Mechanisms of somatic embryogenesis in cell cultures: Physiology,biochemistry, and molecular biology

Summary One of the most characteristic cell functions in plants is totipotency. Somatic embryogenesis can be regarded as a model system for the investigation of mechanisms of totipotency, because a high frequency and synchronous embryogenic system from single somatic cells has been established in carrot suspension cultures. Four phases are recognized in this process, and several molecular markers, viz. polypeptides, mRNAs, antigens against monoclonal antibodies, can be detected during the expression of totipotency, but they disappear during its loss. Four organ-specific genes have been isolated from hypocotyls and roots by differential screening. They were expressed preferentially after the globular-heart stages of embryogenesis, and were strongly suppressed by auxin. A CEM 1 gene was isolated by differential screening of embryogenic cell clusters. This gene was expressed strongly and transiently during the proglobular and globular stages. The sequence of CEM 1 was found to encode a polypeptide showing high homology to the elongation factor isolated from eucaryotic cells. Thus good progress is being made in understanding the basic mechanisms of somatic embryogenesis. Presented in the Session-in-Depth Developmental Biology of Embryogenesis at the 1991 World Congress on Cell and Tissue Culture, Anaheim, California, June 16–20, 1991.     

Intracellular accumulation and oligosaccharide processing of alkaline phosphatase under disassembly of the Golgi complex caused by brefeldin A

Electron microscopic observations showed that the fungal metabolite brefeldin A caused disassembly of the Golgi complex in human choriocarcinoma cells and accumulation of alkaline phosphatase (ALP) in the endoplasmic reticulum (ER) and nuclear envelope, where ALP was not apparently detectable in control cells. Pulse/chase experiments with [35S]methionine demonstrated that in the control cells, ALP synthesized as a 63-kDa precursor form was rapidly converted to a 66-kDa form, by processing of its N-linked oligosaccharides from the high-mannose type to the complex type, which was expressed on the cell surface after 30 min of chase. In contrast, in the brefeldin-A-treated cells the precursor was gradually converted to a 65-kDa form, slightly smaller than the control mature form, which was not expressed on the cell surface even after a prolonged time of chase. Kinetics of the ALP processing in the brefeldin-A-treated cells demonstrated that the precursor was initially converted to an intermediate form, partially sensitive to endo-beta-N-acetylglucosaminidase H (endo H), then to an endo-H-resistant 65-kDa form. In addition, this form was found to be sensitive to neuraminidase digestion, though its sialylation was not so complete as that of the control mature form. Taken together, these results suggest that under disassembly of the Golgi complex caused by brefeldin A, oligosaccharide-processing enzymes including sialyltransferase, an enzyme in the trans Golgi cisterna(e) and/or the trans Golgi network, might be redistributed into the ER and involved in processing of the oligosaccharides of ALP accumulating there.     

cDNA cloning and sequencing of component C9 of proteasomes from rat hepatoma cells

The nucleotide sequence of component C9 of rat proteasomes (multicatalytic proteinase complexes) has been determined from a recombinant cDNA clone isolated by screening a Reuber H4TG hepatoma cell cDNA library using synthetic oligodeoxynucleotide probes corresponding to partial amino acid sequences of the protein. The predicted sequence of C9 consists of 261 amino acid residues with a calculated molecular weight of 29,496. The C9 component is a novel protein, differing from known proteins, but its primary structure resembles those of other proteasome components, including C2, C3 and C5, although its similarity to C5 is relatively low, suggesting that proteasomes consist of a family of proteins that have evolved from a common ancestor.     

Suppression mutations in the defective beta subunit of F1-ATPase from Escherichia coli

The Escherichia coli mutant of the proton-translocating ATPase KF11 (Kanazawa, H., Horiuchi, Y., Takagi, M., Ishino, Y., and Futai, M. (1980) J. Biochem. (Tokyo) 88, 695-703) has a defective beta subunit with serine being replaced by phenylalanine at codon 174. Four suppression mutants (RE10, RE17, RE18, and RE20) from this strain capable of growth on minimal plate agar supplemented by succinate were isolated. The original point mutation at codon 174 was intact in these strains. Additional point mutations, Ala-295 to Thr, Gly-149 to Ser, Leu-400 to Gln, Ala-295 to Pro, for RE10, RE17, RE18, and RE20, respectively, were identified by the polymerase chain reaction and sequencing. These mutations, except for RE10, were confirmed as a single mutation conferring a suppressive phenotype by genetic suppression assay using KF11 as the host cells. The results indicated that Ser-174 has functional interaction with Gly-149, Ala-295, and Leu-400. The residues are located within the previously estimated catalytic domain of the beta subunit, indicating that this domain is indeed folded for the active site of catalytic function. Growth rates of the revertants in the minimal medium with succinate increased compared with that of KF11, showing that ATP synthesis recovered to some extent. The ATP hydrolytic activity in the revertant membranes increased in RE17 and RE20 but did not in RE10 and RE18, suggesting that synthesis and hydrolysis are not necessarily reversible in the proton-translocating ATPase (F1F0).     

Three-dimensional cytoarchitecture of the media of arteriovenous anastomoses in the rabbit ear

Arteriovenous anastomoses in the rabbit ear were examined with scanning electron microscopy to elucidate the structural differentiation of the media of the shunt. Arterial, intermediate, and venous segments in the shunt and two layers of the media in the intermediate segment were differentiated based on cell shape and cell organization. In the arterial segment, smooth muscle cells were spindle-shaped, either elongated or short, with a few branches, and were arranged circularly or diagonally with respect to the vessel's long axis. There were also stellate muscle cells with radiating processes. In the intermediate segment, the smooth muscle cells of the outer layer of the media were also arranged circularly and resembled the elongated cells in the arterial segments, but they were more irregular in shape and had more processes than those of the arterial segment. The epithelioid cells of the inner layer of the media were oval or polygonal and oriented irregularly with respect to the vessel's long axis, clustering to form longitudinal plicae. The smooth muscle cells of the venous segment were flat with many lateral processes and formed a thin, discontinuous layer. The smooth muscle cells in the arterial segment and those of the outer layer of the intermediate segment exhibited a highly rugged surface texture, indicating their strong contractility; the epithelioid cells and the smooth muscle cells in the venous segment exhibited a generally smooth surface, indicating less contractility. The intermediate segments were supplied with a dense nerve plexus. The intermediate segments, therefore, may be actively involved in the regulation of blood flow under neuronal influence.     

Dental agenesis in the Dariusleut Hutterite Brethren: comparisons to selected Caucasoid population surveys

We report the results of a study of the prevalence of nonsyndromic dental agenesis among a sample of 208 individuals (105 females, 103 males) between the ages of 15 and 29 years from a North American religious and genetic isolate, the Dariusleut Hutterites of Western Canada. Direct examination of dental casts, oral examination reports, dental treatment records, and a limited number of dental radiographs reveals congenital absence and/or obvious morphometric reduction of at least one tooth (excluding third molars) in 98 subjects (55 females, 43 males), yielding a prevalence estimate of approximately 47%. This estimate is nearly four times those reported for nonisolate Caucasoid populations of European descent and substantially higher than the elevated prevalences observed in several other isolated populations. Although the prevalence of dental agenesis in the Dariusleut is indeed high, neither the incidence of bilateral agenesis (exhibited at least once in 58% of affected dentitions), number of affected teeth per person (mean, 2.4), morphologic tooth classes affected, or combinations of tooth classes affected ostensibly distinguish them from other populations with similar geographic origins. We conclude that the dental agenesis observed in this North American genetic isolate does not represent a private polymorphism or rare developmental variant. Consequently, the results of further study in these Dariusleut Brethren will be directly relevant to critically testing as yet unresolved hypotheses for the mode of gene action and the relative contributions of hereditary and environmental factors to the reduction of tooth numbers in human dentitions.     

Respiration and Motility in Starfish Spermatozoa at Various Times in the Breeding Season

In the spermatozoa of Asterias amurensis , patterns of changes in the respiratory rate and motility following dilution of dry sperm in sea water varied among batches and were classified into three types. The type I spermatozoa were immotile and exhibited quite low respiratory rate. Ethylenediamine tetraacetic acid made the type I spermatozoa motile and elevated their respiratory rate. The type II spermatozoa were also immotile and the respiratory rate remained quite low for about 5 min after the dilution. Thereafter, they spontaneously became motile and the respiratory rate increased. The type III spermatozoa became motile upon their dilution and exhibited high respiratory rate. Differences in the motility and respiratory rate of spermatozoa among batches probably result from degree of their maturation. In moving spermatozoa, the ADP and AMP levels increased at the expense of ATP. 2,4-Dinitrophenol elevated the respiratory rate only in immotile spermatozoa, which showed a high ATP level and quite low ADP level, but did not made them motile. Oligomycin inhibited the respiration of both motile and immotile spermatozoa. Probably, the respiratory rate is made low by a shortage of ADP in immotile spermatozoa and is enhanced by ADP production due to the initiation of their movement.     

The random walk description for isotope exchange in a polypeptide

Isotope exchange in a polypeptide is considered from the point of view in which the boundary point between helix and coil regions of a polypeptide behaves like a weakly asymmetric random walker. We assume that the boundary point is reflected completely at the ends of a polypeptide. The equilibrium fraction of helix region is obtained under this assumption, and this is also confirmed by computer simulation. The experimental results of isotope exchange can be explained in this situation. On the other hand. the rate constant of exchange of a residue given by experiments can also be explained by another assumption, as considered before (M. Fujiwara and N. Saitô, Polym. J. 9 (1977) 625.), in which the nucleations of coil states take place in the helix region. Which of the two is of major importance is left to further studies.     

Primary and secondary structure of 5.8S rRNA from the silkgland of Bombyx mori.

Nucleotide sequence of 5.8S rRNA of the silkworm, Bombyx mori has been determined by gel sequencing methods. The 5.8S rRNA was the longest so far reported, with the 5'-terminal sequence several nucleotides longer than those of the other organisms. Upon constructing the secondary structure in accordance with the "burp gun" model (12), the Bombyx 5.8S rRNA formed a wide-open "muzzle" due to several unpaired bases at the ends. The overall structure also appeared less stable with less G . C pairs and more unpaired bases than that of the HeLa 5.8S rRNA. These structural features may be essential for those 5.8S rRNAs which interact with 28S rRNAs containing the hidden break to form a stable complex.     

Primary and secondary structures of Tetrahymena and aphid 5.8S rRNAs: structural features of 5.8S rRNA which interacts with the 28S rRNA containing the hidden break

The Tetrahymena 5.8S rRNA is 154 nucleotides long, the shortest so far reported except for the split 5.8S rRNAs of Diptera (m5.8S plus 2S rRNA). In this molecule several nucleotides are deleted in the helix e (GC-rich stem) region. Upon constructing the secondary structure in accordance with "burp-gun" model, the Tetrahymena 5.8S rRNA forms a wide-open "muzzle" of the terminal regions due to both extra nucleotides and several unpaired bases. The aphid 5.8S rRNA consists of 161 nucleotides and can form stable helices in both terminal and helix e regions. As a whole, the secondary structure of Tetrahymena 5.8S rRNA resembles that of Bombyx 5.8S molecule while the aphid 5.8S rRNA shares several structural features with the HeLa 5.8S molecule. Likely, the 5.8S rRNA attached to the 28S rRNA with the hidden break differs in structure from those interacting with the 28S partners without the break. Nucleotide sequences of 5.8S rRNA in insects as well as in protozoans are not so conservative evolutionarily as in vertebrates.