place in the pedigree of S. coelicolor derivatives. IS110 did not hybridise to any known S. coelicolor plasmid, nor to any of several other IS-like elements previously described in other Streptomyces plasmids or phages. It hybridised strongly to DNA from only a small minority of other Streptomyces species and was absent from S. lividans, a close relative of S. coelicolor.     

Identification of a defective transposable element in tobacco

A putative defective transposable element has been identified in tobacco. This element has been found and characterised in two separate parts of the tobacco genome, specifically within the 3rd intron of the pollen-specific polygalacturonase gene (Npg1) and upstream of the endochitinase gene (Chn50). The element is ca. 0.4 kb in length and is bounded by conserved inverted repeats and putative target site duplications. It appears to fall into the category of non-autonomous transposable elements.     

Molecular and functional characterization of Slide,anAc-like autonomous transposable element from tobacco

A new transposable element of tobacco, Slide, was isolated from thetl mutant line, which shows somatic instability, after its transposition into a locus encoding nitrate reductase (NR). The Slide-124 element is 3733 bp long and its coding sequences show similarities with conserved domains of the transposases ofAc, Tam3 andhobo. Excision from the NR locus is detectable in somatic leaf tissues and Slide mobility is triggered by in vitro tissue culture. Slide excision events create footprints similar to those left byAc and Tam3. Tobacco lines derived from thetl mutant line seem characterized by unmethylated copies of a few members of the highly repetitive Slide family. Slide mobility was monitored in transient expression assays. In wild-type tobacco protoplasts, the complete Slide element, as well as a defective copy, is able to excise. The complete Slide element, but not the defective version, is able to excise in protoplasts of the heterologous species lettuce (Lactuca sativa). These results show that Slide carries the functions required for its own mobility, and represents the first autonomousAc-like element characterized inSolanaceae species.     

Cloning,expression and characterization of the esterase estUT1 from Ureibacillus thermosphaericus which belongs to a new lipase family XVIII

A new esterase gene from thermophilic bacteria Ureibacillus thermosphaericus was cloned into the pET32b vector and expressed in Escherichia coli BL21(DE3). Alignment of the estUT1 amino acid sequence revealed the presence of a novel canonical pentapeptide (GVSLG) and 41–47% identity to the closest family of the bacterial lipases XIII. Thus the esterase estUT1 from U. thermosphaericus was assigned as a member of the novel family XVIII. It also showed a strong activity toward short-chain esters (C2–C8), with the highest activity for C2. When p-nitrophenyl butyrate is used as a substrate, the temperature and pH optimum of the enzyme were 70–80 °C and 8.0, respectively. EstUT1 showed high thermostability and 68.9 ± 2.5% residual activity after incubation at 70 °C for 6 h. Homology modeling of the enzyme structure showed the presence of a putative catalytic triad Ser93, Asp192, and His222. The activity of estUT1 was inhibited by PMSF, suggesting that the serine residue is involved in the catalytic activity of the enzyme. The purified enzyme exhibited high stability in organic solvents. EstUT1 retained 85.8 ± 2.4% residual activity in 30% methanol at 50 °C for 6 h. Stability at high temperature and tolerance to organic solvents make estUT1 a promising enzyme for biotechnology application.

     

The nucleotide sequence and protein-coding capability of the transposable element IS5

The nucleotide sequence of IS5, a bacterial insertion sequence, has been determined. It is 1195 bp long and contains an inverted terminal repetition of 16 bp with one mismatch. One open reading frame, spanning nearly the entire length of the element, could encode a polypeptide of 338 amino acids. Upon insertion into a DNA segment, IS5 causes a duplication of 4 bp. Based on seven examples, this site of insertion appears to be nonrandom, and the consensus target site sequence is C . T/A . A . G/A (or C/T . T . A/T . G on the opposite strand). The nucleotide sequences of IS5 insertions into the B and cim genes of bacteriophage Mu have allowed tentative identification of the protein-coding frames of B and cim.     

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.