The Rad50 genes of diploid and polyploid wheat species. Analysis of homologue and homoeologue expression and interactions with Mre11.

The MRN complex plays a central role in the DNA repair pathways of eukaryotic cells and takes part in many other processes, including cell cycle checkpoint signalling, meiosis, DNA replication and telomere maintenance. This complex is formed by the interaction of the products of the Mre11, Rad50 and Nbs1 genes. This paper reports the molecular characterization, expression and interactions of the Rad50 gene in several wheat species with different levels of ploidy. The homoeologous Rad50 wheat genes were found to show a high level of conservation. Most of the RAD50 domains and motifs previously described in other species were also present in wheat RAD50; these proteins are therefore likely to have similar functions. Interactions between the RAD50 wheat proteins and their MRE11 counterparts in the MRN complex were observed. The level of expression of Rad50 in each of the species examined was determined and compared with those previously reported for the Mre11 genes. In some cases similar levels of expression were seen, as expected. The expression of the RAD50 homoeologous genes was assessed in two polyploid wheat species using quantitative PCR. In both cases, an overexpression of the Rad50B gene was detected. Although the results indicate the maintenance of function of these species' three homoeologous Rad50 genes, the biased expression of Rad50B might indicate ongoing silencing of one or both other homoeologues in polyploid wheat. To assess the consequences of such silencing on the formation of the MRN complex, the interactions between individual homoeologues of Rad50 and their genomic counterpart Mre11 genes were examined. The results indicate the inexistence of genomic specificity in the interactions between these genes. This would guarantee the formation of an MRN complex in wheat.

Localization of Rad50, a single-copy gene, on group 5 chromosomes of wheat, using a FISH protocol employing tyramide for signal amplification (Tyr-FISH).

An indirect tyramide signal amplification FISH technique (Tyr-FISH) was adapted to detect single-copy genes in plants. In this study, Rad50 loci (responsible for a protein involved in the repair of breaks in double-stranded DNA) were localized on the chromosomes of homologous group 5 in common wheat (Triticum aestivum). Three loci, Rad50A, Rad50B and Rad50D, none of which have been previously mapped using any kind of molecular approach, were localized at similar positions on the short arm of chromosomes 5A, 5B and 5D, respectively. The loci are all proximal to 5S ribosomal gene clusters. The sensitivity and efficiency of the Tyr-FISH technique was tested using 5 differently sized fragments of the gene Rad50D as probes in FISH involving the chromosomes of the diploid wheat Triticum tauschii (the D-genome donor species of the hexaploid wheats). The procedure, which provided specific signal amplification with low level background, detected target DNA sequences as small as 2 kb with a frequency of 37.5%.

Physical organisation of simple sequence repeats (SSRs) in Triticeae: structural, functional and evolutionary implications.

A significant fraction of the nuclear DNA of all eukaryotes is occupied by simple sequence repeats (SSRs) or microsatellites. This type of sequence has sparked great interest as a means of studying genetic variation, linkage mapping, gene tagging and evolution. Although SSRs at different positions in a gene help determine the regulation of expression and the function of the protein produced, little attention has been paid to the chromosomal organisation and distribution of these sequences, even in model species. This review discusses the main achievements in the characterisation of long-range SSR organisation in the chromosomes of Triticum aestivum L., Secale cereale L., and Hordeum vulgare L. (all members of Triticeae). We have detected SSRs using an improved FISH technique based on the random primer labelling of synthetic oligonucleotides (15-24 bases) in multi-colour experiments. Detailed information on the presence and distribution of AC, AG and all the possible classes of trinucleotide repeats has been acquired. These data have revealed the motif-dependent and non-random chromosome distributions of SSRs in the different genomes, and allowed the correlation of particular SSRs with chromosome areas characterised by specific features (e.g., heterochromatin, euchromatin and centromeres) in all three species. The present review provides a detailed comparative study of the distribution of these SSRs in each of the seven chromosomes of the genomes A, B and D of wheat, H of barley and R of rye. The importance of SSRs in plant breeding and their possible role in chromosome structure, function and evolution is discussed.

Similarities in the chromosomal distribution of AG and AC repeats within and between Drosophila, human and barley chromosomes.

Two simple sequence repeats (SSRs), AG and AC, were mapped directly in the metaphase chromosomes of man and barley (Hordeum vulgare L.), and in the metaphase and polytene chromosomes of Drosophila melanogaster. To this end, synthetic oligonucleotides corresponding to (AG)(12) and (AC)(8) were labelled by the random primer technique and used as probes in fluorescent in situ hybridisation (FISH) under high stringency and strict washing conditions. The distribution and intensity of the signals for the repeat sequences were found to be characteristic of the chromosomes and genomes of the three species analysed. The AC repeat sites were uniformly dispersed along the euchromatic segments of all three genomes; in fact, they were largely excluded from the heterochromatin. The Drosophila genome showed a high density of AC sequences on the X chromosome in both mitotic and polytene nuclei. In contrast, the AG repeats were associated with the euchromatic regions of the polytene chromosomes (and in high density on the X chromosome), but were only seen in specific heterochromatic regions in the mitotic chromosomes of all three species. In Drosophila, the AG repeats were exclusively distributed on the tips of the Y chromosome and near the centromere on both arms of chromosome 2. In barley and man, AG repeats were associated with the centromeres (of all chromosomes) and nucleolar organizer regions, respectively. The conserved chromosome distribution of AC within and between these three phylogenetically distant species, and the association of AG in specific chromosome regions with structural or functional properties, suggests that long clusters of these repeats may have some, as yet unknown, role.

Telomeric DNA localization on dinoflagellate chromosomes: structural and evolutionary implications.

Dinoflagellates are eukaryotic microalgae with distinct chromosomes throughout the cell cycle which lack histones and nucleosomes. The molecular organization of these chromosomes is still poorly understood. We have analysed the presence of telomeres in two evolutionarily distant and heterogeneous dinoflagellate species (Prorocentrum micans and Amphidinium carterae) by FISH with a probe containing the Arabidopsis consensus telomeric sequence. Telomere structures were identified at the chromosome ends of both species during interphase and mitosis and were frequently associated with the nuclear envelope. These results identify for the first time telomere structures in dinoflagellate chromosomes, which are formed in the absence of histones. The presence of telomeres supports the linear nature of dinoflagellate chromosomes.

Replication of 5 S ribosomal genes precedes the appearance of early nuclear replication complexes.

The present work shows that replication of the 5 S ribosomal genes differs in time and 3'deoxyadenosine sensitivity from replication of other nuclear genes, in Allium cepa L. root meristems. Fluorescence in situ hybridization with the pTa794 DNA probe which contains a complete 410 bp 5 S gene from Triticum aestivum allowed to detect four clusters of 5 S genes in these diploid cells (2n = 16), two of them in the short arm of the smallest metacentric chromosomal pair 7. Replication of the 5 S ribosomal genes occurred very early in interphase, as discerned by their resolution as doubled spots only two hours after interphase was initiated in synchronous binucleate cells. Codetection of nuclear replication (by immunodetection of 5-bromo-2'-deoxyuridine incorporation) showed that the replication of the 5 S ribosomal genes occurred before any incorporation of 5-bromo-2'deoxyuridine could be detected in the nuclei. The earliest Br-DNA detected in these cells followed a radial pattern from different foci apparently dispersed along some chromosomal arms. These structures seem to represent early replication complexes, as a result of the displacement of multiple DNA forks from the foci known as pre-replication complexes where the replication machinery of the earliest replicating genes assembles. No consistent positional correlation existed between the formation of the early replication complexes and the already replicated 5 S ribosomal clusters. Finally, nuclear replication but not that of the 5 S genes was prevented by 3'deoxyadenosine, and the earliest replicating 5 S ribosomal gene cluster differed in both sister nuclei resulting from the segregation of one single chromosome in anaphase.

Z-DNA, a new in situ marker for transcription.

Z-DNA forms transiently behind the active RNA polymerases, because of the mechanical torsional stress produced during transcription. In this paper, we explore the possibility that the distribution of Z-DNA stretches signals the sites related to nuclear transcription. To localize transcription, the in situ assay for active RNA polymerases, that allows the elongation of the already initiated transcripts but no initiation of new ones (run-on experiments), was carried out in isolated nuclei of Allium cepa L. root meristems. Both nucleolar and non-nucleolar sites appeared labelled. Nucleoli were most active in transcription than the multiple non-nucleolar foci altogether. In situ immunodetection of Z-DNA provided images that were comparable to those obtained after the run-on assay, with one exception: while Z-DNA and transcription sites were scattered throughout the whole nucleus, Z-DNA also accumulated in the nuclear periphery, where no transcription foci were detected in run-on assays. The peripheral Z-conformation signals might correspond to dsRNA segments present in the pre-mRNA in the process of their export to cytoplasm. The Z-containing structures nearly disappeared when non-nucleolar RNA polymerase II-dependent transcription had been previously abolished by the adenosine analogue DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole). This inhibition selectively decreased the amount of all nucleoplasmic Z structures. On the other hand, the inhibition of the nucleolar RNA polymerase I by cordycepin (3'-deoxyadenosine) prevented the presence of Z-DNA in nucleoli. We propose to use the in situ immunodetection of Z-DNA as a marker of the transcription level in both nucleolus and non-peripheral nucleoplasmic regions of nuclei. Co-detection of Z-DNA and of intermediate filament (IF) proteins, the major components of the nuclear matrix, was also carried out. The IFA antibody recognizes a conserved epitope essential for dimerization of the multiple IF proteins. They co-localized with most nucleolar Z-DNA, but not with the nucleoplasmic ones. In the nuclear periphery, the Z-positive signals were adjacent to the IF proteins constituting the lamina, though both signals did not often co-localize.

Influence of buoyancy-driven convection on protein separation by free-flow electrophoresis.

In free-flow electrophoresis, the stability and reproducibility of the flow field are prerequisites for a satisfactory separation. Due to the presence of the species to be separated and due to mass- and heat-transfer phenomena, non-uniformities in density inevitably appear, both within the carrier buffer and between the carrier buffer and the sample stream. On earth they give rise to buoyancy-driven convection which interferes with the separation. These effects have been quantified by numerical modelling. Agreement has been found between the numerical results and experimental observations.

Fluorescent in situ hybridization and C-banding analyses of highly repetitive DNA sequences in the heterochromatin of rye (Secale montanum Guss.) and wheat incorporating S. montanum chromosome segments.

The molecular characterization of C-banded regions of Secale montanum Guss. by means of in situ hybridization was performed in order to provide new information about their chromosome structure relative to cultivated rye, Secale cereale L. Accurate identification of individual chromosomes was achieved using simultaneous and (or) successive fluorescent in situ hybridization (FISH) and C-banding. FISH identification was performed using total rye DNA, three highly repetitive rye DNA sequences (pSc119.2, pSc74, and pSc34), and the ribosomal RNA probes pTa71 (18S, 5.8S, and 26S rDNA) and pTa794 (5S rDNA). FISH was also used to identify the chromosome segment involved in two spontaneous translocation lines recovered from a 'Chinese Spring'--S. montanum wheat-rye addition line. FISH analysis revealed the exact translocation breakpoints and allowed the identification of the transferred rye segments. The value of this type of analysis is discussed.

Highly repetitive sequences in B chromosomes of Secale cereale revealed by fluorescence in situ hybridization.

An analysis of the presence and distribution of the rye and wheat repeated sequences in rye B chromosomes was carried out by fluorescent in situ hybridization. Probes used consisted of three highly repetitive sequences from rye (pSc119.2, pSc74, and pSc34) and the multigene families for the 25S-5.8S-18S and 5S rDNA from wheat (pTa71 and pTa794, respectively). pSc74 and pSc119.2 showed hybridization signals in the telomeric regions of rye B chromosomes. The remaining DNA clones did not hybridize to the B chromosomes.

Distribution of highly repeated DNA sequences in species of the genus Secale.

The presence and distribution of the most important highly repetitive DNA sequences of rye in cultivated and wild species of the genus Secale were investigated using fluorescence in situ hybridization. Accurate identification of individual chromosomes in the most commonly recognized species or subspecies of the genus Secale (S. cereale, S. ancestrale, S. segetale, S. afghanicum, S. dighoricum, S. montanum, S. montanum ssp. kuprijanovii, S. africanum, S. anatolicum, S. vavilovii, and S. silvestre) was achieved using three highly repetitive rye DNA sequences (probes pSc119.2, pSc74, and pSc34) and the 5S ribosomal DNA sequence pTa794. It is difficult to superimpose trends in the complexity of repetitive DNA during the evolution of the genus on conclusions from other cytogenetic and morphological assays. However, there are two clear groups. The first comprises the self-pollinated annuals S. silvestre and S. vavilovii that have few repeated nucleotide sequences of the main families of 120 and 480 bp. The second group presents amplification and interstitialization of the repeated nucleotide sequences and includes the perennials S. montanum, S. anatolicum, S. africanum, and S. kuprijanovii, as well as the annual and open-pollinated species S. cereale and its related weedy forms. The appearance of a new locus for 5S rRNA in S. cereale and S. ancestrale suggests that cultivated ryes evolved from this wild weedy species.

Mapping and organization of highly-repeated DNA sequences by means of simultaneous and sequential FISH and C-banding in 6x-triticale.

Three families of highly repeated sequences from rye and the rRNA multigenes (NOR and 5S) have been mapped by FISH and C-banding, in chromosomes of triticale. The pSc119.2 probe showed interstitial hybridization in chromosome arms 1RS, 1RL, 4RL, 5RL, 6RS, 6RL, 7RS and 7RL, and is very effective for chromosome identification of rye chromosomes in triticale. This sequence also hybridizes to the 4A, 5A and the seven B-genome wheat chromosomes. Simultaneous hybridization with the pSc119.2 and pTa794 (5S rRNA) is very useful to distinguish the metacentric chromosomes 2R and 3R. The pSc74 probe appears at interstitial sites in the long arm of the most heterobrachial chromosomes (5R and 6R). The three repetitive sequences of 120 bp, 480 bp, and 610 bp hybridize to telomeric regions in rye chromosomes. Different arrangements and complex organizations consisting of arrays of three or more family sequences were found. The results demonstrate a great variation in the relative arrangement of the repetitive sequences in the telomeres of the rye chromosomes. There were quantitative differences in each cytological marker between triticale lines in both in situ labelling and C-banding, probably as the result of differences in the number and/or kind of repeat sequence.

Evolutionary trends of different repetitive DNA sequences during speciation in the genus secale.

The presence and distribution of two simple sequence repeats (SSRs), three highly repetitive sequences from rye, and the 5S rDNA have been investigated in 3 rye cultivars and 10 wild-related species of the genus SECALE: The following conclusions can be drawn in addition to detailed knowledge of the sequence content of chromatin in each accession studied: (1) Every species is unique in either or both the complement and chromosomal distribution of the six repeated sequences analyzed. (2) These sequences reveal multiple landmarks along all the rye chromosomes arms. (3) High polymorphism as well as heterozygosity between homologues in the distribution of the (AAG)(5) and (AAC)(5) was revealed in the outbreeding species of the Secale strictum complex. (4) It is possible to deduce trends in the complexity of repetitive DNA during the evolution of the genus. A possible evolutionary pathway that accounts for the present-day Secale species is presented.

Variation in highly repetitive DNA composition of heterochromatin in rye studied by fluorescence in situ hybridization.

The molecular characterization of heterochromatin in six lines of rye has been performed using fluorescence in situ hybridization (FISH). The highly repetitive rye DNA sequences pSc 119.2, pSc74, and pSc34, and the probes pTa71 and pSc794 containing the 25S-5.8S-18S rDNA (NOR) and the 5S rDNA multigene families, respectively, were used. This allowed the individual identification of all seven rye chromosomes and most chromosome arms in all lines. All varieties showed similar but not identical patterns. A standard in situ hybridization map was constructed following the nomenclature system recommended for C-bands. All FISH sites observed appeared to correspond well with C-band locations, but not all C-banding sites coincided with hybridization sites of the repetitive DNA probes used. Quantitative and qualitative differences between different varieties were found for in situ hybridization response at corresponding sites. Variation between plants and even between homologous chromosomes of the same plant was found in open-pollinated lines. In inbred lines, the in situ pattern of the homologues was practically identical and no variation between plants was detected. The observed quantitative and qualitative differences are consistent with a corresponding variation for C-bands detected both within and between cultivars.

Chromosomal location of structural genes controlling isozymes in Hordeum chilense : 1. 6-Phosphogluconate dehydrogenase and malate dehydrogenase.

A study of 6-phosphogluconate dehydrogenase and malate dehydrogenase isozyme expression in Triticum turgidum conv. durum /Hordeum chilense monosomic addition lines has revealed the location of two structural genes, 6-pgd-H (ch) 2 and Mdh-H (ch) 1, on chromosome 1H(ch) of H. chilense. The homoeology between 1H(ch) and other chromosome of Triticeae related species is discussed on the basis of isozyme gene analysis.

Chromosomal location of structural genes controlling isozymes in Hordeum chilense : 3. Esterases, glutamate oxaloacetate transaminase and phosphoglucomutase.

Polyacrylamide and starch gel electrophoresis of esterase (EST), glutamate oxaloacetate transaminase (GOT) and phosphoglucomutase (PGM) isozymes in Hordeum chilense, Triticum turgidum conv. durum, the amphiploid H. chilense X T. turgidum (Tritordeum), and the durum wheat/H. chilense monosomic addition lines revealed the chromosomal location of one EST locus, two GOT loci and one PGM locus. Loci Est-H (ch)1 and Got-H (ch)2 were found on chromosome 6H(ch),Got-H (ch)3 on chromosome 3H(ch), and Pgm-H (ch)1 on chromosome 4H(ch). These results lend evidence for the assumed homoeology relationships between chromosomes of Triticeae species.

Meiotic pairing and alpha-amylase phenotype in a 5B/5R(m) Triticum aestivum - Secale montanum translocation line in common wheat.

A 5BS/5R(m)S translocation chromosome spontaneously recovered from a 'Chinese Spring' - Secale montanum wheat-rye telocentric 5R(m)S addition line has been identified and cytologically studied using C-banding in somatic and meiotic cells. Analysis of the translocated chromosome showed that a terminal segment of the short arm of 5B had been replaced by a short terminal region of chromosome arm 5R(m)S. The translocation led to the deletion of the genetic system promoting pairing located in 5BS, which is slightly compensated for when doses of 5R(m)S are increased, indicating homoeology to wheat chromosome 5BS. The α-amylase phenotype in 5B/5R(m) translocated material was studied and found to be identical to that of ditelocentric line 5BL of 'Chinese Spring'. An effect on the α-amylase activity was detected as a result of the removal of the terminal region of 5BS, perhaps as a consequence of variation in dormancy period duration.

C-banding in 6x-Triticale x Secale cereale L. hybrid cytogenetics.

The meiotic behaviour of F1 hybrids of hexaploid Triticale that differed in their genotypic or chromosomic constitution, and diploid rye, was investigated. Meiotic analysis were done by Feulgen and C-banding staining methods. A differential desynaptic effect in the hybrids was detected and explained in terms of genetic differences in pairing regulators. The high homoeologous pairing (A-B wheat chromosomes and wheat-rye chromosomes) observed in the hybrids can be explained in terms of an inhibition of the effect of a single dose of the Ph allele of the 5B chromosome produced by two doses of the 5R chromosome. The higher homoeologous pairing detected in the hybrid 188 x 'Canaleja' could be the overall result of the balance between the Ph diploidizing system (1 dose), the pairing promoter of the 5R chromosome (2 doses) and that of the 3D chromosome (1 dose coming from the parental line Triticale with the substitution 3R by 3D).

Characterisation and analysis of new HMW-glutenin alleles encoded by the Glu-R1 locus of Secale cereale.

This work reports the molecular characterisation of new alleles of the previously reported Glu-R1 locus. Wheat lines carrying the chromosome substitution 1R(1D), rye cultivars and related wild species were analysed. Five new x-type and four y-type Glu-R1 glutenin subunits were isolated and characterised. The coding region of the sequences shows the typical structure of the HMW glutenin genes previously described in wheat, with the N and C-terminal domains flanking the central repetitive region. Tri-, hexa- and nona-peptides found in the central repetitive region of wheat glutenin genes were also present in the rye genes. Duplications and deletions of these motifs are responsible for allelic variation at the Glu-R1 locus. Orthologous genes (from different genomes) were more closely related than paralogous genes (x- and y-type), supporting the hypothesis of gene duplication before Triticeae speciation. Differences in the number and position of cysteine residues identified alleles which in wheat are associated with good dough quality. SDS proteins encoded by some characterised alleles were presumptively identified.

Encoding genes for endosperm proteins in Hordeum chilense.

The endosperm proteins encoded by the genome Hch in Hordeum chilense, Tritordeum (amphiploid Hordeum chilense x Triticum turgidum), common wheat-H. chilense addition lines, and the segregating plants resulting from the cross Tritordeum x T. turgidum, were fractionated by three electrophoretical techniques: SDS-PAGE, A-PAGE, and bidimensional PAGE. Prolamin subunits with a high molecular weight (HMW) were well visualized by SDS-PAGE, the A-PAGE technique permitted good resolution for many hordeins and gliadins, and two-dimensional electrophoresis allowed new sets of bands coded by gene complexes from H. chilense chromosomes to be distinguished. The loci Hor-Hch1 (up to 11 subunits belonging to the ω-, ß - and α-hordeins), Glu-Hch1 (one HMW prolamin subunit), Hor-Hch2 (one α-hordein), and Hor-Hch3 (up to four α-hordeins) were located on the H. chilense chromosomes 1Hch, 5Hch, and 7Hch.