Endosymbiotic bacteria in the parasitic ciliate Ichthyophthirius multifiliis.

Endosymbiotic bacteria were identified in the parasitic ciliate Ichthyophthirius multifiliis, a common pathogen of freshwater fish. PCR amplification of DNA prepared from two isolates of I. multifiliis, using primers that bind conserved sequences in bacterial 16S rRNA genes, generated an approximately 1,460-bp DNA product, which was cloned and sequenced. Sequence analysis demonstrated that 16S rRNA gene sequences from three classes of bacteria were present in the PCR product. These included Alphaproteobacteria (Rickettsiales), Sphingobacteria, and Flavobacterium columnare. DAPI (4',6-diamidino-2-phenylindole) staining showed endosymbionts dispersed throughout the cytoplasm of trophonts and, in most, but not all theronts. Endosymbionts were observed by transmission electron microscopy in the cytoplasm, surrounded by a prominent, electron-translucent halo characteristic of Rickettsia. Fluorescence in situ hybridization demonstrated that bacteria from the Rickettsiales and Sphingobacteriales classes are endosymbionts of I. multifiliis, found in the cytoplasm, but not in the macronucleus or micronucleus. In contrast, F. columnare was not detected by fluorescence in situ hybridization. It likely adheres to I. multifiliis through association with cilia. The role that endosymbiotic bacteria play in the life history of I. multifiliis is not known.

Parental expression of the chromodomain protein Pdd1p is required for completion of programmed DNA elimination and nuclear differentiation.

Thousands of DNA elimination events occur during somatic differentiation of many ciliated protozoa. In Tetrahymena, the eliminated DNA aggregates into submacronuclear structures containing the protein Pdd1p, a member of the chromodomain family. We disrupted somatic copies of PDD1, eliminating parental expression of the gene early in the sexual phase of the life cycle. Even though zygotic expression, from the undisrupted germline PDD1 copy, is activated before DNA elimination normally occurs, the somatic knockout cells suffer defects in DNA elimination, genome endoduplication, and nuclear resorption, and eventually die, demonstrating that PDD1 is essential and suggesting Pdd1p is directly involved in establishing a chromatin structure required for DNA elimination.

Evolutionary conservation of sequences directing chromosome breakage and rDNA palindrome formation in tetrahymenine ciliates.

Extensive, programmed chromosome breakage occurs during formation of the somatic macronucleus of ciliated protozoa. The cis-acting signal directing breakage has been most rigorously defined in Tetrahymena thermophila, where it consists of a 15-bp DNA sequence known as Cbs, for chromosome breakage sequence. We have identified sequences identical or nearly identical to the T. thermophila Cbs at sites of breakage flanking the germline micronuclear rDNA locus of six additional species of Tetrahymena as well as members of two related genera. Other general features of the breakage site are also conserved, but surprisingly, the orientation and number of copies of Cbs are not always conserved, suggesting the occurrence of germline rearrangement events over evolutionary time. At one end of the T. thermophila micronuclear rDNA locus, a pair of short inverted repeats adjacent to Cbs directs the formation of a giant palindromic molecule. We have examined the corresponding sequences from two other Tetrahymena species. We find the sequence to be partially conserved, as previously implied from analysis of macronuclear rDNA, but of variable length and organization.

Pdd1p, a novel chromodomain-containing protein, links heterochromatin assembly and DNA elimination in Tetrahymena.

During Tetrahymena conjugation, programmed DNA degradation occurs in two separate nuclei. Thousands of germline-specific deletion elements are removed from the genome of the developing somatic macronucleus, and the old parental macronucleus is degraded by an apoptotic mechanism. An abundant polypeptide, Pdd1p (formerly p65), localizes to both of these nuclei at the time of DNA degradation. Here we report that, in developing macronuclei, Pdd1p localizes to electron-dense, heterochromatic structures that contain germline-specific deletion elements. Pdd1p also associates with parental macronuclei during terminal stages of apoptosis. Sequencing of the PDD1 gene reveals it to be a member of the chromodomain family, suggesting a molecular link between heterochromatin assembly and programmed DNA degradation.

Genome downsizing during ciliate development: nuclear division of labor through chromosome restructuring.

The ciliated protozoa divide the labor of germline and somatic genetic functions between two distinct nuclei. The development of the somatic (macro-) nucleus from the germinal (micro-) nucleus occurs during sexual reproduction and involves large-scale, genetic reorganization including site-specific chromosome breakage and DNA deletion. This intriguing process has been extensively studied in Tetrahymena thermophila. Characterization of cis-acting sequences, putative protein factors, and possible reaction intermediates has begun to shed light on the underlying mechanisms of genome rearrangement. This article summarizes the current understanding of this phenomenon and discusses its origin and biological function. We postulate that ciliate nuclear restructuring serves to segregate the two essential functions of chromosomes: the transmission and expression of genetic information.

Mutational analysis of polymeric immunoglobulin receptor/ligand interactions. Evidence for the involvement of multiple complementarity determining region (CDR)-like loops in receptor domain I.

The polymeric Ig receptor (pIgR) mediates the transport of IgA and IgM across a variety of mucosal epithelia. The ectodomain of this receptor consists of five immunoglobulin-like domains (I-V), the first four being structurally similar to immunoglobulin variable regions, and the fifth to Ig constant regions. This study examines the structural features of the pIgR that participate in binding of the ligand, dimeric IgA (dIgA). Recent evidence suggests that a highly conserved region of the first Ig-like domain (domain I) may be important in this process (Bakos, M.A., Kurosky, A., and Goldblum, R. M. (1991) J. Immunol. 147, 3419-3426). In support of this hypothesis, molecular modeling of domain I places this conserved region in an exposed loop analogous to the CDR1 loop of Ig, suggesting that interactions between dIgA and the pIgR may be similar to those between antibodies and their cognate antigens. To test this hypothesis directly, we performed a mutagenic analysis of all three CDR-like loops in domain I of the pIgR. We found that point mutations in multiple residues of CDR1 produced effects on IgA binding ranging from minimal (90% of control) to profound (7%). In addition, we replaced regions corresponding to the CDR2 and CDR3 loops of domain I with their counterparts from domain II (which does not bind IgA), which in both cases resulted in complete abrogation of IgA binding. Taken together, these data suggest that each of the three CDR-like loops of domain I of the rabbit pIgR participates in the binding of dimeric IgA.

Cell shape and interaction defects in alpha-spectrin mutants of Drosophila melanogaster.

We show that the alpha-spectrin gene is essential for larval survival and development by characterizing several alpha-spectrin mutations in Drosophila. P-element minigene rescue and sequence analysis were used to identify the alpha-spectrin gene as the l(3)dre3 complementation group of the Dras-Roughened-ecdysoneless region of chromosome 3 (Sliter et al., 1988). Germ line transformants carrying an alpha-spectrin cDNA, whose expression is driven by the ubiquitin promoter, fully rescued the first to second instar lethality characteristic of the l(3)dre3 alleles. The molecular defects in two gamma-ray-induced alleles were identified. One of these mutations, which resulted in second instar lethality, contained a 73-bp deletion in alpha-spectrin segment 22 (starting at amino acid residue 2312), producing a premature stop codon between the two EF hands found in this segment. The second mutation, which resulted in first instar lethality, contained a 20 base pair deletion in the middle of segment 1 (at amino acid residue 92), resulting in a premature stop codon. Examination of the spectrin-deficient larvae revealed a loss of contact between epithelial cells of the gut and disruption of cell-substratum interactions. The most pronounced morphological change was seen in tissues of complex cellular architecture such as the middle midgut where a loss of cell contact between cup-shaped cuprophilic cells and neighboring interstitial cells was accompanied by disorganization of the cuprophilic cell brush borders. Our examination of spectrin deficient larvae suggests that an important role of non-erythroid spectrin is to stabilize cell to cell interactions that are critical for the maintenance of cell shape and subcellular organization within tissues.

A leucine zipper domain of the suppressor of Hairy-wing protein mediates its repressive effect on enhancer function.

The suppressor of Hairy-wing [su(Hw)] protein mediates the mutagenic effect of the gypsy retrotransposon by repressing the function of transcriptional enhancers controlling the expression of the mutant gene. A structural and functional analysis of su(Hw) was carried out to identify domains of the protein responsible for its negative effect on enhancer action. Sequence comparison among the su(Hw) proteins from three different species allows the identification of evolutionarily conserved domains with possible functional significance. An acidic domain located in the carboxy-terminal end of the Drosophila melanogaster protein is not present in su(Hw) from other species, suggesting a nonessential role for this part of the protein. A second acidic domain located in the amino-terminal region of su(Hw) is present in all species analyzed. This domain is dispensable in the D. melanogaster protein when the carboxy-terminal acidic domain is present, but the protein is nonfunctional when both regions are simultaneously deleted. Mutations in the zinc fingers result in su(Hw) protein unable to interact with DNA in vivo, indicating a functional role for this region of the protein in DNA binding. Finally, a region of su(Hw) homologous to the leucine zipper motif is necessary for the negative effect of this protein on enhancer function, suggesting that su(Hw) might exert this effect by interacting, directly or indirectly, with transcription factors bound to these enhancers.

The Drosophila su(Hw) gene, which controls the phenotypic effect of the gypsy transposable element, encodes a putative DNA-binding protein.

Homozygous mutations at the suppressor of Hairy-wing [su(Hw)] locus reverse the phenotype of gypsy-induced alleles in a number of genes located throughout the Drosophila genome. To understand the molecular basis of this phenomenon, the su(Hw) locus was isolated by chromosomal walking from a cloned homeo-box-containing sequence. The exact location of the gene was determined by Southern analysis of the DNA alterations associated with several su(Hw) alleles. A 9.5-kb KpnI-SalI fragment, where all the DNA changes associated with su(Hw) mutations were mapped, was able to rescue the su(Hw) mutant phenotype after P-element-mediated germ-line transformation. This DNA fragment encodes a 3.3-kb RNA that is expressed in all stages of Drosophila development; the size or abundance of this RNA is affected in several su(Hw) alleles tested. This transcript encodes a protein that contains a highly acidic region and 12 repeats of the 'Zn finger' domain characteristic of some DNA-binding and transcription-activating proteins, supporting the hypothesis that the su(Hw) locus might encode a transcription factor that plays a role in the expression of the gypsy element.