Hypotrichous Ciliate Stylonychia Lemnae Research Articles

Minichromosomal DNA replication in the macronucleus of the hypotrichous ciliate Stylonychia lemnae is independent of chromosome-internal sequences.

The origins of DNA replication in prokaryotes and eukaryotes are typically defined by cis-acting sequences. However, in ciliates, evidence suggests that the replication of short macronuclear minichromosomes may not require such determinants. In hypotrichous ciliates, macronuclei contain millions of gene-sized minichromosomes, which generally have a single protein-coding region, two short noncoding flanks and, on each end, a short telomere consisting of a double-stranded repeat region and a single-stranded 3' overhang. Electron microscopic studies that showed that replication of minichromosomes initiates at or near telomeres and the discovery of a primase activity synthesizing RNA primers over the whole 3' telomeric overhang in vitro suggested that minichromosome replication starts directly at telomeres. Conversely, many minichromosomes contain an AT-rich, semi-conserved, palindromic sequence motif in their subtelomeric regions and it has been proposed that this motif is involved in regulating minichromosomal replication. To analyze what sequences or structures of the minichromosomes are essential for DNA replication, we stably transfected genetically modified alpha1-tubulin-encoding minichromosomes into the hypotrichous ciliate Stylonychia lemnae. Cotransfection of mutated and control minichromosomes revealed that noncoding regions can be deleted or replaced with unrelated sequences without affecting minichromosome replication efficiency in vegetatively growing cells. Similarly, replacement of the coding region resulted in a minichromosome that was stably maintained in transfected cells at the same high copy number for many months. In contrast, alpha1-tubulin-encoding minichromosomes without telomeres were rapidly lost after transfection. Hence, DNA replication of the alpha1-tubulin-encoding minichromosome does not depend on chromosome-internal sequences but may depend on telomeres.

Both subtelomeric regions are required and sufficient for specific DNA fragmentation during macronuclear development in Stylonychia lemnae.

Programmed DNA-reorganization and DNA-elimination events take place frequently during cellular differentiation. An extreme form of such processes, involving DNA reorganization, DNA elimination and DNA fragmentation, is found during macronuclear differentiation in hypotrichous ciliates. Ciliated protozoa can therefore serve as a model system to analyze the molecular basis of these processes during cellular differentiation in eukaryotic cells. Using a biological approach to identify cis-acting sequences involved in DNA fragmentation, we show that in the hypotrichous ciliate Stylonychia lemnae sequences required for specific DNA processing are localized in the 3'- and the 5'-subtelomeric regions of the macronuclear precursor sequence. They can be present at various positions in the two subtelomeric regions, and an interaction between the two regions seems to occur. Sequence comparison revealed a consensus inverted repeat in both subtelomeric regions that is almost identical to the putative Euplotes chromosome breakage sequence (E-Cbs), also identified by sequence comparison. When this sequence was mutagenized, a processed product could no longer be detected, demonstrating that the sequence plays a crucial role in DNA processing. By injecting a construct into the developing macronucleus, which exclusively contains the subtelomeric regions of the Stylonychia alphal-tubulin gene, we show that subtelomeric regions are not only required but are also sufficient for DNA processing in Stylonychia. Our results indicate that an inverted repeat with the core sequence 5'-TGAA present in both subtelomeric regions acts as a Cbs in Stylonychia. The results allow us to propose a mechanistic model for DNA processing in this ciliate.

A subtelomeric DNA sequence is required for correct processing of the macronuclear DNA sequences during macronuclear development in the hypotrichous ciliate Stylonychia lemnae.

During macronuclear differentiation in ciliated protozoa a series of programed DNA reorganization processes occur. These include the elimination of micronuclear-specific DNA sequences, the specific fragmentation of the genome into small gene-sized DNA molecules, the de novo addition of telomeric sequences to these DNA molecules and the specific amplification of the remaining DNA molecules. Recently we constructed a vector containing the modified micronuclear version of macronuclear destined DNA sequences that was correctly fragmented and telomeres were added de novo after injection into the developing macronucleus. It therefore must contain all the cis- acting sequences required for these processes. We made a series of vectors deleting different sequences from the original vector. It could be shown that at least in the case studied here no micronuclear-specific sequences are required for specific fragmentation of the genome and telomere addition. However, a short subtelomeric sequence at the 3[prime]-end is essential for these processes, whereas no specific cut seems to occur at the 5[prime]-end. In addition, we can show that the processing activity is restricted to a short period of time during macronuclear differentiation and that a preceding transcription is required for correct processing of macronuclear-destined DNA sequences. Possible mechanisms of these processes will be discussed.

Several highly divergent histone H3 genes are present in the hypotrichous ciliate Stylonychia lemnae

In the protozoan Stylonychia lemnae 10 different histone H3 genes were discovered by polymerase chain reaction (PCR) amplification and sequence analysis. One of them is interrupted by a short intron sequence. These genes code for nine divergent histone H3 proteins. The genetic distances between some of these variants are very high. Most of the substitutions, as well as insertions/deletions, were found in the amino-terminal region. One variant shows an extremely elongated and altered N-terminus, which did not allow an unambiguous alignment with other histone H3 variants in this region. Hybridization experiments using the different H3 genes as probes indicate that even more histone H3 variants must exist in this species.

Stable transfection of the hypotrichous ciliate Stylonychia lemnae with tagged α1 tubulin minichromosomes

Summary Macronuclei of Stylonychia lemnae contain millions of gene sized minichromosomes. These minichromosomes in general harbour only one gene. Their nontranscribed regions are usually less than 400 bp long and most likely carry the signals for DNA replication and transcription. Therefore, S. lemnae seems to be an ideal object to study these processes in a protist organism. By microinjecting defined linear minichromosomal DNA directly into macronuclei of vegetative cells, we have developed a suitable in vivo assay system for S. lemnae. Transfections were carried out with α1 tubulin minichromosomes whose gene was internally tagged by a 19 bp-long insertion. Although the endogenous ocl tubulin minichromosome is present in approximately 100,000 copies per cell, clonal cell lines were generated which stably contained tagged minichromosomes in up to a fivefold excess of endogenous copy numbers. As demonstrated by Southern blot analysis, the copy number of endogenous α1 tubulin minichromosomes was not reduced in these cell lines, and transfected minichromosomes were not noticeably rearranged by the cells. Moreover, tagged ocl tubulin genes were unequivocally expressed. Transcription of these genes was initiated correctly, and the amount of transcript present in steady-state RNA correlated with gene copy number.

Characterization of a family of repetitive sequences that is eliminated late during macronuclear development of the hypotrichous ciliate Stylonychia lemnae

Characterization of a family of repetitive sequences that is eliminated late during macronuclear development of the hypotrichous ciliate Stylonychia lemnae

Characterization of a family of repetitive sequences that is eliminated late during macronuclear development of the hypotrichous ciliate Stylonychia lemnae.

A repetitive element from the hypotrichous ciliate Stylonychia lemnae was characterized by restriction and hybridization analysis. This repetitive element is present in about 5,000-7,000 copies per haploid genome in the micronucleus and the macronuclear anlagen. Its DNA sequence is very conserved, but the length of the repetitive sequence blocs is variable. In some cases, it is associated with telomeric sequences and macronucleus-homologous sequences. Restriction analysis of genomic micronuclear and macronuclear anlagen DNA and in situ hybridization showed that the repetitive sequences are amplified during the formation of polytene chromosomes. They are localized in many bands of the polytene chromosomes and are eliminated during the degradation of the polytene chromosomes. Possible functions of the repetitive sequences during macronuclear differentiation are discussed.

The formation of new nucleoli during macronuclear development of the hypotrichous ciliate Stylonychia lemnae visualized by in situ hybridization.

After sexual reproduction in ciliates, the old macronucleus degenerates and a new macronucleus is formed from a micronuclear derivative. Macronuclear development in hypotrichous ciliates includes the formation of polytene chromosomes, degradation of these chromosomes and elimination of DNA, specific fragmentation of macronuclear DNA in short gene-sized DNA molecules and specific amplification of these molecules. After fusion of the two haplid micronuclei, the zygote nucleus divides mitotically; one of the daughter nuclei develops into a new micronucleus and the other into a new macronucleus. A first DNA synthesis phase in the developing macronucleus (macronuclear anlage) leads to the formation of polytene chromosomes. These polytene chromosomes become degraded and up to over 90% of the DNA is eliminated, leading to a DNA-poor stage. During a second DNA synthesis phase, replication bands become visible, and finally the new vegetative macronucleus is formed (for a review see Ammermann et al. 1974, Prescott 1994, Lippe & Eder 1996). As in most other ciliates analysed so far (for a review see Prescott 1994), rDNA occurs as a single-copy gene in the micronucleus but is highly amplified in the vegetative macronucleus (Steinbrück 1990). This amplification of rDNA is accompanied by the formation of many new nucleoli in the course of macronuclear development. In the light microscope, nucleoli become first visible at the beginning of the second DNA synthesis phases and multiply in subsequent rounds of replication (Ammermann et al. 1974). In this report, we describe the amplification of rDNA and the formation of new nucleoli during macronuclear differentiation by in situ hybridization of rDNA to different stages of the developing macronucleus.

Sequential excision of internal eliminated DNA sequences in the differentiating macronucleus of the hypotrichous ciliate Stylonychia lemnae.

Elimination of internal eliminated sequences (IES) during macronuclear development of the hypotrichous ciliate Stylonychia lemnae was analyzed in one cluster of macronuclear precursor DNA sequences. The results indicate that IES elimination is a highly ordered process, it starts very early during macronuclear development and has only finished immediately before DNA fragmentation takes place. It occurs in distinct steps and the IES are eliminated in a specific order, where a defined IES is only removed after complete elimination of other IES. Transfection experiments clearly demonstrate that the structure of the IES itself is not sufficient for its correct excision but other cis-acting sequences or additional structural requirements are needed for IES elimination.

Transcription of the highly amplified tubulin gene family of Stylonychia lemnae

Summary The macronuclear DNA of the hypotrichous ciliate Stylonychia lemnae contains two highly amplified α- and two highly amplified s-tubulin genes with copy numbers between 30,000 and 150,000 molecules per macronucleus. We show that the transcription of these genes is not regulated by their different gene copy numbers. Measurements of the steady-state levels of the corresponding transcripts reveal that the concentrations of the tubulin mRNAs in Stylonychia lemnae are significantly higher for the less amplified genes. We have estimated the concentrations of α- and s-tubulin mRNA after induction of transcription with Concanavalin A. The results indicate that transcript stabilities and probably the promotor strength are the decisive factors for tubulin mRNA concentration. The less amplified α2- and s2-tubulin genes yield more stable transcripts and/or provide a greater efficiency of transcription initiation than their higher amplified α1- and s1-tubulin counterparts. Estimation of the poly(A) tails of the tubulin transcripts indicates a strong correlation between transcript stability and poly(A) tail length: the more stable α2- and s2-tubulin transcripts have longer poly (A) tails than the less stable α1- and s1 -tubulin transcripts.

A gene from the hypotrichous ciliate Stylonychia lemnae coding for a protein with homology to cyclin B

A nucleotide (nt) sequence of a DNA molecule from Stylonychia lemnae with an open reading frame encoding a protein showing homology to cyclin B has been determined. The DNA molecule is 3791-nt long and the deduced 444-amino-acid (aa) sequence shares about 30% identity with the sequences of two yeast cyclin-B homologs over a length of about 210 aa.

Transformation of ciliates with a circular plasmid derived from an overamplified macronuclear DNA of Stylonychia lemnae.

Transformation of ciliates was attempted with a circular plasmid containing a chromosomal DNA of the hypotrichous ciliate Stylonychia lemnae by calcium phosphate-mediated transfection. The plasmid, designated Tubingen-3, was constructed from a bacterial plasmid, a gene for neomycin resistance derived from a vector for the cellular slime mold Dictyostelium, and a 1.2 kb macronuclear chromosomal DNA of Stylonychia. Transformants were obtained at a frequency of one per 104 to 105 of input cells in Stylonychia lemnae and at a lower frequency, one per 10 6, in Tetrahymena thermophila. The vector when simply linearized by digestion with restriction enzymes failed to give transformants, whereas when the vector was linearized so as to have short telomeric sequences at both ends, effective transformation was again observed. The Dictyostelium vector, pCERF DRpl4, with a segment of DNA that contained the putative origin of replication was found, unexpectedly, to be effective in transforming Stylonychia cells. The origin of replication in Stylonychia and Dictyostelium might thus be functionally similar.

The processing of macronuclear DNA sequences during macronuclear development of the hypotrichous ciliate Stylonychia lemnae.

The organization of two macronuclear DNA sequences in the polytene chromosomes of the hypotrichous ciliate Stylonychia lemnae and their processing from the micronucleus via the polytene chromosome stage up to the macronucleus was analyzed. The overall organization of these sequences in the polytene chromosomes resembles that described for the micronucleus of other hypotrichous ciliates, i.e. they are interrupted by internal eliminated sequences and not associated with telomeric sequences. The spacer region between the genes is bordered by direct repeats and inverted repeats are found at the termini of macronuclear sequences and in the spacer region. The organization of these macronuclear DNA sequences in the micronucleus was analyzed by polymerase chain reactions. The results obtained show that in the sequences analyzed no DNA reorganization occurs during polytene chromosome formation.

The organization of internal telomeric repeats in the polytene chromosomes of the hypotrichous ciliate Stylonychia lemnae.

There exist about 1000-1500 internal telomeric sequences per haploid genome in the polytene chromosomes of the hypotrichous ciliate Stylonychia lemnae. All these telomeric repeats are contained in a very conserved element. This element consists of two 2 kb direct repeats flanking a 2.6 kb sequence. Immediately adjacent to one of the repeats a 18mer C4A4C4A4C2 telomeric sequence is localized. Sequences homologous to macronuclear DNA follow 180 bp downstream of the C4A4-bloc. These macronuclear homologous sequences are flanked by the second direct repeat. The possible origin and function of these telomere containing elements is discussed.

Primary structure of a gene-sized DNA encoding calmodulin from the hypotrichous ciliate Stylonychia lemnae

We have isolated and characterized a gene-sized DNA encoding calmodulin (Clm) from macronuclear (MA) DNA of the hypotrichous ciliate, Stylonychia lemnae. The gene has 3500 copies per macronucleus. The length of the gene was deduced by agarose-gel electrophoresis of MA DNA and Southern blot analysis using a Clm cDNA probe from chicken. We then isolated the gene from a MA library. The overall length of the gene is 821 bp with a 450-bp intronless coding region. The deduced amino acid (aa) sequence of ciliate Clm has 149 aa and an M r of 16819. Both ends of the cloned gene have the hypotrichous telomeric C 4A 4 repeat. The coding region is flanked by a 158-bp 5'-leader sequence and a 3'-trailer sequence of 213 bp. S1 analysis was used to locate the transcription start point ( tsp) 49 bp upstream from the start codon. No common eukaryotic transcription signals were found upstream from the tsp. A second gene-sized DNA, detected by its cross-hybridization with the Clm DNA, predicts the existence of a second Ca 2+-binding protein with only one Ca 2+-binding site. It's function and biological significance is yet unknown.

Transcriptionally inactive micronuclei, macronuclear anlagen and transcriptionally active macronuclei differ in histone composition in the hypotrichous ciliateStylonychia lemnae

The chromatin fraction of transcriptionally active macronuclei ofStylonychia lemnae shows a typical core histone pattern after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). However, extracts of transcriptionally inactive micronuclei display only two bands in the molecular weight range of macronuclear core histones, corresponding to macronuclear H2A/H2B and H4. While a band corresponding to macronuclear H3 cannot be detected, the micronuclear fraction contains a prominent Mr 21,000 protein (protein X). We examined the chromatin fractions of the two kinds of nuclei in detail using specific antibodies directed against core histones and synthetic peptides of histones. Both microand macronuclei contain histones that are specifically recognized by antibodies directed against H2A, H2B and H4. Antigenicity in association with core histone H3 is found only in the macronuclear anlagen and in macronuclear extracts. None of the antibodies used react with the micronucleus specific protein X. During macronuclear development protein X disappears while macronuclear H3 appears. Thus we speculate, that protein X may be functionally related to macronuclear H3. The inactive state of the micronucleus is also characterized by the presence of two proteins, Y1 and Y2, with apparent Mrs of 45,000 in SDS-polyacrylamide gels. These proteins are recognized by antibodies raised against the N-terminal peptide of H3 and the C-terminal peptide of H2B respectively. Y1 and Y2 are also present during all of the transcriptionally inactive stages of macronuclear development but are not found in the active macronucleus. By these means chromatin extracts of micronuclei, macronuclear anlagen and macronuclei can be clearly distinguished. The observed differences reveal a sequence of changes in chromatin organization that correlate with the transition from the transcriptionally inactive to the transcriptionally active state of the chromatin.

Identification of an amplification promoting DNA sequence from the hypotrichous cilateStylonychia 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.

Both alpha-tubulin genes are transcriptionally active in Stylonychia lemnae.

Macronuclear DNA of the hypotrichous ciliate Stylonychia lemnae contains two size-classes of molecules coding for alpha-tubulin. Analysis of their coding regions demonstrates that these two size-classes represent two different functional alpha-tubulin genes, alpha 1 and alpha 2; a comparison of these regions shows a 97% homology in their nucleotide sequence and 98.5% in their amino acid sequence. S1-mapping experiments indicate that both alpha 1- and alpha 2-tubulin genes are transcribed. The 5' and 3' noncoding regions flanking the alpha 1- and alpha 2-tubulin genes differ in both length and nucleotide sequence within one strain. When different strains are compared, however, identical non-coding regions and slightly varying coding regions can be found in DNA molecules containing the alpha 1-tubulin genes.

Nucleotide sequence and expression of two β-tubulin genes in Stylonychia lemnae

The gene-sized macronuclear DNA of the hypotrichous ciliate Stylonychia lemnae contains one size class of DNA molecules of 1.85kb (1 kb = 10 3 base-pairs) coding for β-tubulin. These DNA molecules consist of two different β-tubulin genes, β 1 and β 2, which are amplified to about 150,000 ( β 1) and 30,000 ( β 2) copies per macronucleus. Both genes were cloned and sequenced entirely. The coding sequences of the two molecules (1329 base-pairs including TGA) predict identical amino acid sequences for the proteins and show a nucleotide homology of 97.2%. The nucleotide as well as the encoded amino acid sequences are highly conserved, when compared to β-tubulin genes from vertebrates. The ciliatespecific codon TAA specifying glutamine is present only in the β 2-tubulin gene, whereas glutamine is encoded soley by CAA in the β 1-tubulin gene. The 5′ and 3′-non-coding regions of both β-tubulin genes are similar in length, but differ extremely in nucleotide sequence. Both β-tubulin genes are transcriptionally active in S. lemnae, although not all putative transcription-regulatory sequences known from higher eukaryotes can be detected within the non-coding regions. The two transcription products localized by S 1-mapping experiments show a similar length of about 1.40 kb and transcription seems to be regulated differently for β 1 and β 2.

A telomere binding protein in the macronuclei of the hypotrichous ciliate Stylonychia lemnae

Macronuclear proteins from the hypotrichous ciliate Stylonychia lemnae were isolated, separated on SDS-PAGE and electroblotted onto nitrocellulose filters. Filter binding assays with 32P-labeled macronuclear DNA and the synthetic telomere sequence (C 4A 4/G 4T 4) in combination with competition experiments revealed strong preference of a protein with a molecular mass of approx. 40 kDa to the terminal sequence of macronuclear gene-sized molecules.