Legumin of Vicia faba major: accumulation in developing cotyledons,purification, mRNA characterization and chromosomal location of coding genes

Summary Experiments were carried out on Vicia faba major involving (1) determination of the pattern of legumin accumulation during seed development, (2) protein purification from mature cotyledons, (3) the characterization of legumin mRNA, and (4) the chromosomal localization of the genes coding for legumins. In developing cotyledons the synthesis of legumin begins 28 days after petal desiccation (DAPD), and 4 days after initiation of vicilin synthesis. The two subunits (_A and _A) of legumin A appear 2 days earlier than those (_B and _B) of legumin B. While the accumulation of vicilin peaks on the 30th DAPD, that of legumin continues during further seed development, and the synthesis of legumin mRNA peaks on the 37th DAPD. Northern blot hybridizations using two DNA plasmids containing cDNA inserts with sequence homology to the A- and B-type legumin genes, respectively, indicated that legumin mRNAs extracted from cotyledons 36 DAPD band below the 18S RNA band. In addition, a faint band below that of the 25S RNA band can be observed in legumin mRNAs extracted from cotyledons at an earlier developmental stage (30 DAPD). By means of polyacrylamide gel electrophoresis in the presence or absence of SDS and 2-mercaptoethanol, two fractions could be eluted after zonal isoelectric precipitation of the globulins from mature seeds: one fraction contains mainly vicilin, the other, legumin. In situ hybridization showed that legumin genes are arranged in two clusters: the genes coding for legumin A are located in the longer arm of the one between the two shortest subtelocentric chromosome pairs whose centromere is in a less terminal position; those coding for legumin B are located in the non-satellited arm of the longer submetacentric pair.     

Cytogenetics in fruit breeding - localization of ribosomal RNA genes on chromosomes of apple (Malus×domestica Borkh.)

 The localization of rRNA genes was studied by fluorescent in situ hybridization (FISH) on chromosomes of the cultivated apple, M.×domestica ‘Pinova’ (2n=34). The 18S/25S rRNA loci were detected in terminal positions of the short arms of two submetacentric and two metacentric chromosome pairs. One 5S rRNA gene locus was found in the proximal region of the short arm of a small metacentric chromosome pair. Received : 21 June 1996 / Accepted : 28 June 1996     

Karyotypic characterization and genomic organization of the 5S rDNA in <Emphasis Type="Italic">Polypterus senegalus</Emphasis> (Osteichthyes,Polypteridae)

Polypteridae (Cladistia) is a family of archaic fishes, confined to African freshwaters. On account of their primitiveness in anatomical and morphological characters and mosaic relationships among lower Osteichthyans fishes, they constitute an important subject for the study of evolution in vertebrates. Very little is known about the karyological structure of these species. In this article, a cytogenetic analysis on twenty specimens of Polypterus senegalus (Cuvier, 1829) was performed using both classical and molecular techniques. Karyotype (2n = 36; FN = 72), chromosome location of telomeric sequences (TTAGGG) _n , (GATA)₇ repeats and ribosomal 5S and 18S rRNA genes were examined by using Ag-NOR, classical C-banding, CMA₃ staining and FISH. Staining with Ag-NOR showed the presence of two GC rich NORs on the p arm of the chromosome pair no. 1. CMA₃ marked all centromerical and some (no. 1 and no. 14) telomeric regions. FISH with 5S rDNA marked the subtelomeric region of the q arm of the chromosome pair no. 14. FISH with 18S rDNA marked the telomeric region of the p arm of the chromosome pair no. 1, previously marked by Ag-NOR. (GATA)₇ repeats marked the subtelomeric regions of all chromosome pairs, with the exclusion of the no. 1, 3 and 14. Hybridization with telomeric probes (TTAGGG) _n showed bright signals at the end of all chromosomes. After cloning, the 5SrDNA alignment revealed an organization of sequences made up of two different classes of tandem arrays (5S type I and 5S type II) of different lengths.     

Organization and mapping of legumin genes in Pisum

Summary The organization of three classes of legumin gene has been studied by exploiting restriction fragment length polymorphisms. Hybridization of cDNA probes, representing different sub-families of legumin gene, to DNA from F₂ and F₆ plants from selected crosses showed that there was a tight linkage of individual genes within each of two sub-families. One of these sub-families has been mapped to a locus on chromosome 7 close to r, while the other maps to a locus near a on chromosome 1. The results from one of the crosses analyzed indicated that a third class of legumin gene is also linked to a.     

Physical Mapping of rRNA Gene Loci and Inter-specific Relationships in Wild <Emphasis Type="Italic">Lilium</Emphasis> Distributed in Korea

Molecular cytogenetic analyses using fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) were carried out to elucidate inter-specific relationships among wild Lilium species distributed in Korea. FISH revealed four to eight 45S rRNA gene loci, which are located on chromosomes 1–7, 10, and 11 among the different species. In contrast, the 5S rRNA gene locus was conserved on the long arm of chromosome 3, occasionally with two adjacent sites on the same chromosome arm in a few species. The 5S rDNA site was located adjacent to the 45S rDNA site in only three species, Lilium distichum, Lilium hansonii, and Lilium tsingtauense. GISH analysis using genomic DNA probes detected strong hybridization of genomes between diploid and triploid Lilium lancifolium species, demonstrating that triploid plants were derived from diploid L. lancifolium and not from Lilium maximowiczii. Phylogenetic analysis of the ITS and NTS sequences supported the cytogenetic data as well as Comber’s classification of the genus Lilium.     

Physical mapping of the 5S ribosomal RNA genes in Arabidopsis thaliana by multi-color fluorescence in situ hybridization with cosmid clones

The 5S ribosomal RNA genes were mapped to mitotic chromosomes of Arabidopsis thaliana by fluorescence in situ hybridization (FISH). In the ecotype Landsberg erecta, hybridization signals appeared on three pairs of chromosomes, two of which were metacentric and the other acrocentric. Hybridization signals on one pair of metacentric chromosomes were much stronger than those on the acrocentric and the other pair of metacentric chromosomes, probably reflecting the number of copies of the genes on the chromosomes. Other ecotypes, Columbia and Wassilewskija, had similar chromosomal distribution of the genes, but the hybridization signals on one pair of metacentric chromosomes were very weak, and detectable only in chromosomes prepared from young flower buds. The chromosomes and arms carrying the 5S rDNA were identified by multi-color FISH with cosmid clones and a centromeric 180 bp repeat as co-probes. The metacentric chromosome 5 and its L arm carries the largest cluster of the genes, and the short arm of acrocentric chromosome 4 carries a small cluster in all three ecotypes. Chromosome 3 had another small cluster of 5S rRNA genes on its L arm. Chromosomes 1 and 2 had no 5S rDNA cluster, but they are morphologically distinguishable; chromosome 1 is metacentric and 2 acrocentric. Using the 5S rDNA as a probe, therefore, all chromosomes of A. thaliana could be identified by FISH. Chromosome 1 is large and metacentric; chromosome 2 is acrocentric carrying 18S-5.8S-25S rDNA clusters on its short arm; chromosome 3 is metacentric carrying a small cluster of 5S rDNA genes on its L arm; chromosome 4 is acrocentric carrying both 18S-5.8S-25S and 5S rDNAs on its short (L) arm; and chromosome 5 is metacentric carrying a large cluster of 5S rDNA on its L arm.     

Karyotypes of three somaclonal variants and wild plants ofAllium tuberosum by bicolor FISH

Using the fluorescence in situ hybridization (FISH) technique, we conducted karyotype analyses to identify the lost chromosomes in three somaclonal variants obtained from tissue culture of wildAllium tuberosum (2n = 4X = 32). The three lost chromosomes of the At29 variant (2n = 29) were all chromosome 2, the two for At30 (2n = 30) were chromosomes 7 and 8, and At31 was missing chromosome 2. Chromosome compositions of these variants were confirmed as being fixed lines during two years of greenhouse cultivation. The bicolor FISH technique, involving both 5S and 18S–5.8S–26S ribosomal RNA genes as probes, was used to assign chromosomal locations and to confirm whether the lost chromosomes contained any rRNA markers. The 5S rRNA gene signals in all variants as well as the wild type were detected as two sets, one on the intercalary region of the short arm of chromosome 3, the other on the intercalary region of the long arm of chromosome 6. One 18S–5.8S–26S rRNA gene site on the secondary constriction included a flanking satellite and terminal region on the short arm of chromosome 8. Signals of the 18S–5.8S–26S rRNA gene in At30 showpd in only three chromosomes, indicating that one of the lost chromosomes was chromosome 8. Overall, three marker chromosomes were established by FISH, using rRNA multigene families.     

Karyotype analysis in Hyacinthella dalmatica (Hyacinthaceae) reveals vertebrate-type telomere repeats at the chromosome ends.

Chromosome analysis of three different populations of Hyacinthella dalmatica (Lallem.) Trinajsti?, an endemic species of the coastal region of southeastern Europe, showed a unique chromosome number, 2n = 2x = 20, and bimodal karyotype with one large and nine smaller pairs of chromosomes. Staining with fluorochromes CMA3 (chromomycin A3) and DAPI (4,6-diamidino-2-phenylindole) revealed heterochromatic regions associated with NORs, centromeres, and several interstitial heterochromatic bands on the longest chromosome pair. Double-target FISH with two ribosomal DNA probes revealed one locus of 5S rRNA genes in the pericentromeric region of chromosome pair 3 and one locus of 18S-5.8S-26S rRNA genes on the short arm of chromosome pair 4 in all plants and populations analyzed. Southern hybridization analysis and FISH experiments demonstrated that the distal ends of H. dalmatica chromosomes contain the vertebrate telomere (5'-TTAGGG-3') repeat type rather than the Arabidopsis (5'-TTTAGGG-3') heptamer, and so suggest that this Asparagales species along with Aloe and Othocallis contains the vertebrate-type telomere repeat.     

Chromosome analysis by fluorescence in situ hybridization of callus-derived regenerants in Allium cyaneum R.

Investigations were performed to confirm the optimal in vitro culture condition for callus induction and plant regeneration, to observe if somoclonal variation occurs among regenerated plants at the ploidy level and to analyse the chromosomal location of 5S and 18S-26S rRNA gene families using fluorescence in situ hybridization in callus-derived plants of Allium cyaneum. High-est callus initiation was achieved with bulb explants cultured on MS medium supplemented with 2,4-D and BAP at 1 mg l^–1 each. A total of 195 plants was obtained when using MS medium supplemented with 1 mg l^–1 NAA and 5 mg l^–1 BAP; about 92% were diploid having 2n=16; 8% showed a variation in ploidy level. Using digoxigenin-labelled 5S rRNA and biotin-labelled 18S-26S rRNA gene probes, we compared the fluorescence in situ hybridization patterns of autotetraploid plants with the A. cyaneum wild type. The 5S rRNA gene sites were detected on the interstitial region in the short arm of chromosome 4 and on the interstitial region in both arms of chromosome 7. The 18S-26S rRNA gene sites were detected on the terminal region of the short arm, including the satellite of chromosome 5, as well as on a part of chromosome B. The chromosomal location of both rRNA genes in regenerated autotetraploid plants corresponded to those of the wild species. Received: 20 March 1998 / Revision received: 15 June 1998 / Accepted: 8 July 1998     

Dual-color FISH karyotype analyses using rDNAs in three Cucurbitaceae species

Dual-color fluorescence in situ hybridization (FISH) analysis of three Cucurbitaceae species from different genera was conducted using 5S and 45S rDNA probes. In Benincasa hispida (Thunb.) Cogn. (2n=24), the 45S rDNA probe hybridized on two chromosomes, one in the short arm of a medium-sized metacentric chromosome and another at the satellite of a chromosome. The 5S rDNA hybridized at a site proximal to the centromere of the same short arm of the 45S rRNA gene locus that occupied almost the entire short arm. For Citrullus lanatus (Thunb.) Matsum & Nakai (2n=22), the 45S rDNA probe hybridized at sites in the short arms of two chromosomes and the 5S rDNA probe was co-localized with the 45S rRNA locus at the region proximal to the centromere in one chromosome. The 45S rRNA loci occupied almost all of the short arms in both chromosomes. In Cucurbita moschata Duch. (2n=40), the 45S rDNA probe hybridized in five chromosomes in which the 45S rRNA genes occupied almost two-thirds of the chromosomes in two large chromosomes and the entire short arm of a medium-sized chromosome. Two other loci were present in two medium-sized chromosomes, one in the proximal region in the short arm of a chromosome and another at the tip of the long arm of a chromosome. Chromosomes of B. hispida were relatively larger than those of the other two species. The karyotype of B. hispida is composed of two metacentrics and 10 submetacentrics, while that of C. lanatus is composed of seven metacentrics and four submetacentrics and that of C. moschata is composed of 18 metacentrics and two submetacentrics. Comparative chromosome evolution among the three Cucurbitaceae species was attempted using the karyotypes and the chromosomal distribution patterns of the 5S and 45S rDNAs. The results presented herein will be useful in elucidating the phylogenetic relationships among Cucurbitaceae species, and will provide basic data for their breeding programs.     

Detection of 5S and 25S rRNA genes in Sinapis alba, Raphanus sativus and Brassica napus by double fluorescence in situ hybridization

Different ribosomal RNA (5S and 25S) genes were investigated simultaneously by fluorescence in situ hybridization (FISH) in Sinapis alba, Raphanus sativus and Brassica napus. The chromosomes of S. alba carried four 5S and six 25S gene sites, and those of R. sativus four sites of each gene, respectively. These two species have one chromosome pair with both rDNA genes; the two are closely located on a short arm of S. alba, while in R. sativus one is distal on the short arm (5S) and the other more proximal on the long arm (25S). In B. napus we have confirmed 12sites of 25S rDNA. The detection of 5S rDNA genes revealed 14 signals on 12 chromosomes. Of these, six chromosomes had signals for both rDNA genes. The FISH with 5S rDNA probes detected two sites closely adjacent in four chromosomes of B napus. These results are discussed in relation to a probable homoeologous chromosome pair in B. oleracea. Received: 20 July 1999 / Accepted: 8 October 1999     

Chromosomal mapping of the major and minor ribosomal genes, (GATA)n and (TTAGGG)n by one-color and double-color FISH in the toadfish Halobatrachus didactylus (Teleostei: Batrachoididae)

The karyotype of Halobatrachus didactylus presents 46 chromosomes, composed of eight metacentric, 18 submetacentric, four subtelocentric, and 16 acrocentric chromosomes. The results of FISH showed that the major ribosomal genes were located in the terminal position of the short arm of a large submetacentric chromosome. They also showed a high variation in the hybridization signals. The products of amplification of 5S rDNA produced bands of about 420 pb. The PCR labeled products showed hybridization signals in the subcentromeric position of the long arm of a submetacentric chromosome of medium size. Double-color FISH indicated that the two ribosomal families are not co-located since they hybridizated in different chromosomal pairs. Telomeres of all the chromosomes hybridized with the (TTAGGG) _n probe. The GATA probe displayed a strong signal in the long arm of a submetacentric chromosome of medium size, in the subcentromeric position. The double-color FISH showed that the microsatellite GATA and the 5S rDNA gene are located in different chromosomal pairs. The majority presence of GATA probes in one pair of chromosomes is unusual and considering its distribution through different taxa it could be due to evolutionary mechanisms of heterochromatine accumulation, leading to the formation of differentiated sex chromosomes.     

A comparison of sequence resolution on plant chromosomes: PRINS versus FISH

 The resolution of the chromosomal positions of six high- and one low-copy sequences by oligonucleotide-primed in situ (PRINS) labelling was compared with corresponding data obtained after fluorescent in situ hybridization (FISH) on field-bean and barley chromosomes. While PRINS proved to be suitable for the rapid detection of high-copy tandem repeats at the same loci as those revealed by FISH, no clear PRINS signal was obtained for the low-copy family of vicilin genes at their locus on field-bean chromosome II. This indicates that localization of short target sequences by primer extension via Taq polymerase in situ does not yet provide a resolution equal, or superior, to FISH on plant chromosomes. Therefore, the use of a cocktail of chromosome-specific single-copy sequences as primers for PRINS is no alternative for the not as yet feasible chromosome painting in plants. Received: 21 April 1998 / Accepted: 12 May 1998     

Replication of the rRNA and legumin genes in synchronized root cells of pea (Pisum sativum): evidence for transient EcoR I sites in replicating rRNA genes

Summary The temporal pattern of replication of the rRNA and legumin genes differs in synchronized pea root cells. The relative number of rRNA genes replicated hourly during the first five hours of S phase ranges between 5 and 10 percent. In late S phase, during hours six through nine, the number of rRNA genes replicated increases reaching a maximum of about 25 percent at the ninth hour. Unlike the rRNA genes, the legumin genes have a wave-like pattern of replication peaking in early S phase at the third hour and again in late S phase at the eighth hour.Replicating rDNA, isolated by benzoylated naphthoylated DEAE-column chromatography, has EcoR I restriction sites that are absent in non-replicating rDNA sequences. The cleavage of these sites is independent of the time of rDNA replication. The transient nature of the EcoR I sites suggests that they exist in a hemimethylated state in parental DNA.The two Hind III repeat-size classes of rDNA of var. Alaska peas are replicated simultaneously as cells progress through S phase. Thus, even if the 9.0 kb and 8.6 kb repeat classes are located on different chromosomes, their temporal order of replication is the same.     

Ribosomal DNA,tri- and bi-partite pericentromeres in the permanent translocation heterozygote <Emphasis Type="Italic">Rhoeo spathacea</Emphasis>

High- and low-stringency FISH and base-specific fluorescence were performed on the permanent translocation heterozygote Rhoeo spathacea (2n = 12). Our results indicate that 45S rDNA arrays, rDNA-related sequences and other GC-rich DNA fraction(s) are located within the pericentromeric regions of all twelve chromosomes, usually colocalizing with the chromomycin A₃-positive bands. Homogenization of the pericentromeric regions appears to result from the concerted spread of GC-rich sequences, with differential amplification likely. We found new 5S rDNA patterns, which suggest a variability in the breakpoints and in the consequent chromosome reorganizations. It was found that the large 5S rDNA locus residing on each of the 8E and 9E arms consisted of two smaller loci. On each of the two chromosome arms 3b and 4b, in addition to the major subtelomeric 5S rDNA locus, a new minor locus was found interstitially about 40% along the arm length. The arrangement of cytotogenetic landmarks and chromosome arm measurements are discussed with regard to genome repatterning in Rhoeo.     

Localization of 5S rRNA Loci in Three Coregonid Species (Salmonidae)

In the present study the chromosome distribution of the 5S rDNA loci and its relation to the major rDNA genes were investigated in three Coregonid species (Salmonidae): Coregonus lavaretus, Coregonus peled and Coregonus albula, a family which has experienced large karyotype rearrangements along its evolution starting from a tetraploid ancestor. 5S PRINS/CMA₃ sequential staining together with previous data enabled us to locate 5S rRNA genes and nucleolar organizer regions (NORs) in the three species analyzed. PRINS revealed the 5S rDNA cluster at the distal part of the long arm of a similar submetacentric chromosome pair in the three species. Our data indicate that 5S rDNA clusters have probably conserved chromosomal location in the genus Coregonus, whereas 45S rDNA (NOR) sites are clearly differentiated, from a single locus in C. peled, to multiple loci in C. lavaretus and highly polymorphic multichromosomal location in C. albula.     

Molecular-cytogenetic studies of ribosomal genes and heterochromatin reveal conserved genome organization among 11 Quercus species

Genomes of 11 Quercus species were characterized using cytogenetic (Giemsa C-banding, fluorochrome banding), molecular-cytogenetic (fluorescence in situ hybridization, FISH, to ribosomal genes) and molecular (dot-blot for ribosomal gene-copy number assessment) techniques. Ribosomal genes are the first DNA sequences to be physically mapped in oaks, and the copy number of the 18S-5.8S-26 S rRNA genes is estimated for the first time. Oak karyotypes were analysed on the basis of DAPI banding and FISH patterns; five marker chromosomes were found. In addition, chromosomal organization of ribosomal genes with respect to AT- and GC-differentiated heterochromatin was studied. Fluorochrome staining produced very similar CMA/DAPI banding patterns, and the position and number of ribosomal loci were identical for all the species studied. The 18S-5.8S-26 S rRNA genes in oak complements were represented by a major locus at the subterminal secondary constriction (SC) of the only subtelocentric chromosome pair and a minor locus at paracentromeric SC of one metacentric pair. The only 5 S rDNA locus was revealed at the paracentromeric region of the second largest metacentric pair. A striking karyotypic similarity, shown by both fluorochrome banding and FISH patterns, implies close genome relationships among oak species no matter their geographic origin (European or American) or their ecophysiology (deciduous or evergreens). Dot-blot analysis gave preliminary evidence for different copy numbers of 18S-5.8S-26 S rRNA genes in diploid genomes of Q. cerris, Q. ilex, Q. petraea, Q. pubescens and Q. robur (2700, 1300, 2200, 4000 and 2200 copies, respectively) that was correlated with the size polymorphism of the major locus. Received: 26 February 1999 / Accepted: 16 March 1999     

Karyotype of Mesembryanthemum crystallinum (Aizoaceae) studied by chromosome banding,FISH with rDNA probes and immunofluorescence detection of DNA methylation

The karyotype of the common ice plant Mesembryanthemum crystallinum L. (Aizoaceae) was studied using Chromomycin A₃ (CMA)/4′,6-diamidino-2-phenylindole (DAPI) staining, fluorescence in situ hybridization with 5S and 18S–5.8S–25S rDNA probes, DAPI/C-banding and immunodetection of 5-methylcytosine. A single bright CMA-band was revealed on the satellite chromosome, whose location was coincided with a position of a site of 18S–5.8S–25S rRNA genes. A site of 5S rRNA genes was observed on one of the other chromosomes. Relatively large DAPI/C-bands were mainly localized in the pericentromeric regions of the chromosomes. DAPI/C-banding patterns allowed us to identify all the chromosomes in the karyotype of M. crystallinum. The methylation of euchromatic chromosome regions was weaker as compared with heterochromatic DAPI/C-bands, which were hypermethylated. The obtained results may provide opportunities for investigating, at the chromosomal level, the genomic changes occurring in M. crystallinum either under salinization or under the action of other stress factors.