Lateral Protuberance Research Articles

Analysis of the protein–protein interactions between the human acidic ribosomal P-proteins: evaluation by the two hybrid system

The surface acidic ribosomal proteins (P-proteins), together with ribosomal core protein P0 form a multimeric lateral protuberance on the 60 S ribosomal subunit. This structure, also called stalk, is important for efficient translational activity of the ribosome. In order to shed more light on the function of these proteins, we are the first to have precisely analyzed mutual interactions among human P-proteins, employing the two hybrid system. The human acidic ribosomal P-proteins, (P1 or P2,) were fused to the GAL4 binding domain (BD) as well as the activation domain (AD), and analyzed in yeast cells. It is concluded that the heterodimeric complex of the P1/P2 proteins is formed preferentially. Formation of homodimers (P1/P1 and P2/P2) can also be observed, though with much less efficiency. Regarding that, we propose to describe the double heterodimeric complex as a protein configuration which forms the 60 S ribosomal stalk: P0–(P1/P2) 2. Additionally, mutual interactions among human and yeast P-proteins were analyzed. Heterodimer formation could be observed between human P2 and yeast P1 proteins.

Two- and three-dimensional demonstrations of morphological alterations of early anagen hair follicle with special reference to the bulge area.

During late telogen to early anagen secondary hair germ is newly formed by the downgrowth of a clubbed column which is indistinguishable from the bulge. Serial vertical sections demonstrated that the early anagen terminal hair follicle formed the new secondary hair germ associated with a lateral protuberance of basaloid cells which could be considered as the bulge of the new hair follicle. Interestingly, the arrector pili muscle bundle was divided into two branches, one inserted into the original clubbed end and the other into this protuberance of the secondary hair germ. CAM5.2-reactive Merkel cells were present not only in the clubbed ends of the old follicle but also in the protuberance of the new hair germ. The formation of the lateral protuberance of the new hair germ preceded the appearance of CAM5.2-reactive Merkel cells in this location. Ks19.1 immunoreactivity was observed from the clubbed end to the upper half of the new hair germ. These phenomena occurred in early anagen before the club hairs were shed. It is postulated that the early anagen hair follicle formed the area within the new hair germ equivalent to the bulge and Merkel cells either moved from the bulge of the old hair follicle or differentiated de novo from immature epithelial cells. Merkel cells or their products in the bulge may serve as attractants for the readjusting arrector pili muscle to anchor to the bulge of the new hair follicle.

The Large Ribosomal Subunit Stalk as a Regulatory Element of the Eukaryotic Translational Machinery

Publisher Summary This chapter reviews the structural and functional properties of different eukaryotic stalk components, focusing on the Saccharomyces cerevisiae system for which an extensive analysis has been carried out. One of the most characteristic structural features of the large ribosomal subunit is the stalk—a highly flexible lateral protuberance that, in bacterial particles, is formed by a pentameric protein complex of two dimers of proteins, L7 and L12, and one copy of protein L10. Proteins L7 and L12—the N-terminal acetylated and nonacetylated forms of a unique, strongly acidic polypeptide—form in solution stable dimers that have been proposed to have an elongated structure with a globular carboxyl domain and a rigid amino end joined by a flexible “hinge.” The corresponding components of the stalk and the GTPase domain in eukaryotic ribosomes have been identified and characterized. They show characteristics that resemble closely those of their bacterial counterparts, and it is generally accepted that they must play an analogous functional role in the basic protein synthesis machinery, facilitating also the interaction of elongation factors.

Monoclonal antibodies to Escherichia coli ribosomal proteins L9 and L10. Effects on ribosome function and localization of L9 on the surface of the 50 S ribosomal subunit.

Monoclonal antibodies against Escherichia coli ribosomal proteins L9 and L10 were obtained and their specificity confirmed by Western blot analysis of total ribosomal protein. This was particularly important for the L9 antibody, since the immunizing antigen mixture contained predominantly L11. Each antibody recognized both 70 S ribosomes and 50 S subunits. Affinity-purified antibodies were tested for their effect on in vitro assays of ribosome function. Anti-L10 and anti-L9 inhibited poly(U)-directed polyphenylalanine synthesis almost completely. The antibodies had no effect on subunit association or dissociation and neither antibody inhibited peptidyltransferase activity. Both antibodies inhibited the binding of the ternary complex that consisted of aminoacyl-tRNA, guanylyl beta, gamma-methylenediphosphonate, and elongation factor Tu, and the binding of elongation factor G to the ribosome. The intact antibodies were more potent inhibitors than the Fab fragments. In contrast to the previously established location of L10 at the base of the L7/L12 stalk near the factor-binding site, the site of anti-L9 binding to 50 S subunits was shown by immune electron microscopy to be on the L1 lateral protuberance opposite the L7/L12 stalk as viewed in the quasisymmetric projection. The inhibition of factor binding by both antibodies, although consistent with established properties of L10 in the ribosome, suggests a long range effect on subunit structure that is triggered by the binding of anti-L9.

Structural and Functional Equivalence Between Ribosomal Proteins of Escherichia coli L1 and Methanococcus vannielii L6

Summary 50S ribosomes from Escherichia coli mutants lacking r-protein EL1 have been reconstituted with total r-proteins from Methanococcus vannielli , which had been radioactively labelled (either in vivo or in vitro ). A single protein was incorporated, designated ML6 according to the nomenclature of Schmid and Bock ( Schmid, G. and Bock, A . (1982) Zbl. Bakt. Hyg., I. Abt. Orig., C 3, 347–353), indicating that M. vannielli r-protein ML6 is structurally equivalent to E. coli r-protein L1. Protein ML6 was purified to homogeneity and an antiserum was raised which was used to localize protein ML6 by immuno electron microscopy on the surface of 50S subunits from M. vannielii and from mutant ribosomes, that had been reconstituted with ML6. In both cases antibody binding was to the broader lateral protuberance, in the same region where EL 1 has been located in E. coli wild type. Finally it was shown in a poly(U) dependent poly (Phe) in vitro translation system that reconstitution of the mutant 50S subunits with protein ML6 fully restored their activity to wild type level, thereby establishing also the functional equivalence of r-protein ML6 and EL1. We have thus for the first time constructed in vitro a chimeric ribosomal subunit, containing rRNA's and r-proteins from an eubacterium ( E. coli ) and a single r-protein from an archaebacterium ( M. vannielii ). The data suggest that important regions of r-proteins are, as is the case with rRNA's, also conserved between members of these two primary lineages.

In vitro germination and origin of thallus in Griffithella hookeriana (Podostemaceae)

Griffithella hookeriana (Tul.) Warm. (Podostemaceae) is a thalloid plant with no true leaves, stem or root. The seeds are extremely small (1000 seeds weigh 0.809 mg) and can be germinated on moist filter paper, but the resulting seedlings fail to survive beyond 5–6 days. A technique of germinating seeds in vitro on thermocole cubes floated in Murashige and Skoog's liquid medium (MS 1/5 strength with 0.5% sucrose) in continuous light (500–1000 lux) at 27±2°C has been developed. During germination, the radicular pole emerges from the seed coat and elongation of the two cotyledons follows. Rhizoids arise from the radicular pole and secrete a sticky substance that helps the seedling to adhere firmly to the substratum. The plumular apex ceases to funciton after producing 2 or 3 pairs of leaf-like outgrowths. A lateral protuberance is formed from the primary axis below the level of cotyledons and develops into a flat, green and oval, round or tubular (branched or unbranched) thallus (up to 0.5–0.8 cm in diameter) within 65–70 days. There is a high mortality of seedlings and plants at various stages of development: about 20% senesce before the initiation of the plumule, 15–20% after the formation of the leaf-like structures and 2–4% during the thallus stage. Seeds retain 83% viability for more than a year when stored dry at 10–15°C.

Ultrastructure of chemoreceptors in the larvae of the American bollworm Heliothis armigera

The location of olfactory and gustatory sense organs in the larvae of the American bollworm, Heliothis armigera, have been studied using the scanning and transmission electron microscopy. The olfactory chemoreceptors of the larvae are located on the antennae, mostly on the top of the second antennal segment in the form of two trichoid sensilla, two basiconic sensilla, two styloconic sensilla, campaniform sensilla and a uniporous peg sensillum. The third antennal segment bears one basiconic sensillum and two styloconic sensilla. The gustatory sense organs are located on the mouthparts including the labrum, mandibles, hypopharynx, maxillary palps and labium. Trichoid sensilla have been located on the labrum. Epipharyngeal setae as well as long short spines are located in the epipharyngeal membrane. The basal segment of the maxillary palp bears three trichoid sensilla and one campaniform sensilla. The maxillary palp also bears four styloconic sensilla on the distal portion of the lateral protuberance and one styloconic sensillum at the apex of the medial protuberance. Close to the bases of the protuberances, there are three basiconic sensilla and one campaniform sensillum. The third apical segment of the maxillary palp carries three campaniform sensilla on its lateral side, while on its top, eight small basiconic sensilla are distributed.

Localization of L11 on the Escherichia coli ribosome by singlet-singlet energy transfer.

Isolated Escherichia coli ribosomal protein L11 was labeled with maleimidyl derivatives of coumarin or fluorescein at the thiol group of its single cysteine, then reconstituted singly or in pairs with other fluorescently labeled ribosomal components. The characteristics of fluorescence from the labeled protein were studied and its distance to other components was determined by non-radiative energy transfer. The distance between probes on L11 and cysteine residues on other proteins or the 3' end of the ribosomal RNAs were found to be: S1, 7.4-8.3 nm; S21, 7.6 nm; 23S RNA, 6.9 nm; 5S RNA, 7.6 nm; 16S RNA, greater than 8.5 nm. Considered together with previously published results these distances indicate that the location of L11 in the 50S subunit is below the lateral protuberance characterized by L7/L12.

Cross-linking of streptomycin to the 50S subunit of Escherichia coli with phenyldiglyoxal.

[3H]Dihydrostreptomycin was covalently linked to the 50S subunit of Escherichia coli K12A19 with the bifunctional cross-linking reagent phenyldiglyoxal. The cross-linking was abolished under conditions that prevent the specific interaction of streptomycin with the ribosome. The binding primarily involved the ribosomal RNA and also a limited number of proteins, namely, L2, L6, and L17. This suggests that the binding domain for streptomycin is close to the peptidyl transferase center, in the valley between the central protuberance and the wider lateral protuberance of the 50S subunit. This domain faces the binding domain for streptomycin which we have previously characterized on the 30S subunit [Melançon, P., Boileau, G., & Brakier-Gingras, L. (1984) Biochemistry 23, 6697-6703]. Our results indicate that the 50S subunit is involved in the binding of streptomycin to the bacterial ribosome, in addition to the 30S subunit which is generally considered as the specific target of the antibiotic. They are consistent with the occurrence of a single binding site for streptomycin on the ribosome, comprised of regions of both subunits.

Localization of the puromycin binding site on the large ribosomal subunit of Escherichia coli by immunoelectron microscopy.

By using immunoelectron microscopy, we have localized the binding site on 50S Escherichia coli ribosomal subunits for puromycin, an antibiotic that interacts with the ribosomal peptidyltransferase center. This was achieved by affinity-labeling 50S subunits with N-bromoacetyl puromycin and treating the labeled subunits with an antibody specific for the N6,N6-dimethyladenosine moiety of puromycin. The position of the puromycin binding site was then revealed by localizing the attachment sites of the IgG molecules on the surfaces of the 50S subunits under the electron microscope: it was located at the interface between the subunits, on and around the wider lateral protuberance of the 50S subunit. This localizes directly the peptidyl transferase center on the surface of the large ribosomal subunit.

Mutants of Escherichia coli lacking ribosomal protein L1

Two independently isolated mutants of Escherichia coli, RD19 and MV17-10, that appeared to lack protein L1 on their ribosomes, as determined by two-dimensional gels, were subjected to a battery of immunological tests to find if L1 was indeed lacking. The tests involved Ouchterlony double diffusion, modified immunoelectrophoresis, dimer formation on sucrose gradients, and affinity chromatography. By all these criteria, protein L1 was missing from the ribosome in these mutants. Nor was any L1 cross-reacting material detectable in the supernatant. There was, however, a specific two- to fivefold increase in concentrations of protein L11 in the supernatants of the mutants, which was evidence that protein L1 acts as a feedback inhibitor of expression of the operon coding for the genes for proteins L11 and L1. Electron micrographs of ribosomes obtained from these mutants were indistinguishable from those of wild-type strains. 50 S ribosomal subunits from mutants RD19 and MV17-10 were reconstituted with purified L1 from wild-type and investigated by immunoelectron microscopy. The three-dimensional location of ribosomal protein L1 on the surface of the large subunit was determined. L1 is located on the wider lateral protuberance of the 50 S subunit. The position of protein L1 in 50 S subunits reconstituted from mutants RD19 and MV17-10 was indistinguishable from the position in subunits from wild-type.

Welwitschia mirabilis: Fine Structure of the Germinating Seed I. Orientation

Summary In the germinating seed of the gnetalean plant Welwitschia mirabilis a collared zone of the embryo produces a rapidly-developing, positively geotropic, nonvascularized lateral protuberance. This structure, if it did act haustorially or absorptively as a feeder, would appear to have its closest parallel in morphology and function with other plants in the lepidophyte Selaginella. The lower, mucilaginous surface of the protuberance, which reaches its maximum length of approximately 5 mm six days after emergence of the radicle, remains closely cemented to the megagametophyte. The various regions of the gametophyte and the embryonic protuberance - especially their shared interface - that were studied cytochemically and ultrastructurally in order inter alia to clarify the feeder's function, are circumscribed.

The Egg from a Human Case of Schistosomiasis in Laos

A morphological study was made of viable schistosome eggs in feces from a patient who had been infected at Khong Island in southern Laos. The tentative species diagnosis is Schistosoma japonicum; but these eggs are smaller, have a more nipplelike lateral protuberance of the shell, and are nearly spherical. Current literature on the subject is reviewed in light of possible explanations for peculiar differences between the schistosome which is endemic to continental Southeast Asia and S. iaponicum found elsewhere in the Orient. Schistosomiasis in continental Southeast Asia presents a new and enigmatic epidemiological problem. The snail intermediate host is unknown, and species of Oncomelania are not sympatric with the parasite (Ito and Jatanasen, 1961; Kruatrachue and Harinasuta, 1964; Brandt, 1968; Barbier and Brumpt, 1969). Because no adult worms from human cases in Southeast Asia have been described, the taxonomic status of the parasite is still in question. One endemic focus was reported in southern Thailand (Chaiyaporn, Koonvisal, and Dharamadhach, 1959; Koonavisan, Chaiporn, and Dharamatat, 1960; Harinasuta and Kruatrachu, 1960; Harinasuta and Kruatrachue, 1962; Kruatrachue, Bhaibulaya, and Harinasuta, 1964; Harinasuta et al., 1967). Lee, Wykoff, and Beaver (1966) reported two additional foci in Thailand. One of these foci (Ubol) occurs within the Mekong River drainage system (Desowitz et al., 1967). Other endemic areas occur along the Mekong in Laos (Dupont et al., 1957; Barbier, 1966; Brandt, 1968; Barbier and Brumpt, 1969) and in Cambodia (Audebaud et al., 1968). The present report concerns characteristics of eggs in feces from a civilian patient who became infected at Khong Island on the Mekong River in Laos near the Cambodian border between October 1964 and October 1965. Several cases have been previously reported from the same island (Dupont et al., 1957; Barbier, 1966; Brandt, 1968; Barbier and Brumpt,

INITIATION OF LATERAL ROOT PRIMORDIA WITHOUT COMPLETION OF MITOSIS AND WITHOUT CYTOKINESIS IN UNISERIATE PERICYCLE

In wheat, lateral (branch) roots originate always in the pericycle of the parent root between protoxylem poles of 2 successive xylem ridges and facing a sieve tube. Although the endodermis also contributes to the initial lateral protuberance when lateral root primordia develop close to the parent root apex, the pericycle alone gives rise to the lateral root which subsequently develops. Before initiation of lateral root primordia the pericycle and endodermis are each uniseriate. The first cell divisions that are characteristic of the initiation of lateral root primordia are periclinal divisions in the pericycle. When wheat is germinated and grown on colchicine, mitosis is not completed and hence no nuclear and cell divisions occur (i.e., “c‐mitoses,” but no true mitoses, occur). Within the primary roots of these seedlings appear structures, here called “primordiomorphs,” having the shape of lateral root primordia. Each primordiomorph, which develops without cell division, is a radial protuberance in the pericycle accompanied by an associated protuberance of the adjacent endodermis. Primordiomorphs are found only between protoxylem poles of 2 successive xylem ridges and facing a sieve tube. In primordiomorphs some nuclei repeatedly enter, but none complete, mitosis. The primordiomorphs become large enough to compress the cortical parenchyma and make bulges in the epidermis of the parent root. In typical lateral root primordia comparable in size with primordiomorphs, hundreds of cell divisions have occurred that, are absent in the primordiomorphs. After removal of colchicine the primordiomorphs develop with normal cell divisions into polyploid lateral roots as shown by the following findings: (1) Stages of development are detected that are intermediate between primordiomorphs and polyploid lateral roots with typical histological organization. As in normal lateral root initiation close to the parent root apex, the endodermis contributes to the initial protuberance but the pericycle alone gives rise to the lateral root which subsequently develops. (2) The number of these polyploid lateral roots formed can be accounted for by the number of primordiomorphs which disappear. (3) No “c‐mitoses” occur, i.e., the diploid and polyploid nuclei undergo only normal mitoses. Thus, primordiomorphs have 2 definitive morphological characteristics of typical lateral root primordia, viz.: (1) precise identity of histological site of origin, and (2) potentiality (realized after removal of colchicine) to develop into roots having typical histological organization. It is, therefore, concluded that primordiomorphs are lateral root primordia that developed without completion of mitosis and without cytokinesis. Since the parent root pericycle remains uniseriate, even in that region which comprises the primordiomorph, no periclinal cell divisions within the pericycle could have altered the polarization of growth to set up the apical‐basal axis of the primordiomorph. These results demonstrate that the earliest detectable changes in form involved in the initiation of a lateral root primordium can occur without completion of any mitosis and without any cytokinesis.

Development of the Flower and Seed in the Rape Plant, Brassica napus (with resume in English)

Although in past experiments connected with the physiology and ecology of the varieties of the rape plant a number of almost exhaustive investigations have been made of their vegetative organs, particulary of their external characters, the development of their generative organs has received compalatively little attention. While the experiments here referred to will be discussed in greater detail in a later paper, the authors, in this paper, merely attempt to establish criteria, with the aid of which it will be possible to measure the degree of growth of the generative organ in determining its development. Experiments covering this study were conducted during the period from 1939 to 1940 at the Konosu Farm of the Imperial Agricultural Experiment Station, using a variety of rape (Brassica napus L.) "Yokkaiti-Kurotane, " a variety which were preserved at the station. The seeds were sown on Sept. 27, 1939, under normal conditions. Special precautions were taken to have all the cultivating conditions as nearly uniform as possible, in order that the plants sampled from time to time for observation shall be as uniform as possible. Collections were made every third day from Nov. 2, 1939 to May 27, 1940. For the differentiation of inflorescence, a portion of the main stem, including the growing point, was fixed in CARNOY's fluid, and longitudinal or cross sections of from 13 to 17 μ thick were stained with either DELAFIELD's or HEIDENHAIN's iron haematoxylin. As soon as inflorescence became visible, the parts most developed, such as the bud, flower, and either siliqua or seed, that were obtained at the timae of sampling were used for material. The authors followed the extensive morphological changes that took place in the growing point and other parts during the growth of flower to seed, and established criteria by means of which the degrees of development of generative organs were standardized. Consequently, 38 principal stages of development, as shown in the microphotographs, were selected for the purpose. These stages are summarized as follows: 1. Stage of preparation for the first flower bud differentiation (Nov. 10-18) The dome-shaped vegetative corn assumes an elongated and swollen appearance (Pl. I, 1). 2. Stage of first flower bud differentiation, I (Nov. 21-24) Lateral protuberance arising near the growing point in the fundament from which the first flower bud intial originates. This primordium is distinguished from the leaf initials by its blunt and single ridge, its morphological significance marking the transition of growing point of the rape plant from the vegetative to the generative phase (Pl. I, 2). 3. Stage of first flower bud differentiation, II (Nov. 27-Dec. 2) The first flower bud that appeared in the preceding stage elongates upward (Pl. I, 3). 4. Stage of sepal differentiation, I (Dec. 6) Sepal primordia begin to differentiate from the tip of the flower bud, which last grows rapidly (Pl. I, 4). 5. Stage of stamen differentiation, I (Dec. 9) Stemen initials appear as papillae inside the sepals, so that the pistil primordium may be seen although signs of general differentiation are lacking (Pl. II, 5) 6. Stage of sepal and stamen differentiation II (Dec. 12-15) Primordia of sepal, of stamen and of pistil are more advanced than in the preceding stage. Sepals partly overlap the stamens (Pl. II, 6). 7. Stage of sepal and stamen differentiation III (Dec. 18-21). Sepals grow longer and overlap one another above the pistil, and the stamens increase in size. It is notable that the upper region of the pistil initial becomes flattened (Pl. II, 7). 8. Stage of carpel differentiation, I (Dec. 24). Carpel formation is first indicated by the appearance of a protuberance on the margin of the apex of the pistil, marking the end of the pistil concave (Pl. II, 8). 9. Stage of petal primordia differentiation (Dec. [the rest omitted]