Isolation and molecular characterization of the ribosomal protein L6 homolog from Chlamydia trachomatis.

The cloning of a Chlamydia trachomatis eukaryotic cell-binding protein reported earlier from our laboratory (R. Kaul, K. L. Roy, and W. M. Wenman, J. Bacteriol. 169:5152-5156, 1987) represents an artifact generated by nonspecific recombination of chromosomal DNA fragments. However, the amino terminus of this plasmid-encoded fusion product demonstrated significant homology to Escherichia coli ribosomal protein L6. By using a 458-bp PstI-HindIII fragment of recombinant pCT161/18 (representing the 5' end of the cloned gene), we isolated and characterized a C. trachomatis homolog of the ribosomal protein L6 gene of E. coli. Sequence analysis of an 1,194-bp EcoRI-SacI fragment that encodes chlamydial L6 (designated CtaL6e) revealed a 552-bp open reading frame comprising 183 amino acids and encodes a protein with a molecular weight of 19,839. Interestingly, complete gene homology between C. trachomatis serovars L2 and J, each of which exists as a single copy per genome, was observed. Expression of a plasmid-encoded gene product is dependent on the lac promoter, since no product was obtained if the open reading frame was oriented in opposition to the lac promoter. Immunoblotting of purified ribosomes revealed functional, as well as antigenic, homology between the E. coli and C. trachomatis ribosomal L6 proteins.     

Characterization of the S7 ribosomal protein gene in wheat mitochondria

Summary By screening a wheat mitoplast cDNA bank, we have identified an open reading frame of 444 by that has a derived amino acid sequence homologous to bacterial-type S7 ribosomal proteins. This gene, designated rps7, is located upstream of one of two 26S rRNA gene copies in the wheat mitochondrial genome and is expressed as an abundant mRNA of approximately 0.7 kb. Its 5 terminus maps to the end of an 80 by element that is closely related to sequences preceding the wheat coxII, orf25 and atp6 genes. Southern hybridization analysis indicates that rps7-homologous sequences are present in the mitochondria of rice and pea, but not soybean.     

Nucleotide sequences of Bacillus stearothermophilus ribosomal protein genes: part of the ribosomal S10 operon

Restriction fragments from Bacillus stearothermophilus chromosomal DNA were cross-hybridized with the Escherichia coli ribosomal protein L2 gene rplB. A 2-kb EcoRI fragment which showed cross-hybridization was cloned into the M13 phage and sequenced by the dideoxy chain-terminating method. Comparison of the deduced amino-acid sequences with the corresponding sequences of E. coli ribosomal proteins showed that this fragment contains the region encoding the C-terminus of L2, the genes encoding S19, L22, S3 as well as the N-terminus of L16. Thus the organization of this gene cluster is the same as that in the S10 operon of E. coli. The deduced sequences of proteins L22 and S3, which have not been determined so far, were found to have 52% or 55% amino-acid identity, respectively, with those of the corresponding proteins in E. coli. The deduced B. stearothermophilus S19 protein sequence was in accordance with the reinvestigated protein sequence (H. Hirano, personal communication).     

Identification and characterization of the gene for Drosophila S20 ribosomal protein

A cDNA clone that encodes a Drosophila homologue of ribosomal protein S20 was isolated from a Drosophila ovary cDNA library. The Drosophila S20 gene (RpS20) is highly conserved with S20 genes in other organisms. It is a single copy gene and maps to position 92F-93A on polytene chromosomes. No Minute mutation in this location has been reported; at least five essential genes are possible candidates to encode RpS20. RpS20 message is expressed ubiquitously in embryos, but is expressed at high levels in the midgut.     

Structure of the Escherichia coli S10 ribosomal protein operon.

The complete structure of the Escherichia coli S10 ribosomal protein operon is presented. Based on the DNA sequence, the deduced order of the 11 genes in the operon is rpsJ, rplC, rplD, rplW, rplB, rpsS, rplV, rpsC, rplP, rpmC, rpsQ. The estimated transcribed length of the operon is 5181 base pairs. Putative sequences involved in ribosome binding are discussed. The DNA sequence data corrects several errors in previously determined protein sequence data.     

A defined mutation in the protein export gene within the spc ribosomal protein operon of Escherichia coli: isolation and characterization of a new temperature-sensitive secY mutant.

We describe the properties of a temperature-sensitive mutant, ts24, of Escherichia coli. The mutant has a conditional defect in export of periplasmic and outer membrane proteins. At 42 degrees C, precursor forms of these proteins accumulate within the cell where they are protected from digestion by externally added trypsin. The accumulated precursors are secreted and processed very slowly at 42 degrees C. The mutation is complemented by expression of the wild-type secY (or prlA) gene, which has been cloned into a plasmid vector from the promoter-distal part of the spc ribosomal protein operon. The mutant has a single base change in the middle of the secY gene, which would result in the replacement of a glycine residue by aspartic acid in the protein product. These results demonstrate that the gene secY (prlA) is essential for protein translocation across the E. coli cytoplasmic membrane.     

The primary structure of rat ribosomal protein S7

The amino acid sequence of the rat 40S ribosomal subunit protein S7 was deduced from the sequence of nucleotides in two recombinant cDNAs and confirmed from the amino acid sequence of a cyanogen bromide peptide obtained from the protein. Ribosomal protein S7 has 194 amino acids and has a molecular mass of 22,113. Hybridization of the cDNA to digest of nuclear DNA suggests that there are 14-16 copies of the S7 gene. The mRNA for the protein is about 725 nucleotides in length. Rat S7 is homologous with Xenopus laevis S8. The protein contains a possible internal duplication of 10 residues.     

Ultrastructural analysis of chlamydial antigen-containing vesicles everting from the Chlamydia trachomatis inclusion

Several chlamydial antigens have been detected in the infected epithelial cell cytosol and on the host cell surface prior to their presumed natural release at the end of the 72-96 h developmental cycle. These extra-inclusion antigens are proposed to influence vital host cell functions, antigen trafficking and presentation and, ultimately, contribute to a prolonged inflammatory response. To begin to dissect the mechanisms for escape of these antigens from the chlamydial inclusion, which are enhanced on exposure to antibiotics, polarized endometrial epithelial cells (HEC-1B) were infected with Chlamydia trachomatis serovar E for 36 h or 48 h. Infected cells were then exposed to chemotactic human polymorphonuclear neutrophils not loaded or pre-loaded in vitro with the antibiotic azithromycin. Viewed by electron microscopy, the azithromycin-mediated killing of chlamydiae involved an increase in chlamydial outer membrane blebbing followed by the appearance of the blebs in larger vesicles (i) everting from but still associated with the inclusion as well as (ii) external to the inclusion. Evidence that the vesicles originated from the chlamydial inclusion membrane was shown by immuno-localization of inclusion membrane proteins A, F, and G on the vesicular membranes. Chlamydial heat shock protein 60 (chsp60) copies 2 and 3, but not copy 1, were released from RB and incorporated into the everted inclusion membrane vesicles and delivered to the infected cell surface. These data represent direct evidence for one mechanism of early antigen delivery, albeit membrane-bound, beyond the confines of the chlamydial inclusion.     

Mapping the ribosomal protein S7 regulatory binding site on mRNA of the E. coli streptomycin operon

In this work it is shown by deletion analysis that an intercistronic region (ICR) approximately 80 nucleotides in length is necessary for interaction with recombinant E. coli S7 protein (r6hEcoS7). A model is proposed for the interaction of S7 with two ICR sites-region of hairpin bifurcations and Shine-Dalgarno sequence of cistron S7. A de novo RNA binding site for heterologous S7 protein of Thermus thermophilus (r6hTthS7) was constructed by selection of a combinatorial RNA library based on E. coli ICR: it has only a single supposed protein recognition site in the region of bifurcation. The SERW technique was used for selection of two intercistronic RNA libraries in which five nucleotides of a double-stranded region, adjacent to the bifurcation, had the randomized sequence. One library contained an authentic AG (?82/?20) pair, while in the other this pair was replaced by AU. A serwamer capable of specific binding to r6hTthS7 was selected; it appeared to be the RNA68 mutant with eight nucleotide mutations. The serwamer binds to r6hTthS7 with the same affinity as homologous authentic ICR of str mRNA binds to r6hEcoS7; apparent dissociation constants are 89 ± 43 and 50 ± 24 nM, respectively.