Micronuclear Genes Research Articles

The Structure of Elementary Strategies for Gene Assembly in Ciliates

We consider in this paper the assembly of micronuclear genes in stichotrichous ciliates to their macronuclear form. We represent the micronuclear genes and all their intermediate forms from micro-to macro-as signed permutations, where integer i stands for the i-th MDS of the macronuclear gene and ī stands for the inverted form of that MDS; the macronuclear assembled gene is represented as the sorted permutation 1 2... n, while its micronuclear form is an arbitrary signed permutation. We focus on the elementary gene assembly model consisting of two operations on signed permutations: eh elementary hairpin inverting and ed elementary double recombination; gene assembly is modeled in this framework as a permutation sorting process. The general problem we investigate is to give a characterization of all signed permutations that can be sorted by the elementary operations. We make progress towards a full solution for this problem by relating sequences of eh and ed operations applicable to a given permutation to paths in the dependency graph associated to that permutation.

Evolution of the germline actin gene in hypotrichous ciliates: multiple nonscrambled IESs at extremely conserved locations in two urostylids.

In hypotrichous ciliates, macronuclear chromosomes are gene-sized, and micronuclear genes contain short, noncoding internal eliminated segments (IESs) as well as macronuclear-destined segments (MDSs). In the present study, we characterized the complete macronuclear gene and two to three types of micronuclear actin genes of two urostylid species, i.e. Pseudokeronopsis rubra and Uroleptopsis citrina. Our results show that (1) the gain/loss of IES happens frequently in the subclass Hypotrichia (formerly Stichotrichia), and high fragmentation of germline genes does not imply for gene scrambling; and (2) the micronuclear actin gene is scrambled in the order Sporadotrichida but nonscrambled in the orders Urostylida and Stichotrichida, indicating the independent evolution of MIC-actin gene patterns in different orders of hypotrichs; (3) locations of MDS-IES junctions of micronuclear actin gene in coding regions are conserved among closely related species.

A sequence-based analysis of the pointer distribution of stichotrichous ciliates

Micronuclear genes in stichotrichous ciliates are broken into blocks separated by noncoding sequences, sometimes with the blocks in a shuffled order, some even inverted. During reproduction, all blocks are assembled in the correct order and orientation. This process is possible due to the special structure of micronuclear genes: each coding block M ends with a short nucleotide sequence (called pointer) that is repeated at the beginning of the coding block that should follow M in the assembled gene. Many of the pointers have multiple occurrences along both strands of the gene. This yields a very high number of pointer-induced possible divisions into coding and noncoding blocks. We investigate the distribution of pointers for all currently sequenced micronuclear ciliate genes with the goal of identifying what distinguishes the real gene structure among all possible coding/noncoding divisions. We find a sharp criterion in the average a/t-content of the noncoding blocks: the real division has, in most cases, the maximum such content among all possible combinations. Even for pointers as short as two nucleotides, the real division is one of very few with an average a/t-content of its noncoding blocks over 80%. The separation is most clear when the loci of pointers of up to four nucleotides (even three in the case of unscrambled genes) are fixed (e.g., through a template-based recombination mechanism).

Solutions to computational problems through gene assembly

Gene assembly in stichotrichous ciliates is an impressive computational process. They have a unique way of storing their genetic information in two fundamentally different forms within their two types of nuclei. Micronuclear genes are broken into blocks (called MDSs), with MDSs shuffled and separated by non-coding material; some of the MDSs may even be inverted. During gene assembly, all MDSs are sorted in the correct order to yield the transcription-able macronuclear gene. Based on the intramolecular model for gene assembly, we prove in this paper that gene assembly may be used in principle to solve computational problems. We prove that any given instance of the Hamiltonian path problem may be encoded in a suitable way in the form of an `artificial' gene so that gene assembly is successful on that gene-like pattern if and only if the given problem has an affirmative answer.

Template-guided recombination for IES elimination and unscrambling of genes in stichotrichous ciliates

The micronuclear versions of genes in stichotrichous ciliates are interrupted by multiple, short, non-coding DNA segments called internal eliminated segments, or IESs. IESs divide a gene into macronuclear destined segments, or MDSs. In some micronuclear genes MDSs are in a scrambled disorder. During development of a micronucleus into a macronucleus after cell mating the IESs are excised from micronuclear genes and the MDSs are spliced in the sequentially correct order. Pairs of short repeat sequences in the ends of MDSs undergo homologous recombination to excise IESs and splice MDSs. However, the repeat sequences are too short to guide unambiguously their own alignment in preparation for recombination. Based on experiments by others on the distantly related ciliate, Paramecium, we propose a molecular model of template-guided recombination to explain the excision of the 100 000–150 000 IESs and splicing of MDSs, including unscrambling, in the genome of stichotrichous ciliates. The model solves the problem of correct pairing of pointers, precisely identifies MDS–IES junctions, and provides for irreversible recombination.

Characterizing the Micronuclear Gene Patterns in Ciliates

The process of gene assembly in ciliates is one of the most complex examples of DNA processing known in any organism, and it is fascinating from the computational point of view—it is a prime example of DNA computing in vivo. In this paper we continue to investigate the three molecular operations (ld, hi , and dlad ) that were postulated to carry out the gene assembly process in the intramolecular fashion. In particular, we focus on the understanding of the IES/ MDS patterns of micronuclear genes, which is one of the important goals of research on gene assembly in ciliates. We succeed in characterizing for each subset S of the three molecular operations those patterns that can be assembled using operations in S. These results enhance our understanding of the structure of micronuclear genes (and of the nature of molecular operations). They allow one to establish both similarity and complexity measures for micronuclear genes.

Gene assembly through cyclic graph decomposition

We present in this paper a graph theoretical model of gene assembly, where (segments of) genes are distributed over a set of circular molecules. This model is motivated by the process of gene assembly in ciliates, but it is more general. In this model a set of circular DNA molecules is represented by a bicoloured and labelled graph γ consisting of cyclic graphs, and the recombination takes place in two stages: first, by folding γ∗P with respect to a set P of pairs of vertices of the graph (representing pointers in the micronuclear genes of the ciliate), and secondly, by unfolding the so obtained graph to γ⊛P with respect to vertices of higher valency. The final graph γ⊛P is again a set of bicoloured cyclic graphs, where the genes are present as maximal monochromatic paths. Thus, the process of gene assembly corresponds to the dynamic process of changing cyclic graph decompositions. We show that the operation ⊛ is well behaved in many respects, and that there is a sequence of pointer sets P1,…,Pm consisting of one or two pairs such that γ⊛P=(⋯((γ⊛P1)⊛P2)⋯⊛Pm) and each intermediate step γi=(⋯((γ⊛P1)⊛P2)⋯⊛Pi) is intracyclic, that is, the segments of a gene that lie in the same connected component of γi, will lie in the same connected component of the successor graph γi+1.

Evolution of IESs and scrambling in the actin I gene in hypotrichous ciliates.

Germ-line (micronuclear) genes in hypotrichous ciliates are interrupted by numerous, short, noncoding, AT-rich segments called internal eliminated segments, or IESs. IESs divide a gene into macronuclear destined segments, or MDSs. IESs are excised from micronuclear genes, and the MDSs are spliced when a micronuclear genome is processed into a macronuclear genome after cell mating. In the micronuclear version of the actin I gene intramolecular recombination between IESs during evolution has put MDSs into a scrambled disorder in some but not all hypotrichs. Studies using rDNA sequences to define phylogenetic relationships among eight hypotrichs suggests that evolution of the micronuclear actin I gene proceeds by successive addition of IESs in earlier diverging species, without MDS scrambling. Continued addition of IESs and recombination among IESs in later diverging species produced actin I genes with scrambled MDSs. Subsequent to MDS scrambling, additional IESs were inserted into the more recently evolved species. Thus, IES insertions and gene scrambling occur in a progressive manner during species evolution to produce micronuclear actin I genes of increasing structural complexity.

Does ribosomal DNA get out of the micronuclear chromosome in Paramecium tetraurelia by means of a rolling circle?

The macronuclear genes coding for rRNA (ribosomal DNA [rDNA]) of Paramecium tetraurelia, stock 51, are arranged in polymers consisting of units made up of a transcribed coding region and a nontranscribed spacer region. The whole macronuclear polymer ends with a portion of the spacer on either end followed by a telomere. Six kinds of macronuclear units, or genes, were mapped. Spacers were different, and transcribed regions were the same. These genes are found in markedly different numbers in the macronucleus. The most common gene shows two regions in the spacer where a sequence is followed by a direct repeat. The next most common gene is similar but shows a deletion plus a number of base pair substitutions. Although most cosmid clones contain only a single kind of gene, many contain more than one. These are thought to be produced by somatic crossing over. The four micronuclear genes that have been isolated consist of a single central transcribed region and portions of the spacer on either end. Sequencing indicates that the two ends of the molecule are partially redundant. While the spacer region at the right end of the macronuclear polymer is derived from the micronuclear spacer on the right, the spacer at the left end of the macronuclear polymer is derived from regions of the micronuclear spacer on both the right and the left. To account for this situation, a rolling-circle model for generation of the macronuclear rDNA from the micronuclear DNA is proposed.

Evolution of DNA organization in hypotrichous ciliates.

Profound changes have been introduced into the germline (micronuclear) and somatic (macronuclear) genomes of hypotrichous ciliates during evolution. First, multiple, short, unique, noncoding sequences, called IESs, have been inserted into micronuclear genes. IESs are spliced out of each gene, and the gene segments, called MDSs, are ligated during conversion of the micronuclear genome to a macronuclear genome after cell mating. The IESs in a particular gene can change dramatically in number, position, length, and sequence during speciation. Once inserted, IESs can shift along the DNA of a gene 1 or 2 bp at a time by a mutational mechanism that does not alter the coding sequence of the gene. Second, the MDSs in the same genes have been rearranged into random or nonrandom, scrambled disorder. The origin of nonrandom scrambling patterns can be explained by a model of simultaneous insertion of multiple IESs into a germline gene during evolution. Subsequent recombinations among the IESs may change the MDS arrangement from a nonrandomly scrambled to a randomly scrambled pattern, including inversions of MDSs. Third, during the conversion of a micronucleus to a macronucleus after cell mating, MDSs are ligated in the unscrambled, orthodox order in association with IES excision, and the genes are then removed from the chromosomes as individual, short DNA molecules. These gene-size molecules are amplified many-fold to produce a mature macronucleus. All of these phenomena attest to a remarkable fluidity of the hypotrich genome both over evolutionary time and in the conversion of a germline genome to a somatic genome. The significance of this fluidity for the life and evolution of these organisms is still obscure. Recombination among IESs could shuffle MDSs and facilitate faster evolution of new genes.

Evolution of gene scrambling in ciliate micronuclear genes.

Evolution of gene scrambling in ciliate micronuclear genes.

Volatility of internal eliminated segments in germ line genes of hypotrichous ciliates.

Germ line micronuclear genes in ciliated protozoa contain two types of interrupting sequences. Some genes contain introns, but internal eliminated segments (IESs) are much more prevalent. IESs are AT-rich DNA segments that separate macronucleus-destined segments (MDSs) in micronuclear genes. All IESs are excised and destroyed when a micronucleus develops into a macronucleus after each cell mating. IESs have no discernible function. Therefore, an investigation of the behavior of IESs in evolution has been undertaken to assess their possible significance. The IESs in the micronuclear gene encoding the beta-subunit of the telomere-binding protein (beta-TP) are not conserved in number, position, sequence, or length during the evolution of four oxytrichid ciliates. In contrast, the scrambled pattern of MDSs and IESs of the micronuclear actin I gene has been conserved during evolution; however, the precise positions, sequences, and lengths of the IESs differ among species, and in some organisms the actin I gene contains an additional IES and MDS. Corresponding IESs in the actin I genes among the different organisms have shifted positions by 1 to 14 bp, presumably by a mutation-shifting mechanism, creating differences in the repeat sequences flanking IESs. Thus, conservation of a particular repeat sequence among species is not required for IES excision. The changes in IES number and position in the beta-TP genes among ciliates are in sharp contrast to the stability of the intron position. Therefore, IESs are volatile, hypermutable elements that are inserted, removed, shifted, and modified continuously in the germ line through evolutionary time.

Internal eliminated segments (IESs) of Oxytrichidae.

Internal eliminated segments (IESs) are sequences that interrupt coding and noncoding regions of germline (micronuclear) genes of ciliated protozoa. IESs are flanked by short, unique repeat sequences, which are presumably required for precise IES excision during macronuclear development. Coding and noncoding segments of genes separated by IESs are called macronuclear-destined segments, or MDSs. We have compiled the characteristics of 89 individual IESs in 12 micronuclear genes in the Oxytricha and Stylonychia genera to define the IES phenomenon precisely, a first step in determining the origin, function and significance of IESs. Although all 89 IESs among the 12 different genes are AT-rich, they show no other similarity in sequence, length, position or number. Two main types of IESs are present. IESs that separate scrambled MDSs are significantly shorter and more frequent and have longer flanking repeat sequences than IESs that intervene between nonscrambled MDSs. A comparison of the nonscrambled gene encoding beta-telomere binding protein in three species of hypotrichs shows that even in the same gene IESs are not conserved in sequence, length, position, or number from species to species. A comparison of IESs in the scrambled gene encoding actin I in the three species shows that the evolutionary behavior of IESs in a scrambled gene may be more constrained. However, IESs in the scrambled actin I gene have shifted along the DNA molecule during evolution. In total, the various studies show that IESs are hypermutable in sequence and length. They insert, excise, and shift along DNA molecules more or less randomly during evolution, with no discernible function or consequences.

Scrambling of the actin I gene in two Oxytricha species.

The DNA in a germ-line nucleus (a micronucleus) undergoes extensive processing when it develops into a somatic nucleus (a macronucleus) after cell mating in hypotrichous ciliates. Processing includes destruction of a large amount of spacer DNA between genes and excision of gene-sized molecules from chromosomes. Before processing, micronuclear genes are interrupted by numerous noncoding segments called internal eliminated sequences (IESs). The IESs are excised and destroyed, and the retained macro-nuclear-destined sequences (MDSs) are spliced. MDSs in some micronuclear genes are not in proper order and must be reordered during processing to create functional gene-sized molecules for the macronucleus. Here we report that the micronuclear actin I gene in Oxytricha trifallax WR consists of 10 MDSs and 9 IESs compared to the previously reported 9 MDSs and 8 IESs in the micronuclear actin I gene of Oxytricha nova. The MDSs in the actin I gene are scrambled in a similar pattern in the two species, but the positions of MDS-IES junctions are shifted by up to 14 bp for scrambled and 138 bp for the nonscrambled MDSs. The shifts in MDS-IES junctions create differences in the repeat sequences that are believed to guide MDS splicing. Also, the sizes and sequences of IESs in the micronuclear actin I genes are different in the two Oxytricha species. These observations give insight about the possible origins of IES insertion and MDS scrambling in evolution and show the extraordinary malleability of the germ-line DNA in hypotrichs.

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.

Analysis of a scrambled gene: the gene encoding alpha-telomere-binding protein in Oxytricha nova.

Following cell mating in ciliates, a copy of the micronuclear genome is processed into a new macronucleus through massive cutting, reordering, splicing, elimination, and amplification of the DNA. DNA processing includes the deletion of short interrupting elements called internal eliminated sequences (IESs), followed by the splicing of the remaining segments, known as macronuclear destined sequences (MDSs). The MDSs in some micronuclear genes, such as actin I, are scrambled and must be reordered during IES removal and MDS splicing to yield a functional gene. Here, we describe the cloning, sequencing, and characterization of a different scrambling pattern for the gene that encodes the alpha subunit of the telomere-binding protein of Oxytricha nova. The micronuclear gene is made up of 14 MDSs in the scrambled order 1-3-5-7-9-11-2-4-6-8-10-12-13-14. Only the scrambled version is present in the micronucleus, and only the unscrambled version is present in the macronucleus. We propose that unscrambling occurs by homologous recombination guided by pairs of direct repeats at MDS-IES junctions. The patterned array of scrambling may be a clue to the origin of scrambling in this gene.

A genetic screen for vegetative gene expression in the micronucleus of Tetrahymena thermophila.

The presence of a micronucleus with at least a small portion of the micronuclear genome appears to be indispensable for vegetative viability in the ciliate Tetrahymena thermophila. A genetic screen was devised to detect evidence of expression of essential genes in the vegetative micronucleus by identification of thermosensitive-lethal mutations expressed in the absence of nuclear reorganization. Although control experiments demonstrated the efficacy of the method for induction and recovery of thermosensitive lethal mutations in micronuclear genes, no expressed mutations were recovered in the absence of nuclear reorganization. This finding complements the existing lack of convincing biochemical evidence for gene expression in the vegetative micronucleus and suggests that the essential function may involve genomic DNA sequences for which thermosensitive mutant alleles are not recoverable, or perhaps a non-genomic component of the organelle.

The nature of control of oral development by the micronucleus in sexual reproduction of Paramecium tetraurelia

Twelve laser-irradiated cell lines and eight cis. platin-treated cultures possessing defective micronuclei exhibited micronuclear and oral abnormalities during autogamy. Micronuclear abnormalities were characterized by the failure of some of the cells to complete the micronuclear cycle resulting in the absence of either micronuclei or macronuclear anlagen, or both. Oral abnormalities included reduction in the length of the buccal cavity and oral membranelles, abnormal oral membranellar patterns and arrest of oral development at early and late stages. The present study demonstrated a close relationship between micronuclear and stomatogenic abnormalities during sexual reproduction. It is concluded that the micronucleus plays an important role in the specification of a normal oral pattern during sexual reproduction. The participation of postzygotic micronuclear activities in the control of sexual stomatogenesis is discussed. In contrast to the situation in sexual reproduction, the development of the oral apparatus was normal during asexual propagation of the cell lines possessing defective micronuclei. This paradoxical situation forms the basis of speculations on the nature of micronuclear control of oral development in sexual reproduction. It is probable that micronuclear genes are involved.

Formation of stable chromatin structures on the histone H4 gene during differentiation in Tetrahymena thermophila.

The relationship between chromatin structure and the transcriptional activity of the histone H4-I gene of Tetrahymena thermophila was explored. Indirect end-labeling studies demonstrated that major DNase I- and micrococcal nuclease-hypersensitive sites flank the active macronuclear genes but not the inactive micronuclear genes. Runon transcription experiments with isolated macronuclei indicated that histone gene transcription rates decreased when cells were starved. However, macronuclear nuclease-hypersensitive sites persisted upon starvation. Thus, one level of transcriptional control of the H4-I gene results in altered chromatin structure and is established during nuclear differentiation. The rate of transcription is also controlled, but not through hypersensitive site-associated structures.