Transformation of Paramecium tetraurelia by electroporation or particle bombardment.

Methods for mass transformation of Paramecium tetraurelia were established using plasmids bearing neomycin-resistance or calmodulin gene fragments. Phenotypic and molecular analyses showed that, although variable, up to 5% transformation can be achieved by electroporation. Concentrations of divalent cations Ca2+ and Mg2+ in the electroporation medium were crucial for efficient transformation. Strong neomycin-resistance transformation using bioballistic particle bombardment with gold particles was observed. For both methods, hybridization to transformant DNA revealed plasmid signals consistent with macronuclear transformation and correlated with transformed phenotypes. Complementation of a known calmodulin gene mutation was also achieved by mass transformation. Possible sources of variation and the general utility of these methods are discussed.

In vivo Paramecium mutants show that calmodulin orchestrates membrane responses to stimuli.

Paramecium generates a Ca2+ action potential and can be considered a one-cell animal. Rises in internal [Ca2+] open membrane channels that specifically pass K+, or Na+. Mutational and patch-clamp studies showed that these channels, like enzymes, are activated by Ca(2+)-calmodulin. Viable CaM mutants of Paramecium have altered transmembrane currents and easily recognizable eccentricities in their swimming behavior, i.e. in their responses to ionic, chemical, heat, or touch stimuli. Their CaMs have amino-acid substitutions in either C- or N-terminal lobes but not the central helix. Surprisingly, these mutations naturally fall into two classes: C-lobe mutants (S101F, I136T, M145V) have little or no Ca(2+)-dependent K+ currents and thus over-react to stimuli. N-lobe mutants (E54K, G40E+D50N, V35I+D50N) have little or no Ca(2+)-dependent Na+ current and thus under-react to certain stimuli. Each mutation also has pleiotropic effects on other ion currents. These results suggest a bipartite separation of CaM functions, a separation consistent with the recent studies of Ca(2+)-ATPase by Kosk-Kosicka et al. [41, 55]. It appears that a major function of Ca(2+)-calmodulin in vivo is to orchestrate enzymes and channels, at or near the plasma membrane. The orchestrated actions of these effectors are not for vegetative growth at steady state but for transient responses to stimuli epitomized by those of electrically excitable cells.

Efficient transformation of cam2, a behavioral mutant of Paramecium tetraurelia, with the calmodulin gene.

An Ile-136----Thr substitution in calmodulin reduces the Ca(2+)-dependent K+ currents of cam2, a behavioral mutant of Paramecium tetraurelia, and renders it overly susceptible to BaCl2. DNA fragments carrying the wild-type CAM gene injected into cam2 macronuclei reverted these phenotypes in the clonal descendants of the recipients. Tetrahymena telomeric sequences, added in vitro to the fragment termini before injection, enhanced the efficiency and quality of transformation. Five times 10(4) copies of such fragments consistently restored the phenotypes to near normal; even 10(3) or fewer copies could still effect weak transformation. The restored phenotypes were stable for greater than 20 fissions in many clones and were lost after autogamy. We examined the fate of the injected fragments in the transformed clones and discuss the possible application of this efficient transformation in the cloning of other genes of P. tetraurelia.

Efficient expression of the Paramecium calmodulin gene in Escherichia coli after four TAA-to-CAA changes through a series of polymerase chain reactions.

We have expressed the Paramecium calmodulin gene in Escherichia coli by changing the four TAA codons in this gene to CAAs. This was carried out by three polymerase chain reactions (PCRs) and then cloning the product into the expression vector pKK223-3 immediately downstream of its trp-lac hybrid promoter. JM109 strain of E. coli, transformed with the recombinant plasmid harboring the altered Paramecium calmodulin gene, produces a protein judged to be calmodulin. It is recognized by a monoclonal antibody to Paramecium calmodulin; it migrates with the native protein at nearly the same rate in electrophoreses; and it shows a Ca(2+)-dependent shift in electrophoretic pattern. The production of calmodulin is about 170 times as efficient with E. coli as with Paramecium in terms of unit volume of packed cells, and is about 400 times as efficient in unit volume of liquid culture. This method appears useful in site-directed mutageneses and in the heterologous productions of other ciliate proteins. A critique of this method is provided. A calmodulin half-molecule, a by-product of this project, is described.

Circadian distribution of proteins in urine from healthy young men.

Urine samples were collected at 3-hr intervals over a single 24-hr period from each of seven clinically healthy men who ranged in age from 21-25 years. Urines at each collection time were subsequently pooled using 20% of each volume and serially dialyzed against ammonium-barbituric acid buffer (pH 7.35 +/- 0.02), using a cellulose membrane permeable to compounds of less than 12,000-14,000 molecular weight (mw). When the dialyzed portions were then analyzed for total proteins, the sum of proteins in eight pools amounted to 74 mg. A 1 ml aliquot of each pool, representing approximately 50 micrograms of proteins, was concentrated and reconstituted. Approximately 20 micrograms of reconstituted proteins were then subjected to polyacrylamide gel electrophoresis. The stained gel was then scanned by laser densitometry and planimetry. Each aliquot revealed eight segments as identified by Coomassie and silver staining. Their molecular weights, estimated by extrapolation from concurrently run protein standards, and their total protein amounts were: 116,000 mw (9.44 mg), 91,000 mw (3.3 mg), 68,000 mw (11.58 mg), 53,000 mw (2.58 mg), 43,000 mw (9.12 mg), 32,000 mw (7.13 mg), 24,000 mw (4.52 mg) and 20,000 mw (5.27 mg). A statistically significant rhythm (P = 0.022 from ANOVA and 0.011 from Single Cosinor) was found for the excretion of total proteins, with an acrophase in the afternoon (1537) for these diurnally-active subjects.

Comparative study of refractile (R) bodies and their genetic determinants: relationship of type 51 R bodies to R bodies produced by Pseudomonas taeniospiralis.

The relationship of type 51 refractile (R) bodies to R bodies produced by Pseudomonas taeniospiralis was investigated. Proteins associated with type 51 R bodies were not serologically cross-reactive with proteins associated with R bodies from P. taeniospiralis. The genetic determinants for type 51 R bodies did not exhibit close homology with DNA sequences from P. taeniospiralis.

Organization and expression of genetic determinants for synthesis and assembly of type 51 R bodies.

Type 51 R bodies are produced by all bacterial endosymbionts (Caedibacter taeniospiralis) of Paramecium tetraurelia that confer the hump-killer trait upon their hosts. Type 51 R-body synthesis by C. taeniospiralis is required for expression of the hump-killer trait. The genetic determinants for type 51 R-body synthesis by C. taeniospiralis 47 have been cloned and expressed in Escherichia coli. In this communication we describe three species of polypeptides required for R-body synthesis and the organization of their genetic determinants. Each polypeptide species is controlled by a separate gene that is expressed as an independent transcriptional unit possessing regulatory signals that are recognized by E. coli. Two polypeptide species of 10 and 18 kilodaltons are required for R-body synthesis but apparently are not structural subunits. The third polypeptide species (13 kilodaltons) is the major structural subunit. R-body assembly involves polymerization reactions that result in high-molecular-mass polypeptide complexes, primarily composed of the 13-kilodalton polypeptide species, that appear to be the result of covalent cross-linking between structural subunits. The results presented here have been suggested to apply to the assembly and structure of all type 51 R bodies, but not necessarily to other R-body types.

Extrachromosomal elements of extrachromosomal elements of Paramecium and their extrachromosomal elements.

Expression of killer traits in Paramecium is due to a complex interaction between the lower eukaryote host and two or three elements that can be viewed either as extrachromosomal elements or as endosymbionts. In all cases, the determinants of the killer trait are carried by obligate bacterial endosymbionts belonging to the genus Caedibacter. However, the actual genetic determinants for expression of these traits are not an integral part of the symbiont genome. They are located on extrachromosomal genetic elements (plasmids or bacteriophages) which essentially are molecular endosymbionts of Caedibacter. In the case of the plasmids, they are associated with yet another set of extrachromosomal genetic elements, which are transposons. These transposons have been observed to move into new sites in the plasmids and even to disrupt expression of R body production and the killer trait. Thus, the transposons can be considered either as extrachromosomal elements of extrachromosomal elements (plasmids) of extrachromosomal elements (C. taeniospiralis) of paramecia, or as molecular parasites of molecular endosymbionts (plasmids) of bacterial endosymbionts of paramecia.

Instrumental analysis of trace elements in thumbnails of human subjects.

Human thumbnails were analyzed for trace elements by instrumental analysis using thermal neutron activation technique. The average concentration of metals studied in clinically symptom-free adult female and male subjects were: zinc, 184 vs. 153 ppm; chromium, 6.8 vs. 4.2; selenium, 0.9 vs. 0.6; gold, 2.6 vs. 0.4; mercury, 1.9 vs. 0.4; silver, 0.7 vs. 0.3; cobalt, 0.07 vs. 0.04. A summary of literature reported concentration of metals in human nail is also presented.