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PubMed1989

Isolation and structural characterization of cDNA clones encoding the mating pheromone Er-1 secreted by the ciliate Euplotes raikovi.

cDNA clones comprising the entire coding region for the mating pheromone Er-1 of Euplotes raikovi have been isolated by oligonucleotide screening of two cDNA libraries in the vectors lambda gt10 and pUC12. The cDNA sequence contains an open reading frame of 75 amino acids that constitute pre-pro-Er-1. The amino acid sequence of secreted Er-1 starts at aspartic acid-36 of pre-pro-Er-1 and completely matches that known by direct Er-1 protein sequencing. The coding region of Er-1 cDNA ends with codon TAA, which specifies glutamine in other ciliates. The 5'- and 3'-noncoding regions contain, respectively, two and one inverted repeats. The 3'-noncoding-region inverted repeat, which includes the unusual polyadenylylation signal AACAAA, has been related to RNA 3'-terminus formation.

Amino Acid Sequence↗
PubMed1991

UGA is translated as cysteine in pheromone 3 of Euplotes octocarinatus.

Pheromone 3 mRNA of the ciliate Euplotes octocarinatus contains three in-frame UGA codons that are translated as cysteines. This was revealed from cDNA sequencing and from plasma desorption mass spectrometry of cleaved pheromone 3 in connection with pyridylethylation of the fragments. N-terminal sequence analysis of carboxymethylated protein confirmed this conclusion for the first of the three UGA codons. Besides UGA the common cysteine codons UGU and UGC are also used to encode cysteine. UAA functions as a termination codon. No UAG codon was found. In connection with results reported for other ciliates, this suggests that the role of the classic termination codons had not yet been established when the ciliates started to diverge from other eukaryotes.

Amino Acid Sequence↗
PubMed1985

Large scale synchronous mating and the study of macronuclear development in Euplotes crassus.

After conjugation in hypotrichous ciliates, a new macronucleus is produced from a copy of the micronucleus. This transformation involves large-scale reorganization of DNA, with conversion of the chromosomal micronuclear genome into short, gene-sized DNA molecules in the macronucleus. To study directly the changes that occur during this process, we have developed techniques for synchronous mating of large populations of the hypotrichous ciliate Euplotes crassus. Electron microscope studies show that the micronuclear chromosomes are polytenized during the first 20 h of macronuclear development. The polytene chromosomes lack the band-interband organization observed in other hypotrichs and in the Diptera. Polytenization is followed by transectioning of the chromosomes. We isolated DNA at various times of macronuclear development and found that the average molecular weight of the DNA decreases at the time of chromosome transectioning. In addition, we have shown that a small size group of macronuclear DNA molecules (450-550 base pairs) is excised from the chromosomal DNA approximately 10 h later in macronuclear development.

Animals↗
PubMed1986

Examination of the macronuclear replication band in Euplotes eurystomus with monoclonal antibodies.

A panel of eight monoclonal antibodies (MAbs) was prepared from spleen cells of mice immunized with macronuclear replication bands (RBs) isolated from Euplotes eurystomus. Antibodies were investigated with a solid phase radioimmunoassay (RIA) using either soluble chromatin from isolated RBs or from total macronuclei as antigen. The RIA showed that several MAbs recognized antigens present only in the RB or macronucleus, whereas others recognized antigens present in both structures. Specificity of the MAbs was also examined by indirect immunofluorescence. Antibody C10 recognized an antigen in the rear zone of the RB, whereas MAbs G6 and B2 appeared to stain both the forward and rear zones of the RB. Antibody A7 recognized an epitope distributed throughout the macronucleus except in the RB. Cytochemical studies with degradative enzymes suggested that antigens localized by immunofluorescence were composed of proteins. Immunoblots of SDS PAGE permitted identification of a few proteins that reacted with three of the RB-specific MAbs. Monoclonal antibodies that identify the presence or absence of reactivity of specific proteins in the RB could prove useful in the study of chromatin structure and the mechanism of chromatin replication.

Animals↗
PubMed1990

Identification and initial characterization of an autocrine pheromone receptor in the protozoan ciliate Euplotes raikovi.

The polypeptide pheromone Er-1, purified from the ciliate Euplotes raikovi of mating type I and genotype mat-1/mat-1, was iodinated with 125I-Bolton-Hunter reagent to a sp act of 0.45-0.73 mu Ci/microgram of protein. This preparation of 125I-Er-1 bound specifically to high affinity binding sites on the same cells of mating type I. Binding of 125I-Er-1 occurred with an apparent Kd of 4.63 +/- 0.12 X 10(-9) M in cells in early stationary phase. It was estimated that these cells carry a total number of approximately 5 X 10(7) sites/cell, with a site density that falls in the range of 1,600-1,700/microns 2 of cell surface. Unlabeled Er-1, other homologous pheromones such as Er-2 and Er-10, antibodies specific for Er-1, and human IL-2 were shown to act as effective inhibitors of specific binding of 125I-Er-1 to mating type I cells. The "autocrine" nature of the identified specific high affinity binding sites for Er-1 was further substantiated by cross-linking experiments. These experiments revealed that mating type-I cell membranes contain one protein entity of Mr = 28,000 that is capable of reacting specifically with the homodimeric native form of Er-1.

Animals↗
PubMed1966

The syntheses of total macronuclear protein, histone, and DNA during the cell cycle in Euplotes eurystomus.

The syntheses of histone, total protein, and DNA during the cell cycle were measured in the macronucleus of Euplotes eurystomus by assaying the incorporation of tritiated amino acids and tritiated thymidine in groups of 800 to 1000 synchronized cells. The synthesis of DNA begins at 30% completion of the cell cycle, proceeds at a constant rate, and ends very shortly before the beginning of macronuclear division. Histone labeling is absent during G(1), begins in phase with DNA synthesis, continues at an unchanging rate during the S phase, and ends with the completion of DNA synthesis. The results support the view that the syntheses of histone and DNA are closely coupled events. Label in total protein accumulates at a constant rate during G(1) and appears to shift to a slightly higher rate when histone synthesis begins. At division, radioactive DNA, histone, and total protein are distributed equally between the daughter macronuclei without loss of radioactivity. Radioautographic analysis showed that protein labeling occurs throughout the macronucleus during the entire life cycle. There was no clear difference in the degree of protein labeling between replicated and unreplicated regions of the macronucleus. The distribution of label suggests that most of macronuclear protein labeling during the cell cycle is concerned with the events of transcription rather than replication.

Amino Acids↗
PubMed1986

Preparation and characterization of soluble macronuclear chromatin from the hypotrich Euplotes eurystomus.

Euplotes eurystomus is a hypotrichous ciliate containing a transcriptionally active macronucleus (MAC) and a transcriptionally inactive micronucleus. Soluble MAC chromatin contains a normal complement of inner histones, an H1-like protein which is very sensitive to proteolysis, and a considerable proportion of non-histone proteins. A combination of N-Tosyl lysine chloromethyl ketone (TLCK) and PMSF was found to be most effective in preventing proteolysis. Microccocal nuclease digestion yielded an average nucleosome repeat length of 187 +/- 25 bp for soluble chromatin; and 190 +/- 9 bp for isolated macronuclei. Thermal denaturation profiles of MAC chromatin in 0.25 mM EDTA display two main transitions at about 76 and 83 degrees C, resembling the melting of soluble chicken erythrocyte chromatin. Circular dichroic spectra of MAC chromatin were compared to soluble chicken erythrocyte chromatin under the same ionic strength conditions and were found to be very similar. 2D chromatin/DNA agarose gel electrophoresis resulted in a diagonal line of DNA staining, which establishes a strict correlation between DNA and chromatin electrophoretic mobility.

Animals↗
PubMed1986

Subfractionation of soluble macronuclear chromatin and enrichment of specific genes as chromatin from Euplotes eurystomus.

Euplotes eurystomus is a hypotrichous ciliated protozoan possessing within one cytoplasm a transcriptionally-inactive micronucleus with chromosomal-size DNA and a transcriptionally active macronucleus with "gene-size" DNA fragments. The chromatin in the macronucleus can be isolated in a soluble form without prior treatment by nucleases. In this study, macronuclear, soluble chromatin was subfractionated using isokinetic sucrose density gradient ultracentrifugation in a buffer consisting of 50 mM NaCl, 1 mM Na2 EDTA, 1 mM TEA HCl, pH 7.0, 0.1 mM TLCK and 0.1 mM PMSF. Fractions were collected and analyzed by DNA and protein gel electrophoresis, dot blot hybridization with specific gene probes, and modified Miller chromatin spreads. Analysis of the chromatin spreads showed that the sizes of the chromatin fragments in the various fractions correlate with the DNA size of the fragments. When dot blots of the fractions were hybridized with 5S rRNA, tubulin and rRNA gene probes we obtained about a 6 to 14-fold enrichment of hybridizable sequences in individual fractions. There appear to be differences in the non-histones present on each fraction as well as some overall similarities in histone and non-histone proteins.

Animals↗
PubMed1989

The 5S RNA gene minichromosome of Euplotes.

The macronucleus of the ciliated protozoan Euplotes eurystomus contains about 10(6) copies of a single type of 5S ribosomal RNA gene. This 5S gene DNA is only 930 bp long, is flanked by telomeres, and contains a single coding region of 120 bp which serves as a template for transcription in vivo and in vitro. The 5S gene minichromatin possesses four positioned nucleosomes and hypersensitive cleavage sites in the telomeric regions.

Animals↗
PubMed1990

Internal eliminated sequences are removed prior to chromosome fragmentation during development in Euplotes crassus.

The hypotrichous ciliated protozoa undergo a massive genome rearrangement process after their sexual cycle. One frequent type of rearrangement is the removal of DNA sequences (internal eliminated sequences; IESs) from internal regions of DNA molecules. In this study, we characterized the removal of IESs in Euplotes crassus. Southern hybridization analyses combined with cytological observations indicated that IES removal is an early event in macronuclear development, occurring during the polytene chromosome stage and prior to the chromosome fragmentation process. The results are consistent with IES removal occurring via an intramolecular DNA breakage and rejoining process.

Animals↗
PubMed1991

High fidelity developmental excision of Tec1 transposons and internal eliminated sequences in Euplotes crassus.

Following the sexual phase of its life cycle, the hypotrichous ciliate Euplotes crassus transforms a copy of its chromosomal micronucleus into a transcriptionally active macronucleus containing short, linear, gene-sized DNA molecules. Tens of thousands of DNA breakage and joining, or splicing, events occur during macronuclear development. The DNA removed by such events includes transposon-like elements, referred to as Tec1 elements, as well as segments of unique sequence DNA, termed internal eliminated sequences (IESs). Both types of elements are bounded by short direct repeats. In the current study, a polymerase chain reaction (PCR) and DNA sequencing strategy has been used to examine the fidelity of excision of two Tec1 elements and three IESs. In all cases, the vast majority of excision events were found to be precise, with one copy of the terminal direct repeats retained at the empty site in the macronuclear DNA molecule. These results, in combination with previous studies that have characterized the excised DNA elements, indicate that the two products of excision (the free element and the macronuclear DNA molecule) share DNA sequences. This suggests that excision events are initiated by staggered cuts in the chromosomal DNA.

Animals↗
PubMed1995

Consensus inverted terminal repeat sequence of Paramecium IESs: resemblance to termini of Tc1-related and Euplotes Tec transposons.

During the formation of a transcriptionally active macronucleus, ciliated protozoa excise large numbers of interstitial segments of DNA (internal eliminated sequences; IESs) from their chromosomes. In this study we analyze the published sequences of 20 IESs that interrupt surface protein genes of Paramecium and identify a consensus inverted terminal repeat. This sequence is similar to the ends of the Tc1-related transposons found in nematodes and other metazoans, as well as to both the ends of the Tec transposons and at least some of the IESs in the distantly related ciliate Euplotes crassus. The results of these analyses bolster previous proposals that IESs were created by transposition.

Animals↗
PubMed1989

Characterization of chromosome fragmentation in two protozoans and identification of a candidate fragmentation sequence in Euplotes crassus.

Following the sexual cycle, hypotrichous ciliated protozoans fragment a set of their micronuclear chromosomes to generate the thousands of short, linear DNA molecules present in the transcriptionally active macronucleus. We have used a hybrid selection procedure to examine macronuclear DNA molecules for subtelomeric length heterogeneity to determine whether chromosome fragmentation occurs at unique or multiple sites. The results suggest that multiple, but closely spaced, chromosome fragmentation sites are used by Oxytricha nova. In contrast, Euplotes crassus uses unique chromosome fragmentation sites in a reproducible manner to generate the ends of macronuclear DNA molecules. Additional studies compared DNA sequences in the vicinity of chromosome fragmentation sites in an attempt to define cis-acting sequences that direct the fragmentation process. A conserved sequence was found near chromosome fragmentation sites in E. crassus. The location of the conserved sequence suggests that chromosome fragmentation involves staggered cuts of the micronuclear DNA molecules.

Animals↗
PubMed1996

A challenging case for protein crystal structure determination: the mating pheromone Er-1 from Euplotes raikovi.

Four different phasing methods have been applied to the determination of the crystal structure of the 40 amino-acid mating pheromone of the unicellular ciliated protozoan Euplotes raikovi. The difficulties, failures and successes in attempts to solve the structure by: (1) molecular replacement, (2) direct phasing using the 'Shake and Bake' algorithm, (3) isomorphous replacement, and (4) multiple-wavelength anomalous dispersion are described. The structure was first solved by molecular replacement, and then was the first successful structure determination by 'Shake and Bake' without the direct involvement of its authors. A description of the current status of the high-resolution refinement of the structure is also given. The model is refined against 1 A resolution data to an R factor of 12.9%, and includes H atoms and discretely disordered side chains.

Journal Article↗
PubMed1987

The isolation of gamones 3 and 4 of Euplotes octocarinatus.

Gamones 3 and 4 of the ciliate Euplotes octocarinatus were isolated and purified to chromatographic and electrophoretic homogeneity. They are secreted into the culture medium by cells of certain mating types and induce cells of other mating types to unite in pairs and exchange gametic nuclei. The purified gamones are biologically active at concentrations as low as 0.1-1 pM. Both are polypeptides with unusually low pI values of approximately 3.2, and both have a carbohydrate content of less than 2%. Gamone 3 has an Mr of 18,800 and is slightly smaller than gamone 4, which has an Mr of 23,500. Gamone 3 was isolated from the starvation medium of cells homozygous for the mating type allele mt3 and gamone 4 was isolated from the medium of cells homozygous for the mating type allele mt4. Each of the two homozygous cell lines was found to secrete one gamone only.

Animals↗
PubMed1986

Sequence of the small subunit ribosomal RNA gene from the hypotrichous ciliate Euplotes aediculatus.

We have determined the complete nucleotide sequence of the coding region of the small subunit rRNA gene of the hypotrichous ciliate Euplotes aediculatus. It is 1882 nucleotides long and contains several inserts not present in the small subunit rRNA genes of the hypotrichs Oxytricha nova and Stylonychia pustulata. A comparison of the sequences suggests that E. aediculatus is much less closely related to these other two hypotrichs than they are to each other. Although the gene sequence of E. aediculatus is drifting more rapidly than those of these other two species, its faster evolutionary clock is not enough to account for the degree of difference between them.

Animals↗
PubMed1988

Genetic characterization and use of a restriction fragment length variant in the hypotrichous ciliate Euplotes crassus.

Two forms of a macronuclear DNA molecule differing in the presence or absence of a restriction endonuclease recognition site have been detected in the hypotrichous ciliate Euplotes crassus. Through a series of genetic crosses the two forms were shown to be allelic, being derived from a single micronuclear genetic locus. This restriction fragment length variant (RFLV) was used as a genetic marker to determine that the migratory and stationary pronuclei generated during mating can be genetically non-identical. In addition, the RFLV was used to investigate the efficiency of processing of the alternate alleles during macronuclear development and their subsequent transmission during vegetative growth. Little or no bias in the processing and/or amplification of the two alleles was observed during macronuclear development. During vegetative growth, however, changes in the relative amounts of the two alleles were observed.

Alleles↗
PubMed1988

Organization of the Euplotes crassus micronuclear genome.

Euplotes crassus, like other hypotrichous ciliated protozoa, eliminates most of its micronuclear chromosomal DNA in the process of forming the small linear DNA molecules that comprise the macronuclear genome. By characterizing randomly selected lambda phage clones of E. crassus micronuclear DNA, we have determined the distribution of repetitive and unique sequences and the arrangement of macronuclear genes relative to eliminated DNA. This allows us to compare the E. crassus micronuclear genome organization to that of another distantly related hypotrichous ciliate, Oxytricha nova. The clones from E. crassus segregate into three prevalent classes: those containing primarily eliminated repetitive DNA (Class I); those containing macronuclear genes in addition to repetitive sequences (Class II); and those containing only eliminated unique sequence DNA (Class III). All of the repetitive sequences in these clones belong to the same highly abundant repetitive element family. Our results demonstrate that the sequence organization of the E. crassus and O. nova micronuclear genomes is related in that the macronuclear genes are clustered together in the micronuclear genome and the eliminated unique sequences occur in long stretches that are uninterrupted by repetitive sequences. In both organisms a single repetitive element family comprises the majority of the eliminated interspersed middle repetitive DNA and appears to be preferentially associated with the macronuclear sequence clusters. The similarities in the sequence organization in these two organisms suggest that clustering of macronuclear genes plays a role in the chromosome fragmentation process.

Animals↗