Inheritance of the group I rDNA intron in Tetrahymena pigmentosa.

We have previously argued from phylogenetic sequence data that the group I intron in the rRNA genes of Tetrahymena was acquired by different Tetrahymena species at different times during evolution. We have now approached the question of intron mobility experimentally by crossing intron+ and intron- strains looking for a strong polarity in the inheritance of the intron (intron homing). Based on the genetic analysis we find that the intron in T. pigmentosa is inherited as a neutral character and that intron+ and intron- alleles segregate in a Mendelian fashion with no sign of intron homing. In an analysis of vegetatively growing cells containing intron+ and intron- rDNA, initially in the same macronucleus, we similarly find no evidence of intron homing. During the course of this work, we observed to our surprise that progeny clones from some crosses contained three types of rDNA. One possible explanation is that T. pigmentosa has two rdn loci in contrast to the single locus found in T. thermophila. Some of the progeny clones from the genetic analysis were expanded for several hundred generations, and allelic assortment of the rDNA was demonstrated by subcloning analysis.     

Updating rDNA Restriction Enzyme Maps of Tetrahymena Reveals Four New Intron-Containing Species1

The extrachromosomal rDNA molecules from a number of Tetrahymena strains wered racterized by restriction enzyme mapping using three different restriction enzymes combined with gel blotting and hybridization analysis. Strains from four out of six recently described species were found to contain an intron in the 26s rRNA coding region. The evolutionary relationship among the species of the T. pyriformis complex was examined on the basis of the rDNA maps with emphasis on similarities between two of the new species and the widely studied T. thermophila and T. pigmentosa. Examination of a large number of T. pigmentosa strains showed this species to exhibit an unusual polymorphism with respect to its rDNA. It is suggested that recombinational cross-over events play a role in the formation of new rDNA alleles in this species.     

Under-replication of intron+ rDNA cistrons in polyploid nurse cell nuclei of Calliphora erythrocephala

We have examined the possibility that the intron-containing (intron⁺) rDNA cistrons of Dipteran flies are active in the germ-line derived polyploid nurse cell nuclei of the ovarian follicles. Using the organism, Calliphora erythrocephala, we describe here a procedure which yields very pure nurse cell nuclei and compare the intron-free (intron^–) and intron⁺ rDNA cistron contents of nurse cell nuclei prepared by this procedure to those of 2–18 h embryo nuclei and 3 day pupal nuclei. DNA from three preparations of each nuclear type was examined and the intron^– and intron⁺ cistron contents quantitated using a Southern transfer procedure. The number of intron^– and intron⁺ rDNA cistrons per haploid genome in the presumed diploid 2–18 h embryo DNA was first established, and then the intron^– and intron⁺ cistron contents of nurse cell nuclear DNA and 3 day pupal DNA were determined relative to these values.The intron^– cistron content of nurse cell nuclear DNA was indistinguishable from that of embryonic DNA but the intron⁺ cistrons showed an 8-fold under-replication relative to the presumed diploid DNA. A slight under-representation of the intron^– cistrons and 3-fold under-replication of the intron⁺ cistrons were demonstrated for 3 day pupal DNA. These findings strongly suggest that intron⁺ rDNA cistrons are non-functional in nurse cell nuclei and substantiate the generality of this implication for the whole organism during early pupal life.     

The ribosomal DNA of Calliphora erythrocephala: The cistron classes of total genomic DNA

The composition of the genome set of ribosomal DNA cistrons in Calliphora erythrocephala (a Dipteran fly) has been analyzed. In contrast to previously cloned fragments of the rDNA (see Beckingham & White, 1980), the great majority of the rDNA cistrons do not contain introns in the 28 S β coding region. In the strain of flies studied, however, most cistrons fall into two discrete length classes that are present in approximately equal amounts in the genome. These results from distinct size variants of the non-transcribed spacer in the cistron population.The major genome class of intron-containing (intron⁺) rDNA cistrons was found to constitute approximately 5% of all cistrons and to contain introns of 6·1 × 10³ base-pairs. Interestingly, the intron⁺ cistrons were shown to be clustered within the rDNA and to contain a different population of non-transcribed spacer/external transcribed spacer (NTS + ETS) regions to that seen amongst the intron^? cistrons. The implications of these findings in relation to the mechanisms that maintain homogeneity within tandemly repeated gene sets are discussed.Some evidence for the existence of intron sequence DNA outside the rDNA is presented.     

Extrachromosomal ribosomal RNA genes in Tetrahymena: structure and evolution

The macronuclear ribosomal RNA genes from a number of strains within several species of Tetrahymena have been characterized. Restriction enzyme analysis revealed that individual strains all contained entirely homogeneous populations of extrachromosomal palindromic ribosomal DNA, varying in molecular size from 12 × 10⁶ to 14 × 10⁶ in different strains. Considering that the evolutionary distance among some of the species is estimated to be of the order of 10⁶ years, the rDNA from all the species exhibited a strikingly high similarity in the localization of their restriction sites. Nevertheless, differences both inside and outside the gene region were clearly detectable, showing that the rDNA sequences have diverged in all species.Genetic polymorphism with respect to rDNA structure exists in Tetrahymena, but seems to be rare. In only two out of five species examined (T. borealis and T. pigmentosa) interbreeding strains differing in rDNA structure were found. While the differences detected in the T. borealis rDNA were confined to a small size difference located at the non-coding ends of the molecule, several differences were detected in the rDNA from the T. pigmentosa strains. One of the differences was shown to be due to the presence of an intervening sequence within the structural gene for 26 S rRNA in some of the strains. An intervening sequence of similar size located at the same position within the 26 S gene region was found by R-loop mapping in all strains of the species T. thermophila. Restriction enzyme analysis indicates that the rDNA from two other species contains a similar intervening sequence, and we therefore suggest that the size and localization of the intervening sequence is evolutionarily stable. The two intervening sequences examined so far, however, are not identical, as revealed by restriction enzyme mapping.     

Suicide is not the inevitable outcome of “perpetual” selfing in tetrahymenines collected from natural habitats

A significant fraction of the Tetrahymena clones isolated from natural habitats self (mating occurs within a clone). Early attempts to study such clones failed because stable subclones were rarely, if ever, observed, and isolated pairs all died. Isozyme analysis revealed that these wild selfers were a diverse group; some were very similar to T. australis, a species with synclonal mating type determination and to T. elliotti, shown recently to have a karyonidal mating type system. One originally stable clone of T. australis included some selfing clones after a few years in our laboratory. Other clones manifested unique zymograms. Subclones isolated from 18 selfer strains were heterogeneous. All subclones of several selfers mated massively at each transfer through 100 fissions. Selfing among subclones of other selfers was highly variable or not observed. Although 77% of the pairs isolated died, and 9% of the pair cultures selfed, 15 selfers yielded some viable nonselfing “immature” progeny. Additional immature progeny were obtained by isolating pairs from macronuclear retention synclones. Although some “immature” progeny eventually selfed, most remained stable. Giemsa staining revealed macronuclear anlagen in nearly all mating pairs and some anomalies. Crosses among the F1 progeny clones of the T. elliotti selfers yield viability data comparable to those from crosses among normal strains. Perhaps perpetual selfing is a mechanism of getting rid of deleterious combinations of genes and uncovering better combinations in homozygous state by playing genetic roulette. © 1992 Wiley-Liss, Inc.     

A SEX DETERMINING MECHANISM IN THE CLOSTERIUM EHRENBERGII (CHLOROPHYTA) SPECIES COMPLEX1

Homozygous mt^?/mt^? diploid clones of the Closterium ehrenbergii Menegh. ex Ralfs species complex were obtained by hypertonic treatment from minus vegetative cells, and mating type segregation ratios in the F₁ progeny of “triploid” zygospores between wild type mt⁺ haploid and mt^?/mt^? homozygous diploui were analyzed. The ratio of plus to minus individuals was 1:4.8, and the ratio of the pairs of opposite mating types to those of minus mating type was 1:2.1. The results clearly show that mt^? is dominant to mt⁺ and that the mating type inheritance in these zygospores follows the triploid-like pattern. The validity of our assumption that the two mating types are determined by one genetic factor (mt^? allele dominant) was confirmed in B₁ progeny analyses as well. The results suggest that this sex determining mechanism is working effectively in the C. ehrenbergii species complex, in which several biological species have evolved through polyploidization.     

Alteration of inhibitory and activating natural killer cell receptor expression on T cells in human immunodeficiency virus-infected Chinese

T cell expression of NKRs can trigger or inhibit cell‐mediated cytotoxicity. However, few studies on T lymphocyte NKR expression in HIV infection exist. Here, we examined the expression patterns of NKG2D, NKG2A, and KIR3DL1 on CD8⁺ and CD3⁺CD8^? cells by multicolor flow cytometry in groups of patients with HIV, AIDS or HAART‐treated AIDS, as well as HIV‐negative normal controls. Individual analysis of KIR3DL1 on CD3⁺CD8⁺ or CD3⁺CD8^? cells revealed no significant differences among any of the groups (P > 0.05). In contrast, the percentage of NKG2A⁺NKG2D^?CD8⁺ T cells was higher in the AIDS group than in the HIV‐negative normal control group (P < 0.01). Meanwhile, the prevalence of NKG2D⁺NKG2A^?CD8⁺T cells was lower in the AIDS group than in HIV‐negative normal controls (P < 0.001). Similar results were also observed for the percentage of NKG2A⁺NKG2D^? on CD3⁺CD8^?cells. However, in contrast to CD8⁺ T cells, the frequencies of NKG2D⁺NKG2A^? on CD3⁺CD8^? cells were higher in AIDS and HIV patients than in HIV‐negative normal controls (P < 0.01, P < 0.05, respectively). The percentage of NKG2A⁺NKG2D^?CD8⁺ T cells was negatively correlated with CD4⁺ T cell counts (r=?0.499, P < 0.01), while the percentage of NKG2D⁺NKG2A^?CD8⁺ T cells was positively correlated with CD4⁺ T cell counts (r= 0.494, P < 0.01). The percentage of NKG2D⁺NKG2A^?CD3⁺CD8^? T cells was also positively correlated with viral load (r= 0.527, P < 0.01) and negatively correlated with CD4⁺ T cell counts (r=?0.397, P < 0.05). Finally, HAART treatment reversed the changes in NKR expression caused by HIV infection. These results indicate that the expression of NKRs on T cells may be correlated with HIV disease progression.     

Computer Graphics Modelling and Molecular Dynamics Simulation of the Core of the Self Splicing Intervening Sequence of the T4 NrdB mRNA in the Presence of the Guanosine Cofactor

Abstract

The active site of the self splicing intron in the T4 nrdB RNA has been modelled on a graphics workstation on the basis of the suggested 3D arrangement of the Tetrahymena IVS¹. This structure was then subjected to energy minimization and molecular dynamics simulation to relax tensions. The cofactor guanosine was docked to a proposed² binding site where it was found to fit well.     

Na+-independent uptake of nutrients inTetrahymena

Summary Cell multiplication rates in cultures ofTetrahymena pyriformis andT. thermophila were independent of the external Na⁺ concentrations at the levels of 0.5, 10 and 22 mM. The Na_i/Na₀ and Cl_i/Cl₀ ratios were determined at the low and high Na⁺ concentrations, and assuming that Cl^– is distributed passively, the electrochemical Na⁺ gradients for the two situations were calculated. The energy in these gradients allows a net co-transport of nutrients only in the high Na⁺ medium. Since the doubling times are independent of the Na⁺ concentrations we conclude thatTetrahymena can obtain its energy for nutrient uptake from other sources than the electrochemical Na⁺ gradient.     

Group I Introns from Zygomycota: Evolutionary Implications for the Fungal IC1 Intron Subgroup

The origins of fungal group I introns within nuclear small-subunit (nSSU) rDNA are enigmatic. This is partly because they have never been reported in basal fungal phyla (Zygomycota and Chytridiomycota), which are hypothesized to be ancestral to derived phyla (Ascomycota and Basidiomycota). Here we report group I introns from the nSSU rDNA of two zygomycete fungi, Zoophagus insidians (Zoopagales) and Coemansia mojavensis (Kickxellales). Secondary structure analyses predicted that both introns belong to the IC1 subgroup and that they are distantly related to each other, which is also suggested by different insertion sites. Molecular phylogenetic analyses indicated that the IC1 intron of Z. insidians is closely related to the IC1 intron inserted in the LSU rDNA of the basidiomycete fungus Clavicorona taxophila, which strongly suggests interphylum horizontal transfer. The IC1 intron of C. mojavensis has a low phylogenetic affinity to other fungal IC1 introns inserted into site 943 of nSSU rDNA (relative to E. coli 16S rDNA). It is noteworthy that this intron contains a putative ORF containing a His–Cys box motif in the antisense strand, a hallmark for nuclear-encoded homing endonucleases. Overall, molecular phylogenetic analyses do not support the placement of these two introns in basal fungal IC1 intron lineages. This result leads to the suggestion that fungal IC1 introns might have invaded or been transferred laterally after the divergence of the four major fungal phyla. Received: 8 February 2001 / Accepted: 1 November 2001     

Heterochromatin and rDNA patterns in Solanum species of the Morelloid and Dulcamaroid clades (Solanaceae)

The heterochromatin distribution and the position of 18-5.8-26S, and 5S rDNA loci were determined in 13 species of Solanum of the Morelloid and Dulcamaroid clades. The CMA/DAPI staining and FISH were employed. Two types of constitutive heterochromatin were determined: CMA⁺/DAPI^? associated to NOR and CMA⁺/DAPI^? distributed as terminal bands. In the Morelloid clade, CMA⁺/DAPI^? bands were found in five species while in the Dulcamaroid clade, only S. angustifidum presented this feature. In the Morelloid clade, two to four 18-5.8-26S rDNA loci occupied terminal positions and two rDNA 5S loci were found with variable positions (terminal, intercalary, and centromeric). In the Dulcamaroid clade, two terminal 18-5.8-26S rDNA loci were detected with the exception of S. salicifolium which possessed four such loci and two to four 5S rDNA loci. Solanum crispum is the only species possessing the 5S in synteny with 18-5.8-26S rDNA loci. Karyotype features chromosome banding pattern as well as the location of ribosomal genes which varied among the species, reflecting the chromosome differentiation and evolutionary divergence. The findings obtained contributed to the development of tools that can be used for establishing chromosomic homeologies among species and hence to clarify their taxonomic relationships.     

Instability in the ctMR2 strain of Drosophila melanogaster: role of P element functions and structure of revertants

Summary Simultaneous multiple transpositions and longterm genetic instability have been described in the ct ^MR2 strain of Drosophila melanogaster and its derivatives. This strain originated from a cross that was dysgenic in the P-M system. While spontaneous instability declined over 2 years, instability has been reactivated by backcross to the progenitor P element bearing strain MRh12/Cy. We show here using germline transformation that active P factor alone cannot mimic the effect of this cross, suggesting that MRh12/Cy contains some other activator. In addition, we have observed that ct ⁺ exceptional progeny arise in the F1 s well as the F2 generations. Molecular analysis of X chromosomes from some ct ⁺ progeny indicates that phenotypic reversion of the ct mutation can arise through two unrelated mechanisms.     

Identification and characterization of Tetrahymena myosin

Myosin was partially purified from ciliated protozoan Tetrahymena pyriformis. Tetrahymena myosin has a fibrous tail with two globular heads at one end and contains 220-kDa heavy chains. The tail length of the molecule (200 nm) is longer than that of myosins from other animals (approximately 160 nm). A sample after HPLC column chromatography containing 220-kDa peptide showed a myosin-specific K⁺-/NH₄⁺-EDTA-ATPase activity. Polyclonal anti-crayfish myosin heavy chain antibody reacted with Tetrahymena 220-kDa myosin heavy chain, and monoclonal anti-pan myosin antibody reacted with Tetrahymena 180-kDa peptide. The isolated 180-kDa peptide was identified as a clathrin heavy chain.     

Changes in ion and water content of individual shoot organs in a salt-tolerant and a salt-sensitive clone of Agrostis stolonifera L. during and subsequent to treatment with sodium chloride

Abstract Individual leaves and stems were analysed for Na⁺, Cl^?, K⁺ and water content in two clones of Agrostis stolonifera differing in salt resistance, during 14 d of treatment with NaCl, 100 and 200 mol m^?3, and a further 7 d in a salt-free medium. Great differences in ion and water content were revealed between individual organs, and organ-by-organ analysis also emphasized the differences between the clones better than whole shoot analysis. In both clones, Na⁺ and Cl^? accumulated to the greatest degree in the older leaves, but for corresponding organs, the concentrations were lower in the more tolerant clone. In the sensitive clone, the lowest leaves dehydrated in 200 mol m^?3 NaCl and failed to recover, while the plants of the more resistant clone maintained viable water content in all organs. In the resistant clone, K⁺ concentration decreased less in response to salt treatment than in the more sensitive clone. For a full appreciation of the plants' reactions, it was found necessary to express the analytical data on several bases, namely, per unit dry-weight, unit water, and total ion-content.     

GENITIC VARIABILITY IN TOXIN POTENCIES AMONG SEVENTEEN CLONES OF GAMBIERDISCUS TOXICUS (DINOPHYCEAE)1

Seventeen clones of the ciguatera-causing dinoflagellate Gambierdiscus toxicus Adachi and Fukuyo were acclimated to the same environments over several months. Significant variance components were detected between non-acclimated and acclimated cultures for cell potencies, yields and reproduction rates. The resultant variance in acclimated potencies among clones was statistically significant (P < 0.0001), indicating that potency can be used for genetic comparisons. However, cell potency differences for a clone of G. toxicus in the acclimated vs. non-acclimated phases can exceed genetic differences between clones. This stresses the need for a rigorous acclimation process. Caribbean isolates of G. toxicus were inherently more toxic than isolates from other areas. One Caribbean clone yielded 55 × 10^?4 mu (mouse units)·cell^?1 whereas clones Bermuda, the Bahamas, and Florida ranged from only 1.8 × 10^?4 mu·cell^?1 to a maximum of 19.8 × 10^?4 mu·cell^?1. Toxicity decreased with increasing latitude (r =–0.819, P < 0.01), indicating that environmental differences probably influenced the potencies. A comparison of acclimated reproduction rates at four light intensities also indicated that genetic differences among clones existed. The resulting reproduction rate/light slopes overlapped, indicating that the clones may be adapted to specific light regimes.     

Group I introns and associated homing endonuclease genes reveals a clinal structure for <Emphasis Type="Italic">Porphyra spiralis</Emphasis> var. <Emphasis Type="Italic">amplifolia</Emphasis> (Bangiales,Rhodophyta) along the Eastern coast of South America

Background  

Group I introns are found in the nuclear small subunit ribosomal RNA gene (SSU rDNA) of some species of the genus Porphyra (Bangiales, Rhodophyta). Size polymorphisms in group I introns has been interpreted as the result of the degeneration of homing endonuclease genes (HEG) inserted in peripheral loops of intron paired elements. In this study, intron size polymorphisms were characterized for different Porphyra spiralis var. amplifolia (PSA) populations on the Southern Brazilian coast, and were used to infer genetic relationships and genetic structure of these PSA populations, in addition to cox 2-3 and rbc L-S regions. Introns of different sizes were tested qualitatively for in vitro self-splicing.