Evolutionary aspects of accuracy of phenylalanyl-tRNA synthetase. Accuracy of the cytoplasmic and chloroplastic enzymes of a higher plant (Phaseolus vulgaris)

The phenylalanyl-tRNA synthetases from cytoplasm and chloroplasts of bean (Phaseolus vulgaris) leaves employ different strategies with respect to accuracy. The chloroplastic enzyme that is coded for by the nuclear genome follows the pathway of posttransfer proofreading, also characteristic for enzymes from eubacteria and cytoplasm and mitochondria of lower eukaryotic organisms. In contrast, the cytoplasmic enzyme uses pretransfer proofreading in the case of noncognate natural amino acids, characteristic for higher eukaryotic organisms and archaebacteria. Dependent on the nature of the noncognate amino acid, pretransfer proofreading in this case occurs without tRNA stimulation or with tRNA stimulated with no or little effect of the nonaccepting 3'-OH group of the terminal adenosine. The fundamental mechanistic difference in proofreading between the heterotopic intracellular isoenzymes of the plant cell supports the idea of the origin of the chloroplastic gene by gene transfer from a eubacterial endosymbiont to the nucleus. Origin by duplication of the nuclear gene, as indicated for mitochondrial phenylalanyl-tRNA synthetases [Gabius, H.-J., Engelhardt, R., Schroeder, F.R., & Cramer, F. (1983) Biochemistry 22, 5306-5315], appears unlikely. Further analyses of the ATP/PPi pyrophosphate exchange and aminoacylation of tRNAPhe-C-C-A(3'NH2), using 11 phenylalanine analogues, reveal intraspecies and interspecies variability of the architecture of the amino acid binding part within the active site.     

L-Phenylalanine ammonia-lyase from Phaseolus vulgaris. Characterisation and differential induction of multiple forms from elicitor-treated cell suspension cultures

L-Phenylalanine ammonia-lyase (EC 4.3.1.5) has been purified over 200-fold from cell cultures of bean (phaseolus vulgaris L.) exposed to elicitor heat-released from the cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum. Four forms of the enzyme, with identical Mr but differing apparent pI values of 5.4, 5.2, 5.05 and 4.85, were observed following the final chromatofocussing stage of the purification. A preparation (purified 43-fold by ammonium sulphate precipitation, gel-filtration and ion-exchange chromatography) containing all four forms exhibited apparent negative rate cooperativity with respect to substrates. However, the individual forms displayed normal Michaelis-Menten kinetics, with Km values of 0.077 mM, 0.122 mM, 0.256 mM and 0.302 mM in order of decreasing apparent pI value. A preparation purified 200-fold and containing all four forms was used to immunise rabbits for the production of anti-(phenylalanine ammonia-lyase) serum. The antiserum was characterised by: immunotitration experiments; solid phase enzyme-linked immunosorbent assays; comparison of immunoprecipitates of 35S-labelled phenylalanine ammonia-lyase subunits (synthesized both in vivo and in vitro) on both one-dimensional and two-dimensional polyacrylamide gels after immunoprecipitation with the bean antiserum or antisera raised against pea and parsley phenylalanine ammonia-lyase preparations and immune blotting. SDS/polyacrylamide gels and SDS/polyacrylamide gel electrophoresis followed by immune blotting, indicated that the Mr of newly synthesized (in vivo and in vitro) bean phenylalanine ammonia-lyase subunits is 77000; a 70000-Mr form is readily generated as a partial degradation product during purification. Immunoprecipitates of bean phenylalanine ammonia-lyase synthesized both in vivo and in vitro showed the presence of multiple subunit types of identical Mr but differing in pI. Furthermore, treatment of bean cultures with Colletotrichum elicitor resulted in a 10-fold increase in phenylalanine ammonia-lyase extractable activity within 8 h, and chromatofocussing analysis indicated that this was associated with differential increased appearance of the high-pI, low-Km forms as compared to the two higher Km forms. This differential induction was further confirmed by immune blotting of crude extracts subjected to isoelectric focussing.     

Aerobic spore-forming bacteria as detrimental infectants in plant tissue cultures

Summary Aerobic spore-forming bacteria were isolated from plant tissue cultures from a commercial plant cultivation station. Bacillus circulans was found to be a detrimental infectant as a serious consequence of the heat-resistance of the endospores of these bacteria. They were extremely motile, utilized several growth-promoting factors, and could be eliminated by early microscopical identification, killing by heat treatment, or by using antibiotics or disinfecants.Dedicated to Professor Dr. Gustav Kortüm on the occasion of his 80th birthday     

The behaviour of Dutch fresh water ecologists in response to environmental concern

Due to the rise of environmental concern ecologists have been asked for advice more frequently since the late 1960s. This article discusses how the changing relationship between ecologists and society has affected the behaviour of one particular group of ecologists, namely Dutch fresh water ecologists, by focussing on their behaviour towards job market control and public interest activities. The analysis has been based on interviews with 65 Dutch fresh water ecologists together with an analysis of their publications.In contrast with American ecologists, Dutch fresh water ecologists have made little effort to control their job market by formal arrangements such as formulating a code of ethics and certification procedures. Secondly, Dutch fresh water ecologists generally conceive of their own role in contributing to the solution of environmental problems in relation to that of the environmentalists by doing good research and reliable advisory work. Although a significant number of researchers also address lay publics by writing popular articles and/or holding lectures, only a minority actively participates in environmental politics.     

Embryonic development and molting of the antennal coeloconic no pore- and double-walled wall pore sensilla in Locusta migratoria (Insecta,Orthopteroidea)

Summary The embryonic development of antennal coeloconic sensilla was studied at four stages between 132 and 252 h after oviposition in Locusta migratoria. Initially the anlagen of the sensilla consist of 2–4 sensory cells and 3 enveloping cells. Two additional cells contribute later to the formation of socket and pit. The dendritic outer segments of the sensory cells elongate before the trichogen process grows out (ecdysis type I) with exception of one sensory cell in anlagen of poreless (np) sensilla. Other differences between np and double-walled wall pore (dw wp) sensilla are not visible until at least about 220 h after oviposition. Molting, which was studied in four stages, follows ecdysis type I in both sensillum types. The fourth enveloping cell maintains its tight connection to the socket of the sensillum even after apolysis. Its apical portion is torn off and shed together with the old cuticle. The electron-dense material between the dendritic sheath and the cuticular wall of the peg in np sensilla, which is regarded important for stimulus transmission, is not deposited during retraction of the trichogen cell. The concentric walls and spoke channels characteristic of dw wp sensilla result from deposition of cuticular material around wedge-shaped projections of the trichogen cell. The typical trilaminar 15 nm cuticulin layer is produced only on the ridges of these sensilla. The first cuticular lining of the spoke channels is only 7 nm thick and of a different structure. A flocculent material surrounds the outgrowing trichogen process. It is continuous with the filling of the spoke channels and can thus be considered as component of the stimulus-transmitting material in the functioning intermolt dw wp sensilla.     

Dependence of the conformation of a colicin E1 channel-forming peptide on acidic pH and solvent polarity

The secondary structure content of the COOH-terminal tryptic peptide of colicin E1 has been measured by analysis of UV circular dichroism spectra as a function of pH in aqueous medium and in the presence of the nonionic detergents octyl glucoside and Triton X-100. The alpha-helical content of the peptide increased by approximately 10%, from 45-47% to 56-57%, in the presence of the nonionic detergents, but not in aqueous medium, as the pH was decreased from 4.5 to 3.5. This pH dependence of conformation is similar to that reported elsewhere for the in vitro activity and binding of this peptide. A smaller increase in helical content was observed for the peptide in aqueous medium or in Triton X-100 as the pH was decreased from 6.5 to 4.5. The letter change in helical content was not seen in octyl glucoside which was present at a detergent:peptide stoichiometry 100 times that of Triton. The mean residue ellipticity measured at 222 nm for peptide added to asolectin vesicles by a freeze-thaw treatment was slightly larger at pH 3.5, and substantially larger at pH 4.5, than found at these pH values in the detergent solutions. Changes in helical content at the former, but not the latter pH, could be attributed to peptide insertion. It appears that protonation of one or more acidic amino acid residues in the COOH-terminal region of the molecule causes a conformational change that can be attributed to an extra helical domain that is stabilized in a nonpolar environment. From the similar pH dependence of the conformational change and in vitro binding and activity, it is inferred that interaction of this domain with the membrane is essential for binding and insertion.     

Genetic polymorphism of the sixth component (C6) of rat complement

The complement protein C6 has been shown to be genetically polymorphic in the rat. Isoelectric focusing of plasma samples from 19 inbred strains demonstrated two electrophoretically distinguishable migration patterns, each consisting of three bands. Breeding studies with the use of the BN and DA strains showed that the C6 patterns were inherited in a manner consistent with the co-dominant autosomal expression of two alleles (C6 A and C6 B). The distribution of the C6 alleles in a backcross mating was compared with eight independently segregating marker genes: RT1.A, RT2, Gdc -1, Igk-1, Hbb, Svp-1, Fh-1, and Es-6. There was no detectable linkage between C6 and any of these eight loci.     

Identification of two types of keratin polypeptides within the acidic cytokeratin subfamily I

Cytoskeletal filaments of the α-keratin type (cytokeratins) are a characteristic of epithelial cells. In diverse mammals (man, cow and rodents) these cytokeratins consist of a family of approximately 20 polypeptides, which may be divided into the more acidic (I) and the more basic (II) subfamilies. These two subfamilies show only limited amino acid sequence homology. In contrast, nucleic acid hybridization experiments and peptide maps have been interpreted to show that polypeptides of the same subfamily share extended sequence homology.We compare two polypeptides of the acidic cytokeratin subfamily, VIb (M_r 54,000) and VII (M_r 50,000), which are co-expressed in large amounts in bovine epidermal keratinocytes. These two epidermal keratins can be distinguished by specific antibodies and show different patterns of expression among several bovine tissues and cultured cells. In addition, they differ in the stability of their complexes with basic keratin polypeptides and in their tryptic peptide maps. The amino acid sequences deduced from the nucleotide sequences of complementary DNA clones containing the 3′ ends of the messenger RNAs for these keratins are compared with each other and with available amino acid sequences of human, murine and amphibian epidermal keratins. Bovine keratins VIb and VII share considerable sequence homology in the α-helical portion (68% residues identical) but lack significant homology in the extrahelical portion. Bovine keratin VIb shows, in its α-helical region, a pronounced sequence homology (88% identity) to the murine epidermal keratin of M_r 59,000. In addition, the non-helical carboxy-terminal regions of both proteins are glycinerich and contain a canonic sequence GGG^S_GYGG, which may be repeated several times. Moreover, their mRNAs present a highly conserved stretch of 236 nucleotides containing, in the murine sequence, the end of the coding and all of the non-coding region (81% identical nucleotides). Bovine keratin VII is considerably different from the murine M_r 59,000 keratin but is almost identical to the human cytokeratin number 14 of M_r 50,000, both in the α-helical and in the non-α-helical regions of the proteins, and the mRNAs of the human and the bovine keratins also display a high homology in their 3′ non-coding ends.The results show that in the same species keratins of the same subfamily can differ considerably, whereas equivalent keratin polypeptides of different species are readily identified by characteristic sequence homologies in the α-helical and the non-helical regions as well as in the 3′ non-coding portions of their mRNAs. Among the members of the acidic subfamily I of cytokeratin polypeptides that are co-expressed in bovine epidermis, at least two types can be distinguished by their carboxy-terminal sequences. One type is characterized by its abundance of glycine residues, a consensus GGG^S_GYGG heptapeptide sequence, which may be repeated several times, and an extended stretch of high RNA sequence homology in the 3′ non-coding part. The other type shows a predominance of serine and valine residues, a subterminal GGG^S_GYGG sequence (which has been maintained in Xenopus, cow and man) and also a high level of homology in the 3′ non-coding part of the mRNA. The data indicate that individual keratin type specificity overrides species diversity, both at the protein and the mRNA level. We discuss the evolutionary conservation and the tissue distribution of these two types of acidic keratin polypeptides as well as their possible biological functions.