No abstract

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.G enes in micronuclear (germ-line) DNA of hypotrichous ciliates are interrupted by multiple, short, AT-rich, noncoding segments called internal eliminated segments, or IESs. IESs divide a gene into macronuclear destined segments, or MDSs. In some genes intramolecular recombination between IESs during evolution has disordered MDSs-i.e., MDSs have become scrambled. During development of a micronucleus into a new macronucleus after cell mating, IESs are spliced out of genes and MDSs are rearranged and ligated in the orthodox order to produce a transcribable gene (1). After MDS ligation the genes are excised from chromosomal DNA as individual, short DNA molecules, and all of the remaining DNA (spacer DNA), which accounts for Ϸ95% of the nucleotide sequence complexity in the micronuclear genome, is destroyed. Generally, one gene is present per molecule, but a small percentage of molecules may contain two genes (2) or even three genes (D.M.P., J. D. Prescott, and R. M. Prescott, unpublished observation).The significance of IESs in micronuclear genes is not understood, although it has been suggested that they facilitate evolution of new genes; recombination among IESs, which results in shuffling of MDSs, could conceivably create new, intergenic MDS combinations. Thus, the presence of scrambled MDSs in contemporary hypotrichs is a manifestation of intragenic MDS shuffling caught in evolutionary time. In addition, IES position within a gene can change (shift), which alters the sequence composition and size of MDSs in that gene (3), increasing further the versatility of MDS shuffling. At the very least, IES shifting and MDS scrambling by IES recombination, as reaffirmed in the data reported here, attest to an extraordinarily dynamic nature of the germ-line genome in hypotrichs over evolutionary time.This paper discusses the origin and evolution o...

The nucleotide sequences of 16 newly reported and 8 previously reported actin-encoding macronuclear DNA molecules in spirotrichs have been compared. As described for the eight previously reported molecules, the first 50 bases (noncoding) inside the telomere at both 5' strands in additional actin molecules are purine-rich. This anomalous base composition might serve as a signal to identify macronuclear molecules in micronuclear DNA during development. The 50-base segment upstream of the ATG in the 5' leaders of the actin molecules contains extensive, conserved sequence motifs that are possibly promoter elements. The 3' noncoding trailers contain virtually no conserved sequence motifs. With one exception, the 3' trailers contain a second stop codon (TGA) 36 bases on average downstream of the primary stop codon. Excluding Moneuplotes crassus, amino acid identities in actin I range from 78 to 100%, with variations distributed nonrandomly along the sequence. Phylogenetic trees based on the actin nucleotide sequences of 22 spirotrichs define the evolutionary relationships of their actin-encoding molecules. The actin phylogeny, while well supported by posterior probabilities, does not always coincide with the phylogeny defined in rDNA analyses or classical taxonomic classifications.

The lack of a standardized culture environment for establishment of embryonic stem cell lines has hindered the orchestrated differentiation of cells and the application of this technology. Oxygen concentration has a profound effect on proliferation and differentiation of many cell types. This study tested the hypothesis that establishment dynamics, lactate dehydrogenase (LDH) isoforms, and mRNA expression patterns would be affected by the oxygen tension in the culture environment. Recovered (day 4) murine blastocysts were cultured in a gas environment of 6% CO(2) and either 20% or 5% O(2) (balance supplemented with N(2)). More (p < 0.05) blastocysts produced outgrowths in the low (79.3 +/- 0.1%) compared to the high (57.1 +/- 0.1%) O(2) groups, and more (p < 0.05) colonies in the low O(2) group (14/15; 93.3 +/- 0.1%) stained positive for alkaline phosphatase relative to the high O(2) group (9/15; 60.6 +/- 0.1%). Oxygen treatment had no effect on the activity of the oxioreductase lactate dehydrogenase. Interestingly, the stem cell lines in both treatments displayed multiple isoforms (III, IV, and V) of LDH, whereas the outgrowths displayed isoforms I and V. In contrast, two-cell embryos and blastocysts displayed only isoform I, and fibroblasts displayed isoforms IV and V. There were no treatment differences in mRNA expression of LDHalpha in the outgrowths, or established stem cells. LDH transition from the heart (I) to the muscle (V) isoform indicated an increase in glycolytic activity, consistent with the peri-hatching/implantation time period. Reduced O(2) environment had significant positive effects on the establishment and maintenance of murine stem cells, supporting the hypothesis, whereas the LDH isozyme transition was consistent among treatments.