Evolution of Tribolium madens (Insecta,Coleoptera) Satellite DNA Through DNA Inversion and Insertion

Two different satellite DNAs from tenebrionid speciesTribolium madens (Insecta, Coleoptera) have been detected, cloned, and sequenced. Satellite I comprises 30% of the genome; it has a monomer size of 225 by and a high A + T content of 74%. Satellite 11, with a monomer size of 711 by and A + T content of 70%, is less abundant, making 4% of the total DNA. Sequence variability of the monomers relative to consensus sequence is 4.1% and 1.2% for satellite I and II, respectively. Both satellites are localized in the heterochromatic regions of all chromosomes. A search for internal motifs showed that both satellites contain a related subsequences, about 100 by long. The creation of satellite I monomer is explained by duplication of the basic subunit, followed by subsequent divergence by single point mutations, deletions, and gene conversion. Inversion of the subsequence in addition to its duplication has occurred in satellite II. The result of this inversion is possible formation of a long, stable dyad structure. The 408-bp sequence, inserted within satellite II monomer, shares no similarity with a basic subunit. Frequent direct repeats found within the inserted sequence point to its evolution by duplication of shorter motifs. It is proposed that both satellites have been derived from a common ancestral sequence whose duplication played a major role in the formation of satellite I monomer, while insertion of a new sequence together with inversion of an ancestral one induced the occurrence of satellite II. Correspondence to: D. Ugarković     

Long Inversely Oriented Subunits Form a Complex Monomer of Tribolium brevicornis Satellite DNA

Highly abundant satellite DNA named TBREV is detected and characterized in the beetle Tribolium brevicornis (Insecta: Coleoptera). An outstanding peculiarity of the TBREV satellite monomer is its complex structure based on the two 470-bp-long subunits, inversely oriented within a 1061-bp-long monomer sequence. The proposed evolutionary history demonstrates a clear trend toward increased complexity and length of the TBREV satellite monomer. This tendency has been observed on three levels: first as direct and inverted duplications of short sequence motifs, then by inverse duplication of the 470-bp sequence segment, and, finally, by spread of inversely duplicated elements in a higher-order register and formation of extant monomers. Inversely oriented subunits share a similarity of 82% and have a high capacity to form a thermodynamically stable dyad structure that is, to our knowledge, the longest ever described in any satellite monomer. Analysis of divergences between inversely oriented subunits shows a tendency to a further reduction in similarity between them. Except in its centromeric localization, the TBREV satellite does not show similarity to other known Tribolium satellites, either in nucleotide sequence or in monomer length and complexity. However, TBREV shares common features of other Tribolium satellites that might be under functional constraints: nonconstant rate of evolution along the monomer sequence, short inverted repeats in the vicinity of an A+T tract, nonrandom distribution of A or T 3 tracts, and CENP-B box-like motifs. Although long inverted subunits might reinforce structural characteristics of the satellite monomer, their nucleotide sequence does not seem to be under constraints in order to preserve the dyad structure. Reviewing Editor: Dr. Willie Swanson     

Molecular evolution of centromere-associated nucleotide sequences in two species of canids

The major centromeric satellite nt sequences present in the domestic dog (Canis familiaris) and in the grey fox (Urocyon cineroargenteus) have been examined. The dog satellite monomer is 737 bp long and contains 51% G + C; the grey fox satellite monomer is 880 b long and contains 54% G + C. The two satellites share three regions of 78, 92 and 314 bp with 70-80% sequence similarity. Sequence data from 16 monomers of dog satellite and 19 monomers of grey fox satellite demonstrate that the substitution spectra are different in the two canid species. For example, substitutions involving G or C residues are much more common in the grey fox satellite than in the domestic dog satellite despite their similar G + C contents.     

Characterization of a New HpaI Centromeric Satellite DNA in Salmo salar

A highly repeated HpaI DNA family was revealed in Atlantic salmon (Salmo salar) and analyzed by Southern blotting and fluorescence in situ hybridization (FISH). In this report, we describe the nucleotide sequence, genomic structure and chromosomal localization of this HpaI repeat. This novel satellite appeared tandemly arrayed and located at centromeric areas of three acrocentric chromosome pairs as evidenced by FISH. The sequence was characterized by a high AT content (63%), a short consensus motif (A/T)(G/C)AAA(T/C) similar to other centromeric satellites motifs, and by short AT enriched stretches. The presence of this sequence in other salmonid species was also tested by Southern blot hybridization and used to analyze its evolution within this group.     

Characterization and chromosomal distribution of satellite DNA sequences of the water buffalo (Bubalus bubalis).

Satellite DNA sequences were isolated from the water buffalo (Bubalus bubalis) after digestion with two restriction endonucleases, BamHI and StuI. These satellite DNAs of the water buffalo were classified into two types by sequence analysis: one had an approximately 1,400 bp tandem repeat unit with 79% similarity to the bovine satellite I DNA; the other had an approximately 700 bp tandem repeat unit with 81% similarity to the bovine satellite II DNA. The chromosomal distribution of the satellite DNAs were examined in the river-type and the swamp-type buffaloes with direct R-banding fluorescence in situ hybridization. Both the buffalo satellite DNAs were localized to the centromeric regions of all chromosomes in the two types of buffaloes. The hybridization signals with the buffalo satellite I DNA on the acrocentric autosomes and X chromosome were much stronger than that on the biarmed autosomes and Y chromosome, which corresponded to the distribution of C-band-positive centromeric heterochromatin. This centromere-specific satellite DNA also existed in the interstitial region of the long arm of chromosome 1 of the swamp-type buffalo, which was the junction of the telomere-centromere tandem fusion that divided the karyotype in the two types of buffaloes. The intensity of the hybridization signals with buffalo satellite II DNA was almost the same over all the chromosomes, including the Y chromosome, and no additional hybridization signal was found in noncentromeric sites.     

Sequence and phylogenetic analysis of the complete mitochondrial genome of the flour beetle Tribolium castanaeum

We describe the first complete mitochondrial genome sequence from a representative of the insect order Coleoptera, the flour beetle Tribolium castaneum. The 15,881 bp long Tribolium mitochondrial genome encodes 13 putative proteins, two ribosomal RNAs and 22 tRNAs canonical for animal mitochondrial genomes. Their arrangement is identical to that in Drosophila melanogaster, which is considered ancestral for insects and crustaceans (Boore et al., 1998; Hwang, et al., 2001a). Nucleotide composition, amino acid composition, and codon usage fall within the range of values observed in other insect mitochondrial genomes. Most notable features are the use of TCT as tRNA(Ser(AGN)) anticodon instead of GCT, which is used in most other arthropod species, and the relative scarcity of special sequence motifs in the 1431 bp long control region. Phylogenetic analysis confirmed resolving power in the conserved regions of the mitochondrial proteome regarding diversification events, which predate the emergence of pterygote insects, while little resolution was obtained at the level of basal perygote diversification. The partition of faster evolving amino acid sites harbored strong support for joining Lepidoptera with Diptera, which is consistent with a monophyletic Mecopterida.     

The development of PCR-based markers for molecular sex identification in the model insect species Tribolium castaneum

The red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), is a major pest of stored grain and cereal crops. It is also an important model species in genetic, ecological, and evolutionary research. The majority of its genome was recently sequenced and published. However, the genomic sequence of the small Y-chromosome is still undetermined, which hinders the development of molecular sex identification methods. Traditional methods for sexing adult forms of Tribolium beetles are troublesome. Therefore, a method for molecular sex identification in the red flour beetle was developed. One sex-linked randomly amplified polymorphic DNA marker was converted into a sequence-characterized amplified region (SCAR). The SCAR was aligned with the T. castaneum reference whole-genome sequence and fully matched a fragment of a single contig of unknown genomic location. The novelty of the method is that the fragment consists of shorter DNA fragments that are also present at other locations around the genome, but the particular order of these fragments within the sequenced region appeared to be Y-specific and this property was utilized for marker development. A set of three primers for multiplex PCR reaction was designed resulting in amplification of different length Y-specific and not-Y-specific (control) DNA fragments in a single PCR, which allows to distinguish males from females. The primers successfully sexed pre-sexed pupae and adult beetles from six laboratory strains, showing that the order of the repeated fragments is conserved in the species and is not strain-specific.     

Similarity of Structural Features and Evolution of Satellite DNAs from Palorus subdepressus (Coleoptera) and Related Species

A novel highly abundant satellite DNA comprising 20% of the genome has been characterized in Palorus subdepressus (Insecta, Coleoptera). The 72-bp-long monomer sequence is composed of two copies of T₂A₅T octanucleotide alternating with 22-nucleotide-long elements of an inverted repeat. Phylogenetic analysis revealed clustering of monomer sequence variants into two clades. Two types of variants are prevalently organized in an alternating pattern, thus showing a tendency to generate a new complex repeating unit 144 bp in length. Fluorescent in situ hybridization revealed even distribution of the satellite in the region of pericentric heterochromatin of all 20 chromosomes. P. subdepressus satellite sequence is clearly species specific, lacking similarity even with the satellite from congeneric species P. ratzeburgii. However, on the basis of similarity in predicted tertiary structure induced by intrinsic DNA curvature and in repeat length, P. subdepressus satellite can be classified into the same group with satellites from related tenebrionid species P. ratzeburgii, Tenebrio molitor, and T. obscurus. It can be reasonably inferred that repetitive sequences of different origin evolve under constraints to adopt and conserve particular features. Obtained results suggest that the higher-order structure and repeat length, but not the nucleotide sequence itself, are maintained through evolution of these species. Received: 23 April 1997 / Accepted: 11 July 1997     

Satellite DNA repeat sequence variation is low in three species of burying beetles in the genus Nicrophorus (Coleoptera: Silphidae)

Three satellite DNA families were identified in three species of burying beetles, Nicrophorus orbicollis, N. marginatus, and N. americanus. Southern hybridization and nucleotide sequence analysis of individual randomly cloned repeats shows that these satellite DNA families are highly abundant in the genome, are composed of unique repeats, and are species-specific. The repeats do not have identifiable core elements or substructures that are similar in all three families, and most interspecific sequence similarity is confined to homopolymeric runs of A and T. Satellite DNA from N. marginatus and N. americanus show single-base-pair indels among repeats, but single-nucleotide substitutions characterize most of the repeat variability. Although the repeat units are of similar lengths (342, 350, and 354 bp) and A + T composition (65%, 71%, and 71%, respectively), the average nucleotide divergence among sequenced repeats is very low (0.18%, 1.22%, and 0.71%, respectively). Transition/transversion ratios from the consensus sequence are 0.20, 0.69, and 0.70, respectively.      

赤拟谷盗HSP70基因克隆和竞争定量PCR检测

为研究赤拟谷盗Tribolium castaneum受到热胁迫后高度保守的热激蛋白70（heat shock protein 70,HSP70）基因的表达变化,本研究扩增了681 bp的赤拟谷盗hsp70片段,编码227个氨基酸残基,GenBank登录号为HM345948。同源性分析表明：赤拟谷盗hsp70核苷酸序列与马铃薯甲虫Leptinotarsa decemlineata的hsp70（GenBank登录号：AF322911.1）同源性最高,为97%;其推测的蛋白序列与马铃薯甲虫、甘蓝夜蛾Mamestra brassicae、黑腹果蝇Drosophila melanogaster和美洲斑潜蝇Liriomyza sativae的HSP70蛋白均有94%以上的同源性。利用RT-PCR技术得到与赤拟谷盗hsp70进行竞争定量的内部竞争物, 以等量的目标cDNA和一系列稀释的竞争模板进行竞争PCR扩增,构建了hsp70的竞争定量PCR检测体系, 该体系标准曲线的线性方程为Y=1.032X-1.618 (r²=0.975)。这些结果为赤拟谷盗的hsp70定量检测提供了方便,并为热控技术防治害虫提供了基础资料。     

A new family of satellite DNA sequences as a major component of centromeric heterochromatin in owls (Strigiformes)

We isolated a new family of satellite DNA sequences from Hae III- and Eco RI-digested genomic DNA of the Blakistons fish owl ( Ketupa blakistoni). The repetitive sequences were organized in tandem arrays of the 174 bp element, and localized to the centromeric regions of all macrochromosomes, including the Z and W chromosomes, and microchromosomes. This hybridization pattern was consistent with the distribution of C-band-positive centromeric heterochromatin, and the satellite DNA sequences occupied 10% of the total genome as a major component of centromeric heterochromatin. The sequences were homogenized between macro- and microchromosomes in this species, and therefore intraspecific divergence of the nucleotide sequences was low. The 174 bp element cross-hybridized to the genomic DNA of six other Strigidae species, but not to that of the Tytonidae, suggesting that the satellite DNA sequences are conserved in the same family but fairly divergent between the different families in the Strigiformes. Secondly, the centromeric satellite DNAs were cloned from eight Strigidae species, and the nucleotide sequences of 41 monomer fragments were compared within and between species. Molecular phylogenetic relationships of the nucleotide sequences were highly correlated with both the taxonomy based on morphological traits and the phylogenetic tree constructed by DNA-DNA hybridization. These results suggest that the satellite DNA sequence has evolved by concerted evolution in the Strigidae and that it is a good taxonomic and phylogenetic marker to examine genetic diversity between Strigiformes species.An erratum to this article can be found at Communicated by Y. Hiraoka     

Molecular cytogenetic characterization and chromosomal distribution of the satellite DNA in the genome of Oxya hyla intricata (Orthoptera: Catantopidae)

The genomic DNA of the grasshopper (Oxya hyla intricata) was subjected to electrophoresis after digestion with HaeIII, and the result showed two bands of highly repetitive DNA, approximately 200 and 400 bp in length. The 200-bp HaeIII-digested fragment was cloned and characterized by sequencing and fluorescence in situ hybridization (FISH). The results showed the presence of two distinct satellite DNA (stDNA) families: one consisting of a 169-bp repeated element having an A+T content of 60.9% and the other consisting of a 204-bp repeated element having an A+T content of 53.9%. No significant homology between the two stDNA families was observed. FISH showed that the chromosomal locations of these families are different from each other. The 169-bp element was located in the C-band-positive regions of the short arms of most of the chromosomes, whereas the 204-bp element was located in the centromeric regions of three chromosome pairs. These results imply that the origins of these two DNA families are different. The results of zoo-blot hybridization to the genomic DNA from four Oxya species, O. hyla intricata, O. japonica japonica, O. chinensis formosana, and O. yezoensis, suggest that the two stDNA families found in the present study are species-specific for O. hyla intricata.     

赤拟谷盗全基因组和EST中微卫星的丰度

微卫星是近年大力开发的一种分子标记,为了推进赤拟谷盗Tribolium castaneum(Herbst)遗传学相关研究,对赤拟谷盗全基因组和EST中由1~6个碱基重复单元组成的简单序列重复进行分析,进而对其微卫星的丰度和分布进行比较分析。微卫星在赤拟谷盗EST中的分布频率为1/0.87kb,其中单碱基重复序列占71.25%,是最丰富的重复单元,而六、三、四、二,五碱基重复单元序列分别占23.93%,2.94%,1.56%,0.17%,0.15%。全基因组中微卫星的分布频率为1/3.65kb,其中六碱基重复序列占61.96%,是最丰富的重复单元,而三,四,一,五,二碱基重复单元序列分别占14.35%,13.75%,4.68%,3.60%,1.69%。同时发现富含A和T碱基的微卫星占主导地位,富含G和C碱基的微卫星数量较少。进一步的分析显示,微卫星在每条染色体上的丰度存在很大的相似性。     

A new AluI satellite DNA in the root-knot nematode Meloidogyne fallax: relationships with satellites from the sympatric species M. hapla and M. chitwoodi

A highly abundant satellite DNA comprising 20% of the Meloidogyne fallax (Nematoda, Tylenchida) genome was cloned and sequenced. The satellite monomer is 173 bp long and has a high A + T content of 72.3%, with frequent runs of A's and T's. The sequence variability of the monomers is 2.7%, mainly due to random distribution of single-point mutations. A search for evidence of internal repeated subunits in the monomer sequence revealed a 6-bp motif (AAATTT) for which five degenerated repeats, differing by just a single base pair, could be identified. Pairwise comparison of the M. fallax satellite with those from the sympatric species Meloidogyne chitwoodi and Meloidogyne hapla revealed a high sequence similarity (68.39%) with one satellite DNA subfamily in M. chitwoodi, which indicated an unexpected close relationship between them. Given the high copy number and the extreme sequence homogeneity among monomeric units, it may be assumed that the satellite DNA of M. fallax could have evolved through some recent and extensive amplification burst in the nematode genome. In this case, its relatively short life would not yet have allowed the accumulation of random mutations in independent amplified repeats. Considering the morphological resemblance between the two species and their ability to produce interspecific fertile hybrids under controlled conditions, these results indicate that M. fallax may share a common ancestor with M. chitwoodi, from which it could have diverged recently. All these data suggest that M. fallax could be the result of a recent speciation process and show that Meloidogyne satellite DNAs may be of interest to resolve phylogenetic relationships among closely related species from this genus.      

Expression of the Tribolium castaneum （Coleoptera： Tenebrionidae） hsp83 gene and its relation to oogenesis during ovarian maturation

Heat shock proteins (HSP) can protect organisms and cells from thermal damage. In this study, we cloned the full length cDNA encoding the HSP83 protein (the homologue of HSP90) of Tribolium castaneum (red flour beetle). The isolated cDNA contains the full coding sequence, a partial 5′ untranslated region of 55 bp and the complete 3′ untranslated region. We found the hsp83 gene is located on chromosome 5 of the T. castaneum genome. The predicted HSP83 protein sequence has a high similarity (on average 86.77%) with that of other insect species. The expression of the hsp83 gene in the whole body and in the ovary could be induced with heat stress (40°C for 1 h) in newly hatched (within 3 h post emergence) and mature (10 days post emergence) beetles. Under normal conditions, the hsp83 expression in the ovary is about 3-fold higher than in the whole body at both stages. No significant difference in hsp83 expression was observed between the two ovarian developmental stages regardless if the beetles were treated with heat shock or not. The expression of the HSP83 protein in the whole body could also be induced with heat stress in newly hatched and mature beetles. However, in the ovary, HSP83 was only expressed in the follicle cells of mature beetles and not in newly hatched beetles, regardless if the beetles were treated with heat shock or not. Furthermore, the females were not able to produce mature oocytes after knock-down of the hsp83 expression by injecting dsRNA. These results suggest that the HSP83 protein is involved in protection against heat stress and could be involved in oogenesis during ovarian maturation of T. castaneum.     

Evolution of satellite DNAs from the genus Palorus--experimental evidence for the "library" hypothesis

Satellite DNA profiles have been characterized in the congeneric species Palorus ratzeburgii, Palorus subdepressus, Palorus genalis, and Palorus ficicola (Coleoptera, Insecta), each of which contains a single, A + T-rich satellite DNA comprising a considerable portion of the genome (20%-40%). These satellites exhibit insignificant mutual sequence similarity. Using PCR assay, it has been shown that all four sequences are present in each of the tested Palorus species: one of them is amplified into a high copy number or a major satellite, while the three others are in the form of low-copy-number repeats estimated to make up approximately 0.05% of the genome. Each of the four satellites is interspecifically high conserved concerning the sequence, monomer length, and tandem repeat organization. Major, as well as low- copy-number, satellites are colocalized in the regions of pericentromeric heterochromatin on all chromosomes of the complement. The low-copy-number satellites are dispersed between the large arrays of the major satellite over the whole heterochromatic block. Our results explain satellite DNA evolution, confirming the hypothesis that related species share a "library" of conserved satellite sequences, some of which could be amplified into a major satellite. Due to the evolutionary dynamics of satellite DNAs, the content of the "library" is variable; the elimination of some sequences parallels the creation of the new ones. Quantitative changes in satellite DNAs, induced by occasional amplification of satellite repeat from the "library", could possibly occur in the course of the speciation process, thus forming a species-specific profile of satellite DNAs.      

Characterization and chromosomal organization of satellite DNA sequences in Picea abies

Three clones containing satellite DNA sequences were selected from a randomly sheared genomic DNA library of Picea abies (clones PAF1, PAG004P22F (2F), and PAG004E03C (3C)). PAF1 contained 7 repeats that were 37-55 bp in length and had 68.9%-91.9% nucleotide sequence similarity. Two 2F repeats were 305-306 bp in length and had 83% sequence similarity. Two 3C repeats were 193-226 bp in length and had a sequence similarity of 78.6%. The copy number per 1C DNA of PAF1, 2F, and 3C repeats was 2.7 x 106, 2.9 x 105, and 2.9 x 104, respectively. In situ hybridization showed centromeric localization of these sequences in two chromosome pairs with PAF1, all pairs but one with 2F, and three pairs with 3C. Moreover, PAF1 sequences hybridized at secondary constrictions in six pairs, while 2F-related sequences were found at these chromosome regions only in four pairs. These hybridization patterns allow all chromosome pairs to be distinguished. PAF1-related repeats were contained in the intergenic spacer (IGS) of ribosomal cistrons in all six nucleolar organizers of the complement, while sequences related to 2F were found on only one side of the rDNA arrays in four pairs, showing structural diversity between rDNA regions of different chromosomes.     

Localization and characterization of recombinant DNA clones derived from the highly repetitive DNA sequences in the Indian muntjac cells: their presence in the Chinese muntjac

A total of seven, highly repeated, DNA recombinant M13 mp8 clones derived from a Hpa II digest of cultured cells of the Indian muntjac (Muntiacus muntjac vaginalis) were analyzed by restriction enzymes, in situ hybridization, and DNA sequencing. Two of the clones, B1 and B8, contain satellite DNA inserts which are 80% homologous in their DNA sequences. B1 contains 781 nucleotides and consist of tandem repetition of a 31 bp consensus sequence. This consensus sequence, TCCCTGACGCAACTCGAGAGGAATCCTGAGT, has only 3 bp changes, at positions 7, 24, and 27, from the consensus sequence of the 31 bp subrepeats of the bovine 1.715 satellite DNA. The satellite DNA inserts in B1 and B8 hybridize primarily but not specifically to chromosome X, and secondarily to other sites such as the centromeric regions of chromosomes 1 and 2. Under less stringent hybridization conditions, both of them hybridize to the interior of the neck region and all other chromosomes (including chromosomes 3 and Y). The other five DNA clones contain highly repetitive, interdispersed DNA inserts and are distributed throughout the genome except for the neck region of the compound chromosome X+3. Blot hybridization results demonstrate that the satellite DNA component is also present in Chinese muntjac DNA (Muntiacus reevesi) in spite of the very different karyotypes of the Chinese and Indian muntjacs.     

Sequence organization and cytological localization of the minor satellite of mouse.

A complete 120 bp genomic consensus sequence for the mouse minor satellite has been determined from enriched L929 centromeric sequences. The extensive sequence homology existing between the major and minor satellite suggests an evolutionary relationship. Some sequences flanking the minor satellite has also been identified and they provide insight into centromeric DNA organization. Isotopic in situ hybridization analysis of the minor satellite to mouse L929 and Mus musculus metaphase spreads showed that this repetitive DNA class is localized specifically to centromeres of all chromosomes of the karyotype. With the use of high resolution non-isotopic fluorescence in situ hybridization the minor satellite is further localized to the outer surface of the centromere in a discrete region at or immediately adjacent to the kinetochore. Our cytological data suggests that the minor satellite might play a role in the organization of the kinetochore region rather than, as previously suggested, sites for general anchoring of the genome to the nuclear matrix.