Stylonychia Lemnae Research Articles

Estimation of the Number of α-Tubulin Genes in Three Ciliated Protozoa

Summary: In an attempt to study the multitubulin family in ciliated protozoa, we have estimated the number of α-tubulin genes present in the genome of three “Spirotrich” ciliates by using an α-tubulin probe from the ciliate Stylonychia lemnae . Southern hybridization analyses of single or double-digested DNA from ciliates Histriculus cavicola, Euplotes eurystomus and Stentor coeruleus revealed banding patterns consistent with the presence of three, two and at least nine α-tubulin genes in each species, respectively. Comparison of the hybridizing patterns of Histriculus and Euplotes digested DNAs showed a great similarity between both species. The existence of common restriction fragments is an evidence of a sequence homology at the genomic level and might attribute much weight to a close relation between both ciliates. The controversial relationships among “Spirotrich” ciliates are discussed.

Telomerase RNAs of different ciliates have a common secondary structure and a permuted template.

Telomerase is composed of protein and RNA. The RNA serves as a template for telomere DNA synthesis and may also be important for enzyme structure or catalysis. We have used the presence of conserved sequence elements in the promoter and template regions to amplify by PCR the telomerase RNA genes from six different hypotrichous ciliates: Oxytricha nova, Oxytricha trifallax, Stylonychia mytilis, Stylonychia lemnae, Euplotes aediculatus, and Euplotes eurystomus. RNaseH cleavage of the O. nova RNA in extracts by use of a complementary oligonucleotide leads to loss of telomerase activity, supporting the identification. Primary sequence and biochemical experiments suggest that the templates of Oxytricha and Stylonychia are circularly permuted relative to that of E. aediculatus. On the basis of the pause sites, the former two add G4T4 during a single primer elongation cycle, whereas E. aediculatus adds G3T4G. The only primary sequence element outside the template that is conserved between these phylogenetically distant telomerase RNAs is the sequence 5'-(C)UGUCA-3', which precedes the template regions by exactly two bases. We propose a common secondary structure model that is based on nucleotide covariations, a model which resembles that proposed previously for tetrahymenine telomerase RNAs.

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.

A β-tubulin gene of Naegleria encodes a carboxy-terminal tyrosine : Aromatic amino acids are conserved at carboxy termini

A gene that directs the programmed synthesis of flagellar beta-tubulin during the rapid differentiation of Naegleria gruberi from amoebae to flagellates has been cloned and sequenced. The intronless gene is one of 8 to 10 similar but non-identical genes that are dispersed in the genome. beta-Tubulin mRNA homologous to this gene family is expressed transiently during differentiation, and has not been detected in amoebae. The encoded beta-tubulin is strongly conserved, with features that closely resemble the beta-tubulins of diverse organisms, especially organisms that, like Naegleria, use tubulin to assemble flagellar axonemes. In most sequenced alpha-tubulins, the encoded carboxy-terminal amino acid is tyrosine, which undergoes post-translational removal and readdition, conserved processes of unknown function. In N. gruberi, unusually, the terminus of alpha-tubulin is encoded as glutamine while that of beta-tubulin is tyrosine. The presence of these divergent termini on subunits of a conserved tubulin provoked us to re-examine aromatic amino acids at the termini of alpha- and beta-tubulins. Although evolution has tinkered extensively with the carboxy-terminal domains of tubulin subunits, we find an unexpected conservation. In every organism or cell type for which both tubulin subunits have been sequenced, except the ciliate Stylonychia lemnae, at least one tubulin subunit of some or all tubulin heterodimers terminates in an aromatic amino acid, either tyrosine or phenylalanine. This remarkable conservation of carboxy-terminal aromatic amino acids suggests that these residues serve some crucial function.

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.

Non‐Coding DNA in Macronuclear Chromosomes of Hypotrichous Ciliates

Massive elimination of sequences occurs in the development of the macronucleus of hypotrichous ciliates. The surviving sequences are presumed to have functions in the macronucleus; what little is known about non-coding macronuclear sequences is reviewed. The 1.7 kbp macronuclear chromosome that carries a histone H4 gene consists primarily of non-coding DNA 5' of the histone gene. This region is shown by sequence comparison to carry several perfectly conserved sequence blocks up to 14 bp long, scattered amongst regions which have evolved greatly since the divergence of Oxytricha nova and Stylonychia lemnae. This result is consistent with the suggestion of Harper and Jahn [Harper, D. S. & Jahn, C. L. 1989. Actin, tubulin and H4 histone genes in three species of hypotrichous ciliated protozoa. Gene, 75:93-107] that this large non-coding 5' region may be involved in the transcriptional regulation of the histone H4 gene carried on the 1.7 kbp chromosome. Very little is known about transcriptional control in hypotrichs; identification of conserved non-coding sequences of orthologous hypotrich genes promises to provide clues to potential cis-acting control signals.

A comparative study of RNAs from four ciliate groups and a mollusc and data on their poly(A) + RNA in vitro translation

RNAs from the ciliates Euplotes octocarinatus, Paramecium tetraurelia, Stylonychia lemnae, Tetrahymena thermophila and the snail Lymnaea stagnalis were isolated and compared for structural differences. The organisms were found to differ considerably in the size and structure of their rRNAs, the extent of polyadenylation and the length of the respective poly A tails of their mRNAs. The mRNAs of Euplotes, Paramecium, Stylonychia and Lymnaea were also compared for in vitro translatability in the rabbit reticulocyte lysate and the wheat germ translation systems. In accordance with earlier observations for Paramecium [15] and the finding that Paramecium, Stylonychia, Oxytricha and Tetrahymena deviate from the universal genetic code by using the stop codons UAA and UAG to encode glutamine [4, 8,11,12,21] it was found that Paramecium and Stylonychia poly(A)(+) RNAs were poor templates for in vitro translation. Unexpectedly, however, poly(A)(+) RNA of the ciliate Euplotes was readily translated in vitro and yielded a great number of polypeptides with different molecular weights comparable to the data obtained with poly(A)(+) RNA of Lymnaea.

Macronuclear and micronuclear configurations of a gene encoding the protein synthesis elongation factor EF 1α in Stylonychia lemnae

The micronuclear and macronuclear configurations of a gene encoding the protein synthesis elongation factor EF 1 alpha in the hypotrich ciliate Stylonychia lemnae were compared. The two sequences are generally colinear. The coding sequence of the micronuclear gene is, however, interrupted by a 64 bp insert flanked by a 2 bp direct repeat in a gene region which is moderately conserved among EF 1 alpha genes of different organisms. The insertion site is distinct from known intron positions in eukaryotic EF 1 alpha genes. The insert sequence shows inverted repeats at its ends and thus exhibits typical features of an internal eliminated sequence (IES). Comparison with other such sequences in the related organism Oyxtricha nova shows that the IES falls into a new group of such elements. The macronuclear gene exhibits a strikingly limited codon usage, which cannot be simply explained by the overall base composition of the DNA but probably also relates to the very high copy number of the macronuclear gene and the putative high amount of the gene product.

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.

Actin, tubulin and H4 histone genes in three species of hypotrichous ciliated protozoa

In hypotrichous ciliated protozoa, genes are transcribed in the macronucleus where the genome consists of ‘gene-sized’ linear DNA molecules. We have isolated clones of actin, tubulin and H4 histone macronuclear genes from Oxytricha nova, Stylonychia lemnae and Euplotes crassus in an effort to determine if they possess molecules of similar size for a given coding function, and also to determine the size range of non-coding DNA present on these molecules. Our results indicate that while the length of their non-coding DNA can vary slightly, both between different hypotrichs and within the gene family of a single organism, actin and tubulin macronuclear molecules are similarly sized. The sizes observed for these molecules support the hypothesis that each macronuclear molecule encodes a single gene. However, the H4 histone macronuclear molecules show a much wider size range and generally are much longer than necessary to encode the H4 histone. We therefore sequenced a 1700-bp H4 histone macronuclear molecule from O. nova to determine if it might possibly encode additional gene products. Sequence data reveals the presence of nine open reading frames (ORFs) greater than 100 bp in length; however, Northern hybridization analysis of the products of this DNA molecule reveals only a single transcript.

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.

Characterization of the alpha-tubulin gene family of Arabidopsis thaliana.

The genome of Arabidopsis thaliana (Linnaeus) Heynhold was shown to contain an alpha-tubulin gene family consisting of at least four genes and/or pseudogenes. The primary structure of a transcribed alpha-tubulin gene was determined. A comparison of the predicted amino acid sequence of the A. thaliana alpha-tubulin with the predicted amino acid sequences of alpha-tubulins of Chlamydomonas reinhardtii, Stylonychia lemnae, and Homo spaiens reveals a high degree of homology; 90%, 87%, and 83% identity, respectively. Thus, a plant alpha-tubulin exhibits a high degree of homology to the alpha-tubulins of protists and animals. The coding sequence of the A. thaliana alpha-tubulin gene is interrupted by four introns, which occur at positions different from those of the less numerous introns of C. reinhardtii and rat alpha-tubulin genes. S1 nuclease mapping data showed that transcription is initiated 99 +/- 1 base pairs upstream from the translation initiation codon. Both 5' and 3' noncoding gene-specific probes were used to examine the expression of the alpha-tubulin gene in leaves, roots, and flowers by hybridization to total RNA isolated from these tissues. The results showed that the alpha-tubulin gene was transcribed in all three tissues.

Germ line specific DNA and chromosomes of the ciliate Stylonychia lemnae

The variation in DNA content of the micronucleus (germinal nucleus) of Stylonychia lemnae and its relation to the number of chromosomes was examined. Different populations possess similar amounts of micronuclear DNA but there are differences of ±30% between clones of the same population. However, the DNA content varies by about 100% in the micronuclei during the lifetime of a clone. The haploid micronucleus contains 35 or 36 chromosomes which persist in the developing macronucleus anlagen and grow to giant chromosomes. Besides this remaining subset, the micronucleus contains a variable number of germ line restricted chromosomes (mean about 140; range between 100 and 180). The somatic macronucleus eliminates these elements early in its development. The varying number of the germ line restricted chromosomes is responsible for the variation in the micronuclear DNA content.

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.

A linear shuttle vector for yeast and the hypotrichous ciliate Stylonychia

A linear plasnud was constructed in vitro using the telomeres of the rDNA of Tetrahymena pyriformis. These telomeres were added to a yeast circular vector containing an ARS sequence from Dictyostelium, the LEU2 gene of yeast and the neo gene from Escherichia coli Tn 5 fused with a eukaryotic promoter. The resulting plasmid was used to transform yeast. During the replication of the linear plasmid in yeast it was spontaneously modified at the extremity by the addition of 300 bp of yeast telomeric sequence for each end. Total DNA prepared from yeast transformants was used to transform the hypotrichous ciliate Stylonychia lemnae. The same plasmid isolated from Stylonychia can again be replicated in yeast.

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.