Personal Perspectives on Plant Ribosomal RNA Genes Research: From Precursor-rRNA to Molecular Evolution

The history of rDNA research started almost 90 years ago when the geneticist, Barbara McClintock observed that in interphase nuclei of maize the nucleolus was formed in association with a specific region normally located near the end of a chromosome, which she called the nucleolar organizer region (NOR). Cytologists in the twentieth century recognized the nucleolus as a common structure in all eukaryotic cells, using both light and electron microscopy and biochemical and genetic studies identified ribosomes as the subcellular sites of protein synthesis. In the mid- to late 1960s, the synthesis of nuclear-encoded rRNA was the only system in multicellular organisms where transcripts of known function could be isolated, and their synthesis and processing could be studied. Cytogenetic observations of NOR regions with altered structure in plant interspecific hybrids and detailed knowledge of structure and function of rDNA were prerequisites for studies of nucleolar dominance, epistatic interactions of rDNA loci, and epigenetic silencing. In this article, we focus on the early rDNA research in plants, performed mainly at the dawn of molecular biology in the 60 to 80-ties of the last century which presented a prequel to the modern genomic era. We discuss – from a personal view – the topics such as synthesis of rRNA precursor (35S pre-rRNA in plants), processing, and the organization of 35S and 5S rDNA. Cloning and sequencing led to the observation that the transcribed and processed regions of the rRNA genes vary enormously, even between populations and species, in comparison with the more conserved regions coding for the mature rRNAs. Epigenetic phenomena and the impact of hybridization and allopolyploidy on rDNA expression and homogenization are discussed. This historical view of scientific progress and achievements sets the scene for the other articles highlighting the immense progress in rDNA research published in this special issue of Frontiers in Plant Science on “Molecular organization, evolution, and function of ribosomal DNA.”

First report of karyological analysis and heteromorphic nucleolar organizer region of Black Surgeonfish (Acanthurus gahhm, Acanthuridae) in Thailand

This research was the first report on karyological analysis and heteromorphic nucleolar organizer region of black surgeonfish (Acanthurus gahhm, Acanthuridae) in Thailand. The 10 male and 10 female specimens were collected from Phuket Marine Biological Center, and Phang Nga Coastal Research and Development Center, Andaman Sea, Thailand. Mitotic chromosomes were directly prepared from gill and kidney tissues. The chromosomes were stained by conventional Giemsa staining and Ag-NOR banding techniques. Results showed that the diploid chromosomes number of A. gahhm was 2n=48, the fundamental numbers (NF) was 54 in both male and female. The karyotype consist of 6 large acrocentric, 20 large telocentric, 18 medium telocentric and 4 small telocentric chromosomes. None of strange size chromosomes related to sex was found. The heteromorphic nucleolar organizer regions (NORs) were observed on telomeric short arm of first acrocentric which can defined as 1a1b. There is NOR in 1a and not in 1b. The karyotype formula of black surgeon fish was as follows: 2n (48) = La6+Lt20+Mt18+St4

Comparative FISH analysis of Senna tora tandem repeats revealed insights into the chromosome dynamics in Senna

Background DNA tandem repeats (TRs) are often abundant and occupy discrete regions in eukaryotic genomes. These TRs often cause or generate chromosomal rearrangements, which, in turn, drive chromosome evolution and speciation. Tracing the chromosomal distribution of TRs could therefore provide insights into the chromosome dynamics and speciation among closely related taxa. The basic chromosome number in the genus Senna is 2n = 28, but dysploid species like Senna tora have also been observed. Objective To understand the dynamics of these TRs and their impact on S. tora dysploidization. Methods We performed a comparative fluorescence in situ hybridization (FISH) analysis among nine closely related Senna species and compared the chromosomal distribution of these repeats from a cytotaxonomic perspective by using the ITS1-5.8S-ITS2 sequence to infer phylogenetic relationships. Results Of the nine S. tora TRs, two did not show any FISH signal whereas seven TRs showed similar and contrasting patterns to other Senna species. StoTR01_86, which was localized in the pericentromeric regions in all S. tora, but not at the nucleolar organizer region (NOR) site, was colocalized at the NOR site in all species except in S. siamea. StoTR02_7_tel was mostly localized at chromosome termini, but some species had an interstitial telomeric repeat in a few chromosomes. StoTR05_180 was distributed in the subtelomeric region in most species and was highly amplified in the pericentromeric region in some species. StoTR06_159 was either absent or colocalized in the NOR site in some species, and StoIGS_463, which was localized at the NOR site in S. tora, was either absent or localized at the subtelomeric or pericentromeric regions in other species. Conclusions These data suggest that TRs play important roles in S. tora dysploidy and suggest the involvement of 45S rDNA intergenic spacers in “carrying” repeats during genome reshuffling.

The genomic structure of a human chromosome 22 nucleolar organizer region determined by TAR cloning

The rDNA clusters and flanking sequences on human chromosomes 13, 14, 15, 21 and 22 represent large gaps in the current genomic assembly. The organization and the degree of divergence of the human rDNA units within an individual nucleolar organizer region (NOR) are only partially known. To address this lacuna, we previously applied transformation-associated recombination (TAR) cloning to isolate individual rDNA units from chromosome 21. That approach revealed an unexpectedly high level of heterogeneity in human rDNA, raising the possibility of corresponding variations in ribosome dynamics. We have now applied the same strategy to analyze an entire rDNA array end-to-end from a copy of chromosome 22. Sequencing of TAR isolates provided the entire NOR sequence, including proximal and distal junctions that may be involved in nucleolar function. Comparison of the newly sequenced rDNAs to reference sequence for chromosomes 22 and 21 revealed variants that are shared in human rDNA in individuals from different ethnic groups, many of them at high frequency. Analysis infers comparable intra- and inter-individual divergence of rDNA units on the same and different chromosomes, supporting the concerted evolution of rDNA units. The results provide a route to investigate further the role of rDNA variation in nucleolar formation and in the empirical associations of nucleoli with pathology.

The Karyotype of Blainville’s Beaked Whale, Mesoplodon densirostris

The karyotype of the Odontocete whale, <i>Mesoplodon densirostris</i>, has not been previously reported. The chromosome number is determined to be 2n = 42, and the karyotype is presented using G-, C-, and nucleolar organizer region (NOR) banding. The findings include NOR regions on 2 chromosomes, regions of heterochromatic variation, a large block of heterochromatin on the X chromosome, and a relatively large Y chromosome. The karyotype is compared to published karyograms of 2 other species of <i>Mesoplodon</i>.

The Correlate between Argyrophilic Nucleolar Organizer Region Stain and Ki-67 Immnochistochemistry in Diagnostic Breast Cancer

Introduction: This experimental study gauged the value of argyrophilic nucleolar organizer region (AgNOR) staining as a possible technique for the estimation of cell kinetics in conventional histology sections, in benign and malignant breast lesions. Methods: With a silver staining technique and immunohistochemistry, we associated the numbers of AgNORs and Ki67 scores in 30 breast carcinomas and 10 benign breast lesions. Results: The mean values of Ag NORs silver stain dots count for normal, benign, grade II and III were 1.28±0.17, 2.83±0.68, 5.23±0.87 and 7.32±0.92, respectively. There was a statistically significant difference (p≤0.001) was noticed between the all individual groups, among the normal and breast lesion as well as among the GII and GIII. Immunohistochemical Results of Ki-67 protein exhibited homogenous golden-brown color in control case and a positive brown granules or diffuse dark brown color in the nuclei of both benign and malignant cases under the 400X magnify examined under the light microscope. Discussion: AgNOR counts performed on routine formalin-fixed paraffin sections could provide substantial kinetic evidence. Additionally, the difference in AgNOR counts between benign and malignant tumors is such that they may be of diagnostic worth.

First chromosome analysis of Thai pufferfish Pao cochinchinensis (Steindachner, 1866)

Pissaparn M, Phimphan S, Chaiyasan P, Tanoamtong A, Liehr T, Suwannapoom C, Reungsing M, Supiwong W. 2020. First chromosome analysis of Thai pufferfish Pao cochinchinensis (Steindachner, 1866). Biodiversitas 21: 4309-4316. Here first analysis of chromosomes and nucleolar organizer region (NOR) pattern in pufferfish Pao cochinchinensis (Steindachner, 1866) was undertaken. Chromosomal preparations were obtained from kidney of P. cochinchinensis from Chi River basin in Thailand. Chromosomal characteristics were analyzed by Giemsa staining, Ag-NOR banding as well as fluorescence in situ hybridization (FISH) using microsatellites d(CA)15 and d(CGG)10 probes. P. cochinchinensis had 2n = 40 with the fundamental number (NF) 74, both in male and female. The karyotype exhibited 12 metacentric (m), 10 submetacentric (sm), 12 acrocentric (a) and 6 telocentric (t) chromosomes. No differentiated heteromorphic sex chromosomes were observed. NORs were located on short arms adjacent to telomere of the metacentric chromosome pair 4, which coincide with signals of d(CGG)10 probe. FISH with d(CGG)10 sequences were also displayed at the telomeres of most other chromosomes, whereas d(CA)15 repeats highly accumulated throughout almost all entire chromosomes except for centromeric regions. The results of conventional Giemsa staining presented the differentiation even the same genus. The localization of NORs on one pair of chromosomes only is a common characteristic found in many fish groups as well as other vertebrates. Mapping of two distinct microsatellites demonstrated the remarkable chromosomal diversification that characterizes evolution in the genus Pao. Both, conventional and molecular cytogenetics are excellent tools to study, and better understand chromosomal evolution, as well as to uncover biodiversity among fishes.

Detection of antitumoral effects of quercetin through protein synthesis associated with argyrophilic nucleolar-regulating region

Quercetin is a natural flavonoid with potential anticancer properties without significant cytotoxicity in normal tissues. However, the effects of quercetin on Ehrlich ascites carcinoma (EAC) have not yet been clarified. The study aimed to show the antitumoral effect of quercetin through argyrophilic nucleolar organizer region (AgNOR) protein synthesis in mice carrying EAC. Thirty mice were used in the experiment (negative control ( n = 6), tumor control ( n = 8), quercetin 50 mg/kg ( n = 8), and quercetin 100 mg/kg ( n = 8) intravenously). The animals were euthanized on the 14th day, and the solid tumors were removed. Then, the total AgNOR area/nuclear area (TAA/NA) and average AgNOR number were calculated for each mice and the immunoreactivity of the factor VIII protein in tumor was evaluated. Tumor volumes and animal weights were statistically significant compared to the tumor control group ( p < 0.05). Statistically significant differences were observed between the groups in terms of TAA/NA ratio ( p < 0.05). Factor VIII expressions decreased in quercetin groups compared to control tumor tissue ( p < 0.05). The current study showed that quercetin has an important function against cancer development.