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A monoclonal antibody raised against cytochrome b558 reacted specifically with the 22- to 23-Kd protein, the small subunit of this cytochrome. Cytochemical studies showed that the epitope was located on the surfaces of human neutrophils and monocytes. The small subunit of cytochrome b558, therefore, was expressed at least in part on the outer surface of these cells.

Corresponding sites of the Euglena chloroplast and yeast small subunit ribosomal RNAs (rRNAs) show only an insignificant match with each other but show extensive matches with the Euglena chloroplast tRNAarg. The match with the tRNA extends farther toward the 5′ end of the Euglena rRNA and toward the 3′ end of the yeast rRNA. The expected number of such configurations given the number of RNAs searched is about 1 in 100,000. Comparison of two sequences with a third sequence frequently reveals relationships where pairwise comparisons fail to do so.

When the precursor of ribulose bisphosphate carboxylase of Chlamydomonas reinhardtii y-1 is bound to antibodies and treated with the soluble cell fraction, it is cleaved to the mature form (M(r) 16,500) via an intermediate of M(r) 18,500. Although this intermediate has only been observed in vitro, it may be produced during processing of the precursor in vivo.

The RPII15 gene product of Drosophila melanogaster , which has recently been identified by sequence comparison, possesses a high similarity to subunit 9 of yeast RNA polymerase II. Using the polymerase chain reaction the coding region of RPII15 was isolated from genomic DNA of adult flies. Sequence analysis shows four amino acid substitutions in comparison to the previously reported sequence. Antisera were generated against bacterially expressed RPII15 and were used for immunoblotting experiments with RNA polymerase II of Drosophila melanogaster . This analysis identified the M r , 15,000 subunit 9 as gene product of RPII15 .

Fraction 1 protein has been isolated from leaves of a male sterile Nicotiana tabacum plant containing an extra N. debneyichromosome. The extra chromosome induces appearance of a third polypeptide composing the small subunit of fraction 1 protein, which otherwise contains two polypeptides as is shown by analysis of numerous different cultivars of N. tabacum.

The odontoblast layer of the pulp was examined after perfusion fixation with buffered aldehydes. Desmosomal junctions occur between only some of the odontoblasts at the predentinal border. Pale cytoplasmic processes similar in morphology to nerve terminals in the intercellular spaces make specialized junctions on the cell bodies of the odontoblasts which may be small subunit gap-junctions of low electrical resistance.

When free and membrane-bound ribosomes were titrated with N- ethyl [ 14 C]-maleimide or 5,5′-dithio-bis(2-nitrobenzoic acid), 25 free SH groups were detected in polysomes, 10 in the 60 S subunit and 14 in the 40 S subunit. When individual ribosomal proteins were analysed, one large subunit protein L14 was found to be strongly labelled compared to the other ribosomal proteins, in polysomes, monosomes and subunits. However, two additional protein spots from the small subunit were more radioactive in monosomes and subunits than in polysomes. In all these studies, it was found that the distribution of free SH groups in free and membrane-bound ribosomes was identical except for the few proteins which were reported before to be specific for free or membrane-bound ribosomes (Fehlmann, M., Bellemare, G. and Godin, C. (1975) Biochim. Biophys. Acta 378, 119–124 and Fehlmann, M., Bellemare, G. and Godin, C. (1975) FEBS Lett. 59, 8–12).

Genetic, biochemical and physiological analyses of photosynthetic mutants have been used for many years in attempts to broaden our understanding of the structure-function relationships of the photosynthetic apparatus. The recent development of molecular biology has given us some insight into the primary structure of plant genes and is providing the techniques for the investigation of the molecular basis of mutations which were previously only detectable through their phenotypic expression. In spite of these advances we still have little understanding of the mechanisms regulating the expression of these genes. The use of recombinant DNA techniques in directly probing and manipulating the molecular pathways responsible for particular aspects of plant performance, such as photosynthesis, may lead to significant advances in our knowledge and understanding of these processes.

The previous edition of Modern Methods of Plant Analysis contained a summary of characteristics of a “carboxylating enzyme… also known as carboxydismu– tase” (Losada and Arnon 1964). Today, this enzyme is identified as D-ribulose– 1,5-bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxyl-lyase (dimerizing), EC 4.1.1.39]. It was not until 1970 that the synonymy of Fraction I protein with carboxydismutase and RuBP carboxylase1 was documented (Akazawa 1970; Kawashima and Wildman 1970). The reviews illustrated how our perception of Fraction I protein evolved, from a protein presenting the singularly largest component among soluble plant proteins (Singer et al. 1952; Wildman and Bonner 1947), to an enzyme catalyzing the conversion of CO2 and RuBP to D-glycer– ate 3-phosphate in photosynthesis (Jakoby et al. 1956; Racker 1957; Weissbach et al. 1956).

The genes encoding the two polypeptides of ribulose-bisphosphate carboxylase-oxygenase (Rbcase) are located in separate genomes of eukary-otic organisms. The gene for the large subunit (LSU) is found in the chloroplast, while those for the small subunit (SSU) are located in the nucleus. We are interested in the interaction between these two genomes and are studying the expression of the genes for the LSU and SSU of Rbcase as a model system for this interaction.

1. 1. Artiodactyla haptoglobins (Hps), goat, sheep and cattle (family Bovidae), and pig (family Suidae) were structurally characterized. 2. 2. The polymeric Hp systems of goat, sheep and cattle were similar to the polymeric human Hp system, while the monomeric system of pig was more comparable to the monomeric human form. 3. 3. All members of the Artiodactyla (family Bovidae) examined exhibited a large polypeptide subunit, comparable to that of the β subunit of human Hp. 4. 4. In addition, a small subunit, similar in molecular weight to the human α2 subunit, was demonstrated. Pig Hp was shown to have two subunits, one slightly larger than the human β subunit and the other intermediate in size to the human α1 and α2 subunits. 5. 5. Immunoelectrophoretic and immunodiffusion studies indicated complete cross reactivity among the polymeric Artiodactyla Hps. 6. 6. The polymeric Hps do not, however, cross react with the monomeric pig Hp.

The murine major histocompatibility complex (MHC) codes for three groups of identifiable cell-surface proteins, the K, D molecules, the I–A subregion antigens and the I–E subregion antigens. All three groups of molecules display a high degree of serologically detectable polymorphism1 and consist of two noncovalently associated polypeptides2–8. Amino acid sequence and peptide comparisons among allotypes of K, D and I–E molecules reveals that one polypeptide is relatively constant, whereas the other is highly variable9–13. Thus, it is likely that only one of the two polypeptides, the variable component, determines the antigenic specificities recognized by alloantisera. In contrast to the K, D and I–E molecules, both subunits of I–A molecules display substantial structural differences when comparisons among allotypes are made4,14–16. Therefore, we have investigated whether one or both subunits of I–A molecules determine their alloantigenic specificities. Our results, presented here, indicate that only one of the two subunits determines a particular allospecificity recognized by a monoclonal antibody.

Following a single dose of dimethylnitrosamine (DMNA) the phosphorylation of rat liver ribosomes was strongly enhanced. The incorporation of [ 32 P]phosphate into the 40 S subunit was stimulated 5- and 9-fold 2 and 5 hr, respectively, after administration of the drug. The phosphorylation of the large ribosomal subunit was not affected. The enhanced phosphorylation of the small subunit was due to a 20-fold stimulated incorporation of phosphate into ribosomal protein S6, the only one affected by DMNA.

Publisher Summary This chapter elaborates the ribosome structure and functional sites. The structure of the large and small subunits is described. The small subunit is divided into two unequal parts by an indentation, and a region of accumulated negative stain. The parts are referred to as the upper one-third and the lower two-thirds. A region of the subunit, called the platform, extends from the lower two-thirds of the small subunit, and forms a cleft between it, and the upper one third. This cleft is an important functional region of the small subunit, and has been proposed as the site of the codonanticodon interaction, and as a part of the binding site of initiation factors IF1, IF2, and IF3. It is found that since protein locations determined by immunoelectron microscopy critically depend upon the model used to map them, protein locations differ markedly from those of the Berlin group for both small and large subunits, and the functional implications are completely different). The large subunit, like the small subunit, is asymmetric. It consists of a central protuberance and projections inclined at approximately 50° from the central protuberance.

As part of a project pertaining the organization of ribosomal genes in Kinetoplastidae, we have created a data base for published sequences of ribosomal nucleic acids, with information in Spanish. As a first step in their processing, we have written a computer program which introduces the new feature of determining the length of the fragments produced after single or multiple digestion with any of the known restriction enzymes. With this information we have detected conserved SAU 3A sites: (i) at the 5' end of the 5.8S rRNA and at the 3' end of the small subunit rRNA, both included in similar larger sequences; (ii) in the 5.8S rRNA of vertebrates (a second one), which is not present in lower eukaryotes, showing a clear evolutive divergence; and, (iii) at the 5' terminal of the small subunit rRNA, included in a larger conserved sequence. The possible biological importance of these sequences is discussed.

Publisher Summary This chapter describes techniques used for isolating genes encoding the small subunit (SSU) of ribulose-l,5-bisphosphate carboxylase (RuBisCo) from the duckweed Lemna gibba and investigates the expression of those genes. In every higher plant species, SSU is encoded by a small family of nuclear genes. As a result of their multiple copy number, the SSU genes present certain circumstances that would not be encountered when investigating single-copy genes. The chapter discusses the procedures used for cloning the Lemna gibba nuclear genome in the phage λ vector Charon 4. The factors affecting sequence representation in genomic clone libraries are also discussed. The method used for preparing a partial restriction digest for cloning is preferable to simply scaling up a single set of reaction conditions that yield the optimal size range of fragments, because the restriction sites are not distributed randomly throughout the genome, and the rate of cleavage by restriction enzymes varies from site to site. To minimize underrepresentation of sequences delimited by infrequently cleaves sites, the DNA should be subjected to a series of digests at different enzyme concentrations.

A number of immunological methods have been employed to show that the small subunit of HL-A antigens, isolated either after papain treatment or after solubilization with detergent, is identical to β 2 -microglobulin, a protein previously isolated from human urine and shown to be homologous in structure to constant region domains of immunoglobulins. Moreover, quantitative data indicate virtually total identity between the small subunit of HL-A antigens and β 2 -microglobulin. Studies of the turnover of labeled HL-A antigens from the lymphocyte surface indicate that the two subunits turn over at similar rates, although only the small subunit could be detected in the culture medium. The significance of these observations is discussed.

The possible curvature in the light-responsive element (LRE) of the plant gene which codes for the small subunit of ribulose-1,5-bisphosphate carboxylase has been derived from the nucleotide sequence of the LRE on the basis of a theoretical method previously developed in our research group. The oligonucleotides corresponding to regions of higher curvature in the profile, taking also into account biochemical data, have been synthesized and their electrophoretic mobilities have been measured after ligation. Retardation effects have been found, increasing with length of the multimers, in good agreement with theoretical prediction. These results suggest that the curvature of LRE could be the structural determinant in the control of rbcS-3A gene photoactivation.

Leaf rubisco (Fraction 1 protein) was purified from a number of cruciferous species, and subunit polypeptide patterns were examined by isoelectric focusing in the presence of 8 M urea. Both cultivated and wild Brassica species were studied by this method and results suggest that the protein may be a useful taxonomic marker, especially where species may have a possible hybrid origin. One small subunit (designated SS 1) appeared from studies to have a restricted distribution within the Brassicaceae. Antiserum was raised to rubisco from B. oleracea. In double diffusion experiments, all cruciferous species examined gave reactions of complete identity when tested against B. oleracea, but outside the family solely reactions of partial and incomplete identity were noted.

Pyrenoid proteins and RuBPCase were highly purified and their peptide compositions were studied with SDS-polyacrylamide gel electrophoresis. Both proteins had a large subunit of 50 kD and a small subunit of 17 kD. The peptide map of the large subunit of pyrenoid was coincident with that of RuBPCase. The pyrenoid had several minor components which were not found in RuBPCase.

The structure of protein S5 from the small subunit of the Bacillus stearothermophilus ribosome is described to a resolution of 5 Å. The molecular boundary is visible and shows the protein to be essentially compact although slightly elongated in one dimension.

By a method employing centripetal floatation of membranes away from sedimenting particles, the binding capacity of pyrophosphate/citrate-treated rough membranes at 3 o for large ribosomal subunits has been found to be greater than for small subunits. Accurate comparison of the membrane affinity for the 2 subunits is apparently precluded however by greater instability of the small subunit, even in the presence of ribonuclease inhibitor.

The membranes from M. lysodeikticus can readily be disaggregated into a very small subunit form with sodium lauryl sulfate. Reaggregation of these subunits into membranous sheets occurs during dialysis in a menstruum of relatively low ionic strength, in the presence of a divalent cation at near neutral pH. Reducing conditions and a monovalent salt need not be present during dialysis. Effect of several compounds on membranes and subunit reaggregation was studied. Reaggregated membrane sheets are very similar chemically and morphologically to whole membranes; however, a difference was noted. The major bonding mode in membranes appears to involve hydrophobic groups.

1. 1. The molecular weights of the Jensen sarcoma ribosome and its large subunit, calculated from their sedimentation and diffusion coefficients, are 4.3·106 and 2.7·106 respectively. The molecular weights of the ribosomal RNA's, calculated from their sedimentation coefficients and intrinsic viscosities, are 1.64·106 and 0.67·106 respectively. Thus these ribosomes appear to dissociate into two subunits, of approximately two thirds and one third, each containing one strand of structural RNA that makes up half its mass. 2. The large subunit exists in a variety of states whose sedimentation coefficients decrease from 57 S to 30 S as cations are removed. These changes have been attributed to changes in conformation. 3. The small subunit has been found in two forms: 33 S when magnesium is present and 29 S in its absence.

A wild injured Iberian lynx (Lynx pardinus) was taken from the Sierra Morena population. During the health check small intraerythrocytic piroplasms, morphologically indistinguishable from other feline piroplasms, were observed in Wright-Giemsa-stained blood films. Amplification by polymerase chain reaction of a portion of the 18S nuclear small subunit (NSS) rRNA gene and sequencing revealed similarity of the unknown organism with sequences obtained from Pallas's cat from Mongolia and from a domestic cat in Spain. In a retrospective (1993-2003) study of 50 Iberian lynx tissue samples, no amplifications of the 18S NSS rRNA gene of the organism were obtained. This is the first report of a naturally occurring erythroparasitemia in the Iberian lynx and the first documented case of naturally occurring piroplasm infection in a free-ranging felid from Europe.

By introducing the TSAO-resistance mutation 138Glu→Lys in only the p51 subunit of HIV-1 RT, we obtained compelling evidence for a structural and functional role of the p51 subunit in the sensitivity and/or resistance of the enzyme to the TSAO-derivatives.

The European database of the Small Subunit (SSU) Ribosomal RNA is a curated database that strives to collect all information about the primary and secondary structure of completely or nearly-completely sequenced rRNAs. Furthermore, the database compiles additional information such as literature references and taxonomic status of the organism the sequence was derived from. The database can be consulted via the WWW at URL http://rrna.uia.ac.be/ssu/ . Through the WWW, sequences can be easily selected either one by one, by taxonomic group, or by a combination of both, and can be retrieved in different sequence and alignment formats.

High-throughput sequencing (HTS) of gene amplicons is a preferred method of assessing microbial community composition, because it rapidly provides information from a large number of samples at high taxonomic resolution and low costs. However, mock community studies show that HTS data poorly reflect the actual relative abundances of individual phylotypes, casting doubt on the reliability of subsequent statistical analysis and data interpretation. We investigated how accurately HTS data reflect the variability of bacterial and eukaryotic community composition and their relationship with environmental factors in natural samples. For this, we compared results of HTS from three independent aquatic time series (n = 883) with those from an established, quantitative microscopic method (catalyzed reporter deposition-fluorescence in situ hybridization [CARD-FISH]). Relative abundances obtained by CARD-FISH and HTS disagreed for most bacterial and eukaryotic phylotypes. Nevertheless, the two methods identified the same environmental drivers to shape bacterial and eukaryotic communities. Our results show that amplicon data do provide reliable information for their ecological interpretations. Yet, when studying specific phylogenetic groups, it is advisable to combine HTS with quantification using microscopy and/or the addition of internal standards. IMPORTANCE High-throughput sequencing (HTS) of amplified fragments of rRNA genes provides unprecedented insight into the diversity of prokaryotic and eukaryotic microorganisms. Unfortunately, HTS data are prone to quantitative biases, which may lead to an erroneous picture of microbial community composition and thwart efforts to advance its understanding. These concerns motivated us to investigate how accurately HTS data characterize the variability of microbial communities, the relative abundances of specific phylotypes, and their relationships with environmental factors in comparison to an established microscopy-based method. We compared results obtained by HTS and catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) from three independent aquatic time series for both prokaryotic and eukaryotic microorganisms (almost 900 data points, the largest obtained with both methods so far). HTS and CARD-FISH data disagree with regard to relative abundances of bacterial and eukaryotic phylotypes but identify similar environmental drivers shaping bacterial and eukaryotic communities.

Accurate assembly and maturation of human mitochondrial ribosomes is essential for synthesis of the 13 polypeptides encoded by the mitochondrial genome. This process requires the correct integration of 80 proteins, 1 mt (mitochondrial)-tRNA and 2 mt-rRNA species, the latter being post-transcriptionally modified at many sites. Here, we report that human ribosome-binding factor A (RBFA) is a mitochondrial RNA-binding protein that exerts crucial roles in mitoribosome biogenesis. Unlike its bacterial orthologue, RBFA associates mainly with helices 44 and 45 of the 12S rRNA in the mitoribosomal small subunit to promote dimethylation of two highly conserved consecutive adenines. Characterization of RBFA-depleted cells indicates that this dimethylation is not a prerequisite for assembly of the small ribosomal subunit. However, the RBFA-facilitated modification is necessary for completing mt-rRNA maturation and regulating association of the small and large subunits to form a functional monosome implicating RBFA in the quality control of mitoribosome formation.

The N-terminal sequence of the small subunit of Fraction I protein isolated from tobacco was investigated, using an automatic protein sequencer. The amino acid sequence of the first 21 residues is presented.

We have embarked on a series of EM experiments using the dark field mode in an attempt to ascertain how much of a role the ribonucleic acid (RNA) component of the ribosome has in determining the structure of the protein-nucleic acid complex. We have observed that treatment of specimens of the smaller subunit enhances the visibility of band-like structures on the "back" of the subunit. In total, though not necessarily all appearing at once on a single individual, three parallel bands of 20 Å diameter are seen (Fig. 2). We have speculated that these are double stranded sections of the RNA that emerge on the surface. This does not preclude external single stranded sections that are not as prominent, however. The identification of the bands with double stranded external RNA is still subject to further testing. In a very recent experiment we have examined a heated RNAase digestion product that has been shown to lack a section of its RNA.

The complete amino acid sequence of the small subunit of ribulosebisphosphate carboxylase from spinach has been determined. There are 120 amino acids. The N-terminus of the protein is frequently blocked. There is a tyrosine-proline substitution at position 91 and the average molecular weight of the two forms is 13 897. The interest of the sequence data for students of physiology and evolution is discussed.