5S DNA Units Research Articles

Elimination of 5S DNA unit classes in newly formed allopolyploids of the generaAegilopsandTriticum

Two classes of 5S DNA units, namely the short (containing units of 410 bp) and the long (containing units of 500 bp), are recognized in species of the wheat (the genera Aegilops and Triticum) group. While every diploid species of this group contains 2 unit classes, the short and the long, every allopolyploid species contains a smaller number of unit classes than the sum of the unit classes of its parental species. The aim of this study was to determine whether the reduction in these unit classes is due to the process of allopolyploidization, that is, interspecific or intergeneric hybridization followed by chromosome doubling, and whether it occurs during or soon after the formation of the allopolyploids. To study this, the number and types of unit classes were determined in several newly formed allotetraploids, allohexaploids, and an allooctoploid of Aegilops and Triticum. It was found that elimination of unit classes of 5S DNA occurred soon (in the first 3 generations) after the formation of the allopolyploids. This elimination was reproducible, that is, the same unit classes were eliminated in natural and synthetic allopolyploids having the same genomic combinations. No further elimination occurred in the unit classes of the 5S DNA during the life of the allopolyploid. The genetic and evolutionary significance of this elimination as well as the difference in response to allopolyploidization of 5S DNA and rDNA are discussed.

Molecular confirmation of the genomic constitution of Douglasdeweya (Triticeae: Poaceae): demonstration of the utility of the 5S rDNA sequence as a tool for haplome identification

A new genus Douglasdeweya containing the two species, Douglasdeweya deweyi and D. wangii was published in 2005 by Yen et al. based upon the results of cytogenetical and morphological findings. The genome constitution of Douglasdeweya-PPStSt-allowed its segregation from the genus Pseudoroegneria which contains the StSt or StStStSt genomes. Our previous work had demonstrated the utility of using 5S rDNA units, especially the non-transcribed spacer sequence variation, for the resolution of genomes (haplomes) previously established by cytology. Here, we show that sequence analysis of the 5S DNA units from these species strongly supports the proposed species relationships of Yen et al. (Can J Bot 83:413-419, 2005), i.e., the PP genome from Agropyron and the StSt genome from Pseudoroegneria. Analysis of the 5S rDNA units constitutes a powerful tool for genomic research especially in the Triticeae.

The 5S rRNA gene diversity in the genus Secale and determination of its closest haplomes

We analyzed 127 rDNA sequences (5S DNA units) obtained from 23 seed accession samples from more or less 10 taxa in wild and cultivated rye, genus Secale L. The sequences fell into two known groups, here assigned to two unit classes, viz. long R1 and short R1 (designations to reflect on R haplome of rye). The different taxa could not be fully differentiated based on the 5S DNA units. We searched for 5S DNA sequences from known unit classes most closely similar to the long R1 and the short R1. One set with the long R1 unit class contained sequences of the long P1 unit class from Agropyron (P haplome) and from Kengyilia (StYP haplome), long J1 from Thinopyrum (J haplome), whereas the set with the short R1 included the long S1 from Pseudoroegneria (St haplome) and Kengyilia (StYP haplome), the short J1 from Thinopyrum (J haplome) and the short V1 from Dasypyrum (V haplome). Each of the two sets was analyzed separately by maximum likelihood (ML) phylogenetic analysis from which we were able to infer that the 5S DNA units of Secale differentiated in a non-clock fashion and followed the HKY substitution model in the gene tree with the long R1 unit class and the HKY + G in the gene tree with the short R1 unit class. A complementary Bayesian analysis yielded identical tree topologies to the ML ones for each of the two sequence sets. In the tree with the long R1 units the long P1 and long J1 unit classes were closest to the long R1 unit class, whereas in the tree with the short R1 units the long S1 and short J1 unit classes were closest to the short R1 unit class, indicating possibly a close relationship between the St, J and R haplomes.

Inferring genetic relationship among haplomes in Triticeae: The utility of the 5S DNA units with examples

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The South African Hordeum capense is more closely related to some American Hordeum species than to the European Hordeum secalinum: a perspective based on the 5S DNA units (Triticeae: Poaceae)

Several authors have proposed that the European Hordeum secalinum and the morphologically similar South African Hordeum capense are conspecific. In this paper we provide evidence that the two species differ in their 5S DNA unit class composition. We also report on the diversity of 5S DNA units in Hordeum muticum, a South American species. When the 5S rDNA unit class composition for these three species is compared with the unit class composition for all Hordeum species thus far investigated, it appears that H. capense is more closely related to the American Hordeum species containing the long Y2 unit class, than to H. secalinum, which lacks the long Y2 unit class but contains the long X2 unit class found in H. marinum. This analysis suggests H. capense may have originated from a stock common to the South American species, such as H. muticum.Key words: 5S DNA unit class, Hordeum capense, Hordeum secalinum, Hordeum muticum, continental drift.

The specific isolation of complete 5S rDNA units from chromosome 1A of hexaploid, tetraploid, and diploid wheat species using PCR with head-to-head oriented primers

The presence of 5S rDNA units on chromosome 1A of Triticum aestivum was shown by the development of a specific PCR test, using head-to-head oriented primers. This primer set allowed the amplification of complete 5S DNA units and was used to isolate SS-Rrna-A1 sequences from polyploid and diploid wheat species. Multiple-alignment and parsimony analyses of the 132 sequences divided the sequences into four types. The isolates from T. aestivum and the tetraploid species (T. dicoccoides, T. dicoccum, T durum, T. araraticum, and T timopheevi) were all of one type, which was shown to be closely related to the type mainly characteristic for T. urartu. The other two types were isolated exclusively from the diploid species T. monococcum, T aegilopoides, T. thaoudar, and T. sinskajae and the hexaploid species T. zhukovski. Triticum monococcum was the only species for which representatives of each of the four sequence types were found to be present. Further, we discuss the possible multicluster arrangement of the 5S-Rrna-A1 array.

The 5S DNA units of bread wheat (Triticum aestivum)

A collection of 44 cloned 5S DNA units fromTriticum aestivum cv. ‘Chinese Spring’ were grouped into 12 sequence-types based on sequence similarity and the respective consensus sequences were then produced. The relationship between these 12 consensus sequences (T. aestivum S 1-S 8 andT. aestivum L 1-L 4), together with two clones sequenced byGerlach andDyer, and the 5S DNA consensus sequences from diploidTriticum spp. were then determined by numerical methods. Both phenetic and cladistic analyses were carried out. The following wheat 5S DNA sequences were found to group with respective sequences from diploidTriticum spp.:T. aestivum S 4, S 6 withT. tauschii S;T. aestivum S 3 withT. monococcum S andT. monococcum S-Rus 7;T. aestivum L 1 andT. aestivum L-GDT. aestivum L 2, L 3 withT. tauschii L;T. aestivum L 4 withT. monococcum L andT. monococcum L-Rus 12. The analyses suggested that 5 out of the 65S Dna loci present in wheat were identified at the sequence level. The locus that could not be identified in this analysis was the5S Dna-B 1 locus. A group ofT. aestivum sequences (T. aestivum S 1, S 7, S 8, S-G&D) were found to be distinct from the other 5S DNA sequences in the data base. The existence of the distinct group of 5S DNA sequences suggests that there is a gap in our current understanding of wheat evolution with respect to the5S Dna loci.

An overview of evolution in plant 5S DNA

The DNA sequence properties of 5S DNA (5S RNA gene plus spacer) from a wide range of families of plants is reviewed with particular reference to the possibility of using the information for phylogenetic inference. Although the data-base is extremely limited, the available evidence suggests that within a subclass or tribe phylogenetic inference can be made, provided that a knowledge about the number of chromosomal locations of the gene loci (5S Dna loci) is available. The evidence suggests little, if any, exchange occurs between the 5S DNA units at different chromosomal loci and the available data favour a mechanism involving amplification/deletion processes for creating structural changes at the5S Dna loci. Sequences originating from species in the familiesRosaceae, Poaceae, andBrassicaceae tended to group together in cladistic analyses but with low confidence limits. Surprisingly little of the spacer region showed conservation of sequence that may relate to a function in the control of transcription by RNA polymerase III.

The 5S DNA units ofAcacia species (Mimosaceae)

The DNA sequence structure of 5S DNA units inAcacia species, including representatives from the three subgenera ofAcacia, have been determined. The data was interpreted to suggest that at least three lineages of 5S DNA sequences exist inAcacia and the proposal was made that the lineages be named5S Dna-1, 5S Dna-2, and5S Dna-3. The5S Dna-1 lineage was represented by units fromA. boliviana andA. bidwilli, the5S Dna-2 lineage by units fromA. melanoxylon, A. pycnantha, A. ulicifolia, A. boliviana, A. bidwillii, andA. albida, and the5S Dna-3 lineage by units fromA. bidwillii, A. boliviana, andA. senegal. Based on this interpretation of the sequence data, the Australian species of subg.Phyllodineae grouped together as a cluster, quite separate from the subgeneraAculeiferum andAcacia. As expected from the analyses of morphological characters, the 5S DNA units fromAcacia albida (syn.Faidherbia albida) were quite separate from the otherAcacia spp.

Relationships betweenOryza species (Poaceae) based on 5S DNA sequences

Relationships between 9Oryza species, covering 6 different genomes, have been studied using hybridization and nucleotide sequence information from the5S Dna locus. Four to five units of the major size class of 5S DNA in each species, 55 units in all, were cloned and sequenced. Both hybridization and sequence data confirmed the basic differences between the A and B, C, D genome species suggested by morphological and cytological data. The 5S DNA units of the A genome species were very similar, as were the ones from the B, C, and D genome-containing species. The 5S DNA ofO. australiensis (E genome) grouped with the B, C, D cluster, while the units ofO. brachyantha (F genome) were quite different and grouped away from all other species. 5S DNA units fromO. minuta, O. latifolia, O. australiensis, andO. brachyantha hybridized strongly, and preferentially, to the genomic DNA from which the units were isolated and hence could be useful as species/genome specific probes. The 5S DNA units fromO. sativa, O. nivara, andO. rufipogon provided A genome-specific probes as they hybridized preferentially to A genome DNA. The units fromO. punctata andO. officinalis displayed weaker preferential hybridization toO. punctata DNA, possibly reflecting their shared genome (C genome).

Evolutionary change at the5S Dna loci of species in theTriticeae

Previous studies have shown that two separate loci for 5S DNA sequences may exist within a species. Two size classes have been tentatively assigned in the ranges 327–468 bp (chromosome 1), and 469–500 bp (chromosome 5) and the entire data-base was subjected to various numerical taxonomic analyses. The results confirm the existence of two lineages of 5S DNA sequences represented by the two size classes.Hordeum vulgare is separate from the species known also asCritesion and, together withDasypyrum, occupy an intermediate position between the two size classes. The 5S DNA units fromTriticum spp. that have also been namedAegilops spp. in other classifications appear as a distinct group withinTriticum spp. at both loci. The consensus 5S DNA sequence fromPsathyrostachys is remote fromHordeum s.l. in contrast to opinions based on morphology. Other aspects are detailed and discussed, including the inadequacy of the computing methods used as well as the need for more data in view of the large amount of homoplasy. The merit of using the spacer for phylogenetic inference is also discussed.

Phylogenetic relationships of Triticum tauschii, the D-genome donor to hexaploid wheat. 4. Variation and chromosomal location of 5S DNA.

The 5S DNA sequences in Triticum tauschii are organised in large clusters containing units that are primarily either 420 ("short") or 490 base pairs (bp) in length ("long"). The main cluster of short units was shown to be located on chromosome 1D in hexaploid wheat and is designated 5SDna-D1, while the cluster of long units was shown to be on chromosome 5D and is designated 5SDna-D2. The chromosomal locations in hexaploid wheat most likely correspond to those in T. tauschii and this could be shown directly for the 5SDna-D2 locus by using a T. tauschii 5D substitution in 'Chinese Spring' wheat. The sequence alignment of units derived from 5SDna-D1 and 5SDna-D2 revealed three apparent deletions in the noncoding spacer region, which were fixed in units from 5SDna-D1, and one deletion, which was fixed in units from 5SDna-D2. A minor size class, 400 bp long and closely related to the units from 5SDna-D1, was found in 2 of 415 accessions surveyed. A continuous range of quantitative changes in the number of 5S DNA units at the two loci was evident with up to a 10-fold relative abundance level of units being found in some accessions. Triticum tauschii var. typica was particularly noteworthy in that many accessions showed more units at 5SDna-D2 relative to 5SDna-D1. Partial thermal dissociation experiments with radioactive probes, synthesized from either the short or long 5S DNA units, hybridized to genomic DNA showed that the population of units at the respective loci were relatively homogeneous and clearly distinct from each other.(ABSTRACT TRUNCATED AT 250 WORDS)

A second locus for the 5S multigene family in Secale L.: sequence divergence in two lineages of the family

The 5S RNA genes in Secale sp. are arranged as tandem arrays of a 460- and 480-bp repeating sequence. These size classes were initially discovered by restriction endonuclease analysis using BamHI and subsequently by DNA sequencing of cloned units. The length variation between short and long units originated from major deletion-insertion events in the noncoding spacer region of the 5S DNA repeat units. In situ hybridization with [3H]cRNA and biotin-labelled probes synthesized from both the short and long 5S DNA units of S. cereale localized the sites on chromosome 1R and a new site on a chromosome identified as 5R. We propose that the chromosome 1R locus, which has been mapped previously, be named 5SDna-R1 and the second locus, reported in the present paper, be referred to as 5SDna-R2. A preferential hybridization of a probe from the long unit to the 5SDna-R2 locus and of a probe from the short unit to the 5SDna-R1 locus is reported. The clustering of long units in the 5SDna-R2 locus was confirmed by restriction endonuclease digestion of DNA from rye chromosome 5R additions to wheat. Nucleotide sequence alignment of 5S DNA repeat units from a number of Secale species, using both phenetic and cladistic computer programmes, demonstrated that two clear lineages corresponding to the long and short units existed in this genus. The different Secale species could not be unambiguously differentiated using the 5S DNA sequences.

Recombination of a eukaryotic DNA in bacteria

Single, 824 bp repeating units of Xenopus laevis oocyte-type 5S DNA were inserted into the recombination vectors, λ rva and λ rvb. When the inserts had the same orientation with respect to the λ chromosomes, Spi - imm434 recombinants were recovered by selection on a P2, λ double lysogenic host. Because of the structure of the vectors, the crossover point in each recombinant must lie completely within the 5S DNA insert. The physical characteristics of these recombinants were determined by examination of restriction enzyme digests. By use of RecA mutant hosts and the Red - vector, λ rvc, recombination frequencies were measured separately for the bacterial and phage systems. Some of the recombination events resulted in 5S DNA inserts of altered length due to unequal crossovers within repeated sequences in the 5S DNA spacer. The occurrence of just such events in frog 5S DNA had been predicted, based on the structure of 5S DNA and evolutionary considerations.

A specific transcription factor that can bind either the 5S RNA gene or 5S RNA.

5S ribosomal RNA specifically inhibits transcription of cloned repeating units of 5S DNA in a nuclear extract of Xenopus oocytes. The inhibition can be explained by the interaction of 5S RNA with a transcription factor that binds specifically to a control region located within the 5S RNA gene. This transcription factor is identical to an abundant cytoplasmic protein that is known to be complexed with 5S RNA in immature Xenopus oocytes. Thus the presence of large amounts of this protein in these cells can account for both the high rate of synthesis and the subsequent storage of 5S RNA to ribosome synthesis.

Cloned single repeating units of 5S DNA direct accurate transcription of 5S RNA when injected into Xenopus oocytes.

Single and multiple repeating units of three types of Xenopus 5S DNA recombined with the plasmid pMB9 serve as templates for the accurate synthesis of 5S RNA after their injection into Xenopus laevis oocyte nuclei. All 15 cloned single repeating units of X. laevis oocyte 5S DNA that were tested supported 5S RNA synthesis. Three cloned fragments of X. borealis oocyte 5S DNA and one cloned single repeating unit of X. borealis somatic 5S DNA were templates for 5S RNA synthesis. We conclude that the majority of repeating units of 5S DNA in these multigene families contain the information for accurate initiation and termination of 5S RNA synthesis. The ability of this system to detect sequence changes that affect transcription is demonstrated.

The nucleotide sequence of oocyte 5S DNA in xenopus laevis. I. The AT-rich spacer

The primary sequence of the principal spacer region in X. laevis oocyte 5S DNA has been determined. The spacer is AT-rich and comprises half or more of each repeating unit. The sequence is internally repetitious; most of it can be represented by the following set of oligonucleotides: CAA C AGTTTTCAA AA GGTTTG C AA G TTTTT(T) The spacer, which varies in length from about 360 to 570 or more nucleotides, can be subdivided into a region (A 2) which is variable in length in different repeating units, flanked by regions (A 1, A 3, B 1) which are relatively constant in length. The A 2 region consists, on the average, of 5–6 tandem copies of the oligonucleotide CAAAGTTT-GAGTTTT; variation in the redundancy of this oligonucleotide accounts for much of the repeat length variation in the genomic 5S DNA. Most copies of this oligonucleotide are identical, although several differing by 1 or 2 nucleotides have been detected in plasmid-cloned 5S DNA fragments. Regions A 1 and A 3 comprise a linear array of similar, but not identical, oligonucleotides; most repeating units contain very similar A 1 and A 3 sequences. Region B 1 is a sequence of 49 nucleotides immediately adjacent to the 5′ terminus of the 5S rRNA sequence. It is GC-rich, much less repetitive than the remainder of the spacer and contains several palindromes, but no regions of dyad symmetry. This sequence is identical in all six of the single cloned repeating units of 5S DNA analyzed.

The isolation and characterization of a second oocyte 5S DNA from xenopus laevis

A second and minor DNA component containing 5S RNA genes has been purified from the genomic DNA of Xenopus laevis (Xlt 5S DNA). Some of Its physical and chemical characteristics are described. It contains a 5S RNA gene sequence which has some oocyte and some somatic-specific residues, as well as nucleotides which differ from both types of 5S RNA. There are about 2000 of these 5S RNA genes per haploid complement of DNA compared to about 24,000 of the principal oocyte 5S RNA genes. The multiple repeating units of Xlt 5S DNA are homogeneous in length (about 350 base pairs). We present evidence that Xlt 5S RNA is transcribed in ovaries but not in somatic cells; Xlt 5S DNA may therefore be under the same control as the major oocyte 5S DNA.

Adjacent repeating units of xenopus laevis 5S DNA can be heterogeneous in length

The distribution of length heterogeneity in adjacent repeating units of X. laevis 5S DNA has been examined by “cloning” 5S DNA in bacteria. Fragments of 5S DNA produced by partial digestion with Hind III and containing 1, 4, and 5 repeating units have been inserted at the single Hind III site of the tetracycline-resistance plasmid, pSC101, and the hybrid plasmids cloned in E. coli. Adjacent 5S DNA repeats in the cloned multi-repeat fragments can differ in length. This finding rules out some mechanisms which have been proposed to account for the parallel evolution of tandem repeated DNAs. The results are consistent with an unequal crossing-over mechanism and place some constraints on the molecular processes in this recombinatory event.

Repeating units of xenopus laevis oocyte-type 5S DNA are heterogeneous in length

The restriction enzymes Hind III and Hae III cleave Xenopus laevis 5S DNA at one and three sites, respectively, in each repeating unit of approximately 700 base pairs. The cleavage sites for both enzymes have been located within the repeating unit by denaturation mapping of the restriction fragments. The Hind III products and one of the Hae III fragments are variable in length, indicating heterogeneity in the length of the repeating unit in 5S DNA. This length heterogeneity is confined to the major A + T-rich spacer region. Repeating units differ from each other by discrete quanta of approximately 15 base pairs. The A + T-rich spacer has been shown to consist largely of tandem subrepeats of just this size ( Brownlee, Cartwright, and Brown, 1974). We suggest that the repeat-length heterogeneity is due to variable numbers of these subrepeats in the spacer regions of the major repeating units.