Coding properties of macronuclear DNA molecules in Sterkiella nova (Oxytricha nova)

The DNA in the macronucleus of the stichotrichs like Sterkiella nova (formerly Oxytricha nova) occurs in short molecules ranging from approximately 200 bp to approximately 20,000 bp. It has been estimated that there are approximately 24,500 different sized DNA molecules in the macronucleus. Single genes have been assigned to approximately 130 different sized macronuclear molecules in various stichotrichs (12 in Sterkiella nova) and hypotrichs, suggesting that each of the -24,500 different sized molecules encodes a different gene. To test this proposition we sequenced 31 macronuclear molecules picked randomly from a plasmid library of macronuclear DNA and analyzed them for potential gene content. The open reading frames (ORFs) in three short molecules encode amino acid (aa) sequences that do not match sequences in GenBank. They may or may not encode genes. Twenty-eight of the 31 molecules contain ORFs encoding aa sequences with significant matches to sequences in GenBank. Six molecules contain more than one ORF with a significant match to GenBank. These results indicate that almost all, if not all of the -24,500 different molecules encode one or more genes, yielding an estimate of -26,800 genes in the macronucleus of S. nova.     

The DNA of ciliated protozoa.

Ciliates contain two types of nuclei: a micronucleus and a macronucleus. The micronucleus serves as the germ line nucleus but does not express its genes. The macronucleus provides the nuclear RNA for vegetative growth. Mating cells exchange haploid micronuclei, and a new macronucleus develops from a new diploid micronucleus. The old macronucleus is destroyed. This conversion consists of amplification, elimination, fragmentation, and splicing of DNA sequences on a massive scale. Fragmentation produces subchromosomal molecules in Tetrahymena and Paramecium cells and much smaller, gene-sized molecules in hypotrichous ciliates to which telomere sequences are added. These molecules are then amplified, some to higher copy numbers than others. rDNA is differentially amplified to thousands of copies per macronucleus. Eliminated sequences include transposonlike elements and sequences called internal eliminated sequences that interrupt gene coding regions in the micronuclear genome. Some, perhaps all, of these are excised as circular molecules and destroyed. In at least some hypotrichs, segments of some micronuclear genes are scrambled in a nonfunctional order and are recorded during macronuclear development. Vegetatively growing ciliates appear to possess a mechanism for adjusting copy numbers of individual genes, which corrects gene imbalances resulting from random distribution of DNA molecules during amitosis of the macronucleus. Other distinctive features of ciliate DNA include an altered use of the conventional stop codons.     

The DNA of ciliated protozoa.

Ciliates contain two types of nuclei: a micronucleus and a macronucleus. The micronucleus serves as the germ line nucleus but does not express its genes. The macronucleus provides the nuclear RNA for vegetative growth. Mating cells exchange haploid micronuclei, and a new macronucleus develops from a new diploid micronucleus. The old macronucleus is destroyed. This conversion consists of amplification, elimination, fragmentation, and splicing of DNA sequences on a massive scale. Fragmentation produces subchromosomal molecules in Tetrahymena and Paramecium cells and much smaller, gene-sized molecules in hypotrichous ciliates to which telomere sequences are added. These molecules are then amplified, some to higher copy numbers than others. rDNA is differentially amplified to thousands of copies per macronucleus. Eliminated sequences include transposonlike elements and sequences called internal eliminated sequences that interrupt gene coding regions in the micronuclear genome. Some, perhaps all, of these are excised as circular molecules and destroyed. In at least some hypotrichs, segments of some micronuclear genes are scrambled in a nonfunctional order and are recorded during macronuclear development. Vegetatively growing ciliates appear to possess a mechanism for adjusting copy numbers of individual genes, which corrects gene imbalances resulting from random distribution of DNA molecules during amitosis of the macronucleus. Other distinctive features of ciliate DNA include an altered use of the conventional stop codons.     

A simple and rapid PCR-based method to isolate complete small macronuclear minichromosomes from hypotrich ciliates: 5S rDNA and S26 ribosomal protein gene of Oxytricha (Sterkiella) nova

Hypotrich ciliates present a macronuclear genome consisting of gene-sized instead of chromosome-sized DNA molecules. Exploiting this unique eukaryotic genome feature, we introduce, for the first time in ciliates, a rapid and easy PCR method using telomeric primers to isolate small complete macronuclear DNA molecules or minichromosomes. Two presumably abundant macronuclear DNA molecules, containing ribosomal genes, were amplified from the Oxytricha (Sterkiella) nova complete genome after using this method, and then were cloned and sequenced. The 5S rDNA sequence of O. (S.) nova is the third one reported among hypotrich ciliates; its primary and secondary structure is compared with other eukaryotic 5S rRNAs. The ribosomal protein S26 gene is the first one reported among ciliates. This "End-End-PCR" method might be useful to obtain similar gene-sized macronuclear molecules from other hypotrich ciliates, and, therefore, to increase our knowledge on ribosomal genes in these eukaryotic microorganisms.     

A Simple and Rapid PCR-based Method to Isolate Complete Small Macronuclear Minichromosomes from Hypotrich Ciliates: 5S rDNA and S26 Ribosomal Protein Gene of Oxytricha (Sterkiella) nova

Hypotrich ciliates present a macronuclear genome consisting of gene-sized instead of chromosome-sized DNA molecules. Exploiting this unique eukaryotic genome feature, we introduce, for the first time in ciliates, a rapid and easy PCR method using telomeric primers to isolate small complete macronuclear DNA molecules or minichromosomes. Two presumably abundant macronuclear DNA molecules, containing ribosomal genes, were amplified from the Oxytricha (Sterkiella) nova complete genome after using this method, and then were cloned and sequenced. The 5S rDNA sequence of O. (S.) nova is the third one reported among hypotrich ciliates; its primary and secondary structure is compared with other eukaryotic 5S rRNAs. The ribosomal protein S26 gene is the first one reported among ciliates. This “End-End-PCR” method might be useful to obtain similar gene-sized macronuclear molecules from other hypotrich ciliates, and, therefore, to increase our knowledge on ribosomal genes in these eukaryotic microorganisms.     

Invention and Mystery in Hypotrich DNA1

ABSTRACT “The capacity to blunder slightly is the real marvel of DNA. Without this special attribute, we would still be anaerobic bacteria and there would be no music.” Lewis Thomas³ Hypotrichs have evolved extraordinary ways of organizing, manipulating, and replicating the DNA in their micronuclear and macronuclear genomes. Short macronuclear DNA molecules containing single genes are created by excision from chromosomes, accompanied by massive elimination of the germline DNA sequences between genes. Germline genes themselves are interrupted by multiple noncoding segments called internal eliminated segments, or IESs, that divide genes into multiple macronuclear-destined segments, or MDSs. The functional significance of this organization is unknown. Over evolutionary time IESs accumulate mutations rapidly are inserted into or excised from genes, and shift position along DNA molecules. MDSs are ligated to create functional genes when IESs are spliced out of micronuclear DNA during macronuclear development. MDSs in some germline genes are in scrambled disorder and become unscrambled in association with IES elimination. Replication of DNA in the macronucleus is accomplished by organization of replication enzymes and factors into a structure that sweeps through the macronucleus to replicate the many millions of gene-sized DNA molecules. The significance of many of the bizarre DNA phenomena in the evolutionary/functional success of hypotrichs is still unclear.     

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.     

The micronuclear gene encoding a putative aminoacyl-tRNA synthetase cofactor of the ciliate Euplotes octocarinatus is interrupted by two sequences that are removed during macronuclear development.

The micronuclear gene of the ciliated protozoan Euplotes octocarinatus (Eo) syngen 1 encoding the putative aminoacyl-tRNA synthetase cofactor (ARCE), as well as its macronuclear version and the corresponding cDNA, were amplified and sequenced. Analyses of the sequences revealed that the micronuclear gene contains two sequences (430 and 625bp long) that are missing in the macronuclear version of this gene. These sequences are called 'internal eliminated sequences' (IESs) and appear to occur in all ciliates. The two IESs are located in the coding region of the micronuclear gene. One IES is flanked by a pair of dinucleotide 5'-TA-3' direct repeats and the other one by a pair of hepta-nucleotide 5'-TTACTGA-3' direct repeats. Inside the two IESs, several other sequence repeats were found. The macronuclear DNA molecule carrying this gene is 1517bp long and shows characteristics typical of macronuclear chromosomes of hypotrichous ciliates. Copy number determination revealed that the molecule is amplified to only about 750 copies per macronucleus. The deduced protein is a 441-amino-acid (aa) polypeptide with a molecular mass of 50kDa. It shares a conserved endothelial monocyte-activating polypeptide II (EMAP II)-like carboxyl-terminal domain and a hydrophilic central domain containing a KEKE-motif with a group of proteins associated with aminoacyl-tRNA synthetases and tRNAs.     

Macronuclear DNA organization and transcription in Paramecium primaurelia

Macronuclear DNA was isolated from Paramecium primaurelia, stock 168. Although the macronucleus is polyploid to the extent of 840C, in other respect the DNA appears to be simply organized, having neither satellite sequences nor substantial amounts of intermediately repetitive sequence. The sequence complexity of macronuclear DNA is quite low for a eukaryote cell, being approximately 19 times more complex than the genome of Escherichia coli. In addition, the GC content is low (25%) and the isolated DNA molecules have lengths mostly in the range 0.2–5 μm. In these various respects, the macronuclear DNA of Paramecium is similar to that of other ciliates. A clone of Paramecium cultured under controlled conditions contains polyadenylated RNA sequences which are homologous to 5–8% of the macronuclear DNA. Sequence complexity analysis indicates that the polyadenylated RNA contains two abundance classes of molecules, one present at low frequency and transcribed from approximately 10⁴ genes, the other at 100 times greater concentration and transcribed from about 100 genes. The relevance of these results to the control of gene expression in Paramecium is discussed.     

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.     

Molecular structure and flanking nucleotide sequences of the natural chicken ovomucoid gene

Five independent clones containing the natural chicken ovomucoid gene have been isolated from a chicken gene library. One of these clones, CL21, contains the complete ovomucoid gene and includes more than 3 kb of DNA sequences flanking both termini of the gene. Restriction endonuclease mapping, electron microscopy and direct DNA sequencing analyses of this clone have revealed that the ovomucoid gene is 5.6 kb long and codes for a messenger RNA of 821 nucleotides. The structural gene sequence coding Ifor the mature messenger RNA is split into at least eight segments by a minimum of seven intervening sequences of various sizes. The shortest structural gene segment is only 20 nucleotides long. All seven intervening sequences are located within the peptide coding region of the gene, and the sequences at the 5' and 3' untranslated regions of the mRNA are not interrupted by intervening sequences. The DNA sequences of the regions flanking the 5' and 3' termini of the gene have been determined. Thirty nucleotides before the start of the messenger RNA coding sequence is the heptanucleotide TATATAT, which is also present in a similar location relative to the chicken ovalbumin gene and other unique sequence eucaryotic genes. This sequence resembles that of the Pribnow box in procaryotic genes where a promoter function has been implicated. Seven nucleotides past the 3' end of the gene is the tetranucleotide TTGT, a sequence found to be present at identical locations as either TTTT or TTGT in other eucaryotic genes that have been sequenced. These conserved DNA sequences flanking eucaryotic genes may serve some regulator function in the expression of these genes.     

Actin of Histriculus cavicola: characteristics of the highly divergent hypotrich ciliate actins.

A macronuclear gene-sized molecule carrying an actin gene from the hypotrich ciliate, Histriculus cavicola, was characterized. Southern blot analysis using a coding region probe suggested that actin in H. cavicola is encoded by a single gene. A comparison of the promoter regions indicated that the H. cavicola actin gene has a TATA box in the 5' flanking region in a position identical to those in other oxytrich ciliates. The coding sequence of this gene is not interrupted by any introns, and codes for a protein of 375 amino acid residues. This protein shares a high degree of similarity with other oxytrichid actins, and a relatively low similarity with actins from other eukaryotes. Comparative analyses of sequences indicated that most of the amino acid substitutions in hypotrich actins are found in surface loops, while the core structures are well-conserved. The sites that interact with DNase I and several regions involved in actin-actin contact have diverged considerably in hypotrich actins, while nucleotide-binding sites are the best-conserved interaction motif.