Glenn Herrick

The Oxytricha trifallax Macronuclear Genome: A Complex Eukaryotic Genome with 16,000 Tiny Chromosomes

With more chromosomes than any other sequenced genome, the macronuclear genome of Oxytricha trifallax has a unique and complex architecture, including alternative fragmentation and predominantly single-gene chromosomes.

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.

Imprinting and paternal genome elimination in insects.

In many insects and other arthropods, males transmit only maternally inherited chromosomes (White 1973; Brown and Chandra 1977; Nur 1980, 1990a,b,c; Bell 1982; Bull 1983; Lyon and Rastan 1984; Lyon 1993; Wrensch and Ebbert 1993; Brun et al. 1995; Borsa and Kjellberg 1996). This remarkable genetic asymmetry can result from any of three principal systems of paternal genome exclusion, each of which has evolved several times. The most familiar and widespread exclusion system is arrhenotoky, in which fatherless males develop from unfertilized eggs and therefore lack paternal chromosomes at all stages of development. Most arrhenotokous systems are genetically haplodiploid, but a few are based on other modes of inheritance (see Nur 1980, 1990c; Bell 1982; Suomalainen et al. 1987). In the two other kinds of exclusion systems, a male’s paternally inherited chromosomes are actively eliminated: males begin life as seemingly conventional diploid zygotes but then either (1) lose their paternal chromosomes during embryonic development, becoming true maternal haploids (embryonic elimination), or (2) exhibit dramatically non-Mendelian patterns of meiosis and spermiogenesis, such that mature sperm carry only maternal chromosomes (germline elimination). To denote their formal (transmission-genetic) similarity to haplodiploid arrhenotoky, the embryonic and germline elimination systems are often characterized as parahaplodiploid or pseudoarrhenotokous.

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.

Telomeric properties of C4A4-homologous sequences in micronuclear DNA of Oxytricha fallax

The hypotrichous ciliated protozoan Oxytricha fallax has both macronuclei and micronuclei. The short DNA molecules of the macronucleus are terminated with repeats of the octanucleotide C4A4. Micronuclear DNA carries a family of long sequence blocks, which share a consensus restriction map and have homology with the C4A4 repeats. Each block contains a 3 to 6 kb subregion, which is refractory to all restriction enzymes tested and includes the C4A4 homology. We conclude that these refractory sequences are at DNA termini because Sau 3A fragments containing them fail to ligate to form circles, and they are rapidly degraded by Bal 31 nuclease. These terminal C4A4-homologous sequence blocks likely represent micronuclear chromosomal telomeres. Possible relationships between these sequences, internal micronuclear C4A4 repeats, and macronuclear DNA termini are discussed.

Selection on the Genes of Euplotes crassus Tec1 and Tec2 Transposons: Evolutionary Appearance of a Programmed Frameshift in a Tec2 Gene Encoding a Tyrosine Family Site-Specific Recombinase

ABSTRACT The Tec1 and Tec2 transposons of the ciliate Euplotes crassus carry a gene for a tyrosine-type site-specific recombinase. The expression of the Tec2 gene apparently uses a programmed +1 frameshift. To test this hypothesis, we first examined whether this gene has evolved under purifying selection in Tec1 and Tec2. Each element carries three genes, and each has evolved under purifying selection for the function of its encoded protein, as evidenced by a dearth of nonsynonymous changes. This distortion of divergence is apparent in codons both 5′ and 3′ of the frameshift site. Thus, Tec2 transposons have diverged from each other while using a programmed +1 frameshift to produce recombinase, the function of which is under purifying selection. What might this function be? Tyrosine-type site-specific recombinases are extremely rare in eukaryotes, and Tec elements are the first known eukaryotic type II transposons to encode a site-specific recombinase. Tec elements also encode a widespread transposase. The Tec recombinase might function in transposition, resolve products of transposition (bacterial replicative transposons use recombinase or resolvase to separate joined replicons), or provide a function that benefits the ciliate host. Transposons in ciliated protozoa are removed from the macronucleus, and it has been proposed that the transposons provide this “excisase” activity.

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 C 2 H 2 zinc finger/protein kinase. The protein kinase domain of the 57 kDa protein is unusual, lacking a conserved ATP-binding motif.

Elimination of germ-line tandemly repeated sequences from the somatic genome of the ciliate Oxytricha fallax

The ciliated protozoa exhibit nuclear dimorphism. The genome of the somatic macronucleus arises from the germ-line genome of the micronucleus following conjugation. We have studied the fates of highly repetitious sequences in this process. Two cloned, tandemly repeated sequences from the micronucleus of Oxytricha fallax were used as probes in hybridizations to micronuclear and macronuclear DNA. The results of these experiments show: (1) the cloned repeats are members of two apparently unrelated repetitious sequence families, which each appear to comprise a few percent of the micronuclear genome, and (2) the amount of either family in the macronuclei from which our DNA was prepared is about 1/15 that found in an equal number of diploid micronuclei. Most, if not all, of the apparent macronuclear copies of these repeats can be accounted for by micronuclear contamination, which strongly suggests that these sequences are eliminated from the macronuclei and have no vegetiative function.

Characterization and taxonomic validity of the ciliate Oxytricha trifallax (Class Spirotrichea) based on multiple gene sequences: limitations in identifying genera solely by morphology.

Oxytricha trifallax - an established model organism for studying genome rearrangements, chromosome structure, scrambled genes, RNA-mediated epigenetic inheritance, and other phenomena - has been the subject of a nomenclature controversy for several years. Originally isolated as a sibling species of O. fallax, O. trifallax was reclassified in 1999 as Sterkiella histriomuscorum, a previously identified species, based on morphological similarity. The proper identification of O. trifallax is crucial to resolve in order to prevent confusion in both the comparative genomics and the general scientific communities. We analyzed nine conserved nuclear gene sequences between the two given species and several related ciliates. Phylogenetic analyses suggest that O. trifallax and a bona fide S. histriomuscorum have accumulated significant evolutionary divergence from each other relative to other ciliates such that they should be unequivocally classified as separate species. We also describe the original isolation of O. trifallax, including its comparison to O. fallax, and we provide criteria to identify future isolates of O. trifallax.

An IS4 transposition causes a 13-bp duplication of phage λ DNA and results in the constitutive expression of the cI and cro gene products

We have examined the nature of the additional DNA present in lambda hyp- mutants (Eisen et al., 1982). This DNA is an IS4 element in orientation I, in the y region of bacteriophage lambda at nucleotide position 39,139 (see Moore et al., 1979). Our assignment is based on (i) the similarity in size derived from the PstI, AvaI, and HindII restriction pattern and (ii) the DNA sequence of both the left and right lambda-IS4 DNA junctions in phage lambda hyp15rev4. The IS4 integration event resulted in the duplication of 13 bp of lambda DNA in contrast to the 11- and 12-bp duplications previously observed at the sites of IS4 integrations elsewhere (Klaer et al., 1981).