Isolation of eukaryotic ribosomal proteins. Purification and characterization of the 60 S ribosomal subunit proteins L4, L5, L7, L9, L11, L12, L13, L21, L22, L23, L26, L27, L30, L33, L35', L37, and L39.

The proteins of the large subunit of rat liver ribosomes were separated into seven groups by stepwise elution from carboxymethylcellulose with LiCl at pH 6.5. Seventeen proteins (L4, L5, L7, L9, L11, L12, L13, L21, L22, L23, L26, L27, L30, L33, L35', L37, and L39) were isolated from three of the groups (B60, D60, G60) by ion exchange chromatography on carboxymethylcellulose and by filtration through Sephadex. The amount of protein obtained varied from 0.5 to 15 mg. Eight of the proteins (L9, L11, L13, L21, L22, L35', L37 and L39) had no detectable contamination; the impurities in the others were no greater than 9%. The molecular weight of the proteins was estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; the amino acid composition was determined.     

Sequence of the amino-terminal region of rat liver ribosomal proteins S4, S6, S8, L6, L7a,L18, L27, L30, L37, L37a,and L39

The sequence of the amino-terminal region of eleven rat liver ribosomal proteins–S4, S6, S8, L7a, L18, L27, L30, L37a, and L39 - was determined. The analysis confirmed the homogeneity of the proteins and suggests that they are unique, since no extensive common sequences were found. The N-terminal regions of the rat liver proteins were compared with amino acid sequences in Saccharomyces cerevisiae and in Escherichia coli ribosomal proteins. It seems likely that the proteins L37 from rat liver and Y55 from yeast ribosomes are homologous. It is possible that rat liver L7a or L37a or both are related to S cerevisiae Y44, although the similar sequences are at the amino-terminus of the rat liver proteins and in an internal region of Y44. A number of similarities in the sequences of rat liver and E coli ribosomal proteins have been found; however, it is not yet possible to say whether they connote a common ancestry.     

The complete primary structure of ribosomal proteins L1, L14, L15, L23, L24 and L29 from Bacillus stearothermophilus

The amino acid sequences of ribosomal proteins L1, L14, L15, L23, L24 and L29 from Bacillus stearothermophilus have been completely determined. This has been achieved by sequence analyses of peptides derived from enzymatic digestions of the proteins with trypsin, chymotrypsin, pepsin, Staphylococcus aureus protease, and Armillaria mellea protease as well as by chemical cleavage with hydroxylamine and cyanogen bromide. Based on the primary structures of the six proteins, their secondary structures were predicted using four different computer prediction programs. A comparison of the amino acid sequences of the studied proteins from B. stearothermophilus with the homologous proteins from Escherichia coli revealed that in four proteins (L1, L15, L24 and L29) between 40-50% of the residue in the sequences are identical, whereas this value is significantly higher (69%) for L14 and lower (28%) for L23. The distribution of those amino acid residues which are identical in the corresponding proteins from the two bacteria is not random along the protein chain: some regions are highly conserved whereas others are not. This finding indicates that the regions which are conserved during evolution are important for the spatial structure and/or function of the protein.     

Structure and evolution of the L11, L1, L10, and L12 equivalent ribosomal proteins in eubacteria, archaebacteria, and eucaryotes

The genes corresponding to the L11, L1, L10, and L12 equivalent ribosomal proteins (L11e, L1e, L10e, and L12e) of Escherichia coli have been cloned and sequenced from two widely divergent species of archaebacteria, Halobacterium cutirubrum and Sulfolobus solfataricus, and the L10 and four different L12 genes have been cloned and sequenced from the eucaryote Saccharomyces cerevisiae. Alignments between the deduced amino acid sequences of these proteins and to other available homologous proteins of eubacteria and eucaryotes have been made. The data suggest that the archaebacteria are a distinct coherent phylogenetic group. Alignment of the proline-rich L11e proteins reveals that the N-terminal region, believed to be responsible for interaction with release factor 1, is the most highly conserved region and that there is specific conservation of most of the proline residues, which may be important in maintaining the highly elongated structure of the molecule. Although L11 is the most highly methylated protein in the E. coli ribosome, the sites of methylation are not conserved in the archaebacterial L11e proteins. The L1e proteins of eubacteria and archaebacteria show two regions of very high similarity near the center and the carboxy termini of the proteins. The L10e proteins of all kingdoms are colinear and contain approximately three fourths of an L12e protein fused to their carboxy terminus, although much of this fusion has been lost in the truncated eubacterial protein. The archaebacterial and eucaryotic L12e proteins are colinear, whereas the eubacterial protein has suffered a rearrangement through what appear to be gene fusion events. Within the L12e derived region of the L10e proteins there exists a repeated module of 26 amino acids, present in two copies in eucaryotes, three in archaebacteria, and one in eubacteria. This modular sequence is apparently also present in the L12e proteins of all kingdoms and may play a role in L12e dimerization, L10e-L12e complex formation, and the function of the L10e-L12e complex in translation.     

Molecular phylogenies based on ribosomal protein L11, L1, L10, and L12 sequences

Summary Available sequences that correspond to the E. coli ribosomal proteins L11, L1, L10, and L12 from eubacteria, archaebacteria, and eukaryotes have been aligned. The alignments were analyzed qualitatively for shared structural features and for conservation of deletions or insertions. The alignments were further subjected to quantitative phylogenetic analysis, and the amino acid identity between selected pairs of sequences was calculated. In general, eubacteria, archaebacteria, and eukaryotes each form coherent and well-resolved nonoverlapping phylogenetic domains. The degree of diversity of the four proteins between the three groups is not uniform. For L11, the eubacterial and archaebacterial proteins are very similar whereas the eukaryotic L11 is clearly less similar. In contrast, in the case of the L12 proteins and to a lesser extent the L10 proteins, the archaebacterial and eukaryotic proteins are similar whereas the eubacterial proteins are different. The eukaryotic L1 equivalent protein has yet to be identified. If the root of the universal tree is near or within the eubacterial domain, our ribosomal protein-based phylogenies indicate that archaebacteria are monophyletic. The eukaryotic lineage appears to originate either near or within the archaebacterial domain. Correspondence to: P. Dennis     

Draft genome sequences and description of Lactobacillus rhamnosus strains L31, L34, and L35

Lactobacillus rhamnosus is a facultative, lactic acid bacterium in the phylum Firmicutes. Lactobacillus spp. are generally considered beneficial, and specific strains of L. rhamnosus are validated probiotics. We describe the draft genomes of three L. rhamnosus strains (L31, L34, and L35) isolated from the feces of Thai breastfed infants, which exhibit anti-inflammatory properties in vitro. The three genomes range between 2.8 – 2.9 Mb, and contain approximately 2,700 protein coding genes.     

Localization of proteins L4, L5, L20 and L25 on the ribosomal surface by immuno-electron microscopy

Summary Ribosomal proteins L4, L5, L20 and L25 have been localized on the surface of the 50S ribosomal subunit of Escherichia coli by immuno-electron microscopy. The two 5S RNA binding proteins L5 and L25 were both located at the central protuberance extending towards its base, at the interface side of the 50S particle. L5 was localized on the side of the central protuberance that faces the L1 protuberance, whereas L25 was localized on the side that faces the L7/L12 stalk. Proteins L4 and L20 were both located at the back of the 50S subunit; L4 was located in the vicinity of proteins L23 and L29, and protein L20 was localized between proteins L17 and L10 and is thus located below the origin of the L7/L12 stalk.     

Structural relationships and sialylation among meningococcal L1, L8, and L3,7 lipooligosaccharide serotypes

Eighteen of 34 endemic meningococcal case strains were of the L8 lipooligosaccharide (LOS) type; four of these were both L3 and L7 (L3,7), and seven were L1. L1 structures arose by alternative terminal Gal substitutions of lactosyl diheptoside L8 structures, as determined by electrospray ionization and other mass spectrometric techniques, and enzymatic and chemical degradations (Structures L1 and L1a). [see text for structure] The more abundant molecule, designated L1, had a trihexose globosyl alpha chain; the less abundant one, designated L1a, had a beta-lactosyl alpha chain and a parallel alpha-lactosaminyl gamma chain. A P(k) globoside (Galalpha1-->4Galbeta1-->4 Glc-R) monoclonal antibody bound 9/10 L1 strains, but a P(1) globoside (Galalpha1-->4Galbeta1-->4GlcNAc-R) mAb bound none of them. alpha-Galactosidase caused loss of both L1 structures and creation of L8 structures; beta-galactosidase caused loss of the L8 determinant. The L1/P(k) glycose was partially sialylated. Some LOS also had unsubstituted basal beta-GlcNAc additions. These structural relationships explain co-expression of L8, L1, and L3,7 serotypes.     

Isolation of eukaryotic ribosomal proteins. Purification and characterization of the 60 S ribosomal subunit proteins La, Lb, Lf, P1, P2, L13', L14, L18', L20, and L38

The proteins of the large subunit of rat liver ribosomes were separated into seven groups by stepwise elution from carboxymethylcellulose with LiCl at pH 6.5. Ten proteins (La, Lb, Lf, P1, P2, L13', L14, L18', L20, and L38) were isolated from three groups (A60, B60, and D60) by ion exchange chromatography on carboxymethylcellulose and DEAE-cellulose, and by filtration through Sephadex. The amount of protein obtained varied from 0.3 to 3.8 mg. Two of the proteins (La and L18') had no detectable contamination; the impurities in the others were not greater than 8%. The molecular weight of the proteins was estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; the amino acid composition was determined. Several additional acidic proteins were identified: P1a and P1b are phosphorylated derivatives of P1; P2a, P2b, and P2c are phosphorylated derivatives of P2. P1 and P2 are distinct proteins but both have large amounts of alanine (20.4 and 17.5 mol %).     

Purification of Drosophila ribosomal proteins. Isolation of proteins S8, S13, S14, S16, S19, S20/L24, S22/L26, S24, S25/S27, S26, S29, L4, L10/L11, L12, L13, L16, L18, L19, L27, 1, 7/8, 9, and 11

The proteins of Drosophila melanogaster embryonic ribosomes were separated into seven groups (A80 through G80) by stepwise elution from carboxymethylcellulose with lithium chloride at pH 6.5 by procedures previously described [Chooi, W. Y., Sabatini, L. M., MacKlin, M. D., & Fraser, W. (1980) Biochemistry 19, 1425-1433]. Three relatively acidic proteins, S14, S25/S27, and 7/8, have now been isolated from group A80 by ion-exchange chromatog raphy on carboxymethylcellulose eluted with a linear gradient of lithium chloride at pH 4.2. Fractions containing the relatively basic proteins (groups B80 through G80) were furher combined into a total of 24 "pools". The criterion for combination was the migration patterns in one-dimensional polyacrylamide gels containing sodium dodecyl sulfate (NaDodS04) of every fifth fraction from the carboxymethylcellulose column. Each pool contained between 1 and 12 major proteins. Proteins S8, S13, S16, S19, S20/L24, S22/L26, S24, S26, S29, L4, L10/L11, L12, L13, L16, L18, L19, L27, 1, 9, and 11 have now been isolated from selected pools by gel filtration through Sephadix G-100. The amount of each protein recovered from a starting amount of 1.8 g of total 80S proteins varied form 0.2 to 10.8 mg. Five proteins had no detectable contamination, and in each of the others the impurities were no greater than 9%. The amino acid composition of the individual purified proteins was determined. The molecular weights of the proteins were estimated by polyacrylamide gel electrophoresis in NaDodSO4.     

Quinolizidine alkaloid profiles of Lupinus varius orientalis, L. albus albus, L. hartwegii, and L. densiflorus

Alkaloid profiles of two Lupinus species growing naturally in Egypt (L. albus albus [synonym L. termis], L. varius orientalis) in addition to two New World species (L. hartwegii, L. densiflorus) which were cultivated in Egypt were studied by capillary GLC and GLC-mass spectrometry with respect to quinolizidine alkaloids. Altogether 44 quinolizidine, bipiperidyl and proto-indole alkaloids were identified; 29 in L. albus, 13 in L. varius orientalis, 15 in L. hartwegii, 6 in L. densiflorus. Some of these alkaloids were identified for the first time in these plants. The alkaloidal patterns of various plant organs (leaves, flowers, stems, roots, pods and seeds) are documented. Screening for antimicrobial activity of these plant extracts demonstrated substantial activity against Candida albicans, Aspergillus flavus and Bacillus subtilis.     

The use of 2-iminothiolane as an RNA-protein cross-linking agent in Escherichia coli ribosomes, and the localisation on 23S RNA of sites cross-linked to proteins L4, L6, L21, L23, L27 and L29.

When E. coli ribosomal subunits are reacted with 2-iminothiolane and then subjected to a mild ultraviolet irradiation, an RNA-protein cross-linking reaction occurs. About 5% of the total protein in each subunit becomes cross-linked to the RNA, and a specific sub-set of proteins is involved in the reaction. In the case of the 50S subunit, the sites of cross-linking to the 23S RNA have been determined for six of these proteins: protein L4 is cross-linked within an oligonucleotide comprising positions 613-617 in the 23S sequence, L6 within positions 2473-2481, L21 within positions 540-548, L23 within positions 137-141, L27 within positions 2332-2337 and L29 within positions 99-107.     

Chloroplast ribosomal protein L15, like L1, L13 and L21, is significantly larger than its E. coli homologue

The purification and identification by peptide sequence and immunological data of the spinach chloroplast homologue of E. coli L15 is presented. A significant increase in its mass over the E. coli counterpart is shown and is accounted for, in part, by a sequenced 18-residue N-terminal extension. A still larger C-terminal extension or internal insertion(s) is inferred. The migration position of the L15 in a 2D gel pattern of spinach chloroplast 50S subunit proteins is shown. Lack of sequence identity with the known chloroplast genomic data confirms the nuclear coding of this protein, and the N-terminal sequence given here provides the transit peptide cleavage site of the cytoplasmic precursor.     

Shapes of proteins L1, L9, L25, and L30 from the 50S subunit of the Escherichia coli ribosome, determined by hydrodynamic studies.

Proteins L1, L9, L25, and L30, purified by a nondenaturing method from the 50S ribosomal subunit of Escherichia coli A19, have been characterized. The four proteins were studied under conditions which resemble those used for reconstitution experiments. These proteins have S020,W values of 2.0 S, 1.8 S, 1.8 S, and 1.0 S and D20,W values of 8.4 X 10(-7), 9.0 X 10(-7), 14.0 X 10(-7), and 15.0 X 10(-7) cm2/S. Apparent specific volumes at 20 degrees C are 0.738, 0.733, 0.700, and 0.735 mL/g for the four proteins. The respective molecular weights determined by sedimentation equilibrium are 25 000, 17 300, 12 000, and 6500. The intrinsic viscosity values for the four proteins are 4.0, 5.5, 3.6, and 3.2 mL/g. From these hydrodynamic parameters L1 and L9 appear to have globular or at most only slightly elongated shapes, whereas L25 and L30 appear to be definitely globular.     

Sequencing and characterization of telomere and subtelomere regions on rice chromosomes 1S, 2S, 2L, 6L, 7S, 7L and 8S

Telomeres, which are important for chromosome maintenance, are composed of long, repetitive DNA sequences associated with a variety of telomere-binding proteins. We characterized the organization and structure of rice telomeres and adjacent subtelomere regions on the basis of cytogenetic and sequence analyses. The length of the rice telomeres ranged from 5.1 to 10.8 kb, as revealed by both fibre-fluorescent in situ hybridization and terminal restriction-fragment assay. Physical maps of the chromosomal ends were constructed from a fosmid library. This facilitated sequencing of the telomere regions of chromosomes 1S, 2S, 2L, 6L, 7S, 7L and 8S. The resulting sequences contained conserved TTTAGGG telomere repeats, which indicates that the physical maps partly covered the telomere regions of the respective chromosome arms. These repeats were organized in the order of 5'-TTTAGGG-3' from the chromosome-specific region, except in chromosome 7S, in which seven inverted copies also existed in tandem array. Analysis of the telomere-flanking regions revealed the occurrence of deletions, insertions, or chromosome-specific substitutions of single nucleotides within the repeat sequences at the junction between the telomere and subtelomere. The sequences of the 500-kb regions of the seven chromosome ends were analysed in detail. A total of 598 genes were predicted in the telomeric regions. In addition, repetitive sequences derived from various kinds of retrotransposon were identified. No significant evidence for segmental duplication could be detected within or among the subtelomere regions. These results indicate that the rice chromosome ends are heterogeneous in both sequence and characterization.