Characterization of ribosomal RNA synthesis in a gene dosage mutant: the relationship of topoisomerase I and chromatin structure to transcriptional activity.

The genes encoding 18S, 5.8S, and 28S ribosomal RNA (rRNA) are tandemly repeated at the nucleolus organizer region (NOR). The NORs in the chicken map to one pair of microchromosomes. A line of chickens that contains individuals that are either disomic, trisomic, or tetrasomic for this chromosome, and have two, three, or four nucleoli and NORs, per cell, respectively, has been described previously. Aneuploid animals display a proportional increase in the rRNA gene copy number per cell. But, despite an increase in rDNA dosage, the levels of mature rRNA are regulated to normal levels in cells from aneuploid chickens (Muscarella, D.E., V.M. Vogt, and S.E. Bloom, 1985, J. Cell Biol., 101:1749-1756). This paper addresses the question of how regulation of mature rRNA synthesis occurs in cells with elevated levels of rDNA. An analysis of rRNA transcription in chicken embryo fibroblasts (CEFs) revealed that the relative rates of rRNA synthesis and processing and the amounts of precursor rRNA per cell are similar for all three genotypes. A comparison of chromatin structure, as determined by sensitivity of rDNA in nuclei from CEFs to digestion by DNase I, revealed that some of the rRNA genes from aneuploid cells are more resistant to digestion than corresponding sequences in the disomic cells. A determination of the distribution of topoisomerase I on rDNA has also been performed using the compound camptothecin, which introduces single- and double-strand breaks in topoisomerase-DNA complexes. Quantitation of camptothecin-induced cleavages revealed that a larger proportion of the rRNA genes in aneuploid cells was resistant to cleavage than in disomic cells, and therefore have no detectable amounts of topoisomerase I. These results suggest that the regulation of rRNA synthesis in CEFs with elevated levels of rDNA is achieved by the use of a subset of the rRNA genes.

The ribosomal RNA gene cluster in aneuploid chickens: evidence for increased gene dosage and regulation of gene expression.

In the chicken, the nucleolus organizer regions, or sites of the genes encoding 18S, 5.8S, and 28S ribosomal RNA (rRNA), map to one pair of microchromosomes that can be identified by silver nitrate cytochemistry. This nucleolar organizer chromosome also contains the major histocompatibility complex. Chickens aneuploid for this chromosome have been identified and reproduced for over seven generations. Crossing two trisomic parents results in the production of viable disomic, trisomic, and tetrasomic progeny, showing two, three, and four nucleoli and nucleolar organizers per cell, respectively. A molecular analysis of rRNA genes was undertaken to establish the gene copy numbers in the aneuploid genotypes, and to determine if elevated numbers of rRNA genes are stably maintained and inherited over multiple generations. Gene copy numbers were determined using hybridization analysis of erythrocyte DNA obtained from individuals comprising a family which segregated disomic, trisomic, and tetrasomic genotypes. The values obtained were 290, 420, and 570 rDNA repeats per cell for disomic, trisomic, and tetrasomic animals, respectively. These results provide molecular confirmation of the two aneuploid states and show that elevated gene copy numbers have been maintained over multiple generations. Fibroblasts derived from disomic and tetrasomic embryos were found to grow at similar rates in culture, and mature rRNA levels in chicken embryo fibroblasts from disomic, trisomic and tetrasomic embryos were also found to have similar levels of mature rRNA. Therefore, despite the increase in rDNA content, the level of rRNA is regulated to diploid amounts in aneuploid fibroblasts.

A mobile group I intron from Physarum polycephalum can insert itself and induce point mutations in the nuclear ribosomal DNA of saccharomyces cerevisiae.

Pp LSU3 is a mobile group I intron in the extrachromosomal nuclear ribosomal DNA (rDNA) of Physarum polycephalum. As found for other mobile introns, Pp LSU3 encodes a site-specific endonuclease, I-Ppo, which mediates "homing" to unoccupied target sites in Physarum rDNA. The recognition sequence for this enzyme is conserved in all eucaryotic nuclear rDNAs. We have introduced this intron into a heterologous species, Saccharomyces cerevisiae, in which nuclear group I introns have not been detected. The expression of Pp LSU3, under control of the inducible GAL10 promoter, was found to be lethal as a consequence of double-strand breaks in the rDNA. However, surviving colonies that are resistant to the lethal effects of I-Ppo because of alterations in the rDNA at the cleavage site were recovered readily. These survivors are of two classes. The first comprises cells that acquired one of three types of point mutations. The second comprises cells in which Pp LSU3 became inserted into the rDNA. In both cases, each resistant survivor appears to carry the same alterations in all approximately 150 rDNA repeats. When it is embedded in yeast rDNA, Pp LSU3 leads to the synthesis of I-Ppo and appears to be mobile in appropriate genetic crosses. The existence of yeast cells carrying a mobile intron should allow dissection of the steps that allow expression of the highly unusual I-Ppo gene.

Characterization of I-Ppo, an intron-encoded endonuclease that mediates homing of a group I intron in the ribosomal DNA of Physarum polycephalum.

A novel and only recently recognized class of enzymes is composed of the site-specific endonucleases encoded by some group I introns. We have characterized several aspects of I-Ppo, the endonuclease that mediates the mobility of intron 3 in the ribosomal DNA of Physarum polycephalum. This intron is unique among mobile group I introns in that it is located in nuclear DNA. We found that I-Ppo is encoded by an open reading frame in the 5' half of intron 3, upstream of the sequences required for self-splicing of group I introns. Either of two AUG initiation codons could start this reading frame, one near the beginning of the intron and the other in the upstream exon, leading to predicted polypeptides of 138 and 160 amino acid residues. The longer polypeptide was the major form translated in vitro in a reticulocyte extract. From nuclease assays of proteins synthesized in vitro with partially deleted DNAs, we conclude that both polypeptides possess endonuclease activity. We also have expressed I-Ppo in Escherichia coli, using a bacteriophage T7 RNA polymerase expression system. The longer polypeptide also was the predominant form made in this system. It showed enzymatic activity in bacteria in vivo, as demonstrated by the cleavage of a plasmid carrying the target site. Like several other intron-encoded endonucleases, I-Ppo makes a four-base staggered cut in its ribosomal DNA target sequence, very near the site where intron 3 becomes integrated in crosses of intron 3-containing and intron 3-lacking Physarum strains.

Cell death in the avian blastoderm: resistance to stress-induced apoptosis and expression of anti-apoptotic genes.

We investigated the expression of an apoptotic cell death program in blastodermal cells prior to gastrulation and the susceptibility of these cells to stress-induced cell death. A low frequency (3.1%) of apoptotic blastodermal cells was observed in Hoechst 33342-vitally stained cytological preparations of complete blastoderms from unincubated eggs. These cells showed the stereotypic features of apoptosis including a progression of nuclear changes, cell shrinkage and blebbing, and the formation of apoptotic bodies. Prolonged storage of eggs at 12 degrees C induced apoptosis in blastodermal cells (14%). A modest amount of apoptosis (10%) was also induced at the heat shock temperature of 48 degrees C, but not at 45 degrees C. Etoposide and other potent cytotoxic drugs failed to induce apoptosis in the blastodermal cells after 4 h of exposure. Progressively more apoptosis was induced at 8 and 24 h, but it did not exceed 35% of the cells. We detected transcripts for the anti-apoptotic genes bcl-2, bcl-xL, and hsp70. The developmental expression of these genes, especially hsp70, correlated with the delayed and limited stress-induction of apoptosis. These studies reveal the capacity of pre-streak blastodermal cells to engage in apoptosis and their relative resistance to stress conditions. This may be due to the prominent expression of hsp70 and/or multiple cell death genes which primarily antagonize cell death.

Involvement of gene-specific DNA damage and apoptosis in the differential toxicity of mitomycin C analogs towards B-lineage versus T-lineage lymphoma cells.

Avian and mammalian B- and T-lineage lymphocytes display differential sensitivity to a variety of genotoxic agents. Specifically, T-lineage cells show a high degree of resistance to the toxic effects of exposure to chemotherapeutic drugs, whereas B-lineage cells show a high degree of sensitivity. We used a model system consisting of virally transformed B- and T-lymphoma cell lines to further define the cellular and molecular mechanisms responsible for the differential toxicity of two chemotherapeutic drugs that induce DNA-interstrand cross-links to different degrees, mitomycin C (MMC) and its aminodisulfide analog, BMY 25067. Quantification of the number of cross-links introduced in the transcriptionally active ribosomal RNA gene cluster revealed that similar levels of DNA damage were induced in B- and T-lymphoma cell lines. However, B-lymphoma cells were highly sensitive to induction of apoptosis and inhibition of growth compared with the more resistant T-lymphoma cells for both compounds. BMY 25067 induced approximately 2-fold more cross-links in rDNA than did MMC, along with a concurrent enhanced induction of apoptosis in both B- and T-lymphoma cell lines. An analysis of the persistence of DNA lesions over multiple cell cycles revealed that neither B- nor T-lymphoma cells repaired DNA cross-links to a significant extent. These data suggest that differences in the extent or persistence of DNA-interstrand cross-links are not responsible for the differential toxicity of MMC and its analog towards B- versus T-lineage cells. Rather, differential drug toxicity involves early and extensive entry into apoptosis in B-lymphoma cells contrasted to the delayed and minimal apoptotic induction in T-lymphoma cells.

A mobile group I intron in the nuclear rDNA of Physarum polycephalum.

We have shown that a strain-specific group I intron (intron 3) in the nuclear extrachromosomal rDNA or Physarum polycephalum is a mobile element. Shortly after mating of amoebae from intron-lacking and intron-containing strains, intron 3 transposes in a site-specific manner into all available recipient molecules. The transposition appears to occur by gene conversion, as evidence by the co-conversion of adjacent sequences and by double strand breakage observed in some of the recipient rDNA molecules. We infer that the double strand break is induced by an endonuclease encoded by intron 3, since in vitro transcription and translation of the cloned intron leads to the synthesis of an enzymatically active, site-specific nuclease. This is the first demonstration of the transposition of a nuclear intron in an experimental setting, and provides a rare example of a protein encoded by an RNA polymerase I transcript.

The longevity of chemically induced sister chromatid exchanges in Chinese hamster ovary cells.

The persistence of the lesions leading to sister chromatid exchanges (SCEs) following acute exposure of Chinese hamster ovary (CHO) cells to direct-acting chemical mutagens was measured. The results revealed that these lesions (and consequently SCEs) are rapidly eliminated from cells. SCE levels fell to near control values by the third or fourth day (six and eight cell cycles, respectively) following exposure of CHO cells to quinacrine mustard (QM) and mitomycin C (MMC). In contrast, cells exposed to methyl methanesulfonate (MMS) showed a small but significant increase in SCE level over control up to and including the fifth day following exposure (approximately ten cell cycles), suggesting that the behavior of these lesions in cells is influenced, at least in part, by the mechanism by which a specific agent interacts with DNA. The possibility that the decline in SCE level was due to the loss of cell populations with high numbers of exchanges was eliminated by the assessment of cloning efficiencies of treated and control cultures.

Formation of nucleolar polymorphisms in trisomic chickens and subsequent microevolution of rRNA gene clusters in diploids.

Variations in nucleolar size are common in animals and man, yet the basis and significance of this variation are not well understood. In this report, we describe the generation de novo of individuals that express nucleolar size variations (polymorphisms) and the underlying basis for this phenotype in a vertebrate animal system (Gallus domesticus). Individuals that express nucleolar size polymorphisms were produced from mating chickens trisomic for the nucleolar organizer (NO) chromosome; 10%-18% of progeny demonstrated nucleolar polymorphisms. These progeny were incorporated into a diploid genetic line in which the polymorphic trait was observed to segregate in Mendelian fashion. An even more dramatic nucleolar size polymorphism (one macro- plus one micronucleolus) evolved in one diploid family over the course of only two generations. These individuals were used to ascertain that the polymorphic-nucleoli phenotype was expressed in tissues derived from the three primary embryonic cell layers in embryos and neonates. Image analysis was conducted on cells of these birds to quantitate the size differences between macro- and micronucleoli (5 mu2 versus 1 mu2, respectively). Finally, these birds were studied with the technique of in situ hybridization, which showed that gene number differences between homologous NO chromosomes (i.e., heterozygosity for rRNA gene copy number), underlies the polymorphic-nucleoli phenotype. Thus, the chicken emerges as an experimental system through which heterozygosity for the rRNA gene copy number can be induced, easily identified, transmitted, and expressed in all somatic tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of the genotoxic and embryotoxic potential of chlorpyrifos and its metabolites in vivo and in vitro.

The genotoxicity and embryotoxicity of chlorpyrifos (CPF) and two metabolites were evaluated using the chick embryo, Chinese hamster ovary cells, and by examining blastocysts from superovulated cows crossed to chlorpyrifos-treated bulls. Chlorpyrifos and metabolites were dissolved in acetone and administered to 3-day embryos by the air cell method. The LD50 was 1,500 micrograms/embryo when mortality was checked through and including 17 days of development. The metabolites were more embryotoxic than the parent compound, CPF. Chlorpyrifos and metabolites did not increase the sister chromatid exchange (SCE) frequency above background at any dosage in the 3-day chick embryo assay. Similarly, none of these compounds increased SCE frequencies in three-point dosage tests (1, 10, 100 micrograms/ml) using Chinese hamster ovary cells. Controls in these assays consisted of the solvent carrier acetone (7.0 +/- 2.5 SCE/cell) and 8.6 micrograms/ml methyl methane sulfonate (30.5 +/- 7.4 SCE/cell). Studies of bovine blastocysts obtained from superovulated cows crossed with Dursban 44 treated bulls did not reveal evidence of chromosome aberrations or developmental anomalies associated with pesticide application. However, reproductive performance of breeders may be subnormal as a result of severe poisoning. This underscores the limitations of short-term assays and emphasizes the need to perform thorough toxicological assays of a chemical according to actual usage patterns in the species of concern.

Effects of rRNA gene copy number and nucleolar variation on early development: inhibition of gastrulation in rDNA-deficient chick embryos.

Because of their structural and catalytic functions during protein synthesis, the 18S, 5.8S, and 28S ribosomal RNAs (rRNAs) are essential for the support of differentiation, development, and growth. The genes encoding these RNAs are present in high copy number in all eukaryotes. Although there is evidence for the existence of variation for rRNA gene copy number within higher vertebrate species, there is little knowledge concerning the effects of such variation, especially reductions, on development and viability of homeothermic vertebrates. The main objective of this study was to determine the developmental potential of chick embryos containing defined deficiencies for rRNA gene copy number in order to assess the contribution of rDNA cluster size variation to embryonic mortality in homeothermic vertebrates. This was achieved by studying a strain of chickens containing nucleolar size polymorphisms that reflect rDNA cluster size polymorphisms. Embryos exhibiting a nucleolar phenotype of one large and one very small nucleolus (Pp) are heterozygous for a reduced rDNA cluster (+/p1) and were shown in the present study to contain about 66% of the complement of rRNA genes in normal individuals (+/+) that show two large equal-sized nucleoli (PP). The +/p1 embryos were found to develop and grow normally. Embryos exhibiting a nucleolar phenotype of two very small nucleoli (pp) are homozygous for the rDNA-deficient cluster (p1/p1) and contained about 45% of the normal rDNA complement of genes. These p1/p1 embryos were arrested in their development during early gastrulation. They exhibited a characteristic morphology consisting of a dorsal invagination that was strikingly different from the primitive streak formed in +/+ and +/p1 individuals.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of cell death regulatory genes and limited apoptosis induction in avian blastodermal cells.

Apoptosis is a well-established cellular mechanism for selective cell deletion during development. However, little is known about the expression of an apoptotic pathway and its role in determining the relative sensitivity of the early, pre-gastrula, avian embryo to stress-induced cell death. We examined the sensitivity of avian blastodermal cells to engage in apoptosis upon exposure to etoposide, a topoisomerase II-inhibitor that rapidly and efficiently induces apoptosis in many cell types. We found that while the blastodermal cells are capable of engaging in apoptosis, they are highly resistant to such induction with respect to both concentration of drug required and length of exposure, even when compared to a tumor cell line with a known multi-drug resistant phenotype. Additionally, we assessed the expression of several candidate regulatory genes in blastodiscs from infertile eggs (i.e., maternal RNA transcripts), blastodermal cells immediately following oviposition, and various stages of early development up to gastrulation. This analysis revealed that several genes whose products have anti-apoptotic activity, including bcl-2, bcl-xL, hsp70, grp78 and the glutathione S-transferases, are expressed as early as the stage 1 embryo in the newly oviposited egg. These transcripts are also found in the infertile blastodisc, suggesting a role for maternally derived transcripts in the protection of the oocyte and zygote. Significantly, constitutive levels of hsp70 mRNA exceeded those of the other anti-apoptotic genes in the blastodermal cells. These results contribute to an emerging picture of stress resistance at the earliest stages of avian embryo development which involves multiple anti-apoptotic genes that act at different regulatory points in the apoptotic cascade.

Differential induction of apoptosis and MAP kinase signaling by mitochondrial toxicants in drug-sensitive compared to drug-resistant B-lineage lymphoid cell lines.

A panel of human B-lineage lymphoma cell lines differing in cancer drug-resistance status and Bcl-2/Bax expression were used to study the contribution of mitochondrial-based perturbations and regulation in differential induction of apoptosis. Mitochondrial dysfunction was induced in cells by the uncoupler carbonyl cyanide m-chlorophenylhydrazone (mClCCP) and the respiratory chain inhibitor antimycin A. Cells were then assayed for early changes in MAP kinase signaling and subsequent induction of apoptosis. The cancer drug-resistant cell lines EW36 and CA46, overexpressing Bcl-2 and deficient in Bax, respectively, were both resistant to mitochondrial toxicant-induced cleavage of poly(ADP-ribose) polymerase (PARP) and morphologically detectable apoptotic cell death. In contrast, cancer drug-sensitive ST486 cell line, with low Bcl-2 expression, was sensitive to PARP cleavage and apoptosis engagement. Interestingly, mClCCP induced twofold more apoptosis than antimycin A in the ST486 cells. Exposure to the mitochondrial toxicants resulted in the early and preferential activation of the ERK and p38 MAP kinase pathways in only the drug-sensitive ST486 cell line, with mClCCP more potent than antimycin A. Specific inhibition of the p38 pathway augmented baseline and mClCCP-induced apoptosis. These results show that multi-drug-resistant and -sensitive B-lineage cells are also resistant and sensitive to compounds inducing mitochondrial dysfunction. The differential sensitivity to mitochondrial toxicant effects involved regulation by MAP kinases, since ERK and p38 were found to be preferentially activated only in the drug-sensitive B-lineage cells. Modulation of the p38 signaling pathway altered the sensitivity of cells to mitochondrial stress and may play a more general role in regulating the sensitivity of B-lineage cells to drugs and environmental toxicants.

Contribution of gene-specific lesions, DNA-replication-associated damage, and subsequent transcriptional inhibition in topoisomerase inhibitor-mediated apoptosis in lymphoma cells.

Lymphoid lineage tumor cells differ widely in their relative sensitivity or resistance to the induction of apoptosis by a variety of chemotherapeutic drugs. We used a model system of virally transformed B- and T-lymphoma cell lines to show that avian T-lymphoma cells are highly resistant, whereas B-lymphoma cells are highly sensitive, to the induction of apoptosis by a wide spectrum of chemotherapeutic drugs that induce different types of lesions in DNA. Among the various drugs examined, the topoisomerase inhibitors, camptothecin, actinomycin D, and etoposide, were the most potent inducers of apoptosis. Examination of the relative contribution of DNA replication and transcriptional inhibition to the differential induction of apoptosis by the topoisomerase inhibitors revealed that the signals initiating the apoptotic response vary, even among compounds with similar cellular targets. Specifically, DNA replication plays a major role in the induction of camptothecin-induced apoptosis, and a lesser role in the induction of apoptosis by etoposide. In contrast, DNA replication is not involved in the induction of apoptosis by actinomycin D. Transcriptional inhibition may provide the major cellular signal for apoptosis induction by this compound. In addition, we determined that the extent of topoisomerase I-cleavable complex inhibition is similar even in genes that are transcribed at different levels and by different RNA polymerases. An overexpressed c-myc gene is no more vulnerable to topoisomerase inhibition than its normally expressed counterpart. In contrast, even under conditions yielding similar amounts of topoisomerase inhibition, rRNA genes are more sensitive to transcriptional inhibition than are the c-myc genes.