Activity Of Ribosomal RNA Genes Research Articles

Histone Deacetylase Inhibition Improves Activation of Ribosomal RNA Genes and Embryonic Nucleolar Reprogramming in Cloned Mouse Embryos1

Our group found that the treatment of embryos with histone deacetylase inhibitors (HDACi), including trichostatin A, Scriptaid, suberoylanilide hydroxamic acid, and oxamflatin, after cloning by somatic cell nuclear transfer (SCNT) resulted in significantly improved efficiency. Although many researchers have investigated the use of HDACi treatment to improve the quality of cloned mouse embryos, the mechanism underlying this treatment has not been completely understood. We believe that the effect of HDACi on embryonic gene activation (EGA) is important for normal development of cloned embryos. In the present study, using highly sensitive fluorescence in situ hybridization (FISH) with probes complementary to mouse rDNA, the effect of Scriptaid on the onset of rRNA synthesis was examined in cloned embryos. In addition, to determine how Scriptaid affects pre-rRNA processing machinery in SCNT embryos with activated rDNA transcription, functional nucleolar formation was analyzed in detail by combined assessment of rRNA synthesis and nucleolar protein allocation in preimplantation embryos. In this experiment, at least part of the rRNA localization by FISH was substituted by 5-bromouridine 5'-triphosphate staining after alpha-amanitin treatment. The results show that in the late 2-cell stage, a number of SCNT embryos initiated transcriptional activation while having one blastomere showing inactivated rRNA transcription and another blastomere showing activated rRNA transcription and despite both nuclei being in interphase. In addition, in some SCNT embryos, the same nuclei contained a mixture of inactively and actively transcribed rRNA, which was rarely observed in intracytoplasmic sperm injection embryos. This asynchronous transcription induced a delay of one cell cycle in SCNT embryo activation of functional nucleoli. Scriptaid can overcome this failure in the timely onset of embryonic gene transcription by activation of rRNA genes and promotion of nucleolar protein allocation during the early phase of EGA.

Activation of ribosomal RNA genes in porcine embryos produced in vitro or by somatic cell nuclear transfer

The onset of ribosomal RNA (rRNA) synthesis occurs during the second half of the third cell cycle, that is, at the four-cell stage, in porcine embryos developed in vivo. In the present study the onset of rRNA synthesis was investigated in porcine embryos produced in vitro (IVP) or by somatic cell nuclear transfer (SCNT) using fluorescence in situ hybridization (FISH) with an rDNA probe and subsequent visualization of the nucleolar proteins by silver staining. In the 205 IVP embryos investigated, all two-cell embryos (n = 34) were categorized as transcriptionally inactive. At the late four-cell stage (n = 45), 38% of the embryos contained 1-3 nuclei with signs of rRNA transcription, indicating an asynchronous transcription initiation. This pattern continued in the following stages, as 78% (n = 47), 47% (n = 42) and 83% (n = 37) of the embryos revealed a mixture of transcriptionally inactive and active cells at the eight-cell, 16-cell and blastocyst stage, respectively. In the 143 SCNT embryos investigated, all two-cell embryos (n = 34) and early four-cell embryos (n = 12) were also transcriptionally inactive. At the late four-cell stage (n = 33) and at the eight-cell stage (n = 24) there were equal proportions of transcriptionally active and inactive embryos and essentially all embryos that developed to the 16-cell stage (n = 21) and further to the blastocyst stage (n = 19) contained only transcriptionally active cells. In conclusion, porcine embryos produced in vitro had an asynchronous pattern of rRNA transcription initiation when compared to SCNT and in vivo developed porcine embryos.

Organization, differential expression and methylation of rDNA in artificial Solanum allopolyploids

Uniparental activity of ribosomal RNA genes (rDNA) in interspecific hybrids is known as nucleolar dominance (ND). To see if difference in rDNA intergenic spacers (IGS) might be correlated with ND, we have used artificial Solanum allopolyploids and back-crossed lines. Combining fluorescence in situ hybridization and quantification of the level of the rRNA precursor by real-time PCR, we demonstrated that an expression hierarchy exists: In leaves, roots, and petals of the respective allopolyploids, rDNA of S lycopersicum (tomato) dominates over rDNA of S. tuberosum (potato), whereas rDNA of S. tuberosum dominates over that of the wild species S. bulbocastanum . Also in a monosomic addition line carrying only one NOR-bearing chromosome of tomato in a potato background the dominance effect was maintained. These results demonstrate that there is possible correlation between transcriptional dominance and number of conservative elements downstream of the transcription start in the Solanum rDNA. In anthers and callus tissues under-dominant rDNA was slightly (S. lycopersicum/S. tuberosum) or strongly (S. tuberosum/S. bulbocastanum) expressed indicating developmental modulation of ND. In leaves and petals, repression of the respective parental rDNA correlated with cytosine methylation at certain sites conserved in the IGS, whereas activation of under-dominant rDNA in anthers and callus tissues was not accompanied by considerable changes of the methylation pattern.

Activation of ribosomal RNA genes in pre-implantation In Vitro-produced and nuclear transfer bovine embryos

Contents Transcription of the ribosomal RNA (rRNA) genes occurs in the nucleolus resulting in ribosome synthesis. The rRNA gene activation and the associated nucleolus formation may be used as a marker for the activation of the embryonic genome in mammalian embryos and, thus, serve to evaluate the developmental potential of embryos originating from different embryo technological procedures. In bovine in-vitro-produced embryos, functional ribosome-synthesizing nucleoli become structurally recognizable towards the end of the fourth post-fertilization cell cycle. A range of important nucleolar proteins (e.g. fibrillarin, nucleophosmin, nucleolin, topoisomerase I, upstream binding factor and RNA polymerase I) become localized to the nucleolar anlage over several cell cycles and this localization is completed towards the end of the fourth cell cycle. In nuclear transfer embryos originating from day 5–6 in-vitro-produced bovine embryos, fully developed nucleoli are apparently detected one cell cycle later, and a substantial proportion of the nuclear transfer embryos display aberrations in protein localization in one or more blastomeres. This information may help to explain the abnormalities observed in a proportion of fetuses and offspring derived from nuclear transfer embryos.

Activation of ribosomal RNA genes in preimplantation cattle and swine embryos

Transcription of ribosomal RNA (rRNA) genes occurs in the nucleolus resulting in ribosome synthesis. In cattle and swine embryos, functional ribosome-synthesizing nucleoli become structurally recognizable towards the end of the fourth and third post-fertilization cell cycle, respectively. In cattle, a range of important nucleolar proteins become localized to the nucleolar anlage over several cell cycles and this localization is apparently completed towards the end of the fourth cell cycle. In swine, the localization of these proteins to the anlage is more synchronous and occurs towards the end of the third cell cycle and is apparently completed at the onset of the fourth. The rRNA gene activation and the associated nucleolus formation may be used as a marker for the activation of the embryonic genome in mammalian embryos and, thus, serve to evaluate the developmental potential of embryos originating from different embryo technological procedures. By this approach, we have demonstrated that in vitro produced porcine embryos display a lack of localization of nucleolar proteins to the nucleolar anlage as compared with in vivo developed counterparts. Similarly, bovine embryos produced by nuclear transfer from morulae display such deviations as compared with in vitro produced counterparts. Collectively, this information may help to explain the appearance of abnormalities seen in a certain proportion of offspring derived from in vitro produced embryos and after cloning.

A longitudinal study of human age-related ribosomal RNA gene activity as detected by silver-stained NORs

The relative frequencies of silver-stained nucleolar organizing regions (Ag-NORs) as a function of age have been analyzed in skin fibroblasts derived from eight adult individuals participating in the Gerontology Research Center (GRO) Longitudinal Study, NIA, Baltimore, MD. Since silver staining of NORs is correlated with rRNA gene activity, we used this cytological method to examine the pattern of rRNA gene activity in specific individuals, each at two different ages. Our results show that the average number of Ag-NORs/cell decreases significantly with advancing age of each individual, presumably indicating a general pattern of age-related decline/alteration in rRNA gene activity and this pattern is individual-specific. The findings of our longitudinal study is consistent with the results of previous cross-sectional (population) studies on rRNA gene activity as detected by Ag-NORs. However, it appears that the relative rate in the age-related decline of rRNA gene activity, as evidenced by lower Ag-NOR frequencies with age, is variable from person to person.

The direct measurement of ribosomal RNA gene activity in wheat–rye hybrids

Cloned rDNA segments, from wheat (Triticum aestivum) and rye (Secale cereale), which included the entire spacer region plus parts of the flanking 18S and 26S rRNA genes, were used to assay transcripts formed in the nuclei from wheat–rye hybrids. The tetraploid triticales (× Triticosecale Wittmack) carrying a complete rye chromosome complement and a combination of wheat chromosomes, as well as wheat and rye per se, were studied. Nuclei isolated from young leaves were incubated in the presence of [32P]UTP for 10 min, after which the total RNA was isolated. Hybridization of this radioactive RNA to the cloned rDNA segments showed that the suppression of rye NOR activity, measured cytologically, can be directly related to a major reduction in rRNA synthesis.Key words: NOR suppression, RNA transcripts, isolated nuclei.

Differential silver-positive nucleolus organizer region activity in normal and malignant murine tissues

Transcriptionally active nucleolus organizer regions (NORs) were analyzed using a silver-staining technique. The levels of silver-positive NOR activity for normal murine bone marrow and thymus cells were established, and significant differences in ribosomal RNA gene activity were observed. When tumor cells originating from these two tissue types were also studied, significant differences were seen not only between the normal and malignant tissues, but between the two tumor types as well. These differences in ribosomal RNA gene activity with respect to cell type and malignancy may be useful diagnostically.

Relationship between the number and function of human ribosomal genes

The relative number of ribosomal RNA genes of the acrocentric chromosomes in one individual was measured by counting grains after in situ hybridization of 3H-labeled human 18S rDNA to fixed metaphase chromosomes. The relative amount of ribosomal RNA gene activity of each of the same chromosomes was estimated by determining the frequency with which the chromosome's nucleolus organizer region (NOR) was silver stained, the size of the silver-stained region, and how often the chromosome was found in satellite association. Results were similar in phytohemagglutinin-stimulated T-lymphocytes, Epstein-Barr virus transformed lymphoblasts, and fibroblasts. One chromosome 21 had few gene copies and low activity. One chromosome 22 had many gene copies but low activity. Both chromosomes 14 had few gene copies but high activity. The level of expression that can be achieved by rRNA gene clusters can, therefore, be determined by factors other than the number of gene copies.

Abundance of protein-bound sulfhydryl and disulfide groups at chromosomal nucleolus organizing regions: a cytochemical study on the selective silver staining of NORs.

Silver stainability of the chromosomal nucleolus organizing regions that contain the structural genes for ribosomal RNA can be abolished by proteolytic and oxidative treatments. Histone extraction has no effect. This indicates that reducing groups of non-histone chromosomal proteins are responsible for silver staining. Treatment with fluorescent sulfhydryl and disulfide specific reagents followed by silver staining demonstrates coincidence of silver dots and brightly fluorescent spots at the short arms of human acrocentric chromosomes where ribosomal RNA-genes are located. After treatment with cupric sulfite reagent in the presence of urea fluorescence and silver staining was no longer possible. Silver staining has been reported to be associated with ribosomal RNA-gene activity. Acrocentric chromosomes that are negative in silver staining also lack the brightly fluorescent spots. Therefore, we conclude that an abundance of protein-bound sulfhydryl and disulfide groups occur at nucleolar organizing regions with active genes. Differentially fluorescing spots could not be observed after staining with fluorescamine. So, either the sulfhydryl reagents used in this study are much more sensitive than fluorescamine to study protein distributions in cytological preparations, or our observations point to a local accumulation of some specific protein(s) rich in sulfhydryls. The presence of many sulfhydryl and disulfide groups at the nucleolus organizing regions seems suggestive of a great flexibility of protein(s) by transition of sulfhydryl groups to disulfide bridges and vice versa at these highly active regions of the genome.

Age-dependent variability of ribosomal RNA-gene activity in man as determined from frequencies of silver staining nucleolus organizing regions on metaphase chromosomes of lymphocytes and fibroblasts

Frequencies of silver staining nucleolus organizing regions (NORs) have been determined in lymphocytes and fibroblasts from very young and from aged persons. Since silver staining of NORs is associated with activity of ribosomal RNA-genes, we used this approach to investigate a possible inactivation of these genes during aging. Our lymphocyte data are based on a study per age-group of 220 metaphases from 10 subjects. Although in both age-groups modal numbers of silver staining chromosomes per metaphase had similar ranges over the subjects, the frequency of metaphases containing the maximal number of staining chromosomes was in the old age-group (80–89 years) significantly lower than in the young age-group (less than 1 year old). In fibroblasts, of which 75 metaphases from 4 subjects were included per age-group, differences were more pronounced. Modal numbers of silver staining chromosomes were for the aged persons (69–83 years) lower than for the young children (less than 1 year old). Highly significant differences were observed between both groups in frequency of metaphases containing the maximal number of positively reacting acrocentric chromosomes and, more in general, in frequencies of silver staining D- and G-group chromosomes, the lower frequencies being found in the old age-group. We propose the term NOR-junctions as distinct from satellite associations for arrangements of acrocentric chromosomes which after silver staining are visibly connected at their NORs. The number of acrocentric chromosomes involved in lymphocyte NOR-junctions of aged people was significantly higher than the number of joined acrocentrics in young children. The frequency of these NOR-junctions themselves, irrespective of the number of chromosomes involved, was higher for aged persons than for young children, although this difference appeared to be statistically not significant. Within each age-group the frequency of NOR-junctions in lymphocytes was significantly higher than in fibroblasts. Also based on qualitative observations from our study we discuss technical and biological problems of our approach to study cell aging in vivo by means of silver staining of NORs. We conclude that in man, reflected by the difference in frequencies of silver staining NORs between young and aged persons, a rather extensive loss of ribosomal RNA-gene activity may occur during aging.

Ag-staining of nucleolus organizer regions on human prematurely condensed chromosomes from cells with different ribosomal RNA gene activity

The Ag-staining of the nucleolus organizer regions (NORs) of prematurely condensed chromosomes (PCCs) of human cells showing different degrees of rRNA-gene activity clearly indicates a close correlation between the positive Ag-staining of NORs and the activity of rRNA genes. The Ag-stain, however, seems insensitive to low rates of rRNA synthesis and obviously follows a threshold reaction. Furthermore it was found that the frequency of Ag-positive chromosomes involved in satellite associations in interphase does not differ from that in metaphase.

Quantitative Regulation of Gene Activity in Plants

The regulation of activity of ribosomal RNA genes has been studied in euploid and aneuploid varieties of hyacinth. The gene/gene product relationship was determined by measuring the specific gene product (stable rRNA) and the total gene product (protein) in varieties with different dosages of rRNA genes. The results show that the cellular rRNA content is positively regulated, that maximum gene utilization is not normally employed, and that the degree of utilization responds to the total genome rather than to the specific rRNA gene dosage. The mechanism of control of this regulation appears to be at the transcriptional level.

The number and activity of ribosomal RNA genes in Xenopus laevis embryos carrying partial deletions in both nucleolar organizers.

The number of ribosomal RNA genes and the rate of ribosomal RNA synthesis have been analyzed in a mutant of Xenopus laevis carrying two partial nucleolar organizers (p1−1/p1−2 nu). The p1−1 and p1−2 nucleolar organizers have fewer ribosomal RNA genes than the normal (+ nu) organizer, and each produces a smaller nucleolus than the + nu organizer in heterozygous embryos (+/p1−1 and +/p1−2). These heterozygotes develop normally and synthesize ribosomal RNA at normal rates. The p1−1/p1−2 nu mutants die at an early stage of development, and they synthesize ribosomal RNA at only 50% of the normal rate even though they have 70% of the haploid number of ribosomal RNA genes. These results show that the ribosomal RNA genes contained in a partial nucleolar organizer are not transcribed with the same efficiency as those found in an intact organizer and suggest the existence of genes, within the nucleolar organizer, which control the transcription of ribosomal RNA.