No dosage effect of recombinational hotspots in the mouse major histocompatibility complex

The sites of meiotic recombination in the proximal region of the mouse major histocompatibility complex (MHC) are clustered at hotspots. Some MHC haplotypes derived from Asian wild mice increase the frequency of recombination at such hotspots when heterozygous with standard laboratory haplotypes. The wm7 and cas3 haplotypes, have a hotspot close to the Lmp-2 gene (Lmp-2 hotspot), and the cas4 haplotype has a hotspot about 100 kilobase (kb) proximal, close to the Pb gene (Pb hotspot). To examine the effect of a double dose of hotspots, we estimated the rate of recombination and determined the location of the breakpoints in crosses of wm7/cas3 and wm7/cas4. In 3570 backcross progeny we identified 29 new recombinants in the H-2K to Ab interval, at a frequency of 0.81%. This frequency is 40-fold higher than in crosses between laboratory haplotypes and very similar to those previously obtained in crosses between these wild and standard laboratory haplotypes. Thus, a double dose of hotspots has no additive effect on the frequency of meiotic recombination. The site-specificity of recombination was also conserved. Twenty-three breakpoints were confined within 5.4 kb in the Lmp-2 hotspot, and six breakpoints from the cas4 cross were located in the Pb hotspot, which we have now confined to a 15 kb segment. Correspondence to: T. Shiroishi.     

Potato chromosomes IX and XI carry genes for resistance to potato virus M

Two new loci for resistance to potato virus M (PVM), Gm and Rm, have been mapped in potato. The gene Gm was derived from Solanum gourlayi, whereas, Solanum megistacrolobum is the source of the gene Rm. Gm confers resistance to PVM infection after mechanical inoculation. Rm induces a hypersensitive response in potato plants. Two diploid populations segregating for Gm and Rm, bulked segregant analysis (BSA) using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR), and available potato molecular maps were instrumental for mapping the resistance loci. The novel locus Gm was mapped to a central region on potato chromosome IX. The locus Rm was placed on the short arm of chromosome XI, close to the marker loci GP250 and GP283, where a hotspot for monogenic and polygenic resistance to diverse pathogens is located in the potato and tomato genome.     

Environmental niche conservatism explains the accumulation of species richness in Mediterranean‐hotspot plant genera

The causes of exceptionally high plant diversity in Mediterranean‐climate biodiversity hotspots are not fully understood. We asked whether a mechanism similar to the tropical niche conservatism hypothesis could explain the diversity of four large genera (Protea, Moraea, Banksia, and Hakea) with distributions within and adjacent to the Greater Cape Floristic Region (South Africa) or the Southwest Floristic Region (Australia). Using phylogenetic and spatial data we estimated the environmental niche of each species, and reconstructed the mode and dynamics of niche evolution, and the geographic history, of each genus. For three genera, there were strong positive relationships between the diversity of clades within a region and their inferred length of occupation of that region. Within genera, there was evidence for strong evolutionary constraint on niche axes associated with climatic seasonality and aridity, with different niche optima for hotspot and nonhotspot clades. Evolutionary transitions away from hotspots were associated with increases in niche breadth and elevated rates of niche evolution. Our results point to a process of “hotspot niche conservatism” whereby the accumulation of plant diversity in Mediterranean‐type ecosystems results from longer time for speciation, with dispersal away from hotspots limited by narrow and phylogenetically conserved environmental niches.     

Recombinational hotspot specific to female meiosis in the mouse major histocompatibility complex

Thewm7 haplotype of the major histocompatibility complex (MHC), derived from the Japanese wild mouseMus musculus molossinus, enhances recombination specific to female meiosis in theK/A interval of the MHC. We have mapped crossover points of fifteen independent recombinants from genetic crosses of thewm7 and laboratory haplotypes. Most of them were confined to a short segment of approximately 1 kilobase (kb) of DNA between theA 3 andA 2 genes, indicating the presence of a female-specific recombinational hotspot. Its location overlaps with a sex-independent hotspot previously identified in theMus musculus castaneus CAS3 haplotype. We have cloned and sequenced DNA fragments surrounding the hotspot from thewm7 haplotype and the corresponding regions from the hotspot-negative B10.A and C57BL/10 strains. There is no significant difference between the sequences of these three strains, or between these and the published sequences of the CAS3 and C57BL/6 strains. However, a comparison of this A3/A2 hotspot with a previously characterized hotspot in theE gene revealed that they have a very similar molecular organization. Each hotspot consists of two elements, the consensus sequence of the mouse middle repetitive MT family and the tetrameric repeated sequences, which are separated by 1 kb of DNA.The nucleotide sequence data reported in this paper have been submitted to the DNA Data Bank of Japan nucleotide sequence database and have been assigned the accession numbers d90007-9. Offprint requests to: T. Shiroishi.     

Amazonian invaders in an Asian biodiversity hotspot: Understanding demographics for the management of the armoured sailfin catfish,Pterygoplichthys pardalis in Kerala,India

Invasion of armoured catfish, Pterygoplichthys spp., is a major threat to global aquatic biodiversity, and developing effective strategies for their control and eradication is both a concern and a research priority. A length-based population assessment of invasive Pterygoplichthys pardalis in southern India, a hotspot for endemic aquatic biodiversity, indicated that rapid growth, high growth performance index and continuous recruitment have aided their successful invasion. Increasing fishing pressure on the adults is not adequate for population management, and only targeting young individuals (<30 cm) will result in overexploitation and population collapse.     

Complete chloroplast genomes of Liliaceae (s.l.) species: comparative genomic and phylogenetic analyses

We first report the complete chloroplast (cp) genome of Fritillaria taipaiensis and determine its characteristics, sequence divergence and phylogenetic relationships by comparing it with complete cp genomes of Liliaceae s.l. (including e.g. Nartheciaceae, Amaryllidaceae and Asparagaceae) species obtained from NCBI Genbank. We show that the ycf1, ycf15 and infA genes have become pseudogenes or are lost in some of the seventeen Liliaceae species, and that dispersed repeats are prevailing among the four types of repeats (dispersed, palindromic, complement and tandem repeats). The number of simple sequence repeats ranged from 53 to 84 in the seventeen species, with mononucleotide repeats being the most abundant, followed by dinucleotides. A total of nine genes with positive selection sites were identified (atpB, atpE, ndhF, ndhH, petB, rpl2, rpl20, rpl22 and ycf2). Furthermore, we examined 19 mutational hotspot regions, including three coding regions (rps16, infA and rpl22) and sixteen non-coding regions. A phylogenetic analysis of the complete cp genomes and protein-coding sequences showed that Fritillaria is most closely related to Lilium. Moreover, Asparagus and Polygonatum, Hosta and Yucca are closely related to the Liliaceae. These results will contribute to further study of evolutionary patterns and phylogenetic relationships in Liliaceae s.l.     

An integrative bioinformatics pipeline shows that honeybee-associated microbiomes are driven primarily by pollen composition

The microbiomes associated with bee nests influence colony health through various mechanisms, although it is not yet clear how honeybee congeners differ in microbiome assembly processes, in particular the degrees to which floral visitations and the environment contribute to different aspects of diversity. We used DNA metabarcoding to sequence bacterial 16S rRNA from honey and stored pollen from nests of 4 honeybee species (Apis cerana, A. dorsata, A. florea, and A. laboriosa) sampled throughout Yunnan, China, a global biodiversity hotspot. We developed a computational pipeline integrating multiple databases for quantifying key facets of diversity, including compositional, taxonomic, phylogenetic, and functional ones. Further, we assessed candidate drivers of observed microbiome dissimilarity, particularly differences in floral visitations, habitat disturbance, and other key environmental variables. Analyses revealed that microbiome alpha diversity was broadly equivalent across the study sites and between bee species, apart from functional diversity which was very low in nests of the reclusive A. laboriosa. Turnover in microbiome composition across Yunnan was driven predominantly by pollen composition. Human disturbance negatively impacted both compositional and phylogenetic alpha diversity of nest microbiomes, but did not correlate with microbial turnover. We herein make progress in understanding microbiome diversity associated with key pollinators in a biodiversity hotspot, and provide a model for the use of a comprehensive informatics framework in assessing pattern and drivers of diversity, which enables the inclusion of explanatory variables both subtly and fundamentally different and enables elucidation of emergent or unexpected drivers.     

Genome composition and origin of the polyploid Aegean grass Avenula agropyroides (Poaceae)

Aim The Aegean is a hotspot of plant biodiversity, with its island biota harbouring a large number of endemic taxa. To investigate the relationship between biogeography, polyploid speciation and genomics in the Aegean we used the biogeographically isolated highly polyploid eastern Mediterranean grass species Avenula agropyroides (2n = 70) as an example of complicated polyploid origin. Location Mediterranean, Aegean. Methods To clarify the origin of A. agropyroides, we conducted chromosome studies using repetitive DNAs as hybridization probes in fluorescent in situ hybridization experiments, chromosome banding methods and DNA sequence analyses of plasmid‐cloned nuclear ribosomal (nr) ITS1–5.8S–ITS2 DNA. Results Decaploid A. agropyroides had near‐autopolyploid karyotype structure and contained characteristic sequence motifs of nrDNA repeats not encountered in any of the diploids studied. Special repeat types found in one of its accessions (Crete) showed that A. agropyroides originated from a diploid species with a hybrid background. One of the genomes involved was close to both that of extant species (Avenula aetolica, Avenula compressa, Avenula hookeri, Avenula schelliana, Avenula versicolor) distributed mostly in the eastern Mediterranean to Asia and North America and also to the west Mediterranean (Avenula bromoides). The other resembled that of exclusively western Mediterranean species (Avenula albinervis, Avenula levis, Avenula marginata, Avenula sulcata). Main conclusions Avenula agropyroides represents a remarkable polyploid in the eastern Mediterranean, conserving the genome structure of a diploid species that no longer exists. This highlights how the Aegean has been less affected than other Eurasian regions by the repeated shifts of climatic zones and vegetation belts since the Late Tertiary.     

Diploid hybrid origin of Ostryopsis intermedia (Betulaceae) in the Qinghai‐Tibet Plateau triggered by Quaternary climate change

Despite the well‐known effects that Quaternary climate oscillations had on shaping intraspecific diversity, their role in driving homoploid hybrid speciation is less clear. Here, we examine their importance in the putative homoploid hybrid origin and evolution of Ostryopsis intermedia, a diploid species occurring in the Qinghai‐Tibet Plateau (QTP), a biodiversity hotspot. We investigated interspecific relationships between this species and its only other congeners, O. davidiana and O. nobilis, based on four sets of nuclear and chloroplast population genetic data and tested alternative speciation hypotheses. All nuclear data distinguished the three species clearly and supported a close relationship between O. intermedia and the disjunctly distributed O. davidiana. Chloroplast DNA sequence variation identified two tentative lineages, which distinguished O. intermedia from O. davidiana; however, both were present in O. nobilis. Admixture analyses of genetic polymorphisms at 20 SSR loci and sequence variation at 11 nuclear loci and approximate Bayesian computation (ABC) tests supported the hypothesis that O. intermedia originated by homoploid hybrid speciation from O. davidiana and O. nobilis. We further estimated that O. davidiana and O. nobilis diverged 6–11 Ma, while O. intermedia originated 0.5–1.2 Ma when O. davidiana is believed to have migrated southward, contacted and hybridized with O. nobilis possibly during the largest Quaternary glaciation that occurred in this region. Our findings highlight the importance of Quaternary climate change in the QTP in causing hybrid speciation in this important biodiversity hotspot.     

Gambusia quadruncus (Cyprinodontiformes: Poeciliidae): a new species of mosquitofish from east‐central México

Gambusia quadruncus n. sp., the llanos mosquitofish, is described from east‐central México. The region inhabited by the species represents a hotspot of diversity of Gambusia, and G. quadruncus sometimes coexists with at least three congeners. The species differs from its closest relative, Gambusia affinis, in several characteristics with plausible effects on reproductive isolation, e.g. body size, body and fin morphology, male genital morphology (distal tip of gonopodium) and female anal spot morphology (colouration near the urogenital sinus). Moreover, combined analysis of mitochondrial and nuclear gene sequence data (c. 2158 total base pairs) indicates reciprocal monophyly of G. quadruncus and its sister species G. affinis, with levels of genetic divergence suggesting the two species diverged from one another over a million years ago. The origin of G. quadruncus may reflect a vicariant event associated with Pliocene orogenesis in the Tamaulipas Arch and a frontal section of the Sierra Madre Oriental (Lleran Mesas). Gambusia quadruncus inhabits a variety of freshwater habitats across several river drainages, with its range spanning at least 350 km from north to south, covering over 25 000 km². A key to aid identification of the species is provided.     

Comparative Analyses and Phylogenetic Relationships between <i>Cryptomeria fortunei</i> and Related Species Based on Complete Chloroplast Genomes

Cryptomeria fortunei (Chinese cedar) is a highly adaptable woody species and one of the main forest plantation trees in subtropical high-altitude areas in China. However, there are few studies on its chloroplast (cp) genome. In this study, the complete cp genome of C. fortunei was sequenced and evaluated via comparative analyses with those of related species (formerly the Taxodiaceae) in Cupressaceae. The C. fortunei cp genome was 131,580 bp in length, and the GC content of the whole genome was 35.38%. It lost one relevant large inverted repeat and contained 114 unique genes, including 82 protein-coding genes, 28 tRNAs and 4 rRNAs. The relative synonymous codon usage (RSCU) of codons ending with A/U was more than twice that of codons ending with G/C. Thirty long repeat structures (LRSs) and 213 simple sequence repeat (SSR) loci were detected in the C. fortunei cp genome. Comparative analyses of 10 cp genomes revealed that substantial rearrangements occurred in the gene organization. Additionally, 6 cp hotspot regions (trnS-GGA, ycf1, trnP-GGG, trnC-GCA, psbZ and accD) were identified, and 4 genes (petL, psbM, rpl22 and psaM) had likely underwent positive selection. Phylogenetic analysis showed that Cupressaceae, Taxaceae and Cephalotaxaceae clustered to form a clade and that C. fortunei was most closely related to C. japonica (Japanese cedar), C. japonica cv. Wogon Hort and Taxodium distichum (baldcypress). These results provide references for future studies of population genetics, phylogenetic status and molecular markers among Cupressaceae species and for the cultivation of improved varieties.     

Variation in thermal tolerances of native freshwater fishes in South Africa's Cape Fold Ecoregion: examining the east–west gradient in species' sensitivity to climate warming

The Cape Fold Ecoregion (CFE) is a biodiversity hotspot with high levels of endemism in its freshwater fish fauna. This study examined inter and intra-specific variation in critical thermal maxima (T_Cmax) for eight native species of freshwater fish from the CFE. Cape galaxias Galaxias zebratus, Breede River redfin Pseudobarbus burchelli, Berg River redfin Pseudobarbus burgi, Clanwilliam redfin Pseudobarbus calidus and fiery redfin Pseudobarbus phlegethon were the most thermally sensitive (T_Cmax = 29.8–32.8°C). Clanwilliam rock-catfish Austroglanis gilli, Eastern Cape redfin Pseudobarbus afer and Cape kurper Sandelia capensis were moderately sensitive (T_Cmax = 33.0–36.8°C). An increase in intra-specific thermal sensitivity of S. capensis was observed from east to west. The results were related to in situ water temperature, which influenced T_Cmax for all species, suggesting that thermal history is a major driver of variation in thermal tolerance amongst populations. These thermal tolerance data for freshwater fishes in the CFE demonstrate that resilience to climate warming follows a geographical cline and that the more sensitive western species and regions are conservation priorities.     

Molecular analysis of a recombinational hotspot adjacent to Lmp2 gene in the mouse MHC: Fine location and chromatin structure

Meiotic recombinations in the proximal region of the mouse major histocompatibility complex (MHC) are clustered within certain segments of chromosome, known as hotspots. In this study, we found that one of such hotspots, previously mapped between the Pb and Ob genes, is located very close to the 3′ end of the Lmp2 gene, which encodes a subunit of a proteolytic proteasome. To analyze the molecular basis of the site specificity of hotspots, we examined the structure of the chromatin around this Lmp2 hotspot and another one located in the MHC class II Eb gene, by monitoring DNase I-hypersensitive sites (DHSSs) of the chromatin. DHSSs were detected at the both hotspots in the somatic cells. In the meiotic cells, DHSS was detected within the Eb hotspot, as previously reported, but not in the Lmp2 hotspot. Thus, open structure of chromatin during meiosis, as monitored by hypersensitivity to DNase I, is not a general feature of mouse recombinational hotspots, contrasting the case of the lower eukaryote, S. cerevisiae, in which hotspots are always associated with DHSSs. Received: 25 January 1996 / Accepted: 21 March 1996     

Mutation and expression analysis of the IDH1, IDH2, DNMT3A, and MYD88 genes in colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of death around the world. Its genetic mechanism was intensively investigated in the past decades with findings of a number of canonical oncogenes and tumor-suppressor genes such as APC, KRAS, and TP53. Recent genome-wide association and sequencing studies have identified a series of promising oncogenes including IDH1, IDH2, DNMT3A, and MYD88 in hematologic malignancies. However, whether these genes are involved in CRC remains unknown. In this study, we screened the hotspot mutations of these four genes in 305 CRC samples from Han Chinese by direct sequencing. mRNA expression levels of these genes were quantified by quantitative real-time PCR (RT-qPCR) in paired cancerous and paracancerous tissues. Association analyses between mRNA expression levels and different cancerous stages were performed. Except for one patient harboring IDH1 mutation p.I99M, we identified no previously reported hotspot mutations in colorectal cancer tissues. mRNA expression levels of IDH1, DNMT3A, and MYD88, but not IDH2, were significantly decreased in the cancerous tissues comparing with the paired paracancerous normal tissues. Taken together, the hotspot mutations of IDH1, IDH2, DNMT3A, and MYD88 gene were absent in CRC. Aberrant mRNA expression of IDH1, DNMT3A, and MYD88 gene might be actively involved in the development of CRC.     

Insertions of transposable elements in the promoter proximal region of the gene cluster for Escherichia coli H+-ATPase: 8 base pair repeat generated by insertion of IS1

Summary A plasmid pKY159 (Yamaguchi and Yamaguchi 1983) carrying a promoter proximal portion of the gene cluster of the proton-translocating ATPase (H⁺-ATPase) of Escherichia coli causes growth inhibition of wild-type cells. Insertion of a transposable element in this plasmid released this inhibitory effect. In analyzing this inhibitory effect, we determined the insertion points at the nucleotidesequence level of transposable elements on 30 independent derivatives of pKY159. Insertions of IS1, IS5, and were found between the promoter and the gene for a possible component of 14,000 daltons of the H⁺-ATPase. Of 31 insertions, 26 were of IS1 and were located at the same site, indicating that this site is a hotspot for IS1 insertion and that IS1 insertion is much more frequent than that of IS5 of in this region. Four different sites for IS1 insertion were found; in two of these an 8 base pair (bp) duplicate of the target sequence (AAAAACGT and AAACGTTG) was generated, while in the other two a 9 bp duplicate was found. In all cases in this study the nucleotide sequence of IS1 was the same as that of IS1-K. In the two cases with an 8 bp duplicate in different sites, a common 6 bp sequence (AAACGT) was found. These results suggested that generation of the 8 bp duplicate is related to the common sequence rather than a mutation in IS1 suggested by Iida et al. (1981) and also suggested that the essential length of the duplicate is 8 bp or less than 8 bp. A 6 bp sequence (GTGATG) homologous to the end portion of IS1 was found at the hotspot, but not at other sites, suggesting that this homology contributed to the high frequency of IS1 insertion. The direction of IS1 insertion at the hotspot was the same in 25 of 26 instances, suggesting that the direction of IS1 insertion is determined by the structure of the target and/or its nearby sequence.Abbreviations bp base pairs - 14 K protein a possible component of the H⁺-ATPase with molecular weight of 14,000 (see Kanazawa and Futai 1982 for details) - EDTA ethylenediaminetetraacetate     

A reanalysis of Parabuthus (Scorpiones: Buthidae) phylogeny with descriptions of two new Parabuthus species endemic to the Central Namib gravel plains,Namibia

Two new thick‐tail scorpions in the genus Parabuthus Pocock, 1890 are described from the gravel plains of the Central Namib Desert, Namibia: Parabuthus glabrimanus sp. nov. ; Parabuthus setiventer sp. nov. The two new species occupy discrete distributional ranges, allopatric with the closely related species Parabuthus gracilis Lamoral, 1979 and Parabuthus nanus Lamoral, 1979. The distributions of the four species are mapped and a key provided for their identification. Revised diagnoses are provided for P. gracilis and P. nanus. The two new species are added to a previously published morphological character matrix for Parabuthus species and their phylogenetic positions determined in a reanalysis of Parabuthus phylogeny. Parabuthus setiventer sp. nov. is found to be the sister species of P. nanus, whereas P. glabrimanus sp. nov. is sister to a monophyletic group comprising P. gracilis, P. nanus, and P. setiventer sp. nov. The discovery of two new scorpion species endemic to the Central Namib gravel plains contributes to a growing body of evidence that this barren and desolate region is a hotspot of arachnid species richness and endemism. © 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 159 , 673–710.     

DNA barcoding of Corydalis,the most taxonomically complicated genus of Papaveraceae

The genus Corydalis is recognized as one of the most taxonomically challenging plant taxa. It is mainly distributed in the Himalaya–Hengduan Mountains, a global biodiversity hotspot. To date, no effective solution for species discrimination and taxonomic assignment in Corydalis has been developed. In this study, five nuclear and chloroplast DNA regions, ITS, ITS2, matK, rbcL, and psbA‐trnH, were preliminarily assessed based on their ability to discriminate Corydalis to eliminate inefficient regions, and the three regions showing good performance (ITS, ITS2 and matK) were then evaluated in 131 samples representing 28 species of 11 sections of four subgenera in Corydalis using three analytical methods (NJ, ML, MP tree; K2P‐distance and BLAST). The results showed that the various approaches exhibit different species identification power and that BLAST shows the best performance among the tested approaches. A comparison of different barcodes indicated that among the single barcodes, ITS (65.2%) exhibited the highest identification success rate and that the combination of ITS + matK (69.6%) provided the highest species resolution among all single barcodes and their combinations. Three Pharmacopoeia‐recorded medicinal plants and their materia medica were identified successfully based on the ITS and ITS2 regions. In the phylogenetic analysis, the sections Thalictrifoliae, Sophorocapnos, Racemosae, Aulacostigma, and Corydalis formed well‐supported separate lineages. We thus hypothesize that the five sections should be classified as an independent subgenus and that the genus should be divided into three subgenera. In this study, DNA barcoding provided relatively high species discrimination power, indicating that it can be used for species discrimination in this taxonomically complicated genus and as a potential tool for the authentication of materia medica belonging to Corydalis.     

中国西藏兰科植物新资料

东喜马拉雅地区是全球生物多样性的热点区域,西藏为其重要组成单元,明确该地区本底植物资源及地理分布对区域植物区系研究有着重要意义。该文报道了笔者在西藏考察中发现的西藏兰科(Orchidaceae)植物5个新记录属,即美柱兰属(Callostylis Blume)、异型兰属(Chiloschista Lindl.)、蛇舌兰属(Diploprora Hook. f.)、带叶兰属(Taeniophyllum Blume)和宽距兰属(Yoania Maxim.)。对应的5个新记录种,即美柱兰(Callostylis rigida Bl.)、异型兰(Chiloschista yunnanensis Schlechter)、蛇舌兰 [Diploprora championii(Lindl.)Hook. f.]、毛莛带叶兰(Taeniophyllum retrospiculatum King & Pantl.)和印度宽距兰(Yoania prainii King & Pantl.)。该文还附有新记录属、种的形态描述和特征图片。该研究结果进一步丰富了中国植物区系资料,拓宽了西藏兰科植物的记录,并对西藏及毗邻地区兰科植物多样性保护具有重要价值。     

A crossover hotspot near his-3 in Neurospora crassa is a preferential recombination termination site

During analysis of 148 unselected Neurospora crassa octads, an above average rate of crossing over was detected within a 360-base region near the 3′ end of his-3, suggesting a hotspot for crossing over about 1.8 kb away from the recombination initiation site within cog. Homozygous deletion of the 360-base region increases exchanges in his-3 and on the far side of his-3 from cog, with the heterozygote showing an intermediate increase. We conclude that recombination events initiated at cog terminate within the 360-base sequence more often than in other sections of the cog–his-3 interval and, since some of these terminations will be resolved as crossovers, a cluster of crossovers at this location is the outcome. Removal of this termination site increases the chance that an event will reach his-3, resulting in recombination within the gene, or extend past it to yield a crossover on the other side of his-3. The deleted sequence has substantial predicted secondary structure, including a complex predicted stem-loop, suggesting that DNA secondary structure may be responsible for the termination.