Carolyn L. Jahn

Developmentally controlled genomic rearrangements in ciliated protozoa

The ciliated protozoa undergo an extensive genome reorganization during the course of forming a transcriptionally active macronucleus. The process includes numerous chromosome fragmentation and DNA breakage and rejoining events. Recent work indicates that transposable elements play a role in the process.

Structures of the Euplotes crassusTec1 and Tec2 elements: identification of putative transposase coding regions

The Tec1 and Tec2 transposon-like element families of Euplotes crassus are highly unusual in that all 30,000 copies of each family are excised from the genome during a discrete time period of macronuclear development. Complete nucleotide sequences were generated for the Tec1-1 and Tec2-1 elements, representing the Tec1 and Tec2 families. Open reading frames (ORFs) are conserved in position and sequence between the two elements, although sequences that comprise one ORF (ORF2) of Tec1-1 are split into two overlapping ORFs (ORFs 2A and 2B) in Tec2-1. ORF1 in Tec1-1, its homolog in Tec2-1 and one of the overlapping ORFs from Tec2-1 (ORF2B) contain TGA codons, which may be translated as Cys, as observed for two other Euplotid genes. Sequence analyses of ORFs from other members of each element family indicate that the families are distinct from each other and are highly conserved within each family. Computer searches of sequence databases have revealed sequence similarity between Tec ORF1s and the previously described Tc1-IS630 family of transposases which includes ORFs from bacterial, nematode and insect transposons.

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.

A Phenotypic Spectrum of Sexual Development in Dax1 (Nr0b1)-Deficient Mice: Consequence of the C57BL/6J Strain on Sex Determination

Abstract Nuclear receptor subfamily 0, group B, member 1 (Nr0b1; hereafter referred to as Dax1) is an orphan nuclear receptor that regulates adrenal and gonadal development. Dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 (Dax1) mutations in the mouse are sensitive to genetic background. In this report, a spectrum of impaired gonadal differentiation was observed as a result of crossing the Dax1 knockout on the 129SvIm/J strain onto the C57BL/6J strain over two generations of breeding. Dax1-mutant XY mice of a mixed genetic background (129;B6Dax1−/Y [101 total]) developed gonads that were predominantly testislike (n = 61), ovarianlike (n = 27), or as intersex (n = 13). During embryonic development, Sox9 expression in the gonads of 129;B6Dax1−/Y mutants was distributed across a wide quantitative range, and a threshold level of Sox9 (>0.4-fold of wild-type) was associated with testis development. Germ cell fate also varied widely, with meiotic germ cells being more prevalent in the ovarianlike regions of embryonic gonads, but also observed within testicular tissue. Ptgds, a gene associated with Sox9 expression and Sertoli cell development, was markedly downregulated in Dax1−/Y mice. Stra8, a gene associated with germ cell meiosis, was upregulated in Dax1−/Y mice. In both cases, the changes in gene expression also occurred in pure 129 mice but were amplified in the B6 genetic background. Sertoli cell apoptosis was prevalent in 129;B6Dax1−/Y gonads. In summary, Dax1 deficiency on a partial B6 genetic background results in further modulation of gene expression changes that affect both Sertoli cell and germ cell fate, leading to a phenotypic spectrum of gonadal differentiation.

SEQUENCE OF THE MACRONUCLEAR DNA ENCODING LARGE SUBUNIT RIBOSOMAL PROTEIN 29 (L29) IN EUPLOTES CRASSUS AND CYCLOHEXIMIDE SENSITIVITY

As a first step towards developing a DNA transformation method for the ciliated protozoan Euplotes crassus we determined the minimum inhibitory concentration (MIC) for cell division in the presence of cycloheximide (Chx) for several cell lines and the range of Chx sensitivity for 106 different progeny cell lines derived by mating two lines. All of the cell lines are highly sensitive to Chx. Progeny cell lines show a wider range of sensitivities than the parental lines. Because site-directed mutagenesis of the RPL29 gene encoding the large subunit ribosomal protein 29 (L29) has been used to generate a Chx-resistance marker (ChxR) for another ciliate, Tetrahymena thermophila [Yao and Yao, Proc. Natl. Acad. Sci. USA 88 (1991) 9493-9497], we isolated and sequenced the entire E. crassus macronuclear DNA carrying RPL29. The encoded peptide is 52-73% identical in sequence to L29 sequences from organisms ranging from T. thermophila and Saccharomyces cerevisiae to mouse. In E. crassus, the codon that has been mutated to confer Chx resistance in both S. cerevisiae and T. thermophila already encodes the amino-acid residue of one of the mutant forms identified in these other organisms. Thus, E. crassus RPL29 is not a convenient source of a selectable marker. Notable features of the macronuclear DNA carrying RPL29 are its extremely short non-coding regions and a TAG stop codon.

Characterization of the Euplotes crassus Macronuclear rDNA and its Potential as a DNA Transformation Vehicle

ABSTRACT We have cloned the macronuclear linear DNA molecule carrying the ribosomal RNA genes from the ciliated protozoan Euplotes crassus. DNA sequence analysis was carried out to locate coding regions and to determine whether sequences that have been mutated to confer antibiotic resistance are conserved in the E. crassus genes. The beginning and end of the primary transcript were mapped. In order to determine whether conserved sequences that might serve as replication origins were present, the 5′ and 3′ non‐coding sequences from E. crassus were compared to the corresponding sequences from the macronuclear linear rDNA molecules from the following euplotid species: Euplotes vannus, Euplotes minuta, Euplotes raikovii and Euplotes rariseta. A DNA transformation construct was made by generating a putative anisomycin resistant mutation along with a mutation generating a restriction site polymorphism. Microinjection of the construct into the developing macronucleus of mated cells resulted in exconjugant cell lines with increased resistance to anisomycin. The injected rDNA with the restriction site polymorphism is detectable in the anisomycin resistant cells and appears to represent a minor fraction of the rDNA.

Origin Usage during Euplotes Ribosomal DNA Amplification

ABSTRACT The macronuclear genome of the ciliate Euplotes is comprised of millions of small linear DNA molecules that have telomeres on each end. These molecules are generated during the sexual stage of the life cycle, when the new macronucleus is formed by a series of DNA processing events and multiple rounds of DNA amplification. We have used two-dimensional gels to compare the location of the replication origins used during vegetative growth and the two periods during macronuclear development when DNA amplification takes place. When we examined the pattern of ribosomal DNA (rDNA) replication intermediates, we observed almost identical Y arcs regardless of when in the Euplotes life cycle the DNA was isolated. No bubble or bubble-to-Y arcs could be detected. This indicates that replication of the macronuclear rDNA initiates at or near the telomere even when these molecules are being differentially amplified. Since replication rarely initiated from both ends of the rDNA, we examined the direction of replication fork movement to determine which end of the rDNA served as the origin. Fork movement gels indicated that replication initiated at the 5′ end. As transcription also starts near the telomere at the 5′ end, our findings suggest that the telomere and the promoter region cooperate to recruit Euplotes replication initiation complexes.

Differential expression of linker histone variants in Euplotes crassus

Two genes have been cloned from the ciliate Euplotes crassus that encode proteins with sequence similarity to the linker histones from a variety of organisms. One gene, H1-1, is present on a 1.3-kb macronuclear DNA molecule and encodes a 16.2- kDa protein. The second gene, H1-2, is present on a 0.7-kb DNA molecule and encodes an 18.8-kDa protein. Both H1-1 and H1-2 are expressed in vegetative cells, but the two genes exhibit very different patterns of expression during macronuclear development. H1-1 transcripts accumulate during conjugation and during the final rounds of DNA amplification. H1-2 transcripts accumulate after the onset of polytene chromosome formation and remain high throughout the remainder of macronuclear development. H1-1 is the major perchloric-acid-soluble protein from macronuclei. The pattern of gene expression and the macronuclear location of the H1-1 protein indicate that H1-1 is the predominant linker histone in vegetative macronuclei.