A short internal eliminated sequence with central conserved sequences interrupting the LA-MSC gene of the 81 locus in the hypotrichous ciliates Oxytricha fallax and O. trifallax.

IES-LA is a short Internal Eliminated Sequence interrupting LA-MSC, a protein-coding gene of the 81 locus of Oxytricha fallax and O. trifallax. IES-LA is precisely excised from the gene during development of the macronucleus. The internal eliminated sequence is bounded by CAAT ... AATG, and thereby resembles a TBE1 transposon internal eliminated sequence insertion that is grossly shortened (4.1 kbp to 52-64 bp), consistent with the hypothesis that short IESs are degenerated ancient transposons. The pattern of sequence conservation between five alleles of IES-LA shows that it differs from previously characterized classes of ciliate short IESs: while many short IESs have conserved ends and diverged centers, IES-LA is more conserved in its center and its ends are diverged. This implies a excision mechanism for IES-LA that is distinct from those for other known Oxytricha IESs.

Selection on the protein-coding genes of the TBE1 family of transposable elements in the ciliates Oxytricha fallax and O. trifallax.

TBE1s are "cut-and-paste" transposable elements found in high copy number in the germline genomes of the ciliates Oxytricha fallax and O. trifallax. TBE1 "family" sequence (sequence of mixed polymerase chain reaction products generated using primers that match roughly half the TBE1s in host whole-cell DNA) was obtained from both host species. Although family sequence autoradiograms represent thousands of different elements, they are as legible as those representing corresponding sequences of a single TBE1, implying that ideal polymorphisms are rare within the genes examined. Nucleotide polymorphisms among TBE1s (indicated by ambiguities in family sequence) are far more common at third than at first or second positions of codons of genes, implying that selection has conserved the amino acid sequences of these genes in the majority of TBE1s. Portions of the transposase gene and another TBE1 gene have been sequenced from 10 individual TBE1s. None of these portions is interrupted by stop codons or frameshifts, and, for both genes, pairwise comparisons of these sequences show that nonsynonymous differences are significantly less common than synonymous differences, again implicating conservative selection Phylogenetic analysis shows that multiple divergent lineages of TBE1s have evolved under this selection within O. fallax. All these results are unexpected for cut-and-paste transposons in eukaryotic hosts: since transposase encoded by intact elements presumably acts in trans, it can duplicate mutant copies (those that do not encode functional transposase) found in the same genome, and thus no selection is expected to maintain the transposase gene. The selection demonstrated here could act at transposition (if functional TBE1s are preferentially transposed) or at the level of the host (if the host's fitness depends on functional TBE1 genes). TBE1-encoded proteins might be responsible for the precise excision of TBE1s that occurs during development of the host somatic nucleus; selection on hosts for uninterrupted somatic genes would then translate into selection for TBE1 protein-coding competence. We suggest a method for distinguishing between these two classes of explanations by finding and analyzing divergent alleles of ancestral transposable element insertions.

Two two-gene macronuclear chromosomes of the hypotrichous ciliates Oxytricha fallax and O. trifallax generated by alternative processing of the 81 locus.

We describe the first know macronuclear chromosomes that carry more than one gene in hypotrichous ciliated protozoa. These 4.9- and 2.8-kbp chromosomes each consist almost exclusively of two protein-coding genes, which are conserved and transcribed. The two chromosomes share a common region that consists of a gene that is a member of the family of mitochondrial solute carrier genes (CR-MSC; [Williams and Herrick (1991): Nucleic Acids Res 19:4717-4724]. Each chromosome also carries another gene appended to its common region: The 4.9-kbp chromosome also carries a gene that encodes a protein that is rich in glutamine and charged amino acids and bears regions of heptad repeats characteristic of coiled-coils. Its function is unknown. The second gene of the 2.8 kbp chromosome is a mitochondrial solute carrier gene (LA-MSC); thus, the 2.8-kbp chromosomes consists of two mitochondrial solute carrier paralogs. Phylogenetic analysis indicates that the two genes were duplicated before ciliates diverged from the main eukaryotic lineage and were subsequently juxtaposed. The CR- and LA-MSC genes are each interrupted by three introns. The introns are not in homologous positions, suggesting that they may have originated from multiple group II intron transpositions. These chromosomes and their genes are encoded in the Oxytricha germline by the 81 locus. This locus is alternatively processed to generate a nested set of three macronuclear chromosomes, the 4.9- and 2.8-kbp chromosomes and a third (1.6 kbp) which consists almost exclusively of the shared common gene, CR-MSC. Such alternative processing is common in macronuclear development of O. fallax [Cartinhour and Herrick (1984): Mol Cell Biol 4:931-938]. Possible functions for alternative processing are considered; e.g., it may serve to physically link genes to allow co-regulation or co-replication by a common cis-acting sequence.

Conserved features of TBE1 transposons in ciliated protozoa.

The complete sequences of four TBE1 transposons from Oxytricha fallax and O. trifallax are presented and analyzed. Although two TBE1s are 98% identical to each other at the nucleotide level, the remaining two TBE1s are only 90% identical both to each other and to the other two. This large evolutionary divergence allows us to identify conserved TBE1 features. TBE1 transposons are 4.1 kbp long and are flanked by 3 bp target-site repeats. The elements consist of 78 bp inverted terminal repeats, of which the 17 terminal base pairs are Oxytricha telomere repeats; a central conserved section of 550 bp that includes a set of nested direct and inverted sequence repeats; and 3 open reading frames conserved for encoded amino acid sequence. The three open reading frames encode a 22 kDa basic protein of unknown function, a 42 kDa 'D,D35E' transposase, and a 57 kDa chimeric C2H2 zinc finger/protein kinase. The protein kinase domain of the 57 kDa protein is unusual, lacking a conserved ATP-binding motif.

Internal eliminated sequences interrupting the Oxytricha 81 locus: allelic divergence, conservation, conversions, and possible transposon origins.

Internal eliminated sequences (IESs) often interrupt ciliate genes in the silent germline nucleus but are exactly excised and eliminated from the developing somatic nucleus from which genes are then expressed. Some long IESs are transposons, supporting the hypothesis that short IESs are ancient transposon relics. In light of that hypothesis and to explore the evolutionary history of a collection of IESs, we have compared various alleles of a particular locus (the 81 locus) of the ciliated protozoa Oxytricha trifallax and O. fallax. Three short IESs that interrupt two genes of the locus are found in alleles from both species, and thus must be relatively ancient, consistent with the hypothesis that short IESs are transposon relics. In contrast, TBE1 transposon interruptions of the locus are allele-specific and probably the results of recent transpositions. These IESs (and the TBE1s) are precisely excised from the DNA of the developing somatic macronucleus. Each IES interrupts a highly conserved sequence. A few nucleotides at the ends of each IES are also conserved, suggesting that they interact critically with IES excision machinery. However, most IES nucleotide positions have evolved at high rates, showing little or no selective constraint for function. Nonetheless, the length of each IES has been maintained (+/- 3 bp). While one IES is approximately 33 bp long, three other IESs have very similar sizes, approximately 70 bp long. Two IESs are surrounded by direct repeats of the sequence TTCTT. No other sequence similarities were found between any of the four IESs. However, the ends of one IES do match the inverted terminal repeat consensus sequence of the "TA" IESs of Paramecium. Three O. trifallax alleles appear to have been recipients in recent conversion events that could have been provoked by double-strand breaks associated with IES ends subsequent to IES transposition. Our findings support the hypothesis that short IESs evolved from ancient transposons that have lost most of their sequences, except those necessary for precise excision during macronuclear development.

Effects of the transcription inhibitor actinomycin D on postzygotic development of Tetrahymena thermophila conjugants.

During Tetrahymena thermophila conjugation, new somatic macronuclei develop from a common zygotic nucleus derived from meiotic products of the germline, and the old parental somatic nucleus is destroyed. The transcription inhibitor actinomycin D disrupts many events of postzygotic conjugation (cycloheximide causes indistinguishable effects). Early treatment causes a block of all postzygotic development, suggesting a transcription requirement for conjugants to pass a checkpoint, allowing entry into postzygotic development. Thereafter, pair separation, resorption of the old macronucleus, and elimination of one of the new micronuclei are blocked if actinomycin D is added at least 1.5 hr before each of these events normally occurs. Treatment just before DNA rearrangements in the developing macronuclei (anlagen) causes aberrant anlage DNA loss, suggesting that this DNA loss may be caused by inhibition of gene expression involved in genome rearrangements. DNA loss, and correlated lethality, appear to require previous gene expression, since actinomycin D added earlier causes cells to arrest in development without anlage DNA loss, and these conjugants can (at some frequency) complete conjugation and make viable progeny once actinomycin D is removed. The old macronucleus already had been inactivated before most actinomycin D treatments were initiated, indicating that the various induced defects we observed are the result of inhibition of postzygotic gene expression, presumably in anlagen. The defects induced by actinomycin D are similar to defects previously observed in conjugants harboring nullisomic germline deficiencies but proficient old macronuclei.

Effects of nullisomic chromosome deficiencies on conjugation events in Tetrahymena thermophila: insufficiency of the parental macronucleus to direct postzygotic development.

Conjugation fails postzygotically after mating of Tetrahymena cells that have wild-type parental macronuclei but harbor noncomplementing nullisomic parental germline deficiencies. Failures begin shortly after formation of the new macronuclear precursor (anlage) and completion of the first step in elimination of the parental macronucleus (pycnosis). Conjugants fail to complete pair separation, to eliminate one new micronucleus, and to amplify anlage DNA, and they eventually die. Some deficiencies block resorption of the pycnotic parental macronucleus, but we find no evidence for its regeneration. Some deficiencies cause aberrant anlage DNA loss. Those that do not cause DNA loss are epistatic to those that do, indicating that normal anlage development requires the dependent function of at least two types of genes. The possibility that these genes are involved in developmentally regulated anlage DNA rearrangements is discussed. Each observed conjugation defect indicates insufficiency of the parental macronucleus to direct postzygotic development and can be explained by the deficiency of essential conjugation genes that are expressed from the anlage. The failure of nullisomic conjugants to complete pair separation indicates a requirement for gene products, expressed from the early anlage or its precursors, soon after anlage first differentiate.

Consensus inverted terminal repeat sequence of Paramecium IESs: resemblance to termini of Tc1-related and Euplotes Tec transposons.

During the formation of a transcriptionally active macronucleus, ciliated protozoa excise large numbers of interstitial segments of DNA (internal eliminated sequences; IESs) from their chromosomes. In this study we analyze the published sequences of 20 IESs that interrupt surface protein genes of Paramecium and identify a consensus inverted terminal repeat. This sequence is similar to the ends of the Tc1-related transposons found in nematodes and other metazoans, as well as to both the ends of the Tec transposons and at least some of the IESs in the distantly related ciliate Euplotes crassus. The results of these analyses bolster previous proposals that IESs were created by transposition.

A proposed superfamily of transposase genes: transposon-like elements in ciliated protozoa and a common "D35E" motif.

The transposon-like elements TBE1, Tec1, and Tec2 of hypotrichous ciliated protozoa appear to encode a protein that belongs to the IS630-Tc1 family of transposases. The Anabaena IS895 transposase also is placed in this family. We note that most family members transpose into the dinucleotide target, TA, and that members with eukaryotic hosts have a tendency for somatic excision that is carried to an extreme by the ciliate elements. Alignments including the additional members, and also mariner elements, show that transposases of this family share strongly conserved residues in a large C-terminal portion, including a fully conserved dipeptide, Asp-Glu (DE), and a block consisting of a fully conserved Asp and highly conserved Glu, separated by 34 or 35 residues (D35E). This D35E motif likely is homologous to the previously characterized D35E motif of the family of retroviral-retrotransposon integrases and IS3-like transposases. Because it is known that the IS3-retroposon D35E region is a critical portion of a domain capable of various in vitro transposition-related reactions, the results suggest that the two families share homologous catalytic transposase domains and that members of both families may share a common transposition mechanism.

Developmental precise excision of Oxytricha trifallax telomere-bearing elements and formation of circles closed by a copy of the flanking target duplication.

The 4.1 kbp TBE1 elements of Oxytricha fallax and Oxytricha trifallax are deduced to transpose into a centrisymmetric target, CAnTG, and to duplicate the central AnT. Despite conserved C(A4C4)2 telomeric repeats at their tips, free TBE1s found during macronuclear development are not linear but 4.1 kbp circles closed on one copy of the AnT target duplication. The macronucleus-destined flanks are rejoined to regenerate the target, effecting efficient and precise somatic reversion of the germline transpositional mutation. A model is presented in which transposase catalyzes concerted precise rejoining of the flanks and cyclization of the excised element.

Programmed nuclear death: apoptotic-like degradation of specific nuclei in conjugating Tetrahymena.

During conjugation in the ciliated protozoan Tetrahymena, new macronuclei differentiate from germinal zygotic micronuclei while parental (old) macronuclei are eliminated in two stages, condensation or pycnosis coincident with cessation of transcription followed by resorption. We show that pycnosis is accompanied by degradation of old macronuclear DNA into oligonucleosome-sized fragments, a hallmark of programmed cell death, or apoptosis, in a variety of eukaryotic systems. As expected, oligonucleosome formation does not occur in the new micro- and macronuclei, confirming the coordination of different developmental fates for different nuclei in a common cytoplasm. NULLI 3 conjugants have wild-type old macronuclei but lack chromosome 3 germinally and hence in the new macronucleus. In NULLI 3 conjugants, old macronuclear pycnosis and oligonucleosome fragmentation occur normally but the resorption step fails, and the pycnotic old macronucleus is retained, demonstrating that the two steps are genetically separable and thus distinct and implying that genes on chromosome 3 in the new macronucleus are required for the resorption step. Comparison of whole cell polypeptides synthesized during stages of macronuclear development in both wild-type and NULLI 3 crosses reveal similar profiles. However, a polypeptide (apparent M(r) of 53 kDa) synthesized during old macronuclear elimination is not observed in NULLI 3 conjugants; its role, if any, in elimination of the old macronucleus is unknown. The results show that the old macronucleus is selectively destroyed by a mechanism which is remarkably similar to apoptosis in other eukaryotes and that the zygotic genome is required for the resorption step.

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.

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.

Winter desiccation and injury of subalpine red spruce.

Montane red spruce (Picea rubens Sarg.) in the northeastern United States has undergone a decline during the past two decades. One symptom associated with the decline syndrome is the episodic browning of first-year foliage in early spring. To examine the potential role of winter desiccation in this browning, the water relations of red spruce foliage in a subalpine forest on Mt. Moosilauke, New Hampshire, USA, were monitored from January to May, 1989. All sampled trees lost water during the winter and the first-year foliage on some trees turned brown in early spring. The relative water content of first-year shoots during the winter was a significant predictor of spring browning; red spruce trees that showed browning had desiccated faster and reached lower relative water contents. Damaged trees also had more closely packed needles and lower cuticular resistances to water loss. The first-year shoots had a significantly lower average relative water content than older shoots before and after browning. Cuticular resistance to water loss decreased with elevation. Sun-exposed shoots lost more water than shaded shoots because of solar heating of needles. Winter desiccation can occur before the decline-related spring browning of red spruce foliage.

Precise excision of telomere-bearing transposons during Oxytricha fallax macronuclear development.

In ciliated protozoa, development of the macronucleus from a copy of the germ line micronucleus involves elimination of a large number of sequences by DNA splicing akin to precise excision of transposons. The known examples of such internal eliminated sequences (IESs) are not repetitive. The telomere-bearing elements (TBE1s) of Oxytricha fallax are a family of transposons. We show that two particular TBE1s are also IESs. TBE1-1 and TBE1-2 disrupt a micronuclear region that codes for macronuclear DNA. A variety of tests indicates that each TBE1 and one copy of the flanking target repeat is absent from most, if not all, molecules of the macronuclear DNA, as if the TBE1s were precisely excised during macronuclear development. Three alternative explanations for the absence of TBE1-1 and TBE1-2 from the macronuclear DNA were tested. First, because two other highly homologous versions of that DNA are also found in the macronucleus, recombination between versions during or after macronuclear development could have bypassed the elements. Recombination in the regions flanking the elements was not detected. Second, micronuclear DNA blots show no evidence of a micronuclear counterpart of the macronuclear region that lacks TBE1-1. Third, TBE1-2 was demonstrated in two sexually independent cell lines. This shows that it pre-existed in the germ line, as opposed to having transposed into the micronuclear DNA subsequent to the generation of the macronucleus of the vegetative line that is usually studied. We conclude that TBE1-1 and TBE1-2, and possibly many of the other approximately 1900 micronucleus-limited TBE1s are excised as IESs during macronuclear development. These transposons appear to enjoy the luxury of relaxed constraints on family expansion, because they are removed from the genome before it is expressed. We discuss the possibility that all IESs are transposon-derived, that all are excised by transposition machinery, and that linear excision products are early intermediates in transposition.

Alternative processing during development of a macronuclear chromosome family in Oxytricha fallax.

Macronuclear chromosomes in Oxytricha fallax, a hypotrichous ciliate, are very short. They often belong to small families of cross-hybridizing chromosomes of two or three different sizes. For example, the 81-MAC family consists of three sizes of macronuclear chromosomes (4.9, 2.9, and 1.6 kbp) (Cartinhour and Herrick 1984). We show that the family actually consists of two closely related sets of three each and that the two sets are independently created by alternative processing of two separate precursor (micronuclear) versions. Chromosomes of a set share a common 1.6-kbp region, which contains a transcribed gene coding for a 25-kD protein. Different-sized macronuclear chromosomes of a set result from alternative choices of positions for telomere formation. All six members of the family are reproducibly generated in each developing macronucleus, and their copy numbers are stably maintained during vegetative replication of the macronucleus (Herrick et al. 1987). Here we argue for the existence of three distinct copy control elements in the 81-MAC family chromosomes. A model is discussed in which, following polytenization of the micronuclear chromosomes, different chromatids are processed differently, and, subsequently, replication-competent macronuclear chromosome products are amplified under the influence of the vegetative copy control elements.

Multiple sequence versions of the Oxytricha fallax 81-MAC alternate processing family.

The 81-MAC family consists of three sizes of macronuclear chromosomes in Oxytricha fallax. Clones of these and of micronuclear homologs have been classified according to DNA sequence into three highly homologous (95.9-97.9%), but distinct versions. Version A is represented by a micronuclear clone and by clones of two different-sized macronuclear chromosomes, showing that alternate processing of micronuclear DNA is responsible for the variety of sizes of macronuclear chromosomes. Three Internal Eliminated Sequences (IES's) are demonstrated in Version A micronuclear DNA. Two have been sequenced and show short, flanking direct repeats but no inverted terminal repeats. Version C micronuclear DNA has interruptions in the macronuclear homology which correspond closely to the Version A IES's. Whether they are true IES's is unknown because no Version C macronuclear DNA has been demonstrated. Version C micronuclear DNA may be "macronuclear-homologous" but "micronucleus-limited" and not "macronucleus-destined." Version B is represented by macronuclear DNA clones, but no micronuclear clones. Vegetative micronuclear aneuploidy is suggested. The possible role of micronuclear defects in somatic karyonidal senescence is discussed in light of the precise macronuclear chromosome copy controls demonstrated within the 81-MAC family. These controls apparently operate throughout karyonidal life to maintain 1) a constant absolute amount of 81-MAC sequences in the macronucleus and 2) a constant stoichiometry within the family, both according to version and chromosome size.