Electrophoretic and immunological studies on ribosomal proteins of 100 Escherichia coli revertants from streptomycin dependence

Summary Revertants from streptomycin dependence to independence were isolated as single step mutants from six different streptomycin dependent strains. The ribosomal proteins from 100 such mutants were analyzed by two-dimensional polyacrylamide gel electrophoresis and some of them were also examined by immunological techniques. Altered proteins were found in 40 mutants, 24 in protein S4 and 16 in protein S5. No change in any other protein was detected.Altered S5 proteins migrated into five different positions on the polyacrylamide plate and it can be concluded that the mutant proteins differ from the wild type probably by single amino acid replacements. The altered S4 proteins migrated into 17 different positions on the plate. Extensive changes of length, both shorter and longer than wild type S4 protein, are postulated for many of the mutant S4 proteins.Analysis of the ribosomal proteins of four ram mutants revealed altered S4 protein in two of them. The alterations in these mutant proteins are probably very similar to those found in streptomycin independent mutants.Among the revertants there was no apparent correlation between the protein alteration and the particular response to streptomycin.These studies suggest a strong interaction between protein S12, which confers streptomycin dependence, and protein S4 or S5, which can suppress this dependence.Paper No. 60 on Ribosomal Proteins. Preceding paper is by B. Wittmann-Liebold, Hoppe-Seyler's Z. physiol. Chemie, in press.     

Amino acid replacements in proteins S5 and S12 of two Escherichia coli revertants from streptomycin dependence to independence

Summary Ribosomes were isolated from two E. coli revertants from streptomycin dependence to independence, N660 and d1023. After separation of subunits, proteins were extracted from ribosomal 30S subunits and separated by CM-cellulose column chromatography and gel filtration. Pure S5 and S12 proteins of the two mutants were digested with trypsin and all resulting peptides were isolated by column and paper chromatography. The amino acid compositions of the peptides from the four mutant proteins were compared with the corresponding peptides of the wild type strain A19. The amino acid sequences of non-identical peptides were determined.The following amino acid replacements were found: Glycine by arginine in peptide T2 of protein S5 from mutant N660 and glycine by aspartic acid in peptide T15 of protein S12 from the same mutant. In the other mutant, d1023, arginine in peptide T2 of protein S5 was replaced by leucine and furthermore arginine by serine in peptide T10 of protein S12. Besides the single amino acid replacements mentioned above which are compatible with alterations of single nucleotides, a rather drastic difference between peptides T15 of proteins S12 isolated from strain A19 and mutant d1023 has been detected.The results presented in this paper are compared with amino acid replacements in proteins S5 and S12 from other ribosomal mutants of E. coli.Paper No. 62 on Ribosomal Proteins. Preceding paper is by Wittmann et al., Molec. gen. Genet., in press.     

Pleiotropic effects of ribosomal mutations for cycloheximide resistance in a double-resistant homocaryon of Neurospora crassa.

Pleiotropic effects of two ribosomal mutations for cycloheximide resistance were studied in a double-resistant homocaryon. The results obtained indicated that the combination of the two ribosomal mutations results in: (i) morphological abnormalities which suggest a severe distortion in the extension of cell walls and membranes; (ii) disturbance in the normal 60S-40S subunit ratio; (iii) decreased rate of cell mass production not necessarily associated with inbalance in the 60S-40S ratio; and (iv) cold sensitivity which does not interfere with mycelial mass production.     

Analysis of revertants of a ribosomal mutation in Podospora anserina: Evidence for new ribosomal mutations which confer hypersensitivity to paromomycin

This paper describes the analysis of cold-resistant revertants of a cold-sensitive mutant. Pm1-1 is a ribosomal mutation screened for its paromomycin resistance. Suppression of its cold sensitivity occurs with two kinds of external mutations localized in two different loci. One of them, PmB, is assumed to be a ribosomal gene. PmB mutations confer hypersensitivity to paromomycin in vivo as well as in vitro in a cell-free protein synthesis system.This work was supported by DGRST Grant MRM/P240 and NATO Grant 1637.     

Comparative analysis of bacterial-origin genes for plant mitochondrial ribosomal proteins

Mitochondrial ribosomes contain bacterial-type proteins reflecting their endosymbiotic heritage, and a subset of these genes is retained within the mitochondrion in land plants. Variation in gene location is observed, however, because migration to the nucleus is still an ongoing evolutionary process in plants. To gain insights into adaptation events related to successful gene transfer, we have compiled data for bacterial-origin mitochondrial-type ribosomal protein genes from the completely sequenced Arabidopsis and rice genomes. Approximately 75% of such nuclear-located genes encode amino-terminal extensions relative to their Escherichia coli counterparts, and of that set, only about 30% have introns at (or near) the junction in support of an exon shuffling-type recruitment of upstream expression/targeting signals. We find that genes that were transferred to the nucleus early in eukaryotic evolution have, on average, about twofold higher density of introns within the core ribosomal protein sequences than do those that moved to the nucleus more recently. About 20% of such introns are at positions identical to those in human orthologs, consistent with their ancestral presence. Plant mitochondrial-type ribosomal protein genes have dispersed chromosomal locations in the nucleus, and about 20% of them are present in multiple unlinked copies. This study provides new insights into the evolutionary history of endosymbiotic bacterial-type genes that have been transferred from the mitochondrion to the nucleus.     

Pleiotropic effects of pigmentation genes in horses

Horses are valued for the beauty and variety of colouration and coat patterning. To date, eleven different genes have been characterized that contribute to the variation observed in the horse. Unfortunately, mutations involving pigmentation often lead to deleterious effects in other systems, some of which have been described in the horse. This review focuses on six such pleiotropic effects or associations with pigmentation genes. These include neurological defects (lethal white foal syndrome and lavender foal syndrome), hearing defects, eye disorders (congenital stationary night blindness and multiple congenital ocular anomalies), as well as horse-specific melanoma. The pigmentation phenotype, disorder phenotype, mode of inheritance, genetic or genomic methods utilized to identify the genes involved and, if known, the causative mutations, molecular interactions and other susceptibility loci are discussed. As our understanding of pigmentation in the horse increases, through the use of novel genomic tools, we are likely to unravel yet unknown pleiotropic effects and determine additional interactions between previously discovered loci.     

Pleiotropic effects of beneficial mutations in Escherichia coli

Micromutational models of adaptation have placed considerable weight on antagonistic pleiotropy as a mechanism that prevents mutations of large effect from achieving fixation. However, there are few empirical studies of the distribution of pleiotropic effects, and no studies that have examined this distribution for a large number of adaptive mutations. Here we examine the form and extent of pleiotropy associated with beneficial mutations in Escherichia coli. To do so, we used a collection of independently evolved genotypes, each of which contains a beneficial mutation that confers increased fitness in a glucose-limited environment. To determine the pleiotropic effects of these mutations, we examined the fitnesses of the mutants in five novel resource environments. Our results show that the majority of mutations had significant fitness effects in alternative resources, such that pleiotropy was common. The predominant form of this pleiotropy was positive--that is, most mutations that conferred increased fitness in glucose also conferred increased fitness in novel resources. We did detect some deleterious pleiotropic effects, but they were primarily limited to one of the five resources, and within this resource, to only a subset of mutants. Although pleiotropic effects were generally positive, fitness levels were lower and more variable on resources that differed most in their mechanisms of uptake and catabolism from that of glucose. Positive pleiotropic effects were strongly correlated in magnitude with their direct effects, but no such correlation was found among mutants with deleterious pleiotropic effects. Whereas previous studies of populations evolved on glucose for longer periods of time showed consistent declines on some of the resources used here, our results suggest that deleterious pleiotropic effects were limited to only a subset of the beneficial mutations available.     

Analysis of ribosomal proteins in streptomycin resistant and dependent mutants isolated from streptomycin independent Escherichia coli strains

Summary Mutants resistant to (Str-R) or dependent on streptomycin (Str-D) were isolated from several streptomycin independent (Str-I) strains of Escherichia coli. From 90 of these mutants ribosomes were isolated and the ribosomal proteins analyzed by two-dimensional polyacrylamide gel electrophoresis. The results which are summarized in Tables 1-4 led to the following conclusions:a) The phenotype (Str-R or Str-D) of the mutants isolated from the Str-I strains strongly depends on the parental strain. b) No other ribosomal proteins than S4, S5 and S12 seem to be altered by mutations leading to dependence on, independence from or resistance to streptomycin. c) The S4 proteins of the analyzed mutants belong to three groups. The ratio between the groups depends more on the origin of the mutants than on their phenotype. d) Eight new types of altered S4 proteins were detected. It is very likely that many, if not all, of the altered S4 proteins originated by frame shift mutations. e) Some of the mutants differ from the wild type by alterations in three ribosomal proteins (S4, S5 and S12). The alteration in one protein, S4, apparently compensates for that in another protein, S5, in such a way that the original phenotype is expressed. These mutants are therefore an excellent tool for studies at the molecular level on the interaction of ribosomal components within the particle.     

Tandem duplications resulting from recombination between ribosomal RNA genes in Escherichia coli.

A set of Escherichia coli K12 mutants, which carry a tandem duplication of the glyT purD region, have been analyzed. Three types of duplications have occurred repeatedly, and we show that they were generated by recombination between the ribosomal RNA gene, rrnE, which lies to one side of the glyT purD region and one of threerrn genes which occur as direct repetitions on the other side of this region. Characterization of these duplication mutants has involved the isolation of the duplicated material in the form of a DNA circle. Class I duplications, which extend from rrnE to rrnE, are 39,500 base-pairs long, class II duplications, which extend from rrnA to rrnE, are 164,000 base-pairs long, and class III duplications, which extend from rrnC to rrnE, are 258,000 base-pairs long.     

Cloning and analysis of the nuclear genes for two mitochondrial ribosomal proteins in yeast

Summary Two mitochondrial ribosomal proteins of yeast (Saccharomyces cerevisiae) were purified and their N-terminal amino acid sequences determined. The sequence data were used for the synthesis of oligonucleotide probes to clone the corresponding genes. Thus, the genes for two proteins, termed YMR-31 and YMR-44, were cloned and their nucleotide sequences determined. From the nucleotide sequence data, the coding region of the gene for protein YMR-31 was found to be composed of 369 nucleotide pairs. Comparison of the amino acid sequence of protein YMR-31 and the one deduced from the nucleotide sequence of its gene suggests that it contains an octapeptide leader sequence. The calculated molecular weight of protein YMR-31 without the leader sequence is 12792 dalton. The gene for protein YMR-44 was found to contain a 147 bp intron which contains two sequences conserved among yeast introns. The length of the two exons flanking the intron totals 294 nucleotide pairs which can encode a protein with a calculated molecular weight of 11476 dalton. The gene for protein YMR-31 is located on chromosome VI, while the gene for protein YMR-44 is located on either chromosome XIII or XVI.     

Pleiotropic effects of ribosomal protein s4 studied in Escherichia coli mutants

Summary A temperature sensitive mutant of Escherichia coli was found to have two mutational alterations of its ribosomes: one of these was a streptomycin dependent mutation and the other was a suppressor alteration of S4, with a marked structural change. The altered form of S4 was studied in a strain that was constructed so that this alteration was the only one effecting the structure of the ribosome. Here, it was shown that the mutant form of S4 cause a temperature sensitive defect in the assembly of 30S subunits in vivo which is reflected in the inability of this mutant to properly process ribosomal RNA at the restrictive temperatures. An analysis of both transductants and revertants of this mutant show that the suppression of the streptomycin dependence phenotype, temperature sensitivity, and a defect in RNA processing all have their origin in a single mutational event effecting the structural gene for S4.     

Mouse models of genetic diseases resulting from mutations in elastic fiber proteins.

The inability to study appropriate human tissues at various stages of development has precluded the elaboration of a thorough understanding of the pathogenic mechanisms leading to diseases linked to mutations in genes for elastic fiber proteins. Recently, new insights have been gained by studying mice harboring targeted mutations in the genes that encode fibrillin-1 and elastin. These genes have been linked to Marfan syndrome (MFS) and supravalvular aortic stenosis (SVAS), respectively. For fibrillin-1, mouse models have revealed that phenotype is determined by the degree of functional impairment. The haploinsufficiency state or the expression of low levels of a product with dominant-negative potential from one allele is associated with mild phenotypes with a predominance of skeletal features. Exuberant expression of a dominant-negative-acting protein leads to the more severe MFS phenotype. Mice harboring targeted deletion of the elastin gene (ELN) show many of the features of SVAS in humans, including abnormalities in the vascular wall and altered hemodynamics associated with changes in wall compliance. The genetically altered mice suggest that SVAS is predominantly a disease of haploinsufficiency. These studies have underscored the prominent role of the elastic matrix in the morphogenesis and homeostasis of the vessel wall.