Nucleotide sequence divergence in the A+T-rich region of mitochondrial DNA in Drosophila simulans and Drosophila mauritiana

We have determined the nucleotide sequences of two regions within the A+T-rich region of mitochondrial DNA (mtDNA) in the siIII type of Drosophila simulans and the maI type of D. mauritiana. The sequences of the two regions in siIII and maI are almost identical. The sequences include elements corresponding to the type I and type II repeats elements and the T-stretches as reported in D. melanogaster; an approximately 340-bp region (A region) adjacent to the tRNA(Ile) gene includes a part of the type II repeat element, and an approximately 440- bp region (B region) includes a central portion of the A+T-rich region between the type I and type II repeat arrays. Each sequence of the two species was compared with those of D. melanogaster and D. yakuba. The sequences of the A region are relatively well conserved among the four species. The alignment of the two sequences of the B region with those of D. melanogaster and D. yakuba requires numerous insertions/deletions. For both regions, nucleotide differences between D. simulans or D. mauritiana and D. melanogaster are similar to those between the two and D. yakuba. The tendency is obvious in a subregion within the type II repeat element in the A region. These findings suggest that the rate of nucleotide substitution in the subregion is accelerated in the lineage leading to D. melanogaster. Loss of functional constraint in the stem-loop-forming sequence is proposed for this acceleration.      

Complete mitochondrial genome of the seven-spotted lady beetle, Coccinella septempunctata (Coleoptera: Coccinellidae)

The complete mitochondrial genome (mitogenome) of the seven-spotted lady beetle, Coccinella septempunctata (Coleoptera: Coccinellidae), which is one of the best known insects capable of predation, is described with an emphasis on the noteworthy composition of the A+T-rich region. The C. septempunctata genome consists of 2 rRNAs, 22 tRNAs, 13 protein-coding genes, and 1 control region, designated as the A+T-rich region in insects. Along with an unusually long A+T-rich region (4469 bp), the 18,965-bp long C. septempunctata mitogenome was the largest in Coleoptera. The A+T-rich region is composed of a 2214-bp long non-repeat region composed of 80.17% A/T nucleotides and a 2256-bp long repeat region composed of 65.71% A/T nucleotides. The repeat region harbors 32 identical 70-bp long tandem repeats plus one 15-bp long incomplete first repeat. These repeat sequences may possibly have been caused by slipped-strand mispairing and unequal crossing-over events during DNA replication.     

Molecular characterization of a repeat element causing large-scale size variation in the mitochondrial DNA of the sea scallop Placopecten magellanicus

The scallop Placopecten magellanicus has the largest reported animal mitochondrial DNA (average 35 kb) and exhibits large inter- and intraindividual length variation owing to the varying copy number of a repeated element. We have characterized the repeat array by using restriction mapping and sequence analysis. The repeated element consists of 1,442 bp flanked on either side by the sequence ACTTTCC in a direct orientation. The array contains two to eight copies of the repeated element arranged in a direct orientation and in tandem. Only complete copies of the element are present in the array. The repeat element contains three regions with characteristic nucleotide sequences: a 10-bp inverted repeat shown to extrude into a cruciform in a supercoiled DNA plasmid, a 120-bp tract rich in G/C (70%) and adjacent to the inverted repeat, and periodically interspersed homopolymer runs of A and T occurring near the middle of the element which induce DNA curvature in dimeric constructs of the element. The element appears to be unique to P. magellanicus. The structural properties of the repeat element and its organization in an array of repeats may be important in explaining the generation and maintenance of large-scale mitochondrial DNA size variation observed in many animal species.     

Insect mitochondrial genomics: the complete mitochondrial genome sequence of the meadow spittlebug Philaenus spumarius (Hemiptera: Auchenorrhyncha: Cercopoidae).

We present the complete mitochondrial genome sequence of the meadow spittlebug Philaenus spumarius (Auchenorrhyncha: Cercopoidae). This contribution represents the second mitochondrial genome from the Hemiptera and the second of the three hemipteran suborders sampled. The genome is a circular molecule of 16 324 bp with a total A+T content of 77.0% and 76.7% for coding regions only. The gene content, order, and structure are consistent with the Drosophila yakuba genome structure (Clary and Wolstenholme 1985) and the hypothesized ancestral arthropod genome arrangement (Crease 1999). Nucleotide composition and codon usage are near the means observed in other insect mitochondria sequenced to date but have a higher A+T richness compared with the other hemipteran example, the kissing bug Triatoma dimidiata (Dotson and Beard. 2001. Insect Mol. Biol. 10: 205-215). The major noncoding region (the A+T rich region or putative control region) between the small ribosomal subunit and the tRNAIle gene includes two extensive repeat regions. The first repeat region includes 19 tandem repeats of a 46-bp sequence, whereas the second contains a longer sequence (146 bp) tandemly repeated four times.     

Simian virus 40 DNA replication: functional organization of regulatory elements.

The efficiency of simian virus 40 (SV40) DNA replication is dependent on the structural organization of the regulatory region. The enhancing effect of the G + C-rich 21-base-pair (bp) repeats on SV40 DNA replication is position and dose dependent and to some extent orientation dependent. The inverted orientation is about 50% as effective as the normal orientation of the 21-bp repeat region. Movement of the 21-bp repeat region 180 or 370 bp upstream of the ori sequence abolishes its enhancing effect, whereas no replication is detected if the 21-bp repeat region is placed downstream of the ori sequence. The dose-dependent enhancement of the 21-bp repeat of SV40 DNA replication as first described in single transfection by Bergsma et al. (D. J. Bergsma, D. M. Olive, S. W. Hartzell, and K. N. Subramanian, Proc. Natl. Acad. Sci. USA 79:381-385, 1982) is dramatically amplified in mixed transfection. In the presence of the 21-bp repeat region, the 72-bp repeat region can enhance SV40 DNA replication. In the presence of the 21-bp repeats and a competitive environment, the 72-bp repeat region exhibits a cis-acting inhibitory effect on SV40 DNA replication.     

Complete nucleotide sequence of the A+T-rich region of Drosophila mauritiana mitochondrial DNA

We determined the complete nucleotide sequence of the A+T-rich region of the maII type of mtDNA in D. mauritiana. The nucleotide sequence was found to contain 3,206 bp. Three types of conserved element, i.e., type I element, type II element, and T-stretch, were included in this sequence, as reported for D. melanogaster. Comparison between the two species revealed that the type I elements were less conserved than the type II elements. However, each of these type I elements contained a G-stretch within a loop of a putative stem-loop-forming sequence, which has also been observed in D. melanogaster. Moreover, in both type I and type II repeat arrays, the elements closest to the T-stretch diverged the most, due to nucleotide substitution and/or the insertion of short repeats. Sequence comparison of the two complete sequences of the A+T-rich region of D. melanogaster and the maII type of D. mauritiana, as well as comparison of partial sequences in other types of mtDNA within the melanogaster complex, suggested that the A+T-rich region in this complex has been maintained by concerted evolution after the duplication of two types of element, i.e., type I and type II.     

A 5.9-kb tandem repeat at the euchromatin-heterochromatin boundary of the X chromosome of Drosophila melanogaster

We present an analysis of a chromosomal walk in the region of the euchromatin-heterochromatin transition at the base of the X chromosome of Drosophila melanogaster. This region is difficult to analyse because of the presence of repeated sequences, and we have used cosmids to walk from the last euchromatic gene, suppressor of forked, towards the pericentric heterochromatin. The proximal 30-kb sequence we have isolated consists of repetitive DNA, including four tandem copies of a 5.9-kb sequence. This tandem repeat is itself a mosaic of other, mostly repeated, sequences, including part of a retrotransposon without long terminal repeats, a simple-sequence region of TAA repeats and part of a retrotransposon with long terminal repeats that has not been previously described. Although sequences homologous to these components are found elsewhere in the genome, this arrangement of repeated sequences is only found at the base of the X chromosome. It is conserved in D. melanogaster strains of different geographic origin, but is not conserved in even closely related species.     

Molecular cloning and characterization of novel centromeric repetitive DNA sequences in the blue-breasted quail (Coturnix chinensis,Galliformes)

A new family of centromeric highly repetitive DNA sequences was isolated from EcoRI-digested genomic DNA of the blue-breasted quail (Coturnix chinensis, Galliformes), and characterized by filter hybridization and chromosome in situ hybridization. The repeated elements were divided into two types by nucleotide length and chromosomal distribution; the 578-bp element predominantly localized to microchromosomes and the 1,524-bp element localized to chromosomes 1 and 2. The 578-bp element represented tandem arrays and did not hybridize to genomic DNAs of other Galliformes species, chicken (Gallus gallus), Japanese quail (Coturnix japonica) and guinea fowl (Numida meleagris). On the other hand, the 1,524-bp element was not organized in tandem arrays, and did hybridize to the genomic DNAs of three other Galliformes species, suggesting that the 1,524-bp element is highly conserved in the Galliformes. The 578-bp element was composed of basic 20-bp internal repeats, and the consensus nucleotide sequence of the internal repeats had homologies to the 41-42 bp CNM repeat and the XHOI family repeat of chicken. Our data suggest that the microchromosome-specific highly repetitive sequences of the blue-breasted quail and chicken were derived from a common ancestral sequence, and that they are one of the major and essential components of chromosomal heterochromatin in Galliformes species.     

Single-stranded structures are present within plasmids containing the Epstein-Barr virus latent origin of replication.

The Epstein-Barr virus (EBV) latent origin of plasmid replication (oriP) contains two essential regions, a family of repeats with 20 imperfect copies of a 30-bp sequence and a dyad symmetry element with four similar 30-bp repeats. Each of the repeats has an internal palindromic sequence and can bind EBNA 1, a protein that together with oriP constitutes the only viral element necessary for EBV maintenance and replication. Using single-strand-specific nucleases, we have probed plasmids containing oriP-derived sequences for the presence of secondary structural elements. Multiple single-stranded structures were detected within the oriP region. Of the two essential elements of oriP, the family of repeats seemed to extrude these structures at a much higher frequency than did sequences within the dyad symmetry region. Though negative supercoiling was found to stabilize the single-stranded structures, they showed significant stability even after linearization of the oriP plasmids. Two major single-stranded structures detected involved approximately 12 bp of DNA. These loci could be transiently unwound regions that form because of negative supercoiling and the high A + T content of this region of DNA, or they could be cruciform structures extruded within the palindromic sequences of oriP that may be important sites for protein-DNA interactions in the EBV oriP.     

Tnat1 and Tnat2 from Arabidopsis thaliana: novel transposable elements with tandem repeat sequences.

A computer-aided homology search of databases found that the nucleotide sequences flanking ATLN44, a non-LTR retrotransposon (LINE) from Arabidopsis thaliana, are repeated in the A. thaliana genome. These sequences are homologous to flanking sequences of 664 bp with terminal inverted repeat sequences of about 70 bp. The 664-bp sequence and most of the 14 homologues identified were flanked by direct repeat sequences of 9 bp. These findings indicate that the repeated sequence, named Tnat1, is a transposable element that duplicates a 9-bp sequence at the target site on transposition and that ATLN44 is inserted in one Tnat1 member. Interestingly, all of the Tnat1 members had tandem repeats comprised of several units of a 60-bp sequence, the number of repeats differing among Tnat1 members. Of the Tnat1 members identified, one was inserted into another sequence repeated in the A. thaliana genome: that sequence is about 770 bp long and has terminal inverted repeat sequences of about 110 bp. The sequence is flanked by direct repeats of a 9-bp sequence, indicating that it is another transposable element, named Tnat2, from A. thaliana. Moreover, Tnat2 members had a tandem repeat about 240 bp long. Tnat1 and Tnat2 with tandem repeats in their internal regions show no homology to each other or to any of the elements identified previously; therefore they appear to be novel transposable elements.     

鸮形目4种鸟类线粒体调控区全序列的测定与比较研究

利用Long-PCR和Primer Walking的方法对鸮形目的短耳鸮、长耳鸮、纵纹腹小鸮、灰林鸮4种鸟类的线粒体调控区进行了全序列测定。结果表明：短耳鸮的调控区跃度为3290bp;长耳鸮为2848bp;纵纹腹小鸮为2444bp;灰林鸮为1771bp。短耳鸮的调控区长度是4种鸮中最大的,并且是目前已知最大的鸟类线粒体调控区。这4种鸮类调控区的基本结构和其他鸟类相似,按照碱基变化速率的不同可以分为3个区：碱基变化速率较快的外围区域Ⅰ、Ⅲ和保守的中间区域Ⅱ。这4种鸟类调控区的3’端均存在大量的串联重复序列,短耳鸮为126bp单元重复7次和78bp单元重复14次;长耳鸮为127bp单元重复8次和78bp单几重复6次;纵纹腹小鸮有3个重复单元,分别为89bp单元重复3次、77bp单元重复4次和71bp单元重复6次;灰林鸮仅有1个单元的串联重复为78bp重复5次。调控区中串联重复序列可能是由链的滑动错配产生,另外这些重复序列都能形成热力学稳定的多重茎环二级结构,而且在重复序列中还发现一些保守基序,这说明重复序列可能具有一定的生理功能,影响调控区的调重控功能从而影响线粒体基因组的复制和转录。     

Multicomponent origin of cytomegalovirus lytic-phase DNA replication.

Cytomegalovirus (CMV) lytic-phase DNA replication requires both trans-acting factors, such as the virus-coded DNA polymerase, and a previously undefined cis-acting element, the origin, within which initiation occurs. We have located a candidate origin of CMV lytic-phase DNA replication, oriLyt, in both simian and human strains by assessing the ability of cloned restriction fragments to mediate phosphonoformic acid-sensitive DNA replication after transfection into human fibroblasts when required trans-acting factors were supplied by infection. In initial experiments the simian CMV-like strain Colburn EcoRI D fragment directed DNA replication; this fragment contains all of the single-stranded DNA-binding protein gene (dbp) and about 7 kbp of upstream sequence. A larger region upstream of human CMV dbp also mediated replication in transient assays. Subsequent subcloning and deletion analyses defined a CMV strain Colburn region sufficient for origin function, spanning about 1,300 bp in the apparently noncoding region upstream of dbp. The nucleotide sequence of this region revealed four distinct domains, containing (i) a 9-bp repeated sequence, (ii) an A+T-rich segment, (iii) an 11-bp direct repeat, and (iv) a 47-bp direct repeat. At least some part of each of these domains was required for origin function. Therefore, like the Epstein-Barr virus lytic-phase origin of DNA replication, CMV oriLyt appears to be structurally complex.     

The histidine tRNA genes of yeast

Yeast has at least seven nuclear histidine tRNA genes although there is a single tRNAHis. We have sequenced three of the histidine tRNA genes. The genes have identical coding sequences and the DNA anti-codon sequence GTG corresponds to the GUG anti-codon in tRNAHis. None of the three yeast histidine tRNA genes has an intervening sequence. Two of the three genes contain repeated DNA elements in the region adjacent to the 5' end of the histidine tRNA gene. One of the elements, sigma, is 18 base pairs (bp) from the 5' end of each of these genes, sigma elements are highly conserved and flanked by 5-bp repeats. The other element, delta, is at variable distances from the tRNA gene; one is 439 bp from a histidine tRNA gene and the other is 52 bp from a histidine tRNA gene. These solo delta elements are quite divergent when compared with delta s associated with transposon yeast elements and are not flanked by 5-bp repeats.     

Complete mitochondrial genome of Oxuderces dentatus (Perciformes, Gobioidei)

The complete mitochondrial genome (mitogenome) of Oxuderces dentatus was determined first. The genome was 17,116?bp in length and consisted of 13 protein-coding genes, 22 tRNA genes, 2 ribosomal RNA genes, and 2 main non-coding regions [the control region (CR) and the origin of the light strand replication], the gene composition and order of which was similar to most other vertebrates. The overall base composition of the heavy strand was T 27.9%, C 26.8%, A 30.2%, and G 15.1%, with a slight A+T bias of 58.1%. In addition to the discrete and conserved sequence blocks, unusual long tandem repeat unit (three 150-bp tandem repeat units and an incomplete copy of 146?bp) was also detected within CR. This mitogenome sequence data would play an important role in population genetics and phylogenetic analysis of the Gobioidei.     

Mutational Analysis of Meiotic and Mitotic Centromere Function in Saccharomyces cerevisiae

A centromere (CEN) in Saccharomyces cerevisiae consists of approximately 150 bp of DNA and contains 3 conserved sequence elements: a high A + T region 78-86 bp in length (element II), flanked on the left by a conserved 8-bp element I sequence (PuTCACPuTG), and on the right by a conserved 25-bp element III sequence. We have carried out a structure-function analysis of the element I and II regions of CEN3 by constructing mutations in these sequences and subsequently determining their effect on mitotic and meiotic chromosome segregation. We have also examined the mitotic and meiotic segregation behavior of ARS plasmids containing the structurally altered CEN3 sequences. Replacing the periodic tracts of A residues within element II with random A + T sequences of equal length increases the frequency of mitotic chromosome nondisjunction only 4-fold; whereas, reducing the A + T content of element II while preserving the length results in a 40-fold increase in the frequence of chromosome nondisjunction. Structural alterations in the element II region that do not decrease the overall length have little effect on the meiotic segregation behavior of the altered chromosomes. Centromeres containing a deletion of element I or a portion of element II retain considerable mitotic activity, yet plasmids carrying these same mutations segregate randomly during meiosis I, indicating these sequences to be essential for maintaining attachment of the replicated sister chromatids during the first meiotic division. The presence of an intact element I sequence properly spaced from the element III region is absolutely essential for proper meiotic function of the centromere.     

Highly repeated DNA sequences in birds: the structure and evolution of an abundant, tandemly repeated 190-bp DNA fragment in parrots.

Up to 6.8% of the parrot (Psittaciformes) genome consists of a tandemly repeated, 190-bp sequence (P1) located in the centromere of many if not all chromosomes. Monomer repeats from 10 different psittacine species representing four subfamilies were isolated and cloned. The intraspecific sequence variation ranged from 1.5 to 7%. The interspecific sequence variation ranged from less than 3% between two species of cockatoos to approximately 45% between cockatoos and other parrots. The monomer sequences of all 10 parrot species contained several conserved (> 90%) sequence elements at identical locations within the repeat. A comparison with tandemly repeated DNA sequences in other avian species showed that several of these conserved elements were also present at similar locations within the 184-bp repeat of the Chilean flamingo (Phoenicopterus chilensis), suggesting a great antiquity of the repeat. One of the elements was also found in the tandemly repeated sequences of the crane (Gruidae) and falcon (Falconidae) families. The data were used for the construction of a partial most parsimonious relationship that supports a regional subdivision of the Psittaciformes.     

The heterochromatin of grasshoppers from the Caledia captiva species complex. I. Sequence evolution and conservation in a highly repeated DNA family

The restriction enzyme TaqI digests 0.2% of the genomic DNA from the grasshopper Caledia captiva to a family of sequences 168 bp in length (length of consensus sequence). The sequence variation of this "Taq family" of repeat units was examined among four races from C. captiva to assay the pattern of evolution within this highly repeated DNA. The Taq-family repeats are located in C-banded heterochromatin on at least one member of each homologous pair of chromosomes; the locations range from centromeric to telomeric. Thirty-nine cloned repeats isolated from two population 1A individuals along with 11 clones from seven populations taken from three of the races demonstrated sequence variation at 72 positions. Pairwise comparisons of the cloned repeats, both within an individual and between different races, indicate that levels of intraspecific divergence, as measured by reproductive incompatibility, do not correlate with sequence divergence among the 168-bp repeats. A number of subsequences within the repeat remain unchanged among all 50 clones; the longest of these is 18 bp. That the same 18-bp subsequence is present in all clones examined is a finding that departs significantly (P less than 0.01) from what would be expected to occur at random. Two other cloned repeats, from a reproductively isolated race of C. captiva, have sequences that show 56% identity with this 18-bp conserved region. An analysis showed that the frequency of occurrence of an RsaI recognition site within the 168- bp repeat in the entire Taq family agreed with that found in the cloned sequences. These data, along with a partial sequence for the entire Taq family obtained by sequencing uncloned repeats, suggest that the consensus sequence from the cloned copies is representative of this highly repeated family and is not a biased sample resulting from the cloning procedure. The 18-bp conserved sequence is part of a 42-bp sequence that possesses dyad symmetry typical of protein-binding sites. We speculate that this may be significant in the evolution of the Taq family of sequences.