\u3cem\u3eASI1\u3c/em\u3e, a Gene Encoding a Novel Leucine Zipper Protein, Is Induced during Development of the Macronucleus in \u3cem\u3eTetrahymena\u3c/em\u3e

Sexual reproduction in the ciliate Tetrahymena follows a complex developmental program involving the sequential regulation of dozens of genes. Genes that are up-regulated during post-zygotic development in Tetrahymena were isolated by subtractive hybridization. Anlagen stage induced gene 1 (ASI1) encodes a 2.8 kb transcript that contains a single intron and is induced during macronuclear development. ASI1 is a single copy gene in both the micronucleus and the macronucleus. It encodes a 95 kDa conceptual protein with a leucine zipper near the amino terminu

Progeny of Germ Line Knockouts of \u3cem\u3eASI2\u3c/em\u3e, a Gene Encoding a Putative Signal Transduction Receptor in \u3cem\u3eTetrahymena Thermophila\u3c/em\u3e, Fail to Make the Transition from Sexual Reproduction to Vegetative Growth

The ciliated protozoan Tetrahymena has two nuclei: a germ line micronucleus and a somatic macronucleus. The transcriptionally active macronucleus has about 50 copies of each chromosome. At sexual reproduction (conjugation), the parental macronucleus is degraded and new macronucleus develops from a mitotic product of the zygotic micronucleus. Development of the macronucleus involves massive genome remodeling, including deletion of about 6000 specific internal eliminated sequences (IES) and multiple rounds of DNA replication. A gene encoding a putative signal transduction receptor, ASI2, (anlagen stage induced 2) is up-regulated during development of the new macronuclei (anlagen). Macronuclear ASI2 is nonessential for vegetative growth. Homozygous ASI2 germ line knockout cells with wild type parental macronuclei proceed through mating but arrest at late macronuclear anlagen development and die before the first post-conjugation fission. IES elimination occurs in these cells. Two rounds of postzygotic DNA replication occur normally in progeny of ASI2 germ line knockouts, but endoreduplication of the macronuclear genome is arrested. The germ line ASI2 null phenotype is rescued in a mating of a knockout strain with wild type cells

Progeny of germ line knockouts of ASI2, a gene encoding a putative signal transduction receptor in Tetrahymena thermophila, fail to make the transition from sexual reproduction to vegetative growth

AbstractThe ciliated protozoan Tetrahymena has two nuclei: a germ line micronucleus and a somatic macronucleus. The transcriptionally active macronucleus has about 50 copies of each chromosome. At sexual reproduction (conjugation), the parental macronucleus is degraded and new macronucleus develops from a mitotic product of the zygotic micronucleus. Development of the macronucleus involves massive genome remodeling, including deletion of about 6000 specific internal eliminated sequences (IES) and multiple rounds of DNA replication. A gene encoding a putative signal transduction receptor, ASI2, (anlagen stage induced 2) is up-regulated during development of the new macronuclei (anlagen). Macronuclear ASI2 is nonessential for vegetative growth. Homozygous ASI2 germ line knockout cells with wild type parental macronuclei proceed through mating but arrest at late macronuclear anlagen development and die before the first post-conjugation fission. IES elimination occurs in these cells. Two rounds of postzygotic DNA replication occur normally in progeny of ASI2 germ line knockouts, but endoreduplication of the macronuclear genome is arrested. The germ line ASI2 null phenotype is rescued in a mating of a knockout strain with wild type cells

Isolation and Characterization of Genes Induced During Macronuclear Development in Tetrahymena Thermophila

The ciliated protozoan Tetrahymena is a unicellular eukaryote that contains two nuclei, a germline micronucleus and a somatic macronucleus. During conjugation, the sexual phase of the life cycle, the old macronucleus is degraded and the new macronucleus develops from the zygotic micronucleus. The formation of new macronucleus involves extensive DNA rearrangement and elimination. This process is a complex developmental program involving sequential regulation of dozens of genes. The technique of subtractive hybridization was applied to isolate genes that are specifically transcribed during postzygotic development. A library enriched for cDNA transcripts that are regulated during macronuclear development was constructed by subtraction of a cDNA library, complementary to RNA from conjugating cells at 12hr post-mixing of the two mating types, with mRNA from starved cells. A total of fourteen clones were obtained from the subtracted cDNA library. The insert size of the clones ranged from 150bp to 500bp. A cross-hybridization analysis showed each clone to be unique. Northern analysis of four ASI (a[barbelow]nlagen s[barbelow]tage i[barbelow]nduced) clones confirmed the isolation of genes that are transcribed during macronuclear development. The ASI1 gene was chosen for further analysis because it encodes a 2.8kb transcript, which is specifically induced during the period of macronuclear anlagen development. Southern analysis showed ASI1 is a single copy gene in both the micronucleus and the macronucleus. The ASI1 gene encodes a 95kd conceptual protein containing one intron and a leucine zipper near the amino terminus. The presence of a leucine zipper suggests the ASI1 protein may function as homodimer or heterodimer during the period of macronucleus development in Tetrahymena thermophila . The ASI2 gene encodes a 3.0kb transcript, which is induced during determination and differentiation of the new macronucleus. Southern analysis indicates it is a single copy gene in both the micronucleus and the macronucleus. The ASI3 gene encodes a transcript of 2kb induced during macronuclear anlagen development. BlastX analysis of ASI3 revealed significant homology to long-chain fatty-acid-CoA ligase. The ASI4 gene encodes a transcript of 0.85kb induced during the period of anlagen determination and differentiation. A low level expression is also observed in vegetatively growing cells

Alternate Junctions and Microheterogeneity of Tlr1, a Developmentally Regulated DNA Rearrangement in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

A large number of developmentally regulated DNA rearrangements occur during the development of the macronucleus in Tetrahymena thermophila, Tlr1 is a deletion element which has large inverted repeats near the rearrangement junctions and deletes more than 13 kbp of internal DNA. Previous analysis of caryonidal lines revealed alternate left junctions for the Tlr1 rearrangement in B strain cells. We show here that C2 strain Tetrahymena also use alternate rearrangement junctions. We have mapped and sequenced two additional rearrangement variants and find that both the left and right can vary over a range of approximately 200 bp. We also demonstrate the presence of sequence microheterogeneity in the most commonly found Tlr1 rearrangement product