Subrepeat Region Research Articles

Nucleotide sequence, structural organization and length heterogeneity of ribosomal DNA intergenic spacer in Quercus petraea (Matt.) Liebl. and Q. robur L.

18S-5.8S-26S rDNA family comprises tandemly arranged, repeating units separated by an intergenic spacer (IGS) that contains transcription initiation/termination signals and usually repeating elements. In this study, we performed for the first time thorough sequence analysis of rDNA IGS region in two dominant European oaks, Quercus petraea and Q. robur, in order to investigate (1) if IGS sequence composition allows discrimination between these two species, and (2) if there is an rDNA length heterogeneity arising from IGS sequence. Two spacer length variants (slvs), 2 and 4 kb in length, were found in the genomes of both species. Inter-comparison of both slvs revealed no species-specificity in sequence or structural organization. Both slvs could be divided into four subregions; (1) the subrepeat region containing three repeated elements, (2) the AT-rich region containing matrix attachment sites and putative origin of replication, (3) the promoter region containing putative transcription initiation site and (4) the 5'ETS region. In the 4-kb slvs all four subregions are extended, and the subrepeat, AT-rich and promoter regions are duplicated. This is unique compared to other known IGS sequences where the variation in number of subrepeats is responsible for slvs creation. We also propose a possible evolutionary scenario to explain the formation of the subrepeat region in oak IGS. Results obtained in this work add to the previous picture of low-genetic differentiation of the two oaks and provide important data for further analyses of the function of IGS in control of rRNA gene expression.

A 3D model of Reelin subrepeat regions predicts Reelin binding to carbohydrates

Reelin is a large molecule of the extracellular matrix (ECM) which regulates neuronal positioning during the early stages of cortical development in vertebrate species. The Reelin molecule can be subdivided into a smaller N-terminal domain, showing homology with F-spondin, and a larger C-terminal region containing 8 EGF-like repeats. The localization of Reelin in the ECM, its large dimensions and the modular organization of its primary structure led us to suppose a structure of its modules similar to domains commonly found in ECM proteins such as Agrin, laminins and thrombospondins. We therefore performed a sequence alignment and molecular modeling analysis to study the three-dimensional fold of the Reelin subrepeat regions. Our analysis produces a tentative model of the core region of the Reelin subrepeat sequences and suggests the presence in this 3D model of structural features common to polysaccharide-binding modules which are often found on proteoglycans of the ECM. These findings provide a conceptual framework for further experiments aimed at testing the functions of the EGF-like repeat regions of Reelin.

Molecular characterization of ribosomal intergenic spacer in the tadpole shrimpTriops cancriformis(Crustacea, Branchiopoda, Notostraca)

Nuclear ribosomal DNA constitutes a multigene family, with tandemly arranged units linked by an intergenic spacer (IGS), which contains initiation/termination transcription signals and usually tandemly arranged subrepeats. The structure and variability of the IGS region are analyzed here in hermaphroditic and parthenogenetic populations of the "living fossil" Triops cancriformis (Branchiopoda, Notostraca). The results indicate the presence of concerted evolution at the population level for this G+C-rich IGS region as a whole, with the major amount of genetic variability found outside the subrepeat region. The subrepeats region is composed of 3 complete repeats (a, c, d) intermingled with 3 repeat fragments (b, e, f) and unrelated sequences. The most striking datum is the absolute identity of subrepeats (except type d) occupying the same position in different individuals/populations. A putative promoter sequence is present upstream of the 18S rRNA gene, but not in subrepeats, which is at variance with other arthropod IGSs. The absence of a promoter sequence in the subrepeats and subrepeat sequence conservation suggests that this region acts as an enhancer simply by its repetitive nature, as observed in some vertebrates. The putative external transcribed spacer (840 bp) shows hairpin structures, as in yeasts, protozoans, Drosophila, and vertebrates.

Characterization of Subrepeat Regions within rDNA Intergenic Spacers of the Edible BasidiomyceteLentinula edodes

For 16 commercial cultivars of Lentinula edodes, DNA fragments for the nuclear rDNA intergenic spacers IGS1 and IGS2 were amplified and analyzed. IGS1 contained a subrepeat region, named SR1, and IGS2 contained a pair of direct repeats and a subrepeat region, named SR2. Three and five types of subrepeats were found in SR1 and SR2, respectively. Heterogeneity in the lengths of IGS1 and IGS2 arose mainly from the number of different kinds of subrepeats within SR1 and SR2. The DNA fingerprints from the PCR products targeting SR1 and SR2 were specific for each of the 16 cultivars, and had enough variation for discrimination among the cultivars. This result suggests that the DNA fingerprints targeting SR1 and SR2 are useful for investigations of L. edodes cultivars.

Separation of strains of the yeasts Xanthophyllomyces dendrorhousand Phaffia rhodozyma based on rDNA IGS and ITS sequence analysis.

The type strains of the anamorph Phaffia rhodozyma (CBS 5905) and the teleomorph Xanthophyllomyces dendrorhous (VKM Y-2786) were analyzed by nucleotide sequence analysis and compared to the sequences found in three additional strains (ATCC 24228, ATCC 24230 and CBS 6938). The results of ribosomal DNA Internal transcribed spacer (ITS) and Intergenic spacer (IGS) region analyses indicate that P. rhodozyma, which was isolated from a beech tree, is a distinct species from the other four strains. The latter that were collected from birch trees are considered to be strains of X. dendrorhous. These individual strains of X. dendrorhous, which have geographically distinct isolation sources, can be distinguished by nucleotide substitutions and deletion/insertion gaps in sub-repeat regions of the Intergenic spacer. The conclusions demonstrate that differences in the IGS region provide molecular markers for denoting strains that may differ in their biochemical and physiological capabilities. The hypothesis is presented that strain differences in the IGS region may be useful to demonstrate geographic and host specificity.

Sub-repeat sequences in the ribosomal RNA intergenic regions of Verticillium alboatrum and V. dahliae

Most eukaryotic organisms have sub-repeat sequences in the intergenic regions (IGRs) of their rRNA gene clusters but such subrepeats are unusual in fungi. Verticillium alboatrum and V. dahliae (hyphomycetes) have been shown to have imperfect sub-repeat sequences in their IGRs which probably did not arise by simple reiteration. Although the consensus sequences derived from the subrepeats of V. alboatrum , of haploid isolates of V. dahliae and of diploid isolates of this species were all related, the overall structures of the sub-repeat regions in the three were clearly distinct. Considerable variation in the total length of these regions was found between haploid isolates of V. dahliae but this was not correlated with any identified character, including restriction fragment length polymorphism (RFLP) group. Earlier work on RFLPs, using random genomic clones as probes, showed that the isolates of V. dahliae studied, most of which came from the U.K., could be divided into clearly defined groups. Within-isolate length variation was seen only in a few cases. A single base difference, occurring at the same position in each repeat, was characteristic for V. dahliae RFLP group B or groups A and M combined. A second difference gave similar differentiation but was not present in all repeats. No length variation was seen between V. alboatrum isolates or between two diploid isolates of V. dahliae . The extent of the differences in structures of sub-repeat regions suggest that haploid and diploid isolates of V. dahliae would be better regarded as separate species, along with V. alboatrum , but the lack of correlation between sub-repeat length and any other known character means that sub-repeat length cannot be used for rapid identification of sub-specific taxa.

Secondary structure of synthetic peptides derived from the repeating unit of a giant secretory protein from Chironomus tentans.

The secretory proteins of Chironomus tentans larvae, which are used to construct underwater feeding and pupation tubes, assemble into complexes in vitro. Members of a family of 1000 kDa proteins, the spIs, appear to form the fibrous backbone of the assembled complexes. The spIs consist of a core of tandemly repeating units of 60 to 90 amino acids that can be subdivided into two regions: the subrepeat region, made up of short internal repeats, and the constant region, which lacks simple subrepeats. We have synthesized peptides representative of the constant and subrepeat regions of one of the spIs, and have examined their secondary structure using Fourier transform IR and CD spectroscopy. The IR spectrum of the constant peptide indicates that this peptide has alpha-helical regions and beta-turns. The CD spectrum confirms this. The IR spectrum of the subrepeat peptide is similar to that of the poly(Gly)II helix, and also may indicate the presence of beta-turns. The CD spectrum is consistent with this helical structure. Extrapolation of these results to intact spIs is in agreement with secondary structure prediction and modeling studies. Our results indicate that the alpha-helices and poly(Gly)II-like helices are not arranged as coiled-coils, which are often found in fibrous proteins. We suggest that these structural elements may be in an unusual arrangement in the spIs, organized as alternating alpha-helices and poly(Gly)II or collagen-like helices, interspersed with beta-turns.

Terminal repeats in long repeat arrays are likely to reflect the early evolution of Balbiani ring genes

Balbiani ring (BR) genes in Chironomus tentans are 35 to 40 kb (1 kb = 10 3 bases or base-pairs) in length and encode secretory proteins of exceptional size. Each gene contains a large homogeneous core block consisting of approximately 100 tandemly arranged, highly homologous repeat units. The repeat unit has a constant (C) region and a subrepeat (SR) region. The various BR genes exhibit similar C regions, while the SR regions differ as to sequence, length and number of subrepeats. To study early steps in the evolution of the coding repeat arrays of the BR genes we have analyzed the 3′ ends of the four BR genes in C. tentans: BRI, BR2.1, BR2.2 and BR6. In each gene the very end of the core block consists of two or three repeat unit variants; in each variant repeat the C region is linked to a Cys region, replacing the SR region. Sequence comparisons between the C regions of the closely related BRI and BR2 genes show that during evolution the terminal repeat unit variants have to a large extent been isolated from the remainder of the core block and have probably been more conserved than the interior repeat units. Detailed analysis of the structure of the variant repeat units further supports this latter notion and suggests that the BR core blocks have evolved from an array of a simple 36 base-pair long sequence; larger, more complex repeat units containing subrepeats were gradually formed and spread in the block, mainly by homologous unequal recombination events. During this evolution the interior of the core blocks evolved as a homogeneous repetitive structure, while ancestor repeat units remained as sequence relicts in the terminal parts.

Rapid and Concerted Evolution of Repeat Units in a Balbiani Ring Gene

The Balbiani ring (BR) genes in the midge Chironomus, a genus belonging to Diptera, code for large secretory proteins, used to construct the larval tube. The 15-23-kb long core block in each gene consists of an array of tandemly arranged approximately 200-bp long repeat units, where a single repeat unit is composed of a constant and a subrepeat region. In order to investigate the evolutionary fate of highly repetitive coding DNA, the BR1gamma core block in Chironomus pallidivittatus was characterized and compared to the orthologous core block in the sibling species Chironomus tentans. We find that the 75-100 repeat units in the BR1gamma core block have evolved in an unusual fashion. In all repeat units the constant regions display an expected high degree of homology between the two species, 94% at the nucleotide level. In contrast, the subrepeat regions in all repeat units have diverged concertedly, both as to length, number and sequence of the subrepeats. The observed changes in all repeat units of the core block probably have occurred after speciation of C. pallidivittatus and C. tentans. These findings demonstrate that a tandemly reiterated coding sequence can rapidly and concertedly convert into a related sequence, much in the same way as has been described for satellite DNA.

Correlated changes in steady-state levels of Balbiani ring mRNAs and secretory polypeptides in salivary glands of Chironomus tentans.

Balbiani rings (BRs) on polytenized chromosomes in Chironomid salivary glands contain members of a homologous multigene family that encodes a family (the sp-I family) of high Mr secretory polypeptides. Each of these BR genes is comprised largely of tandemly duplicated core repeat sequences consisting of related constant (C) regions and intergenically divergent subrepeat (SR) regions. A set of oligodeoxyribonucleotide probes were synthesized that correspond to the transcribed strand of the SR region of BR1, BR2 alpha, BR2 beta, and BR6 core repeats. Under a defined set of conditions, it was possible to show that each oligonucleotide probe hybridized exclusively to its cognate repeat type without hybridization to other repeat types in cloned DNA templates. These BR probes were then used in dot-blot hybridization experiments to simultaneously follow alterations in the steady-state level of BR mRNAs in response to prolonged exposure of larvae to galactose. The results indicated that the relative amounts of these four BR mRNAs may change in a noncoordinate manner. These BR probes were also used in experiments to compare simultaneously the salivary gland content of sp-I components and specific BR mRNAs in larvae that exhibited naturally occurring or induced alterations in BR gene expression. A correlation was found which suggested that sp-Ia is encoded in a gene comprised of BR1 repeats, sp-Ib is encoded by BR2 beta repeats, sp-Ic is encoded by BR6 repeats and sp-Id is encoded by BR2 alpha repeats.

Different evolutionary behavior of structurally related, repetitive sequences occurring in the same Balbiani ring gene in Chironomus tentans

The Balbiani ring 2 (BR 2) gene in Chironomus tentans is highly internally repeated. Two types of related repeat units-the alpha and beta types-are tandemly arranged in separate blocks, which together are likely to form the major part of the gene. Every repeat unit has one constant region and one subrepeat region. Here we analyze the length and sequence of a number of repeat units of both types and compare the units within and between the blocks. The approximately 100 alpha repeat units are essentially invariant regarding length and sequence. In contrast, when the approximately 70 beta repeat units are compared, six length variants are found, four of which have been sequenced. The length variations reside in the subrepeat regions and are due to different numbers of whole or half subrepeats. Furthermore, the subrepeat regions differ by several base-pair substitutions, many of which change the amino acid sequence. On the other hand, all beta-type constant regions are of equal length and are virtually homogeneous in sequence. The observed length distributions in combination with analysis of the basepair substitutions in the alpha-and beta-type constant and subrepeat regions suggest that the alpha and beta blocks are of different age, that seemingly homologous repeated regions may play different functional roles at the protein level, and that sequence correction mechanisms are likely to operate to different extents on the constant and subrepeat regions within the beta block.

Balbiani ring 6 gene in Chironomus tentans: a diverged member of the Balbiani ring gene family.

We describe the internal organization of a large part of the Balbiani ring (BR) 6 gene in Chironomus tentans. The BR6 gene is a diverged member of the BR gene family. It displays the characteristic hierarchic organization of repetitive sequences, but in the constant region of the repeat units the overall sequence homology is only 49% when compared to other BR genes. All four cysteines are among the few amino acids conserved in the constant region. In the subrepeat region the central part is built from a repeated tripeptide, Pro-Glu--Arg+. A similar charge distribution adjacent to prolines is found in other BR gene subrepeat regions, most pronouncedly in the BR2-encoded protein. These conserved properties of the BR gene products are relevant to the issue how the various BR gene products interact to form a supramolecular structure, the larval tube, and how functional demands influence the evolution of a eucaryotic gene family.