Control of rRNA synthesis in Escherichia coli at increased rrn gene dosage. Role of guanosine tetraphosphate and ribosome feedback.

The effects of extra, plasmid-borne rRNA genes on the synthesis rate of rRNA in Escherichia coli were examined by measuring the fraction of total RNA synthesis that is rRNA and tRNA (rs/rt), the cytoplasmic concentration of guanosine tetraphosphate (ppGpp), and the absolute rates of RNA and protein synthesis. Experiments were carried out in different growth media and with two different strains of E. coli, B/r and K-12. The results indicated: 1) increased rrn gene dosage from either intact or defective rrn genes reduced bacterial growth rates and ribosome activity (protein synthesis rate/average ribosome), and increased rs/rt. 2) Extra intact, but not extra defective, plasmid-borne rrn genes caused the level of ppGpp to be increased in comparison to the pBR322-carrying control strain. 3) As a function of ppGpp, rs/rt was increased with either intact or defective rrn genes. 4) The rRNA synthesis rate/rrn gene was reduced in the presence of extra rrn genes; this reduction in gene activity was greater with intact than with defective rrn genes. An analysis of these results showed that they are consistent with the ppGpp hypothesis of rRNA control but not with a feedback effector role of translating ribosomes.     

Gene dosage effects on the synthesis of maltase in yeast.

Inbred strains of Saccharomyces cerevisiae carrying MAL1, MAL2, or MAL6 in a common background were used to construct (i) homo- or heterozygous diploids carrying one or two active alleles of a single MAL locus (MAL1, MAL2, or MAL6) and (ii) triploids carrying one, two, or three active alleles of MAL2. The diploid and triploid strains were used to investigate gene dosage effects of the differential rate of maltase synthesis (delta enzyme activity/delta growth) and the kinetics of induction (for MAL2). All three MAL loci exhibited a gene dosage effect on the differential rate of maltase synthesis; MAL2 also exhibited a gene dosage effect on the kinetics of induction. The dosage effects of the MAL1 and MAL6 loci were additive, but the effects of the MAL2 locus were not; the magnitude of the MAL2 gene dosage effect decreased with increasing dosage. These results are compatible with the current genetic evidence that the MAL genes are regulatory loci if the product(s) of the MAL1 and MAL6 locus is produced in limiting amounts but the product(s) of the MAL2 locus is produced in excess, except at very low genes dosages.     

Euploidy in Ricinus. I. Euploidy and gene dosage effects on cellular proteins

Investigation of an euploid series of castor bean, Ricinus communis L., consisting of haploid, diploid, and tetraploid individuals, was performed to determine the value of such a series in studying the biochemical consequences of genome multiplication. The effects of euploidization of the nuclear genome on the biosynthesis of cellular proteins were examined. Extracts of total soluble proteins from 10-day-old leaves of all three ploidy levels examined by electrophoresis on polyacrylamide gels revealed no difference in the complement of proteins present; however, differences in intensity of several protein bands were detected. Analysis of esterase isozyme activity by isoelectric focusing revealed both increases and decreases in the activity levels of individual isozyme variants in response to changes in ploidy levels. Results from this analysis are discussed in terms of possible regulatory mechanisms active in the regulation of duplicated genes.     

Polyploidy and gene dosage effects on chloroplasts of fern gametophytes

Increasing the complement of nuclear genes in fern gametophytes through polyploidy results in an increase in cell volume and dry weight of plants. The concentration of chlorophyll per cell remains constant but as ploidy increases the chloroplasts also increase in size primarily from the accumulation of starch. Studies of ¹⁴CO₂ fixation support the thesis that the increased nuclear gene dosage is without direct effect on photosynthesis.     

Unbalanced oxidant-antioxidant status and its effects in pediatric diseases

Oxidative stress results from a disparity between the generation of reactive oxygen species and the antioxidant ability of the organism. The alteration of the oxidant-antioxidant system brings in adults an effective state of imbalance, which may influence the pathogenesis of many diseases. Oxidative stress also plays a pivotal role in the progression of various pathologies in childhood, through a manipulation of regulatory proteins. In fact, several studies have demonstrated that an unbalanced oxidant-antioxidant status is able to determine toxic effects even during infancy. Therefore, the aim of this review was to summarize current knowledge about the dynamic relationship between oxidative stress and systemic diseases during childhood. In order to better understand these complex mechanisms, a comprehensive review of the literature was done, focusing mainly on pre-pubertal children. In fact, this age-group offers a unique opportunity to exclude confounding factors, especially those related to the metabolic effects induced by puberty. Early identification of these very young patients should be aimed at minimizing the degree of oxidative damage. Only by achieving early diagnosis, will it be possible to identify those children who could benefit from specific therapeutic approaches targeting oxidative stress.     

Effect of RP51 gene dosage alterations on ribosome synthesis in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae ribosomal protein rp51 is encoded by two interchangeable genes, RP51A and RP51B. We altered the RP51 gene dose by creating deletions of the RP51A or RP51B genes or both. Deletions of both genes led to spore inviability, indicating that rp51 is an essential ribosomal protein. From single deletion studies in haploid cells, we concluded that there was no intergenic dosage compensation at the level of mRNA abundance or mRNA utilization (translational efficiency), although phenotypic analysis had previously indicated a small compensation effect on growth rate. Similarly, deletions in diploid strains indicated that no strong mechanisms exist for intragenic dosage compensation; in all cases, a decreased dose of RP51 genes was characterized by a slow growth phenotype. A decreased dose of RP51 genes also led to insufficient amounts of 40S ribosomal subunits, as evidenced by a dramatic accumulation of excess 60S ribosomal subunits. We conclude that inhibition of 40S synthesis had little or no effect on the synthesis of the 60S subunit components. Addition of extra copies of rp51 genes led to extra rp51 protein synthesis. The additional rp51 protein was rapidly degraded. We propose that rp51 and perhaps many ribosomal proteins are normally oversynthesized, but the unassembled excess is degraded, and that the apparent compensation seen in haploids, i.e., the fact that the growth rate of mutant strains is less depressed than the actual reduction in mRNA, is a consequence of this excess which is spared from proteolysis under this circumstance.     

Opposite effects on facial morphology due to gene dosage sensitivity

Sequencing technology is increasingly demonstrating the impact of genomic copy number variation (CNV) on phenotypes. Opposing variation in growth, head size, cognition and behaviour is known to result from deletions and reciprocal duplications of some genomic regions. We propose normative inversion of face shape, opposing difference from a matched norm, as a basis for investigating the effects of gene dosage on craniofacial development. We use dense surface modelling techniques to match any face (or part of a face) to a facial norm of unaffected individuals of matched age, sex and ethnicity and then we reverse the individual’s face shape differences from the matched norm to produce the normative inversion. We demonstrate for five genomic regions, 4p16.3, 7q11.23, 11p15, 16p13.3 and 17p11.2, that such inversion for individuals with a duplication or (epi)-mutation produces facial forms remarkably similar to those associated with a deletion or opposite (epi-)mutation of the same region, and vice versa. The ability to visualise and quantify face shape effects of gene dosage is of major benefit for determining whether a CNV is the cause of the phenotype of an individual and for predicting reciprocal consequences. It enables face shape to be used as a relatively simple and inexpensive functional analysis of the gene(s) involved.     

Gene dosage effects on polyhydroxyalkanoates synthesis from n-alcohols in Paracoccus denitrificans

Putative promoters of polyhydroxyalkanoate (PHA)-synthetic genes of Paracoccus denitrificans were identified. Gene dosage effects for PHA synthesis were investigated in recombinants of P. denitrificans with increased expression levels of each PHA synthetic enzyme. In the cultivation of shake flasks using ethanol or n-pentanol as carbon source, a self-cloning recombinant of the phaC-encoding PHA synthase showed the highest contents [(g PHA). (g total biomass)-1] and the highest rates of PHA accumulation [(g PHA). (g residual biomass)-1. h-1] among these recombinants. The PHA content and PHA accumulation rate (g PHA/g residual biomass. h-1) of the self-cloning recombinant was 2 and 2.7 times higher, respectively, than that of the wild strain. This result strongly suggests that the step of PHA synthase is limited in in vivo PHA synthesis from n-pentanol via 3-ketovaleryl-CoA through beta-oxidation, and from ethanol via acetyl-CoA. Studies on fed-batch cultures keeping the alcohol concentration constant (0.02%) in a 5-L bioreactor showed that the ability of PHA biosynthesis was improved by the gene dosage of PHA synthase, although the growth rate of cells during the growth-associated PHA synthesis phase was retarded. The molecular weight of the polymer isolated from the strain, dosed by the PHA synthase gene, was lower than that of the polymer from the wild strain, indicating that the amount of PHA synthase in vivo affects the molecular weight of the polymer.     

Measuring the toxic effects of high gene dosage on yeast cells

 A novel method, which is rapid, reliable and quantitative, is presented for measuring the toxic effects on yeast cells of high dosage of any given gene. It is based on the possibility of monitoring the presence in cells of a plasmid carrying the ADE2 gene from Saccharomyces cerevisiae by direct observation of colonies, the construction of this particular plasmid being easily made by marked homologous recombination in yeast. Four yeast regulatory genes tested were found to result in various degrees of toxicity at high dosage. Possible implications of the measurement of gene toxicity for eukaryotic cell regulatory mechanisms and for the use of novel general approaches to gene selection, such as the gene-gene interference method, are discussed. Received: 18 March 1996 / Accepted: 23 August 1996     

A concerted DNA methylation/histone methylation switch regulates rRNA gene dosage control and nucleolar dominance

Eukaryotes regulate the effective dosage of their ribosomal RNA (rRNA) genes, expressing fewer than half of the genes at any one time. Likewise, genetic hybrids displaying nucleolar dominance transcribe rRNA genes inherited from one parent but silence the other parental set. We show that rRNA gene dosage control and nucleolar dominance utilize a common mechanism. Central to the mechanism is an epigenetic switch in which concerted changes in promoter cytosine methylation density and specific histone modifications dictate the on and off states of the rRNA genes. A key component of the off switch is HDT1, a plant-specific histone deacetylase that localizes to the nucleolus and is required for H3 lysine 9 deacetylation and subsequent H3 lysine 9 methylation. Collectively, the data support a model in which cytosine methylation and histone deacetylation are each upstream of one another in a self-reinforcing repression cycle.     

Nucleotide sequence of Physarum polycephalum 5.8S rRNA gene and its flanking regions.

The nucleotide sequence of Physarum polycephalum 5.8S rRNA gene and its flanking regions has been determined. The homologies of the 5.8S rRNA sequence with those of Saccharomyces, Chlamydomonas and Xenopus were 56%, 50% and 52%, respectively. In spite of these relatively low homologies, its possible secondary structure was very similar to those of other species.     

Conditional expression of RPA190, the gene encoding the largest subunit of yeast RNA polymerase I: effects of decreased rRNA synthesis on ribosomal protein synthesis.

The synthesis of ribosomal proteins (r proteins) under the conditions of greatly reduced RNA synthesis were studied by using a strain of the yeast Saccharomyces cerevisiae in which the production of the largest subunit (RPA190) of RNA polymerase I was controlled by the galactose promoter. Although growth on galactose medium was normal, the strain was unable to sustain growth when shifted to glucose medium. This growth defect was shown to be due to a preferential decrease in RNA synthesis caused by deprivation of RNA polymerase I. Under these conditions, the accumulation of r proteins decreased to match the rRNA synthesis rate. When proteins were pulse-labeled for short periods, no or only a weak decrease was observed in the differential synthesis rate of several r proteins (L5, L39, L29 and/or L28, L27 and/or S21) relative to those of control cells synthesizing RPA190 from the normal promoter. Degradation of these r proteins synthesized in excess was observed during subsequent chase periods. Analysis of the amounts of mRNAs for L3 and L29 and their locations in polysomes also suggested that the synthesis of these proteins relative to other cellular proteins were comparable to those observed in control cells. However, Northern analysis of several r-protein mRNAs revealed that the unspliced precursor mRNA for r-protein L32 accumulated when rRNA synthesis rates were decreased. This result supports the feedback regulation model in which excess L32 protein inhibits the splicing of its own precursor mRNA, as proposed by previous workers (M. D. Dabeva, M. A. Post-Beittenmiller, and J. R. Warner, Proc. Natl. Acad. Sci. USA 83:5854-5857, 1986).     

Feedback regulation of rRNA synthesis. A mutational alteration in the anti-Shine-Dalgarno region of the 16 S rRNA gene abolishes regulation

It was shown that induction of rRNA (and ribosome) synthesis from the lambda PL promoter/operator by temperature shift-up causes a repression of rRNA and tRNA synthesis from chromosomal genes. We have carried out experiments using a similar conditional rRNA gene expression system in which a mutational alteration was introduced in the anti-Shine-Dalgarno region at the 3'-end of the 16 S rRNA gene. It was found that the repression observed with the wild-type gene was largely abolished by the mutation. It appears that ribosomes inefficient in translational initiation are unable to cause feedback regulation of rRNA synthesis. It is suggested that the cell regulates rRNA (and tRNA) synthesis by monitoring the production of ribosomes, and that this monitoring is apparently carried out through their activity in the initiation (and perhaps subsequent steps) of translation.     

Some thoughts on a possible relationship between known gene dosage effects and neoplastic transformation

An unbalanced gene load resulting from viral infection, oncogenic chemicals, or ionizing radiation can include excess regulatory genes which produce an increased concentration of repressors and thereby cause a decreased production of some regulated proteins. If these proteins are involved in the feedback loop for mitotic inhibition or in the maintainance of cell membrane properties needed to maintain the intracellular concentration of mitotic inhibitor, then their loss or decreased production as a result of the unbalanced gene load can lead to the loss of control over cell growth. A discussion of known gene dosage effects which support the model described above is given.     

The effects of MHC gene dosage and allelic variation on T cell receptor selection

In a T cell receptor transgenic mouse model of thymic selection, the efficiency of selection of the transgenic alpha beta heterodimer is significantly enhanced in animals that express higher densities of the relevant major histocompatibility complex molecule (I-Ek/b). These results imply that there is a stochastic component to positive selection in the thymus. Allelic variants of the original selecting I-Ek molecule are either less efficient (E alpha k:E beta b) or incapable (E alpha k:E beta s and I-Ed) of mediating the selection of transgenic alpha beta + T cells. Two of these three I-E variants appear to differ from I-Ek in amino acid residues of the peptide binding site and not in residues capable of contacting the T cell receptor, suggesting that specific peptides, or conformations of peptides, play a role in positive selection. In contrast, mice transgenic for only the beta chain of this T cell receptor show selection for CD4+ T cells in the presence of all four I-E variants tested.