Itaru Usuki

Structure and evolution of Paramecium hemoglobin genes

Hemoglobin (Hb) genes have been cloned from three different species of ciliated protists, P. multimicronucleatum, P. triaurelia and P. jenningsi. Southern blotting of the genomic DNAs using the P. caudatum Hb cDNA showed both intraspecies variation in different stocks of P. caudatum and interspecies variation within the genus Paramecium. The isolated Hb genes were composed of 118, 117 and 117 codons, and interrupted by a short intron with 27, 29 and 29 bp at the same position, in P. multimicronucleatum, P. triaurelia and P. jenningsi, respectively. This suggests that the one-intron and two-exon structure has been conserved in the Hb genes in this genus. The amino acid sequences of the Paramecium Hbs were more than 87% identical to one another and homologous to those from the other ciliated protists Tetrahymena thermophila and T. pyriformis, the green alga Chlamydomonas eugametos, and the cyanobacterium Nostoc commune Hbs, all of which consist of about 120 amino acid residues (120-aa group). In particular, the amino acid sequences of the P. triaurelia and P. jenningsi Hbs were the same, although there were 20 nucleotide differences between the coding regions in the two genes. A maximum likelihood inference as to the phylogenetic relationships among these genes suggests that the Paramecium Hbs genes have evolved more rapidly than the other genes in the 120-aa group, and that P. triaurelia and P. genningsi are sibling species and the P. aurelia complex became a small cell after it separated from P. jenningsi.

Species-specificity of Chlorella for establishment of symbiotic association with Paramecium bursaria — Does infectivity depend upon sugar components of the cell wall?

Summary The symbiotic association between Paramecium bursaria and Chlorella was studied by means of infection experiments, using 20 strains taken from 9 different species of Chlorella and 5 stocks of algae-free P. bursaria which originated from different localities. At the time of infection, the ciliate had ingested numerous algal cells in all stocks that had been examined. Although the ingested algal cells in 5 species completely disappeared from the ciliate within several days, the cells of C. vulgaris, C. kessleri, C. sorokiniana and zoochlorellae were retained in all stocks of the ciliate even after a period of 2 months. It has been confirmed that all “infection-capable” Chlorella species including zoochlorella are distinguished by the presence of glucosamine as a chemical component in their rigid walls, whereas the rigid walls of “infection-incapable” species contain glucose and mannose. When these facts are taken into consideration, it may be shown that the various species of “Chlorella” hold the key to determine the selection of alga by the ciliate. Moreover, the presence of glucosamine in the rigid wall of alga would seem to be a prerequisite in determining the symbiotic association between Paramecium bursaria and Chlorella.

Disparity of native oxyhemoglobin components isolated from Paramecium caudatum and Paramecium primaurelia

Abstract 1. 1. Native oxyhemoglobin components were isolated chromatographically from Paramecium caudatum and Paramecium primaurelia, and some properties of the isolated components were investigated. 2. 2. P. caudatum was endowed with one homogeneous hemoglobin component, while the hemoglobin in P. primaurelia was resolved into three heterogeneous components being two main and one minor. 3. 3. Spectral properties of the isolated hemoglobin components were quite similar to each other. The isolated components, however, were distinctly different in electrophoretic mobilities. 4. 4. Molecular weight of the isolated hemoglobin components was estimated to be about 11,000.

Zinc as modulator of oxygenation function and stabilizer of quaternary structure in earthworm hemoglobin

Blood of the earthworm Pheretima hilgendorfi contains 10.7 mM Ca, 2.0 mM Mg, 75.5 mM Na, 5.9 mM K, 0.9 mM Zn and 0.3 mM Mn. Some of these cations cannot be removed completely from the blood by dialysis, and 36 atoms of Ca, 1-3 atoms of Mg and 1-2 atoms of Zn per 164 atoms of Fe were retained in purified preparation of the hemoglobin (Hb). At physiological pH, oxygen affinity and the Hill coefficient at half saturation (n1/2 value) of the Hb increased in the presence of 100 mM of Ca, Mg or Na. These effects were in the order of Ca > Mg > Na. At physiological concentration, however, the effect of each of these three cations on oxygenation was rather small. On the other hand, Zn gave a remarkable effect at less than 1 mM. This suggests a possibility that Zn acts as a primal modulator for the oxygenation function of the Hb in vivo. Oxygenation data at various pH values in the presence of each cation strongly suggest that Zn binds to a site different from those for the other three cations. Zn at a concentration of only 1 mM protected considerably the dissociation of the whole molecule to smaller components at alkaline pH and Zn thus contributes to the stabilization of the quaternary structure of the Hb.

Molecular cloning of the cDNA for the major hemoglobin component from paramecium caudatum

Nucleotide sequence of the cDNA for the major hemoglobin component of Paramecium caudatum was determined. An oligonucleotide was synthesized on the basis of the amino acid sequence, and the Paramecium cDNA library constructed in phage lambda gt11 was screened with it. Three positive clones, of which insert sizes were 0.4, 0.6, and 0.9 kbp, were obtained. Sequence analysis made clear that the 0.4-kbp cDNA retains a full length of the nucleotides encoding 116 amino acid residues, and that in the coding region it contains four TAA codons which are known to encode glutamine.

Inter- and intrasyngenic variation of Paramecium hemoglobin—I. Paramecium aurelia complex

Abstract 1. 1. Hemoglobins (Hbs) from Paramecium aurelia complex showed a considerable degree of inter- and intrasyngenic variations by polyacrylamide gel electrophoresis (PAGE). 2. 2. All of the Hbs from various stocks of different syngens were resolved into definite 12 variants, which gave expression to a difference in the mobility by PAGE. 3. 3. An argument that the multiplicity of Paramecium Hb may be an artifact caused by endogenous proteases, was not supported by the facts obtained on a crude Hb sample prepared under the presence of antipain, leupeptin, pepstatin and phosphoramidon in sufficient concentrations. 4. 4. The Hb variants can be subdivided into two groups by their molecular weights, taking either value of 11,000 and 13,000.

Reinvestigation of the hemoglobins from Paramedium jenningsi, P. multimicronucleatum and P. caudatum

Abstract 1. 1. The Hb from Paramecium jenningsi was resolved into five to six variants by PAGE. The Hb profile distinctly differed from that of P. caudatum , but it was similar to those of some stocks in P. multimicronucleatum . 2. 2. A reliable discrimination between the Hbs from P. jenningsi and P. multimicronucleatum was achieved by IEF, in which the major variant in the former species showed a lower pI value than that in the latter one and the presence of a unique minor variant in the former species was demonstrated at the position corresponding to pI 8.8. 3. 3. Relative concentration of the Hb variants showed a noticeable variation among the stock in P. multimicronucleatum . 4. 4. Besides the major and minor Hb variants with acidic pIs, one or two basic variants measuring as high a pI as 9.8 or more were detected in P. caudatum .

Inter- and intrasyngenic variation of paramecium hemoglobin—II. Paramecium caudatum, paramecium jenningsi and Paramecium multimicronucleatum

Abstract 1. 1. Paramecium caudatum contains a multiple form of hemoglobin (Hb), which is composed of high concentration of a major variant and only a small amount of several minor variants. 2. 2. The electrophoretic mobility and molecular weight of the major variant in P. caudatum are identical to a well-characterized variant, Hb10, in P. aurelia complex. 3. 3. P. caudatum shows no intersyngenic variation of the Hb between syngen 1 and syngen 3. Intrasyngenic variation is not significant, although some deviations in the variant concentrations among different stocks are recognized. 4. 4. P. jenningsi generates similar Hbs to those of P. caudatum. 5. 5. The Hb in P. multimicronucleatum is characterized with a major variant corresponding to Hb11 and several minor variants corresponding to Hb2 and Hb12 in P. aurelia complex.

Physicochemical properties of the extracellular hemoglobin from the planorbid snail, Indoplanorbis exustus

Abstract 1. 1. Physicochemical properties of the extracellular hemoglobin from the planorbid snail Indoplanorbis exustus were investigated. 2. 2. The wavelengths of absorption maxima and millimolar absorption coefficients of the hemoglobin derivatives were determined. Amino acid composition of the hemoglobin was also examined. 3. 3. The native hemoglobin showed a sedimentation constant of 35.5S, and its molecular weight which was determined by sedimentation equilibrium was 2.04 × 10 6 . 4. 4. Iron and heme contents of the hemoglobin were 0.300% and 2.89%, which corresponded to a minimum molecular weight of 18,600 and 21,300, respectively. 5. 5. SDS-PAGE of the hemoglobin gave one main band corresponding to 280 kDa in the unreduced state and one main band corresponding to 170 KDa in the reduced one. 6. 6. The results obtained may indicate that the native hemoglobin is composed of 8 submultiples of 2.8 × 10 5 daltons or 12 polypeptide chains of 1.7 × 10 5 daltons.

Respiratory inhibition by cyanide and salicylhydroxamic acid on the three species of Paramecium in stationary growth phase

Abstract 1. 1. Paramecia in stationary growth phase consumed oxygen at a constant rate, exhibiting 21.5 nM/min/mg protein of the cells at 27°C. 2. 2. Respiratory inhibition by 1 mM KCN was significantly high in the following order: P . caudatum (96%) > P . multimicronucleatum (87%) > P . tetraurelia (77%). 3. 3. In the presence of cyanide (CN), 1 mM salicylhydroxamic acid (SHAM) suppressed a large part of the CN-insensitive respiration in the cells, and the cumulative inhibition by CN and SHAM reached approx. 95% in all of the Paramecium species examined. SHAM in the absence of CN caused no detectable change in the respiration of the cells. 4. 4. Oxygen affinity for the CN-sensitive respiration was far stronger than that for the CN-insensitive respiration in the whole cells of Paramecium .