Comparison of the evolutionary distances among syngens and sibling species of Paramecium

The morphospecies of the genus Paramecium have several mating type groups, so-called syngens, composed of cells of complementary mating types. The Paramecium aurelia complex is composed of 15 sibling species assigned to the species from the syngen. To increase our understanding of the evolutionary relationships among syngen and sibling species of the genus Paramecium, we investigated the gene sequences of cytosol-type hsp70 from 7 syngens of Paramecium caudatum and 15 sibling species of P. aurelia. Molecular phylogenetic trees indicated that the P. aurelia complex could be divided into four lineages and separated into each sibling species. However, we did not find any obvious genetic distance among syngens of P. caudatum, and they could only be separated into two closely related groups. These results indicated that the concept of syngens in P. caudatum differs quite markedly from that of the P. aurelia complex. In addition, we also discuss the relationships among these species and other species, Paramecium jenningsi and Paramecium multimicronucleatum, which were once classified as varieties of P. aurelia.     

Characterization and purification of a soluble protein controlling Ca-channel activity in paramecium

The analysis of the voltage-sensitive Ca++ channel of the unicellular eucaryote, Paramecium has been extended to a biochemical level based on recent observations that the transfer of cytoplasm from wild-type cells into mutants lacking Ca++-channel function ("pawn" in P. tetraurelia and "CNR" in P. caudatum) causes mutant cells to regain Ca++-channel function. We have microinjected various cytoplasmic fractions into mutant cells and measured the restored Ca++-channel function using a convenient behavioral assay. Following the "curing" activity, we characterized and purified the component from wild-type cytoplasm that can restore the function missing in cells carrying mutations in the cnrC gene. The curing factor is not an RNA, but a heat-labile, -SH-containing protein that appears to affect existing mutant channels on the ciliary membrane. We have purified this factor over 500-fold from the soluble cytoplasm using conventional techniques. The protein is of low apparent molecular weight (less than 30,000 daltons), acidic, soluble, and does not have the properties of calmodulin.     

Electrokaryotypes of macronuclei of several Paramecium species

A comparative study of macronuclear DNA molecules from the following Paramecium species: the P. aurelia complex, P. caudatum, P. bursaria, P. putrinum and P. multimicronucleatum was performed using pulsed-field gel electrophoresis. The electrophoretic pattern was constant and unique for each species, and is referred to herein as its electrokaryotype. Large differences were observed between Paramecium species according to the range and major size of macronuclear DNA fragments, while different strains of the same species, even belonging to different syngens, were characterized by the same electrokaryotype. In this respect sibling species from the P. aurelia complex are as similar as syngens in other Paramecium species, but are unlike conventional species. The principles and value of electrokaryotype analysis for application to ciliates are discussed.     

Relationships of new sibling species of Paramecium jenningsi based on sequences of the histone H4 gene fragment

Paramecium jenningsi Diller & Earl, 1958 belongs to the "aurelia" subgroup of the genus, together with Paramecium caudatum, Paramecium multimicronucleatum, Paramecium schewiakoffi and species of the Paramecium aurelia complex. The original assumption that the morphospecies P. jenningsi was a single genetic species was questioned because a comparison of genome analyses suggested the possibility that this morphospecies contained two sibling species. To refine understanding of relationships between the strains of P. jenningsi, a molecular phylogenetic analysis was conducted using H4 gene sequences. Some polymorphic sites were found among the compared sequences, and specific patterns of single nucleotide polymorphism (SNP) markers characterize two groups of strains of P. jenningsi. Phylogenetic trees constructed by different methods identified two clearly different groups (from Japan and mainland Asia) whatever the method used. The sequences of the H4 gene analyzed in the present study are closely related, and provide a good subject for phylogenetic analysis. The presence of two isolated groups of strains in the P. jenningsi group can reveal the evolutionary relationship between them; it confirms the presence of two sibling species among the known strains of P. jenningsi, and the close relationships between them and species of the P. aurelia complex.     

ARDRA and RAPD-fingerprinting reject the sibling species concept for the ciliate Paramecium caudatum (Ciliophora, Protoctista)

Morphologically indistinguishable sibling species also known as syngens are a characteristic taxonomic feature of the ciliate genus Paramecium . This has been convincingly demonstrated for the P. aurelia species complex. For a long time this feature has also been assumed for P. caudatum . Classical morphology based techniques of taxonomic analysis are often inefficient to study sibling specie. We therefore investigated 14 P. caudatum strains of seven supposedly different syngens using random amplified polymorphic DNA (RAPD)-fingerprinting and amplified ribosomal DNA restriction analyses (ARDRA, Riboprinting). The RAPD patterns revealed by five different random primers were similar between the different strains of the same syngen (similarity index ranging from 73 to 91%) and also between strains of supposedly different syngens (similarity index ranging from 67 to 91%). The amplified 18S rRNA-fragments of supposedly different syngens, as well as the restriction patterns of these fragments digested by five different endonucleases, were identical for all investigated P. caudatum stains. Consequently we reject the sibling species hypothesis for P. caudatum . According to our molecular analysis, P. caudatum is not a species complex, but just one single species.     

Intraspecific genetic variation in Paramecium revealed by mitochondrial cytochrome C oxidase I sequences

Studies of intraspecific genetic diversity of ciliates, such as population genetics and biogeography, are particularly hampered by the lack of suitable DNA markers. For example, sequences of the non-coding ribosomal internal transcribed spacer (ITS) regions are often too conserved for intraspecific analyses. We have therefore identified primers for the mitochondrial cytochrome c oxidase I (COI) gene and applied them for intraspecific investigations in Paramecium caudatum and Paramecium multimicronucleatum. Furthermore, we obtained sequences of the ITS regions from the same strains and carried out comparative sequence analyses of both data sets. The mitochondrial sequences revealed substantially higher variation in both Paramecium species, with intraspecific divergences up to 7% in P. caudatum and 9.5% in P. multimicronucleatum. Moreover, an initial survey of the population structure discovered different mitochondrial haplotypes of P. caudatum in one pond, thereby demonstrating the potential of this genetic marker for population genetic analyses. Our primers successfully amplified the COI gene of other Paramecium. This is the first report of intraspecific variation in free-living protozoans based on mitochondrial sequence data. Our results show that the high variation in mitochondrial DNA makes it a suitable marker for intraspecific and population genetic studies.     

Screening procedure for complementation-dependent mutants of vesicular stomatitis virus.

To isolate new types of vesicular stomatitis virus (VSV) mutants, a four-stage screen was developed which identifies and characterizes mutants capable of complementing the defect in the VSV temperature-sensitive mutant tsG11. Two types of mutants of VSV, Indiana serotype, have been found by using the screen; they are new temperature-sensitive mutants which are, of necessity, not in complementation group I and mutants which do not produce plaques under conditions of single infection at 31 C (the normal permissive temperature) and are, therefore, called complementation-dependent mutants. The newly isolated, temperature-sensitive mutants fall into three complementation groups, two of which are congruent with known complementation groups; the newly identified group extends to six the number of complementation groups of VSV Indiana. The nature of the complementation-dependent mutants has not been established, but one was shown to not contain a significant deletion in its nucleic acid.     

Transfection of the cloned human excision repair gene ERCC-1 to UV-sensitive CHO mutants only corrects the repair defect in complementation group-2 mutants

The human DNA-excision repair gene ERCC-1 is cloned by its ability to correct the excision-repair defect of the ultraviolet light- and mitomycin-C-sensitive CHO mutant cell line 43-3B. This mutant is assigned to complementation group 2 of the excision-repair-deficient CHO mutants. In order to establish whether the correction by ERCC-1 is confined to CHO mutants of one complementation group, the cloned repair gene, present on cosmid 43-34, was transfected to representative cell lines of the 6 complementation groups that have been identified to date. Following transfection, mycophenolic acid was used to select for transferants expressing the dominant marker gene Ecogpt, also present on cosmid 43-34. Cotransfer of the ERCC-1 gene was shown by Southern blot analysis of DNA from pooled (500-2000 independent colonies) transformants of each mutant. UV survival and UV-induced UDS showed that only mutants belonging to complementation group 2 and no mutants of other groups were corrected by the ERCC-1 gene. This demonstrates that ERCC-1 does not provide an aspecific bypass of excision-repair defects in CHO mutants and supports the assumption that the complementation analysis is based on mutations in different repair genes.     

Protein phosphatase 2C is involved in the cAMP-dependent ciliary control in Paramecium caudatum

Forward swimming of the Triton-extracted model of Paramecium is stimulated by cAMP. Backward swimming of the model induced by Ca(2+) is depressed by cAMP. Cyclic AMP and Ca(2+) act antagonistically in setting the direction of the ciliary beat. Some ciliary axonemal proteins from Paramecium caudatum are phosphorylated in a cAMP-dependent manner. In the presence of cAMP, axonemal 29- and 65-kDa polypeptides were phosphorylated by endogenous A-kinase in vitro. These phosphoproteins, however, were not dephosphorylated after in vitro phosphorylation, presumably because of the low endogenous phosphoprotein phosphatase activity associated with isolated axonemes. We purified the protein phosphatase that specifically dephosphorylated the 29- and 65-kDa phosphoproteins from Paramecium caudatum. The molecular weight of the protein phosphatase was 33 kDa. The protein phosphatase had common characteristics as protein phosphatase 2C (PP2C). The characteristics of the protein phosphatase were the same as those of the PP2C from Paramecium tetraurelia (PtPP2C) [Grothe et al., 1998: J. Biol. Chem. 273:19167-19172]. We concluded that the phosphoprotein phosphatase is the PP2C from Paramecium caudatum (PcPP2C). The PcPP2C markedly accelerated the backward swimming of the Triton-extracted model in the presence of Ca(2+). On the other hand, the PcPP2C slightly depressed the forward swimming speed. This indicates that the PP2C plays a role in the cAMP-dependent regulation of ciliary movement in Paramecium caudatum through dephosphorylation of 29- and/or 65-kDa regulatory phosphoproteins by terminating the action of cAMP.     

Isolation and Characterization of Temperature-Sensitive Mutants of Vesicular Stomatitis Virus, New Jersey Serotype

Forty-eight temperature-sensitive (ts) mutants have been isolated from a wild-type strain of the New Jersey serotype of vesicular stomatitis virus (VSV) after exposure to the base analogue mutagen 5-fluorouracil. Of these mutants, 47 have been classified into 6 nonoverlapping complementation groups containing 21, 17, 4, 3, 2, and 1 mutant, respectively (1 mutant remaining unallocated). The ribonucleic acid (RNA) phenotype of 23 of these mutants has been established. Four of the six groups contain one or more mutants unable to synthesize detectable amounts of viral RNA under restrictive conditions (39 C). No complementation was observed in mixed infection with ts mutants from the five established complementation groups of the Indiana serotype of VSV.     

Isolation and characterization of temperature-sensitive mutants of adenovirus type 7.

Fifty temperature-sensitive mutants, which replicate at 32 degrees C but not at 39.5 degrees C, were isolated after mutagenesis of the vaccine strain of adenovirus type 7 with hydroxylamine (mutation frequency of 9.0%) or nitrous acid (mutation frequency of 3.8%). Intratypic complementation analyses separated 46 of these mutants into seven groups. Intertypic complementation tests with temperature-sensitive mutants of adenovirus type 5 showed that the mutant in complementation group A failed to complement H5ts125 (a DNA-binding protein mutant), that mutants in group B and C did not complement adenovirus type 5 hexon mutants, and that none of the mutants was defective in fiber production. Further phenotypic characterization showed that at the nonpermissive temperature the mutant in group A failed to make immunologically reactive DNA-binding protein, mutants in groups B and C were defective in transport of trimeric hexons to the nucleus, mutants in groups D, E, and F assembled empty capsids, and mutants in group G assembled DNA-containing capsids as well as empty capsids. The mutants of the complementation groups were physically mapped by marker rescue, and the mutations were localized between the following map coordinates: groups B and C between 50.4 and 60.2 map units (m.u.), groups D and E between 29.6 and 36.7 m.u., and group G between 36.7 and 42.0 m.u. or 44.0 and 47.0 m.u. The mutant in group A proved to be a double mutant.     

Genetic complementation of propionyl-CoA carboxylase deficiency in cultured human fibroblasts.

Propionyl-CoA carboxylase (PCC) deficiency is an inherited metabolic disorder showing considerable variability of expression. We have investigated the possibility that there is a genetic basis for the clinical heterogeneity in this disorder by examining complementation in Sendai virus mediated heterokaryons of mutant fibroblast strains. Restoration of PCC activity was monitored in individual multinucleate cells in situ using a radioautographic procedure which detects the incorporation of 14C-propionate into trichloracetic acid precipitable material. Each mutant strain incorporated negligible amounts of radioactivity compared to control strains. Activity was not restored when different mutants were mixed without virus or when homokaryons were produced by self-fusion. Seven mutant strains were fused in all pairwise combinations and examined for increased 14C-propionate incorporation in heterokaryons. Two main complementation groups were revealed. One group was composed of three mutants. The other was a complex group composed of four mutants in which intragroup complementation was demonstrated. Two mutants showing excellent complementation by radioautography were examined for complementation by the direct assay of PCC ACTIVITY. The enzyme activity of virus-treated preparations with 23% multinucleate cells was 183 U (pmol/min/mg protein) compared to 16 U for the untreated mixture (normal range 450-850 u). We conclude that PCC deficiency resulted from mutations of heterogeneous origin, although the classification of mutants into complementation groups did not correlate with patterns of clinical heterogeneity.     

Progeny resulting from complementation between mutants of vesicular stomatitis virus.

The complementation properties of the virus progeny released from cells mixedly infected with mutants of vesicular stomatitis virus belonging to four different complementation groups have been examined. The group IV mutant, tsW16B, was tested in combinations with three group I mutants (tsW4, tsW28, and tsG11), one group II mutant (tsG22), and one group III mutant (tsW29). Virus stocks were grown from isolated plaques appearing on the cell monolayers used to assay the mixed infection yields and tested, in a second series of mixed infections, for their ability to complement each of the two parents. It was found that the virus harvested from each one of the first series of mixed infections contained mutants of both parental types.     

Mutants of Paramecium Defective in Chemokinesis to Folate

Ten mutant lines of Paramecium tetraurelia defective in attraction to folate were isolated and examined. All mutants were normal in response to other attractants and repellents tested. One mutant was able to accumulate in folate given sufficient time. All mutations were recessive and behaved as single site Mendelian lesions. Complementation tests indicate that the mutants fall into three complementation groups. Mutants of Group 2 fall into two phenotypic classes and probably represent two alleles of the mutated fol² gene. Possible sites of the mutants'' blocks in chemoresponse are discussed.     

Genetic Map of Bacteriophage α

Temperature-sensitive mutants of phage alpha were obtained by means of various mutagens and assigned to 25 complementation groups. Temperature-sensitive mutants belonging to 21 complementation groups and a mutant giving turbid plaques were used to perform two- and three-factor crosses. Seventeen of the cistrons and the turbid mutant were shown to belong to the same linear linkage group, which showed no signs of circularity. The remaining four unlinked cistrons showed peculiarities in their recombination properties. Genes which are known to be expressed earlier apear to be grouped together in a terminal segment of the linkage group.     

Induction of Conjugation by Methyl Cellulose in Paramecium

ABSTRACT An improved method has been developed for the induction of selfing conjugation in Paramecium. Methyl cellulose induces selfing conjugation simply and efficiently in all species examined. Induction of conjugation by methyl cellulose was characterized in P. caudatum , where it occurred only in sexually mature, mating reactive cells. Conjugation produced sexual offspring and the time course of nuclear processes was substantially the same as that in natural conjugation between cells of complementary mating types. The method is useful for genetic studies in a wide variety of Paramecium species including P. caudatum, P. tetraurelia, P. multimicronucleatum and P. bursaria.     

A Female Sterile Screen of the Drosophila Melanogaster X Chromosome Using Hybrid Dysgenesis: Identification and Characterization of Egg Morphology Mutants

We have conducted a hybrid dysgenic screen of the X chromosome for mutations affecting female fertility, with particular attention to those causing abnormal egg and eggshell morphology. In a screen of 4017 dysgenic strains, 398 mutants derived from 168 different germ lines were isolated and assigned to eight classes according to their diverse phenotypes. One interesting class consists of mutants that block oogenesis at specific stages. Our analysis has focused on mutations affecting eggshell formation, including mutants that lay morphologically abnormal sterile eggs as well as those that lay no eggs but exhibit blocks in the late stages of oogenesis. A subset of 48 mutants was assorted into 30 allelic groups by inter se complementation and genetically localized by interval mapping. Two multiallele complementation groups, de1 (7 alleles) and ne1 (8 alleles), were identified as well as five two-allele complementation groups. A search for alleles among mutants generated in other female sterile screens was unsuccessful, pointing to the distinctive nature of the dysgenic mutant collection. The single case of allelism determined in this study was one with a lethal allele of the Broad-Complex, l(1)npr, suggesting a possible involvement of ecdysone in choriogenesis. A subset of 18 dysgenic strains was analyzed for P element hybridization and 16 of these were found to have hybridization signals in the appropriate cytogenetic interval. By examining these signals in two or more alleles of the same complementation group, we have been able to tentatively localize two mutations. Light and electron microscopy of the eggshell in 43 different strains has revealed a variety of effects. The respiratory appendages were defective in 27 of these mutants. Effects on the ultrastructure of the main body of the endochorion were not strongly correlated with the appendage defects, and could be classified as minor (14 mutants) or major (16 mutants). Although 13 mutants showed no ultrastructural chorion defects, six of these had defective respiratory appendages.     

Isolation and preliminary characterization of temperature-sensitive mutants of Newcastle disease virus.

Temperature-sensitive (ts) mutants of Newcastle disease virus have been isolated and characterized genetically (complementation), biochemically (RNA synthesis) and biologically (fusion from within and hemadsorption). Fifteen of these mutants have been divided into five complementation groups. Groups A (five mutants) and E (one mutant) are ts for RNA synthesis (RNA-) as well as for the other functions. Group B contains four RNA+ mutants of which one is ts for fusion, one for hemadsorption and two for neither function. Group C contains one RNA+ mutant which is a poor cell fuser. Group D contains two RNA+ mutants which are ts for fusion. In addition, two noncomplementing mutants (group BC) fail to complement both group B and group C mutants while exhibiting complementation with mutants in groups A, D, and E.     

Identification of New Genes Required for Meiotic Recombination in Saccharomyces Cerevisiae

Mutants defective in meiotic recombination were isolated from a disomic haploid strain of Saccharomyces cerevisiae by examining recombination within the leu2 and his4 heteroalleles located on chromosome III. The mutants were classified into two new complementation groups (MRE2 and MRE11) and eight previously identified groups, which include SPO11, HOP1, REC114, MRE4/MEK1 and genes in the RAD52 epistasis group. All of the mutants, in which the mutations in the new complementation groups are homozygous and diploid, can undergo premeiotic DNA synthesis and produce spores. The spores are, however, not viable. The mre2 and mre11 mutants produce viable spores in a spo13 background, in which meiosis I is bypassed, suggesting that these mutants are blocked at an early step in meiotic recombination. The mre2 mutant does not exhibit any unusual phenotype during mitosis and it is, thus, considered to have a mutation in a meiosis-specific gene. By contrast, the mre11 mutant is sensitive to damage to DNA by methyl methanesulfonate and exhibits a hyperrecombination phenotype in mitosis. Among six alleles of HOP1 that were isolated, an unusual pattern of intragenic complementation was observed.