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PubMed1984

Tetrahymena H4 genes: structure, evolution and organization in macro- and micronuclei.

The ciliated protozoan Tetrahymena thermophila contains two types of H4 histone genes (H4-I and H4-II). Southern blotting and analysis of DNA from nullisomic strains indicate that H4-I and H4-II are on different chromosomes and that only H4-II is closely linked to an H3 gene. No DNA sequence rearrangements are observed for either of the H4 genes when the transcriptionally inert, germ line, micronucleus is compared to the transcriptionally active, somatic macronucleus. Comparison of the H4-I gene and its flanking sequences to H4 gene sequences of other organisms indicates that there are evolutionary constraints on coding nucleotides that are unrelated to their protein coding function and that these evolutionary pressures operate at the level of translation.

Amino Acid Sequence↗
PubMed1984

Sequence organization within and flanking clusters of 5S ribosomal RNA genes in Tetrahymena.

Macro- and micronuclei of Tetrahymena thermophila each contain approximately 30 clusters of 5S genes per haploid genome. Structural changes in DNA sequences associated with some of these clusters occur during the development of the transcriptionally active macronucleus from the transcriptionally inert micronucleus. Exonuclease digestion indicates that DNA fragmentation is not responsible for these changes, making it likely that sequence rearrangements occur near some 5S genes during macronuclear development. These rearrangements appear to be random in location with respect to the 5S genes and do not alter either the tandem repeat organization of the genes, the average number (five) or the range in number (one to about 16) of genes per cluster. The 5S gene clusters are not closely linked and are not flanked by common repeating elements large enough to cross-hybridize. Sequence analysis of one tandem repeat suggests that Tetrahymena 5S genes have intragenic promoters. These observations indicate that features other than DNA rearrangements or DNA sequence per se are responsible for the transcriptional activation of 5S genes during macronuclear development.

Animals↗
PubMed1984

Physical structure of gene-sized chromatin from the protozoan Oxytricha.

Oxytricha nova is a hypotrichous ciliate containing a transcriptionally active macronucleus and a transcriptionally inactive micronucleus. Two-dimensional gel electrophoresis shows that macronuclei contain a normal complement of inner histones. However, despite extensive efforts, no classical H1-like protein has been detected. Micrococcal nuclease digestion indicates a nucleosome repeat length of approximately 220 bp for macronuclear chromatin. Thermal denaturation profiles of macronuclear chromatin in 0.2 mM EDTA display four transitions at about 46, 57, 64, and 79 degrees C. The lowest of these shifts to higher temperature as the ionic strength is raised to 3-5 mM Na phosphate. These results are consistent with the absence of H1 and a nucleosome repeat of 220-230 bp. Circular dichroism (CD) results agree with these findings. By contrast, micronuclear chromatin displays a much smaller premelt and a more suppressed DNA CD signal at 285 nm, consistent with a micronuclear chromatin repeat of 165-185 bp as determined by micrococcal nuclease digestion.

Animals↗
PubMed1985

DNA synthesis, methylation and degradation during conjugation in Tetrahymena thermophila.

We have investigated the timing of DNA synthesis, methylation and degradation during macronuclear development in the ciliate, Tetrahymena thermophila. DNA synthesis was first detected in the anlagen early in macronuclear development, but the majority of DNA synthesis occurred later, after pair separation. Anlagen DNA was first detectably methylated at GATC sites 3-5 hours after its synthesis. Once initiated, de novo methylation was rapid and complete, occurring between 13.5 and 15 hours of conjugation. The level of methylation of GATC sites was constant throughout the remainder of conjugation, and was similar to that in mock-conjugated cells. Degradation of DNA in the old macronucleus and DNA synthesis in the anlagen began at about the same time. Upon pair separation, less than 20% of old macronuclear DNA remained. A small percentage of nucleotides prelabeled prior to conjugation were recycled in the developing anlagen.

Cell Nucleus↗
PubMed1985

Nucleotide sequence of a macronuclear DNA molecule coding for alpha-tubulin from the ciliate Stylonychia lemnae. Special codon usage: TAA is not a translation termination codon.

The gene-sized macronuclear DNA of the hypotrichous ciliate Stylonychia lemnae contains two size classes of DNA molecules (1.85 and 1.73 kbp) coding for alpha-tubulin. Each macronucleus contains about 55000 copies of the 1.85 kbp molecules and about 17000 copies of the 1.73 kbp DNA molecules. Five macronuclear molecules of these sequences were cloned and sequenced, one, from the 1.85 kbp size class in its entirety. The 5 sequences fell into two classes suggesting that Stylonychia lemnae contains at least two different alpha-tubulin genes. All 5 clones show the codon TAA in the same nucleotide positions of the coding region. In this position the TAA codon cannot function as a translational stop codon and we suggest that this codon codes for the amino acid glutamine. The nucleotide sequence of the coding region as well as the encoded amino acid sequence is highly conserved compared to alpha-tubulin genes from vertebrates. The noncoding regions show several putative transcription-regulatory sequences as well as sequences presumably functioning as replication origins.

Base Sequence↗
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

A family of DNA sequences is reproducibly rearranged in the somatic nucleus of Tetrahymena.

A small family of DNA sequences is rearranged during the development of the somatic nucleus in Tetrahymena. The family is defined by 266 bp of highly conserved sequence which restriction mapping, hybridization and sequence analysis have shown is shared by a cloned micronuclear fragment and three sequences which constitute the macronuclear family. Genomic Southern hybridization experiments indicate there are five members of the family in micronuclear DNA. All of the family members are present in whole genome homozygotes and are therefore nonallelic. The three macronuclear sequences are all present in clonal cell lines and are reproducibly generated in every developing macronucleus. The rearrangement event begins 14 hours after conjugation is initiated and is nearly completed by 16 hours.

Animals↗
PubMed1986

Sequence characterization of Tetrahymena macronuclear DNA ends.

Tetrahymena is a ciliated protozoan which has two nuclei: a micronucleus, which maintains the genetic continuity of the cell, and the macronucleus which is derived from the micronucleus after sexual conjugation. A macronuclear DNA library was constructed to contain DNA ends. A probe containing C4A2 repeats which are known to be present at macronuclear DNA ends (1) was used to screen the library. Three clones were characterized by sequencing, restriction enzyme mapping and Bal 31 digestion. The data indicate that these three clones represent macronuclear DNA ends which were generated by DNA fragmentation during macronuclear formation. The sequencing data at the C4A2 repeat junction show a conserved sequence of five nucleotides, TTATT. Sequences further away show no obvious homologies except that they are highly enriched in AT. This structure is quite different from the subtelomeric sequences of other organisms.

Animals↗
PubMed1988

Sequence and properties of the message encoding Tetrahymena hv1, a highly evolutionarily conserved histone H2A variant that is associated with active genes.

hv1 is a histone H2A variant found in the transcriptionally active Tetrahymena macronucleus, but not in the transcriptionally inert micronucleus. hv1 also contains antigenic determinants conserved in the histone complements of representatives of all four eukaryotic kingdoms. A cDNA clone encoding hv1 has been isolated and sequenced. Comparison of the derived protein sequence of hv1 with that of the chicken variant H2A.F and the sea urchin variant H2A.F/Z reveals remarkable homology in all but the extreme amino- and carboxy-termini and a small region in the conserved core. Putative regions of conserved antigenicity are discussed. Evidence is presented that suggests that hv1 is a single-copy, intron-containing gene that encodes a polyadenylated message. Unusual features in the 3' flanking sequence and in codon usage are also described. Evidence is also presented showing that hv1 message amounts are ten-fold greater in growing cells than in starved cells.

Amino Acid Sequence↗
PubMed1988

Alternative use of chromosome fragmentation sites in the ciliated protozoan Oxytricha nova.

During its life cycle, the hypotrichous ciliated protozoan Oxytricha nova transforms a copy of its micronucleus, which contains chromosome-sized DNA, into a macronucleus containing linear, gene-sized DNA molecules. A region of the micronuclear genome has been defined that gives rise to two distinct macronuclear DNA molecules during development. Through analysis of recombinant macronuclear and micronuclear clones, the generation of the two macronuclear DNA molecules was shown to be the result of alternative use of chromosome fragmentation sites. In addition, evidence was obtained that adjacent micronuclear precursors of macronuclear DNA molecules can overlap by a few base pairs. The significance of these findings in relation to developmental chromosome fragmentation is discussed.

Animals↗
PubMed1988

Developmentally regulated telomere addition in Tetrahymena thermophila.

To investigate the developmentally programmed telomere addition that accompanies chromosome fragmentation during macronuclear differentiation in Tetrahymena thermophila, five representative telomeric regions from the macronucleus were cloned and characterized in detail. The sequences adjacent to the telomeric (C4A2:T2G4) repeats on these five macronuclear ends had no significant sequence homology or shared secondary structure. Two developmentally independent examples of one macronuclear telomere had a 5 base pair difference in the position of the junction between the telomeric repeats and the adjacent sequences. A telomere-adjacent sequence, in the form of a synthetic oligonucleotide, was unable to prime the addition of telomeric repeats in vitro. The implications of these results for the mechanisms underlying developmentally programmed chromosome fragmentation and telomere addition in Tetrahymena are discussed.

Animals↗
PubMed1988

Drosophila has a single copy of the gene encoding a highly conserved histone H2A variant of the H2A.F/Z type.

The Tetrahymena histone H2A variant designated hv1 is localized exclusively in the transcriptionally active macronucleus and is absent from the quiescent micronucleus (1). A cDNA clone of the hv1 gene (2) was used to screen a Drosophila cDNA library. A cross-hybridizing clone was recovered and shown by sequence analysis to code for a protein homologous to hv1 as well as to the chicken H2A variant, H2A.F (3), the sea urchin H2A variant, H2A.F/Z (4) and the mammalian H2A variant H2A.Z (5). Southern analysis of Drosophila genomic DNA indicates that the H2AvD (H2A variant Drosophila) gene is present in one copy. In situ hybridization places the locus at 97CD on chromosome 3, while the S-phase regulated histone genes are on chromosome 2 (6). Thus the Drosophila H2A variant should be accessible to genetic analysis, which will enable its function to be determined.

Amino Acid Sequence↗
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↗
PubMed1989

Identification of an amplification promoting DNA sequence from the hypotrichous ciliate Stylonychia lemnae.

The macronucleus of the hypotrichous ciliate Stylonychia lemnae contains a 1218 bp long DNA molecule which becomes highly amplified during vegetative growth due to a continuous overreplication over a long time range. The region which is located upstream the open reading frame of the overamplified 1.2kbp Stylonychia DNA molecule enabled plasmids containing an inefficiently transcribed thymidine kinase gene to persist and amplify upon transfection into mouse L fibroblasts under selective conditions. This region contains long AT-rich stretches. The AT-rich sequences interact with a previously characterized HMG-I like protein from mouse Ehrlich ascites tumour cells. A binding activity for AT-rich stretches could also be identified in macronuclear extracts from Stylonychia lemnae. We suggest a common mechanism for overamplification in Stylonychia macronuclei during vegetative growth and amplification of plasmid DNA in heterologous mouse cells under the influence of a common element.

Amino Acid Sequence↗
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↗
PubMed1991

Expression of the gene encoded by a family of macronuclear chromosomes generated by alternative DNA processing in Oxytricha fallax.

Hypotrichous ciliated protozoa, such as Oxytricha fallax, produce tiny chromosomes during generation of the transcriptionally active macronucleus. The 81-MAC family of macronuclear chromosomes is produced by alternative DNA processing, such that the chromosomes share a common region of 1.6 kbp. Transcription of a 1.3 kb mRNA from the common region has been analyzed. Transcription starts very near the telomere (34 bp), in a 23 bp region of pure A + T DNA. Polyadenylation sites are very near the other telomere (26 bp), also in a region of nearly pure A + T DNA. Three introns are clustered in the first third of the gene. Intron removal can follow polyadenylation, and the order of removal is not fixed. All three known sequence versions of the 81-MAC chromosomes are represented in the mRNA pool, with no evidence of any further versions. The A + T sequences surrounding the transcription starts and polyadenylation sites are conserved among versions. Introns have conserved 5' and 3' ends and a putative branch-point sequence (YYRAT), but otherwise are highly diverged and are AT-rich. A single long open reading frame, interrupted by the three introns, encodes a homolog of known mitochondrial solute carriers, and contains the codon TAA, which does not encode 'stop,' but a conserved glutamine; TAG appears also to encode glutamine. The results significantly enlarge the small data set of transcription start and polyadenylation sites, of intron features, and of translation signals for hypotrichs.

Animals↗
PubMed1992

A repetitive DNA sequence in Paramecium macronuclei is related to the beta subunit of G proteins.

A repeated DNA sequence has been identified in the macronucleus of several Paramecium species. In P.tetraurelia the repeat was identified in the subtelomeric region of four randomly selected telomere clones, as well as downstream of the A type variable surface protein gene. The complete sequence of the A gene linked repeat consists of 15 tandem repeats of exactly 126 nucleotides that contain an open reading frame with significant similarity to the beta subunits of trimeric G proteins. The most striking consensus feature is the amino acid sequence DX omega WD where X is any amino acid and omega is I, L, or V spaced at precise 42 amino acids intervals. This sequence and spacing are found in G-protein beta subunits and other members of this protein motif family. Analysis of the five cloned telomeric restriction fragments showed the repeats can be found in either orientation with respect to the telomere. Poly(A) RNA transcripts containing this sequence have been identified in Paramecium tetraurelia. The conserved presence of this sequence in several species of Paramecium suggests an important physiological function, and the study of this repeat may reveal information about the evolution of this common protein motif.

Amino Acid Sequence↗
PubMed1993

A micronucleus-specific sequence exists in the 5'-upstream region of calmodulin gene in Tetrahymena thermophila.

Tetrahymena thermophila possesses a transcriptionally inactive micronucleus and an active macronucleus. Both nuclei are developed from micronucleus-derived germ nuclei during conjugation. Extensive DNA rearrangement and transcriptional activation are known to be involved in macronuclear development, but little has been known about these processes in a particular functional gene. Therefore the micro- and macronuclear genomic DNAs for calmodulin gene were analyzed. A 1,384 bp micronucleus-specific sequence located about 3.5 kb upstream of calmodulin gene has been found, suggesting DNA rearrangement during macronuclear development. The micronucleus-specific sequence had 85% A + T, no extensive ORF, ATTAs at both ends, and two palindromic structures just outside of both ends. Interestingly, the micronucleus-specific sequence included a T-rich tract, T16CT5, in the middle, and a nearly complementary A-rich tract, A5TA10GA5, existed 7 bp upstream from the initiation codon. In addition, there was a 20 bp repetitive sequence TAAT(TAAC)4 about 100 bp upstream of the micronucleus-specific sequence and also in the promoter region of calmodulin gene. Although the functional significance of the micronucleus-specific sequence remains unclear, T16CT5 and TAAT(TAAC)4 elements might exert an influence on transcription of the calmodulin gene. Stringent Southern hybridization revealed that this micronucleus-specific sequence or very similar sequence(s) were abundant in the Tetrahymena micronuclear genome.

Animals↗