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.     

ISH51: a large, degenerate family of insertion sequence-like elements in the genome of the archaebacterium, Halobacterium volcanii.

We describe a new family of repetitive elements in the genome of the archaebacterium Halobacterium volcanii. There are some 20-30 copies of this element, which we designate ISH51. Sequenced copies show typical insertion sequence characteristics (terminal inverted repeats, direct flanking repeats of "target site" DNA). However, members of the ISH51 family are highly heterogeneous, showing on average only 85% primary sequence homology; and some genomic copies appear to be severely truncated. Some ISH51 elements are clustered together in regions of relatively AT-rich DNA. There are at least five such AT-rich "islands" in the H. volcanii genome. Repetitive sequences homologous to ISH51 are found in the genomes of most Halobacterium and Halococcus species.     

Characterization of a second gene involved in bacterio-opsin gene expression in a halophilic archaebacterium.

Southern blot analysis and nucleotide sequencing of DNA from three bacterio-opsin-deficient mutants of the archaebacterium Halobacterium halobium (M86, W105, and W109) revealed that they each contain an alteration in a region 2,000 to 3,800 base pairs (bp) upstream of the bacterio-opsin gene (bop). Nucleotide sequence analysis of this region, which is also located downstream of the previously characterized brp gene, revealed that it contains an open reading frame (ORF) of 2,022 bp. This 2,022-bp ORF has a start codon which overlaps the stop codon of the brp gene and is read in the same direction. The ORF could encode an acidic protein of 73,334 daltons (674 amino acids) with a predicted secondary structure typical of a soluble protein. Bop mutant M86 contains a 1,883-bp deletion extending from bp 351 of the ORF, to 197 bp beyond the stop codon. Mutant W105 has an ISH2 element integrated at bp 1239 of the ORF, and mutant W109 has an ISH26 element integrated at bp 1889. Our results suggest that the ORF is a gene (designated bat for bacterio-opsin activator gene) involved in bop gene expression.     

Sequence analysis of the insertion element ISH1.8 and of associated structural changes in the genome of phage PhiH of the archaebacterium Halobacterium halobium

We have sequenced the insertion element ISH1.8 which can be present in one or two copies in the genome of phage ΦH of Halobacterium halobium. ISH1.8 is 1895 bp long, has no inverted repeat at its ends, and one only of the two copies is flanked by two 5-bp duplications. An 8-bp sequence composed of 4 bp from each end of ISH1.8 is present in both sites lacking the element. This 8-bp sequence could either be a specific insertion sequence or a part of the element that is left behind upon deletion. The plasmid pΦHL, consisting of the invertible L segment of the phage genome which is, in ΦH2 and ΦH5, flanked by two copies of ISH1.8, contains 112 bp of ISH1.8 and is released from the phage genome by recombination within a direct repeat of 9 bp. This 9-bp sequence (TCCCGCCCT) exists as an inverted repeat in ISH1.8 and therefore as two distinct repeats in phage genomes containing two copies of ISH1.8 in inverted orientation.     

Isolation, characterization, and nucleotide sequence of IS1202, an insertion sequence of Streptococcus pneumoniae.

A comparative hybridization protocol was used to isolate a small segment of DNA present in the Streptococcus pneumoniae type 19F strain SSZ but absent from strain Rx1, a nonencapsulated derivative of the type 2 strain D39. This segment of DNA is a 1,747-bp insertion sequence, designated IS1202, flanked by 23-bp imperfect inverted repeats and containing a single open reading frame sufficient to encode a 54.4-kDa polypeptide. A 27-bp target sequence is duplicated at either end of the element. IS1202 is not related to any of the currently known insertion elements and is the first reported for S. pneumoniae. Although found predominantly in type 19F strains in up to five copies, it has also been shown to be present in the chromosomes of pneumococci belonging to other serotypes. One of the four IS1202 copies in the encapsulated strain SSZ is located 1,009 bp downstream of the dexB gene, and transformation studies reveal that it is also closely linked to the type 19F capsular polysaccharide synthesis (cps) locus.     

Structure analysis at the ends of the intervening DNA sequences in the chloroplast 23S ribosomal genes of C. reinhardii

All of the chloroplast 23S ribosomal genes of C. reinhardii are interrupted by a 0.87 kb sequence (Rochaix and Malnoë, 1978). We have sequenced the DNA across the two ends of this intervening element. In parallel, we have examined the nucleotide sequences in the corresponding part of the 23S ribosomal RNA. This allowed us to locate precisely the boundaries between the coding (that is, transcribed into mature 23S rRNA) and the noncoding DNA. The results show that the intervening sequence is flanked by two identical sets of 3 bp (5′-CGT) oriented as direct repeats. In addition, a sequence of 5 bp (5′-CGTGA) lies exactly next to one end and is found very close (16 bp) to the other end, in the coding part of the gene. These two sets are also oriented as direct repeats. Finally, sequences near one end of the intervening element are found with a few alterations near the other end, but in an inverted orientation. Possible interpretations of these results are discussed.     

Structural and genetic organization of IS232, a new insertion sequence of Bacillus thuringiensis.

In the Bacillus thuringiensis strains toxic for the lepidopteran larvae, the delta-endotoxin genes cryIA are frequently found within a composite transposonlike structure flanked by two inverted repeat sequences. We report that these elements are true insertion sequences and designate them IS232. IS232 is a 2,184-bp element and is delimited by two imperfect inverted repeats (28 of 37 bp are identical). Two adjacent open reading frames, overlapping for three codons, span almost the entire sequence of IS232. The potential encoded polypeptides of 50 and 30-kDa are homologous to the IstA and IstB proteins of the gram-negative insertion sequence IS21. The N-terminal part of the 50-kDa polypeptide contains a helix-turn-helix DNA-binding motif. The junctions at the insertion sites of three IS232 elements were analyzed. Each case was different, with 0, 4, or 6 bp of the target DNA being duplicated. Transposition of IS232 in Escherichia coli was demonstrated by using a genetic marker inserted upstream of the two open reading frames.     

The IS1111 family members IS4321 and IS5075 have subterminal inverted repeats and target the terminal inverted repeats of Tn21 family transposons

IS5075 and IS4321 are closely related (93.1% identical) members of the IS1111 family that target a specific position in the 38-bp terminal inverted repeats of Tn21 family transposons and that are inserted in only one orientation. They are 1,327 bp long and have identical ends consisting of short inverted repeats of 12 bp with an additional 7 bp (TAATGAG) or 6 bp (AATGAG) to the left of the left inverted repeats and 3 bp (AGA) or 4 bp (AGAT) to the right of the right inverted repeat. Circular forms of IS5075 and IS4321 in which the inverted repeats are separated by abutting terminal sequences (AGATAATGAG) were detected. A similar circular product was found for the related ISPa11. Transposition of IS4321 into the 38-bp target site was detected, but a flanking duplication was not generated. The precisely reconstituted target site was also identified. Over 50 members of the IS1111 family were identified. They encode related transposases, have related inverted repeats, and include related bases that lie outside these inverted repeats. In some, the flanking bases number 5 or 6 on the left and 4 or 3 on the right. Specific target sites were found for several of these insertion sequence (IS) elements. IS1111 family members therefore differ from the majority of IS elements, which are characterized by terminal inverted repeats and a target site duplication, and from members of the related IS110 family, which do not have obvious inverted repeats near their termini.     

Complete structure of the chloroplast genome of Arabidopsis thaliana.

The complete nucleotide sequence of the chloroplast genome of Arabidopsis thaliana has been determined. The genome as a circular DNA composed of 154,478 bp containing a pair of inverted repeats of 26,264 bp, which are separated by small and large single copy regions of 17,780 bp and 84,170 bp, respectively. A total of 87 potential protein-coding genes including 8 genes duplicated in the inverted repeat regions, 4 ribosomal RNA genes and 37 tRNA genes (30 gene species) representing 20 amino acid species were assigned to the genome on the basis of similarity to the chloroplast genes previously reported for other species. The translated amino acid sequences from respective potential protein-coding genes showed 63.9% to 100% sequence similarity to those of the corresponding genes in the chloroplast genome of Nicotiana tabacum, indicating the occurrence of significant diversity in the chloroplast genes between two dicot plants. The sequence data and gene information are available on the World Wide Web database KAOS (Kazusa Arabidopsis data Opening Site) at http://www.kazusa.or.jp/arabi/.     

A new transposable element in Chironomus thummi

Summary A 1.7 kb long transposable element called TECth1 was found in the 3 flanking region of aChironomus thummi Balbiani ring gene. As shown by sequence comparison with a second copy, TECthl is characterized by a perfect terminal inverted repeat of 17 by flanked by a duplicated target site of 8 bp, four internal imperfect inverted repeats of 17 to 26 by and terminal regions of about 0.25 kb with a high number of short direct repeats of the consensus sequence ACTTT or permutated and mutated forms such as TTTAC or ACTAT. The terminal inverted repeats and the 8 by target site duplication are reminiscent ofDrosophila P and hobo elements but no long open reading frame starting with ATG is present, suggesting that the two TECthl copies studied represent deletion derivatives of a longer element coding for its own transposase. In situ hybridization revealed about 75 labelled sites distributed over all chromosomes with the Balbiani ring locus most strongly labelled. Fifty percent of the sites are specific for a given individual, and these variable sites are often heterozygous for the element.     

二化螟piggyBac类转座子的克隆与分析

piggyBac转座子是DNA型转座子, 广泛分布于生物体内。基于piggyBac转座子超家族成员IFP2开发的转基因工具载体是目前转基因研究中使用最广泛的载体之一, 因此piggyBac转座子的研究受到广泛的关注和重视。本文是对二化螟Chilo suppressalis内源性piggyBac类转座子（piggyBac-like element, CsuPLE）的首次报道。克隆的CsuPLE（GenBank登录号： JX392388）全长2 537 bp, 包含一个长1 914 bp的完整开放阅读框（open reading frame, ORF）, 编码含637个氨基酸残基的转座酶, 转座酶中含有piggyBac家族保守的“DDD-domain”。CsuPLE全长序列具有完全对称的13 bp反向末端重复序列（inverted terminal repeats, ITRs）以及非完全对称的21 bp内部重复序列（internal repeats, IRs）, 在二化螟基因组上插入在特征性的“TTAA”靶位点重复（target site duplication, TSD）处。在我国地理跨度很大的不同二化螟种群中均存在结构完整的CsuPLE序列。本研究结果为深入研究piggyBac转座子的结构与功能的关系提供了新的素材, 也为评价利用转座子载体系统在二化螟体内进行转基因操作的可行性和安全性提供了重要的理论基础。     

The complete nucleotide sequence of the coffee (Coffea arabica L.) chloroplast genome: organization and implications for biotechnology and phylogenetic relationships amongst angiosperms

The chloroplast genome sequence of Coffea arabica L., the first sequenced member of the fourth largest family of angiosperms, Rubiaceae, is reported. The genome is 155 189 bp in length, including a pair of inverted repeats of 25 943 bp. Of the 130 genes present, 112 are distinct and 18 are duplicated in the inverted repeat. The coding region comprises 79 protein genes, 29 transfer RNA genes, four ribosomal RNA genes and 18 genes containing introns (three with three exons). Repeat analysis revealed five direct and three inverted repeats of 30 bp or longer with a sequence identity of 90% or more. Comparisons of the coffee chloroplast genome with sequenced genomes of the closely related family Solanaceae indicated that coffee has a portion of rps19 duplicated in the inverted repeat and an intact copy of infA . Furthermore, whole-genome comparisons identified large indels (> 500 bp) in several intergenic spacer regions and introns in the Solanaceae, including trnE (UUC)– trnT (GGU) spacer, ycf4 – cemA spacer, trnI (GAU) intron and rrn5 – trnR (ACG) spacer. Phylogenetic analyses based on the DNA sequences of 61 protein-coding genes for 35 taxa, performed using both maximum parsimony and maximum likelihood methods, strongly supported the monophyly of several major clades of angiosperms, including monocots, eudicots, rosids, asterids, eurosids II, and euasterids I and II. Coffea (Rubiaceae, Gentianales) is only the second order sampled from the euasterid I clade. The availability of the complete chloroplast genome of coffee provides regulatory and intergenic spacer sequences for utilization in chloroplast genetic engineering to improve this important crop.     

Characterization of insertion sequence IS892 and related elements from the cyanobacterium Anabaena sp. strain PCC 7120.

IS892, one of the several insertion sequence (IS) elements discovered in Anabaena sp. strain PCC 7120 (Y. Cai and C. P. Wolk, J. Bacteriol. 172:3138-3145, 1990), is 1,675 bp with 24-bp near-perfect inverted terminal repeats and has two open reading frames (ORFs) that could code for proteins of 233 and 137 amino acids. Upon insertion into target sites, this IS generates an 8-bp directly repeated target duplication. A 32-bp sequence in the region between ORF1 and ORF2 is similar to the sequence of the inverted termini. Similar inverted repeats are found within each of those three segments, and the sequences of these repeats bear some similarity to the 11-bp direct repeats flanking the 11-kb insertion interrupting the nifD gene of this strain (J. W. Golden, S. J. Robinson, and R. Haselkorn, Nature [London] 314:419-423, 1985). A sequence similar to that of a binding site for the Escherichia coli integration host factor is found about 120 bp from the left end of IS892. Partial nucleotide sequences of active IS elements IS892N and IS892T, members of the IS892 family from the same Anabaena strain, were shown to be very similar to the sequence of IS892.     

Gene deletions causing human genetic disease: mechanisms of mutagenesis and the role of the local DNA sequence environment

Summary Reports describing short (< 20 bp) gene deletions causing human genetic disease were collated in order to study underlying causative mechanisms. Deletion break-point junction regions were found to be non-random both at the nucleotide and dinucleotide sequence levels, an observation consistent with an endogenous sequencedirected mechanism of mutagenesis. Direct repeats of between 2 bp and 8 bp were found in the immediate vicinity of all but one of the 60 deletions analysed. Direct repeats are a feature of a number of recombination, replication or repair-based models of deletion mutagenesis and the possible contribution of each to the spectrum of mutations examined was assessed. The influence of parameters such as repeat length and lenght of DNA between repeats was studied in relation to the frequency, location and extent of these deletions. Findings were broadly consistent with a slipped mispairing model but the predicted deletion of one whole repeat copy was found only rarely. A modified version of the slipped mispairing hypothesis was therefore proposed and was shown to possess considerable explanatory value for 25% of deletions examined. Whereas the frequency of inverted repeats in the vicinity of gene deletions was not significantly elevated, these elements may nevertheless promote instability by facilitating the formation of secondary structure intermediates. A significant excess of symmetrical sequence elements was however found at sites of single base deletions. A new model to explain the involvement of symmetric elements in frameshift mutagenesis was devised, which successfully accounted for a majority of the single base deletions examined. In general, the loss of one or a few base pairs of DNA was found to be more compatible with a replication-based model of mutagenesis than with a recombination or repair hypothesis. Seven hitherto unrecognized hotspots for deletion were noted in five genes (AT3, F8, HBA, HBB and HPRT). Considerable sequence homology was found between these different sites, and a consensus sequence (TGA/GA/GG/ TA/C) was drawn up. Sequences fitting this consensus (i) were noted in the immediate vicinity of 41% of the other (sporadic) gene deletions, (ii) were found frequently at sites of spontaneous deletion in the hamster APRT gene, (iii) were found to be associated with many larger human gene deletions/translocations, (iv) act as arrest sites for human polymerase a during DNA replication and (v) have been shown by in vitro studies of human polymerase a to be especially prone to frameshift mutation. It is proposed that dissociation of polymerase a at arrest sites may, by providing a stable single stranded substrate, lead to deletion of a DNA sequence either by slipped mispairing via a number of different secondary structure intermediates, or by strand-switching or base misincorporation. Human gene deletions thus appear to be caused by multiple mechanisms whose relative importance is probably governed by local primary and secondary DNA structure. Our ability to predict precisely the location and extent of a gene deletion is however hampered both by this complexity and by the possibility that these mechanisms may often act in combination.