The DNA sequence of the gene rpsA of Escherichia coli coding for ribosomal protein S1.

The DNA sequence of the gene rpsA as well as of its neighboring regions has been determined using the dideoxyribonucleotide method. It was found that there is an "open-reading-frame" of 350 bp which precedes the gene rpsA. Furthermore, an extensive internal repeats of nucleotide sequence have been found in this gene.     

Purification and restriction endonuclease mapping of soybean 18 S and 25 S ribosomal RNA genes

The restriction endonuclease map of the 25 S and 18 S ribosomal RNA genes of a higher plant is presented. Soybean (Glycine max) rDNA was enriched by preparative buoyant density centrifugation in CsCl-actinomycin D gradients. The buoyant density of the rDNA was determined to be 1.6988 g cm^–3 by analytical centrifugation in CsCl. Saturation hybridization showed that 0.1% of the total DNA contains 25 S and 18 S rRNA coding sequences. This is equivalent to 800 rRNA genes per haploid genome (DNA content: 1.29 pg) or 3200 for the tetraploid genome. Restriction endonuclease mapping was performed with Bam H I, Hind III, Eco R I, and BstI. The repeating unit of the soybean ribosomal DNA has a molecular weight of 5.9·10⁶ or approximately 9,000 kb. The 25 S and 18 S rRNA coding sequences were localized within the restriction map of the repeating unit by specific hybridization with either [¹²⁵I]25 S or [¹²⁵I]18 S rRNA. It was demonstrated that there is no heterogeneity even in the spacer region of the soybean rDNA.     

Cloning of rpsO, the gene for ribosomal protein S15 of Escherichia coli

Summary The gene for Escherichia coli ribosomal protein S15 (rpsO) was cloned on the vector pBR322 from F-prime JCH55 DNA. The recombinant plasmid was transformed to Serratia marcescens cells and it was proved that E. coli S15 was synthesized and incorporated into ribosome particles in S. marcescens cells. A DNA fragment containing rpsO was also inserted into the vector pRF3, which changes its copy number depending on the growth temperature in a temperature-sensitive polA host. By use of this recombinant plasmid it was shown that the relative synthesis rate of S15 increased about twice even when the copy number of the plasmid increased more than twenty-fold.     

Deletion and insertion mutants in the structural gene for ribosomal protein S1 from Escherichia coli

Mutants have been constructed by deleting regions of the gene rpsA for ribosomal protein S1, which had been cloned in plasmid pACYC184. The mutant genes were analyzed for their ability to complement an S1 amber mutant containing a temperature-sensitive suppressor. Another series of mutants was constructed using the tac promoter plasmid pKK223-3, and the effect of the mutant proteins was analyzed in a strain wild type for rpsA. The gene products of all mutants were identified by the immunoblotting technique. Plasmids with a mutant rpsA gene which do not or only poorly complement the S1 amber mutation cause drastic growth reduction, whereas the overall protein synthesis is affected to different extents depending on the site of the deletion. Mutants which express S1 fragments comprising at least the NH2-terminal 100 amino acids stimulate or inhibit the synthesis of certain cellular proteins. The amount of chromosomal coded S1 was reduced by each mutant plasmid. Our data suggest that S1 has a general regulatory role during protein biosynthesis.     

Comparative studies on the structural gene for the ribosomal protein S1 in ten bacterial species

Summary Callus protoplasts of a plastome chlorophyll-deficient mutant of tobacco, Nicotiana tabacum, were fused with mesophyll protoplasts from one of the following five sources: cms-analogs of tobacco bearing the cytoplasms of N. suaveolens, N. undulata, N. repanda, and N. plumbaginifolia, respectively, and the wild species N. glauca. In the sixth experiment, callus cells of the tobacco chlorophyll-deficient genome mutant, homozygous for the Su gene, were hybridized with mesophyll protoplasts of the plastome chlorophyll-deficient mutant of tobacco. Individual dividing heteroplasmic fusion products were isolated mechanically and cloned in microdroplets of nutrient medium. Among the regenerants in all parental combinations, though not in all clones, besides pure green and pure chlorophyll-deficient plants, numerous variegated plant forms were obtained. The variegation of cybrid and hybrid plants was connected with their heterozygocity for chloroplast DNA composition, as demonstrated by restriction analysis. In analytical crosses, the variegation was inherited maternally by part of the sexual progeny, and variegated F₁ progeny were also heterozygous for chloroplast DNA composition. As demonstrated by electron microscopic studies, the variegation is connected with the presence of mixed, i.e., heteroplastidic cells in leaves. The results obtained demonstrate that (1) upon somatic cell fusion, plastome genes are inherited biparentally in most fusion products, and (2) despite an evident mitotic segregation process, heterozygosity for plastome genes is a relatively durable state and can be revealed in cell hybrids of different specific combinations of plasmons after a great number of cell generations. In this regard the plastome cytogetes obtained by somatic cell fusion do not differ qualitatively from the cytoplasmic heterozygotes that arise as a result of the mutation process.     

Comparative studies on the structural gene for the ribosomal protein S1 in ten bacterial species

Molecular Genetics and Genomics - By applying the Southern blot technique we compared the structural gene rpsA for ribosomal protein S1 and its preceding sequence from Escherichia coli with nine...     

Cloning and characterization of a gene from Rhizobium melilotii 2011 coding for ribosomal protein S1.

A 7 kb chromosomal DNA fragment from R. melilotii was cloned, which complemented temperature-sensitivity of an E. coli amber mutant in rpsA, the gene for ribosomal protein S1 (ES1). From complementation and maxicell analysis a 58 kd protein was identified as the homolog of protein S1 (RS1). DNA sequence analysis of the R. melilotii rpsA gene identified a protein of 568 amino acids, which showed 47% identical amino acid homology to protein S1 from E. coli. The RS1 protein lacked the two Cys residues which had been reported to play an important role for the function of ES1. Two repeats containing Shine-Dalgarno sequences were identified upstream of the structural gene. Binding studies with RNA polymerase from E. coli and Pseudomonas putida located one RNA-polymerase binding site close to the RS1 gene and another one several hundred basepairs upstream. One possible promoter was also identified by DNA sequence comparison with the corresponding E. coli promoter.     

Construction and restriction endonuclease mapping of hybrid plasmids containing Saccharomyces cerevisiae ribosomal DNA.

Summary Fragments produced by partial digestion of Saccharomyces cerevisiae ribosomal DNA (rDNA) with the restriction endonuclease EcoRI were ligated in vitro to the bacterial plasmid RSF2124. The resulting hybrid plasmids were cloned in Escherichia coli. Three hybrid plasmids which contain at least one intact repetitive unit of the multiple, tandem sequences of the yeast rDNA genes have been further characterized. These plasmids have been used to construct a map of the EcoRI, SmaI, HindII and HindIII restriction sites in the individual repetitive units of yeast rDNA.     

Cloning and mapping of infA, the gene for protein synthesis initiation factor IF1.

The gene for translation initiation factor IF1, infA, has been identified by using two synthetic oligonucleotides to screen a Charon 30 library of Escherichia coli DNA. A recombinant lambda phage, C1921, was purified from a plaque positive for both probes. A 2.8 kb BglII fragment and a 2.0 kb HindIII fragment isolated from C1921 were subcloned into the BamHI and HindIII sites of pBR322 to yield pTB7 and pTH2 respectively. Synthesis of IF1 in maxicells transformed with pTB7 or pTH2 indicates the presence of inf A in both inserts. This was confirmed by DNA sequencing: a region was found that codes for a 8,119 dalton protein with an amino acid sequence corresponding to IF1. The chromosomal location of inf A was determined by mapping the closely linked beta-lactamase gene (Ampr) in pTB7 and pTH2. pTB7 and pTH2 were transformed into polA Hfr hosts, and integration of the plasmid by homologous recombination near inf A was selected on the basis of ampicillin resistance. The site of integration was confirmed by Southern blot analysis of restriction nuclease digested wild type and transformed genomic DNA. The Ampr marker (and therefore inf A) was mapped to about 20 minutes by Hfr interrupted matings and P1 transduction experiments. The structure and regulation of the inf A operon currently are being investigated.