Maize Transposable Elements in Development and Evolution

Transposable elements were first discovered in maize by BarbaraMcClintock more than 40 years ago. Today it is apparent thattransposable elements are a common component of the geneticmaterial in virtually all organisms. The best studied maizetransposable elements belong to the Activator-Dissociation andSuppressor-mutator families. They are short DNA sequences thatconsist of genes required for mobility and regulation. Boththe expression and the mobility of transposable elements areregulated in development by a mechanism that relies on the methylationof element sequences critical for expression. Elements can bestably inactivated by the same mechanism, persisting in thegenome in a cryptic form for long periods. The ability of thehost organism to regulate the highly mutagenic transposableelements may be critical to their survival, as well as theirutility as agents of genomic change.     

A test for nucleotide sequence homology

Two macromolecular sequences which have evolved from a common ancestor sequence will tend to include a large number of elements unaffected by replacement mutations in both sequences, as long as the evolutionary rate is not too high or the divergence time is not too great. The positions of corresponding elements may have changed in either daughter sequence due to deletion/insertion mutations involving other sequence elements, but their order can be expected to be the same in both sequences. These sets of correspondences, called matches, may be computed by a recursive algorithm which incorporates constraints on the number of deletion/insertion mutations hypothesized to have occurred. A test is developed which computes the significance of each deletion/insertion hypothesized, based on Monte-Carlo sampling of random sequences with the same base composition as the experimental sequences being tested. Applying the test to 5 S RNAs confirms the relation of Escherichia coli and KB carcinoma 5 S RNAs and establishes the previously undetected homology between Pseudomonas fluorescens and KB 5 S RNAs.     

Cloning and Characterization of New Repetitive Sequences in Field Bean (Vicia fabaL.)

Five new repetitive sequences have been isolated from theViciafabagenome, by cloning bands visible on agarose gel electrophoresisafter digestion of genomic DNA with various restriction enzymes.The sequences were 109 to 584 bp long, their abundance rangingfrom 5x10⁴to 5x10⁵copies per haploid genome. Southern blot andinsituhybridization revealed that four of five newly isolatedrepeats were dispersed in theV. fabagenome. One of the repeats(TIII15) showed tandem organization with several major hybridizationspots on mitotic chromosomesin situ.These sites were distributedin euchromatic as well as in heterochromatic chromosomal regions,and in several loci they were simultaneously localized withpreviously describedFokI repeated elements. The sequence ofTIII15 comprises four 26–27 bp subrepeats, but sharesno homology toFokI elements which have similar sequence organization.All newly described sequences were highly specific forV. faba,withlittle or no hybridization to DNA of otherViciaspecies, andno hybridization to DNA of other legumes tested.Copyright 1999Annals of Botany Company Vicia faba, field bean, repeated DNA sequences, FISH, PRINS, genome organization, copy number.     

Sequence Organization of Simple, Highly Repetitive DNA Elements in Brassica species

The amphidiploid (AACC) nuclear genome of Brassica napus (oil-seedrape) contains c. 5 ? 10⁵ copies of a simple, highly repetitiveDNA element; each repeat is 176 or 177 base pairs long and isdefined by Hind III cutting sites. The diploid (AA) Brassicacampestris (turnip) possesses a very similar repetitive DNA,the consensus sequence of which does not differ from that inB. napus. The 176/177 bp unit consists of three 59 bp sub-units,defined by vestigial EcoRII sites. Analysis of the distributionof variants from consensus in adjacent and non-adjacent unitsprovides evidence for homogenization of sequences by the fixationof independent mutations and for tandem duplication of units.Within units, there is also evidence for inversion and tandemduplication of short (5–8 bp) motifs. Previously published data show that 176/177 base pair repetitiveDNA elements, defined by Hind III cutting sites, are also presentin Sinapis and Raphanus. There is a sequence homology betweenBrassica and Sinapis, and between Brassica and Raphanus, of75%. Sequence homology between Raphanus and Sinapis is 73%. Key words: Repetitive DNA, Brassica, Cruciferae     

The complete mitochondrial genomes of two ghost moths, Thitarodes renzhiensis and Thitarodes yunnanensis: the ancestral gene arrangement in Lepidoptera

ABSTRACT: BACKGROUND: Lepidoptera encompasses more than 160,000 described species that have been classified into 45-48 superfamilies. The previously determined Lepidoptera mitochondrial genomes (mitogenomes) are limited to six superfamilies of the most derived lepidopteran lineage Ditrysia. Compared with the ancestral insect gene order, these mitogenomes all contain a tRNA rearrangement. To gain new insights into Lepidoptera mitogenome evolution, we sequenced the mitogenomes of two ghost moths that belong to primitive lepidopteran lineages and conducted a comparative mitogenomic analysis across Lepidoptera. RESULTS: The mitogenomes of Thitarodes renzhiensis and T. yunnanensis are 16,173 bp and 15,814 bp long with an A+T content of 81.28% and 82.33%, respectively. Different tandem repeats in the A+T-rich region mainly account for the size difference between the two mitogenomes. Both mitogenomes include 13 protein-coding genes, 22 transfer RNA genes, and 2 ribosomal RNA genes. The 1,584-bp sequence from rrnS to nad2 was also determined for Thitarodes sp.QL, which has no repetitive sequence in the A+T-rich region. All three Thitarodes species possess the ancestral gene order with trnI-trnQ-trnM located between the A+T-rich region and nad2, which is different from the gene order trnM-trnI-trnQ in all previously sequenced Lepidoptera species. The formerly identified conserved elements of Lepidoptera mitogenomes (i.e. the motif 'ATAGA' and poly-T stretch in the A+T-rich region and the long intergenic spacer upstream of nad2) are absent in the Thitarodes mitogenomes. The phylogenetic analysis supports that Hepialoidea, represented by T. renzhiensis and T. yunnanensis, occupies a basal position in the currently sampled seven superfamilies. The relationships of the other six superfamilies are (((((Bombycoidea + Geometroidea) + Noctuoidea) + Pyraloidea) + Papilionoidea) + Tortricoidea). CONCLUSION: The mitogenomes of T. renzhiensis and T. yunnanensis exhibit unusual features compared with the previously determined Lepidoptera mitogenomes. Their ancestral gene order indicates that the tRNA rearrangement event occurred after Lepidoptera diverged from other holometabolous insect orders. Phylogenetic analysis based on mitogenome sequences is a power tool for addressing phylogenetic relationships among major Lepidoptera superfamilies. Characterization of the two ghost moth mitogenomes has enriched our knowledge of Lepidoptera mitogenomes and contributed to our understanding of the mechanisms underlying mitogenome evolution, especially gene rearrangements.     

Overlapping redundant septuplets identical with regulatory elements of HIV-1 and SV40.

Overlapping redundant short oligomers in DNA sequences of retroviruses and papovaviruses have been identified. For each sequence, a search procedure determines the 5% short oligomers of the same length with the highest ratios of observed to expected occurrences based on singlet composition of the sequence. These short oligomers are referred to as compositionally-assessed redundant sequence elements (COARSEs). A pair of COARSEs overlapping by at least one base is considered to be a COARSE overlap. Most COARSE overlaps of the 7th order (overlapping septuplets) are found in long terminal repeats of retroviruses and in the regulatory control regions of papovaviruses SV40, BK and JC. Many of the 7th order COARSE overlaps in HIV-1 and SV40 are identical with regulatory elements determined experimentally. On the contrary, very few of the most frequently occurring oligomer overlaps, which are defined differently from COARSE overlaps, are present in the regulatory regions of retroviruses and papovaviruses. Examining DNA sequences of other genomes by the COARSE overlap method may identify putative regulatory regions.     

New transposable elements identified as insertions in rice transposon Tnr1

Tnr1 (235 bp long) is a transposable element in rice. Polymerase chain reactions (PCRs) done with a primer(s) that hybridizes to terminal inverted repeat sequences (TIRs) of Tnr1 detected new Tnr1 members with one or two insertions in rice genomes. Six identified insertion sequences (Tnr4, Tnr5, Tnr11, Tnr12, Tnr13 and RIRE9) did not have extensive homology to known transposable elements, rather they had structural features characteristic of transposable elements. Tnr4 (1767 bp long) had imperfect 64-bp TIRs and appeared to generate duplication of a 9-bp sequence at the target site. However, the TIR sequences were not homologous to those of known transposable elements, indicative that Tnr4 is a new transposable element. Tnr5 (209 bp long) had imperfect 46-bp TIRs and appeared to generate duplication of sequence TTA like that of some elements of the Tourist family. Tnr11 (811 bp long) had 73-bp TIRs with significant homology to those of Tnr1 and Stowaway and appeared to generate duplication of sequence TA, indicative that Tnr11 is a transposable element of the Tnr1/Stowaway family. Tnr12 (2426 bp long) carried perfect 9-bp TIRs, which began with 5'-CACTA- -3' from both ends and appeared to generate duplication of a 3-bp target sequence, indicative that Tnr12 is a transposable element of the En/Spm family. Tnr13 (347 bp long) had 31-bp TIRs and appeared to generate duplication of an 8-bp target sequence. Two sequences, one the transposon-like element Crackle, had partial homology in the Tnr13 ends. All five insertions appear to be defective elements derived from autonomous ones encoding the transposase gene. All had characteristic tandem repeat sequences which may be recognized by transposase. The sixth insertion sequence, named RIRE9 (3852 bp long), which begins with 5'-TG- -3' and ends with 5'- -CA-3', appeared to generate duplication of a 5-bp target sequence. These and other structural features indicate that this insertion is a solo LTR (long terminal repeat) of a retrotransposon. The transposable elements described above could be identified as insertions into Tnr1, which do not deleteriously affect the growth of rice cells.     

昆虫细胞色素P450基因的多样性、进化及表达调控

细胞色素P450单加氧酶（cytochrome P450 monooxygenases, P450s）是由多个功能相关的亚铁血红素 硫醇盐蛋白基因组成的一个基因超家族, 在各种内源和外源物质的代谢中起着主要作用。目前GenBank中注册的昆虫P450基因序列已超过1 000个, 其中双翅目占序列总数的74%, 鳞翅目占序列总数的16%。而昆虫P450基因序列已克隆的全长序列中大部分属于CYP4和CYP6家族, 两个家族成员分别占总数的20%和45%。利用GenBank中现已注册的昆虫P450基因的cDNA全长序列进行比对并绘制进化树, 揭示不同种类昆虫P450的亲缘关系。结果显示基于P450基因的昆虫部分目的进化关系与大部分先前依据其他分子数据或形态分类学得到的昆虫系统进化关系基本吻合。现有研究表明, 细胞色素P450基因的表达可能受顺式作用元件(cis-acting element)、反式作用因子(trans-acting factor)或两者共同调控, 调控可能涉及转录增强的转录机制或mRNA稳定性增加的转录后机制。     

Wolbachia in two insect host–parasitoid communities

Wolbachia form a group of intracellular bacteria that alter reproduction in their arthropod hosts. Two major phylogenetic subdivisions (A and B) of Wolbachia occur. Using a polymerase chain reaction assay we surveyed for the A and B group Wolbachia in 82 insect species from two temperate host–parasitoid communities (food webs) and a general collection of Lepidoptera caught at a light trap. One host–parasitoid community was based around leaf-mining Lepidoptera, and the other around Aphids. We found that: (i) 22.0% of insects sampled were infected with Wolbachia ; and (ii) the prevalence and type (A or B) of Wolbachia infection differed significantly between communities and taxonomic groups. We obtained DNA sequences from the ftsZ gene for the group B Wolbachia found in six leaf-mining species and one of their parasitoids, as well as four of the Lepidoptera caught by a light trap. Taken together, the results of our survey and phylogenetic analyses of the sequence data suggest that host–parasitoid transfer of Wolbachia is not the major route through which the species we have examined become infected. In addition, the Wolbachia strains observed in five leaf-mining species from the same genus were not closely related, indicating that transfer between species has not occurred due to a shared feeding niche or cospeciation.     

Isolation and Characterization of a Water Stress-Specific Genomic Gene, pwsi 18, from Rice

One of the water stress-specific cDNA clones of rice characterisedpreviously, wsi18, was selected for further study. The wsi18gene can be induced by water stress conditions such as mannitol,NaCl, and dryness, but not by ABA, cold, or heat. A genomicclone for wsi18, pwsi18, contained about 1.7 kbp of the 5' upstreamsequence, two introns, and the full coding sequence. The 5'-upstreamsequence of pwsi18 contained putative cis-acting elements, namelyan ABA-responsive element (ABRE), three G-boxes, three E-boxes,a MEF-2 sequence, four direct and two inverted repeats, andfour sequences similar to DRE, which is involved in the dehydrationresponse of Arabi-dopsis genes. The gusA reporter gene underthe control of the pwsi18 promoter showed transient expressionin response to water stress. Deletion of the downstream DRE-likesequence between the distal G-boxes-2 and -3 resulted in ratherlow GUS expression. (Received March 27, 1997; Accepted November 5, 1997)     

褐飞虱肌动蛋白基因3′末端克隆与测序

应用锚定的3′RACE方法,对褐飞虱Nilaparvata lugens (Stal)肌动蛋白基因3′末端进行了扩增、克隆和测序, 并对所得到的5个cDNA片段的不译端的序列进行了比较。结果显示：BPH-A和BPH-D与其他序列不同;BPH-B、BPH-C和PBH-E三条片段的不译端的序列很相似,只是长度不同。另外,所得序列中所含翻译区的氨基酸序列高度保守,根据肌动蛋白基因结构特点可以推断BPH-A和BPH-D属肌肉特异型肌动蛋白基因,BPH-B、BPH-C和BPH-E属细胞质特异型肌动蛋白基因。     

Structural heterogeneity in the R173 family of rye-specific repetitive DNA sequences

The rye-specific R173 family of repeated DNA sequences consists of ca. 15 000 individual copies per diploid rye (Secale cereale) genome and is distributed over all 7 rye chromosomes in a dispersed manner. Individual R173 elements vary in size between 3 and 6 kb, are generally not arranged as tandem repeats and are flanked by both multi-copy and single-copy sequences. DNA sequence analysis of three R173 elements (R173-1, R173-2 and R173-3) demonstrated a high degree of homology in conserved domains. The structure of R173-1 was quite different from the other two elements: long direct repeats, which represent a rye-specific repetitive sequence, were found at the ends and a 600 bp long domain was replaced by an unrelated sequence of approximately equal size. R173-2 and R173-3 were extremely similar to each other with the exception of a terminal truncation of R173-2. No open reading frames for proteins >20 kDa were present and a database search failed to detect significant homologies to published protein sequences. Despite the transposon like genomic organisation of the R173 family, individual elements lacked sequence features frequently associated with transposons and retrotransposons. In contrast, two of the regions flanking R173 elements showed strong DNA homologies to a 850 bp long region of a proposed wheat retrotransposon and to a 300 bp long region downstream of the wheatGlu-D1 gene.     

Distinct characteristics of loop sequences of two Drosophila foldback transposable elements.

A few foldback (FB) transposable elements have, between their long terminal inverted repeats, central loop sequences which have been shown to be different from FB inverted repeat sequences. We have investigated loop sequences from two such FB elements by analyzing their genomic distribution and sequence conservation and, in particular, by determining if they are normally associated with FB elements. One of these FB loop sequences seems to be present in a few conserved copies found adjacent to FB inverted repeat sequences, suggesting that it represents an integral component of some FB elements. The other loop sequence is less well-conserved and not usually associated with FB inverted repeats. This sequence is a member of another family of transposable elements, the HB family, and was found inserted in an FB element only by chance. We compare the complete DNA sequences of two HB elements and examine the ends of four HB elements.     

2.9 A resolution structure of the N-terminal domain of a variant surface glycoprotein from Trypanosoma brucei

The variant surface glycoprotein (VSG) of Trypanosoma brucei forms a coat on the surface of the parasite; by the expression of a series of antigenically distinct VSGs in the surface coat the parasite escapes the host immune response. The 2.9 A resolution crystal structure of the N-terminal domain of one variant, MITat 1.2, has been determined. The structure was solved using data collected from two crystal forms. Initially a partial model was built into an electron density map based on multiple isomorphous replacement phases and improved by phase combination methods. Subsequently this model was used to obtain the molecular replacement solution for a second crystal form, providing starting phases which were refined using 2-fold non-crystallographic symmetry averaging. The current model includes 362 residues and has been refined using X-PLOR to an R value of 0.22 for data between 7 and 2.9 A. The molecule is a dimer, approximately 100 A long, having an asymmetrical cross section with maximum dimensions of approximately 40 A x 60 A. Two long, approximately 70 A, alpha-helices from each monomer pack together to form, with several other helices, a core helix bundle that extends nearly the full length of the molecule. The "top" of the protein, which in the surface coat may be exposed to the external environment, is formed from the ends of the two long helices, a short three-stranded beta-sheet, and a strand having irregular conformation that packs above these secondary structure elements. Two conserved disulfide bridges are in this part of the molecule. Several elements of the MITat 1.2 sequence, which contribute to the formation of the helix bundle structure, have been identified. These elements can be found in the sequences of several different VSGs, suggesting that to some extent the VSG structure is conserved in those variants.     

A 718-kb DNA Sequence of the Escherichia coli K-12 Genome Corresponding to the 12.7-28.0 min Region on the Linkage Map

The 718,122 base pair (bp) sequence of the Escherichia coliK-12 genome corresponding to the region from 12.7 to 28.0 minuteson the genetic map is described. This region contains at least682 potential open reading frames, of which 278 (41%) have beenpreviously identified, 147 (22%) were homologous to other knowngenes, 138 (20%) are identical or similar to the hypotheticalgenes registered in databases, and the remaining 119 (17%) didnot show a significant similarity to any other gene. In thisregion, we assigned a cluster of cit genes encoding multienzymecitrate lyase, two clusters of fimbrial genes and a set of lysogenicphage genes encoding integrase, excisionase and repressor inthe e14 genetic element. In addition, a new valine tRNA gene,designated valZ, and a family of long directly repeated sequences,LDR-A, -B and -C, were found.     

Cloning and characterization of piggyBac- like elements in lepidopteran insects

PiggyBac-like elements (PLE) are widespread in variety of organisms, however, few of them are active or have an intact transposon structure. To further define the distribution PLEs in Lepidoptera, where the original active piggyBac IFP2 was discovered, and potentially isolate new functional elements, a survey for PLEs by PCR amplification and Southern dot blots was performed. Two new PLEs, AyPLE and AaPLE, were successfully isolated from the noctuid species, Agrotis ypsilon and Argyrogramma agnate, respectively. These elements were found to be closely related to each other by sequence similarity, and by sharing the same 16 bp inverted terminal repeat sequences. The AyPLE1.1 and AaPLE1.1 elements are structurally intact having characteristic TTAA target site duplications, inverted terminal repeats and intact open reading frames encoding putative transposases with the presumed piggyBac DDD domains, which are features consistent with autonomous functional transposons. Phylogenetic analysis revealed that AyPLE1.1 and AaPLE1.1 cluster with another noctuid species element, HaPLE1.1, suggesting a common ancestor for the three types of PLEs. This contributes to our understanding of the distribution and evolution of piggyBac in Lepidoptera.     

The Spirulina platensis Adenylate Cyclase Gene, cyaC, Encodes a Novel Signal Transduction Protein

A cyaC gene encoding an adenylate cyclase of the filamentouscyanobacterium Spirulina platensis was se-quenced. The predictedamino acid sequence of the C-ter-minal region of cyaC is similarto the catalytic domains of adenylate cyclases in other cyanobacteriaand eukaryotes. The sequences of other regions are similar tothose of proteins consisting of the bacterial two-componentsignal transduction system: the sensory kinase and the responseregulator. The predicted gene product of cyaC contains, fromthe N-terminal end, a receiver domain of the response regulatorprotein (Rl), a domain similar to the ETR1 of Arabi-dopsis thaliana,a transmitter domain of the sensory kinase protein, a receiverdomain of the response regulator protein (R2), and a catalyticdomain of adenylate cyclase. The cyaC gene was expressed asan affinity-tagged protein in Escherichia coli, and the recombinantprotein was purified. The purified protein had adenylate cyclaseactivity which was activated by Mn²+. The results of Westernblotting using an anti-CyaC antiserum and the S. platensis cellextract confirmed that cyaC gene is expressed in S. platensis (Received February 27, 1997; Accepted April 26, 1997)     

Organization of the human myoglobin gene.

Cross-hybridization of the grey seal myoglobin gene to human DNA detected a single human myoglobin gene plus an extensive family of sequences apparently related to the central exon of this gene. The functional human gene is 10.4 kb long and has a haemoglobin-like three exon/two intron structure with long non-coding regions similar to its seal homologue. At least 300 bp of 5'-flanking region are closely homologous between the two genes, with the exception of a divergent purine-rich region 68-114 bp upstream of the cap site. A diverged tandem repetitive sequence based on (GGAT)165 is located 1100-1750 bp upstream from the gene; internal homology units within this sequence suggest sequence homogenization by gene microconversions. A second 33-bp tandem repeat element in the first intron is flanked by a 9-bp direct repeat, shares homology with other tandem repetitive elements in the human genome and may represent a novel form of transposable element.