Electron Microscopy of the Hypotrich Ciliate Oxytricha platystoma, with Consideration of its Macronuclear Organization

SYNOPSIS. The body of Oxytricha platystoma is covered with 2 membranes, the outer or pellicular membrane and the inner or cytoplasmic membrane. Each cirrus consists of a bundle of 25-50 cilia. There are 2 macronuclei and 2 micronuclei. The micronucleus is a very small body containing dense Feulgen-positive material. The macronucleus is an elongated body surrounded by a double membrane. The dense Feulgen-positive material is embedded in a less dense nucleoplasm. Some larger bodies found inside the nucleus may be nucleoli. There is a single reorganization band in each macronucleus. It consists of a solution plane and a reconstruction plane, and has a total thickness of 1.5 μ. The macronucleus divides amitotically, whereas the micronucleus divides mitotically.     

Lack of Polymorphism on the Drosophila Fourth Chromosome Resulting from Selection

Evolutionary processes can be inferred from comparisons of intraspecific polymorphism and interspecific divergence. We sequenced a 1.1-kb fragment of the cubitus interruptus Dominant (ciD) locus located on the nonrecombining fourth chromosome for ten natural lines of Drosophila melanogaster and nine of Drosophila simulans. We found no polymorphism within D. melanogaster and a single polymorphism within D. simulans; divergence between the species was about 5%. Comparison with the alcohol dehydrogenase gene and its two flanking regions in D. melanogaster, for which comparable data are available, revealed a statistically significant departure from neutrality in all three tests. This lack of polymorphism in the ciD locus may reflect recent positive selective sweeps on the fourth chromosome with extreme hitchhiking generated by the lack of recombination. By simulation, we estimate there to be a 50% chance that the selective sweeps occurred within the past 30,000 years in D. melanogaster and 75,000 in D. simulans.     

A Molecularly Defined Duplication Set for the X Chromosome of Drosophila melanogaster

We describe a molecularly defined duplication kit for the X chromosome of Drosophila melanogaster. A set of 408 overlapping P[acman] BAC clones was used to create small duplications (average length 88 kb) covering the 22-Mb sequenced portion of the chromosome. The BAC clones were inserted into an attP docking site on chromosome 3L using ΦC31 integrase, allowing direct comparison of different transgenes. The insertions complement 92% of the essential and viable mutations and deletions tested, demonstrating that almost all Drosophila genes are compact and that the current annotations of the genome are reasonably accurate. Moreover, almost all genes are tolerated at twice the normal dosage. Finally, we more precisely mapped two regions at which duplications cause diplo-lethality in males. This collection comprises the first molecularly defined duplication set to cover a whole chromosome in a multicellular organism. The work presented removes a long-standing barrier to genetic analysis of the Drosophila X chromosome, will greatly facilitate functional assays of X-linked genes in vivo, and provides a model for functional analyses of entire chromosomes in other species.THE X chromosome of Drosophila melanogaster contains ∼2300 protein-coding genes or ∼15% of such genes in the genome. It contains 22 Mb of euchromatic DNA (Adams et al. 2000). About one-third of these genes are predicted to be mutable to a phenotype that can be scored, e.g., lethality, sterility, or abnormal behavior (Peter et al. 2002). However, most molecularly recognized X-linked genes have not been associated with mutations or studied in any detail (http://flybase.org) (Drysdale 2008). Indeed, one hallmark of the X chromosome in D. melanogaster and many other species is that it is haploid in males. In addition, the presence of one copy of the X in an otherwise diploid animal leads to the phenomenon of dosage compensation, a process that essentially doubles the expression of X-linked genes in Drosophila males (Gelbart and Kuroda 2009).The presence of a single X chromosome in males facilitates screens for behavioral or visible mutant phenotypes in the hemizygous male progeny of a single-generation cross. For this reason, the X chromosome has been well saturated for viable mutations. However, many of these mutations have not been mapped since existing methods are tedious. Moreover, mutations in essential genes and genes required for male fertility cannot be propagated and genetically characterized unless they are complemented with a duplication maintained in the male. Hence, the X chromosome has been significantly less studied than the autosomes for mutations in essential and male fertility genes. For many of those mutations, the genes associated with these phenotypes have been elusive due to the lack of appropriate genetic reagents. Thus, X-linked genes in critical developmental and regulatory pathways are underrepresented in reported analyses as compared to similar classes of genes on the autosomes.Mutations in essential and male fertility genes on the X chromosome can be mapped using a variety of techniques. One approach is to rely on recombination in females and perform meiotic mapping against visible markers (Lindsley and Zimm 1992), P-element insertions (Zhai et al. 2003), or SNPs (Berger et al. 2001; Hoskins et al. 2001; Martin et al. 2001; Nairz et al. 2002; Chen et al. 2008), all of which are labor-intensive strategies or require specialized infrastructure. An alternative is complementation mapping using deficiencies, which requires only a single cross. This approach is possible for viable mutations but not for X-linked lethal and sterile mutations since those cannot be propagated through males. Instead, complementation rescue tests need to be carried out using a segregating duplication, e.g., an X chromosome fragment on the Y chromosome [Dp(1;Y)], an autosome [Dp(1;A)], or a free duplication [Dp(1;f)] (Lindsley and Zimm 1992). Currently, duplications that encompass ∼90% of the X chromosome are available. Only three cytological regions at 13A–13F (∼1 Mb), 16D7–16F4 (∼0.3 Mb), and 18A–18F (∼0.8 Mb) are not covered. Unfortunately, these duplications are typically very large (∼1–1.5 Mb) (http://flybase.org/) (Drysdale 2008), limiting their utility for fine mapping. Moreover, most available duplications were isolated following X-ray mutagenesis, and their breakpoints are poorly defined.Hence, a complete set of small molecularly defined duplications of the X chromosome would be extremely useful for identifying mutations in essential and male fertility genes and for fine-scale mapping of any mutation, including recessive viable mutations. In addition to promoting new genetic screens, a duplication set would allow one to map and assess the numerous, poorly characterized X-linked lethal mutants. Moreover, if molecularly defined genomic DNA clones are used to create the duplication set, then epitope tagging using recombineering would permit determination of expression patterns of genes included in the duplications (Venken et al. 2008, 2009; Ejsmont et al. 2009). Finally, such defined duplications would allow one to carry out structure–function analyses of genes through recombineering by introducing point mutations and small deletions into a gene of interest at unprecedented speed (Sharan et al. 2009).Previously, we created the P[acman] (P/ΦC31 artificial chromosome for manipulation) transgenesis platform (Venken and Bellen 2005, 2007; Venken et al. 2006) for retrieval and manipulation of large DNA fragments in a conditionally amplifiable BAC (Wild et al. 2002). Genomic clones inserted into this vector can be subjected to recombineering (Sharan et al. 2009) and used for transformation of these fragments (up to at least 146 kb) into the genome of flies that carry a defined attP docking site using the ΦC31 integrase system (Groth et al. 2004; Venken et al. 2006; Bischof et al. 2007; Markstein et al. 2008). In a next step, we constructed two genomic BAC libraries, one with an average insert size of 21 kb (CHORI-322) and another with an average insert size of 83 kb (CHORI-321) (Venken et al. 2009). These BAC libraries were end-sequenced and mapped onto the genome sequence and are publicly available (http://pacmanfly.org) and distributed (http://bacpac.chori.org/). Here we bring these resources to a next level: BAC TransgeneOmics (Poser et al. 2008) of an entire chromosome in vivo. The 8.2-fold coverage of the X chromosome in mapped clones from the CHORI-321 library allowed us to select a tiled path of overlapping BACs containing almost all of the annotated genes on this chromosome. Here we describe the creation of the first set of molecularly defined duplications covering an entire chromosome of a multicellular organism, and we illustrate its utility for X-chromosome genetics in several experimental paradigms.     

``break Copy'''' Duplication: A Model for Chromosome Fragment Formation in Saccharomyces Cerevisiae

Introduction of a chromosome fragmentation vector (CFV) into the budding yeast Saccharomyces cerevisiae results in a targeted homologous recombination event that yields an independently segregating chromosome fragment (CF) and an alteration in the strain's karyotype. Fragmentation with an acentric CFV directed in a centromere-proximal orientation generates a CF that contains all sequences proximal to the targeting segment and results in loss of the endogenous targeted chromosome to yield a 2N-1+CF karyotype. In contrast, fragmentation with a centric CFV directed in a centromere-distal orientation generates a CF that contains all sequences distal to the targeting segment and retention of the endogenous targeted chromosome to yield a 2N+CF karyotype. We have termed this phenomenon ``break copy' duplication. Using yeast strains in which the centromere had been transposed to a new location, it was demonstrated that the centromere inhibited break copy duplication. These data suggest that CF formation is the product of an unscheduled DNA replication event initiated by the free end of the CFV and is analogous to a ``half' double-strand break gap-repair reaction. We suggest that break copy duplication may have evolved as a mechanism for maintenance of ploidy following DNA breakage.     

Chromosome Copy Number Variation and Control in the Ciliate Chilodonella uncinata

Copy number variations are widespread in eukaryotes. The unusual genome architecture of ciliates, in particular, with its process of amitosis in macronuclear division, provides a valuable model in which to study copy number variation. The current model of amitosis envisions stochastic distribution of macronuclear chromosomes during asexual reproduction. This suggests that amitosis is likely to result in high levels of copy number variation in ciliates, as dividing daughter cells can have variable copy numbers of chromosomes if chromosomal distribution during amitosis is a stochastic process. We examined chromosomal distribution during amitosis in Chilodonella uncinata, a ciliate with gene-size macronuclear chromosomes. We quantified 4 chromosomes in evolving populations of C. uncinata and modeled the amitotic distribution process. We found that macronuclear chromosomes differ in copy number from one another but that copy number does not change as expected under a stochastic process. The chromosome carrying SSU increased in copy number, which is consistent with selection to increase abundance; however, two other studied chromosomes displayed much lower than expected among-line variance. Our models suggest that balancing selection is sufficient to explain the observed maintenance of chromosome copy during asexual reproduction.     

A Branched-chain Amino Acid Aminotransferase from the Rumen Ciliate Genus Entodinium

A branched-chain amino acid aminotransferase was extracted from rumen ciliates of the genus Entodinium and was partially purified by Sephadex G-200, DEAE-cellulose and DEAE-Sephadex A-50 column chromatography. The purified enzyme was active only with leucine, isoleucine and valine, and required pyridoxal phosphate as cofactor. The amino acids competed with each other as substrates. The enzyme had optimal activity at pH 6.0 in phosphate buffer. The K_m values for the substrates and cofactor are as follows: 1.66 for leucine; 0.90 for isoleucine; 0.79 for valine; 0.29 mM for α-ketoglutarate: and 0.1 μM for pyridoxal phosphate. Enzyme activity was inhibited by p-chloromercuribenzoate and HgCl₂. Gel filtration indicated the enzyme to have a molecular weight of 34,000.     

A Rare Duplication on Chromosome 16p11.2 Is Identified in Patients with Psychosis in Alzheimer's Disease

Epidemiological and genetic studies suggest that schizophrenia and autism may share genetic links. Besides common single nucleotide polymorphisms, recent data suggest that some rare copy number variants (CNVs) are risk factors for both disorders. Because we have previously found that schizophrenia and psychosis in Alzheimer''s disease (AD+P) share some genetic risk, we investigated whether CNVs reported in schizophrenia and autism are also linked to AD+P. We searched for CNVs associated with AD+P in 7 recurrent CNV regions that have been previously identified across autism and schizophrenia, using the Illumina HumanOmni1-Quad BeadChip. A chromosome 16p11.2 duplication CNV (chr16: 29,554,843-30,105,652) was identified in 2 of 440 AD+P subjects, but not in 136 AD subjects without psychosis, or in 593 AD subjects with intermediate psychosis status, or in 855 non-AD individuals. The frequency of this duplication CNV in AD+P (0.46%) was similar to that reported previously in schizophrenia (0.46%). This duplication CNV was further validated using the NanoString nCounter CNV Custom CodeSets. The 16p11.2 duplication has been associated with developmental delay, intellectual disability, behavioral problems, autism, schizophrenia (SCZ), and bipolar disorder. These two AD+P patients had no personal of, nor any identified family history of, SCZ, bipolar disorder and autism. To the best of our knowledge, our case report is the first suggestion that 16p11.2 duplication is also linked to AD+P. Although rare, this CNV may have an important role in the development of psychosis.     

Properties of the telomeric DNA-binding protein from Oxytricha nova

Telomeres of Oxytricha macronuclear DNA exist as discrete DNA-protein complexes. Different regions of each complex display characteristic DNA-protein interactions. In the most terminal region, binding of a 43- and a 55-kDa protein to the telomeric DNA appears to account for all the DNA-protein interactions that can be detected by chemical and nuclease footprinting. We have used gradient sedimentation and protein-protein cross-linking to establish that the 43- and 55-kDa proteins are subunits of a heterodimer. Both subunits are very basic, which is unexpected considering the resistance of the DNA-protein interaction to high concentrations of salt. It is extremely difficult to dissociate the two subunits either from telomeric DNA or from each other. Even after extensive treatment of protein preparations with nuclease, a fragment of the 3' tail from macronuclear DNA remains bound to the protein. A wide range of conditions was screened for dissociation of the subunits from the DNA and/or from each other. Dissociation was only obtained by using conditions that caused some inactivation of the DNA-binding capacity of the protein. The use of reagents that covalently modify sulfydryl groups during the purification procedure facilitates preparation of telomere protein with full DNA-binding activity.     

A Novel Healing Filament in Ciliate Regeneration

The interphase cells of the hypotrich ciliate Paraurostyla weissei possess a complex fibrillar system surrounding basal bodies in the compound ciliary assemblages, cirri and membranelles. During replacement of the ciliature at cell division, transient filaments precede and accompany the development of ciliary primordia and participate in the formation of the fission furrow. Both fibrillar systems are recognized by monoclonal antibody FXXXIX 12G9. We studied regeneration of cellular fragments after transection employing the mAb 12G9 and found a new cytoskeletal structure involved in healing of the excisional wound. The healing filament is formed at the wound edge, distally and in connection with the bases of cirri closest to the wound. It is visible 5 min after transection. Concomitant with development of new ciliary primordia, the healing filament shrinks and finally disappears together with other transient fibers formed in this process. Ultrastructural analysis of immunolabeled regenerating cells revealed that structures recognized by mAb 12G9 contain fine filaments whose packing and arrangement depends on accompanying cytoplasmic elements and the developmental status of a fragment. Assembly of the healing fiber does not depend on microtubules and microfilaments since it develops in cellular fragments exposed to cold, nocodazole, and Cytochalasin D. On Western blots of whole cell and cytoskeletal extracts of P. weissei the 12G9 antibody identified one protein band whose molecular weight corresponds to 60 kDa.     

A Study of Ciliate Protozoa from a Small Polluted Stream in East-Central Illinois

The role of species of ciliate Protozoa in trickling filterwaste treatment processes awaits redefinition. Using the smallfresh-water Salt Fork stream for our study, a stream which receivesall of the discharge from the Champaign-Urbana Waste TreatmentPlant in east-central Illinois, analyses of samples of carefullyidentified ciliates taken from selected sites over a three-yearperiod yielded some interesting results. Four specific situationswere noted: (1) free-swimming microphagous forms feeding selectivelyon primarily attached bacteria, and, in turn, being fed on bycarnivorous ciliates; (2) sessile ciliates (peritrichs) feedingon non-attached bacterial species, but with high selectivity;(3) free-swimming carnivorous gymnostomes feeding on the sessileperitrichs but, again in turn, serving as prey themselves tostalked suctorian species attached to the peduncles of the peritrichs;(4) attached peritrichs and suctorians both serving as foodfor several groups of small invertebrates frequenting the stream.With respect to numbers and diversity of ciliates found, some155 different species, belonging to 93 genera, were identified(including several to be described elsewhere as new species)in the stream and/or the trickling filter settling basin. Thehighest populations and greatest diversity were found at sitesclosest to the waste water influx; such protozoan populationsmight thus be considered as a form of tertiary treatment inthe drainage basin.     

Concordance between Parental Origin of Chromosome 13q Loss and 6p Duplication in Sporadic Retinoblastoma

Two hypotheses are capable of explaining nonrandom loss of one parent's alleles at tumor suppressor loci in sporadic cases of several pediatric cancers, including retinoblastoma—namely, preferential germ-line mutation or chromosome imprinting. We have examined 74 cases of sporadic retinoblastoma for tumors in which at least two genetic events—loss of heterozygosity for chromosome 13q markers and formation of an isochromosome 6p—have occurred. Sixteen cases were found to contain both events. In 13 of 16 such tumors, the chromosomes 13q that were lost and chromosomes 6p that were duplicated are derived from the same parent. These data may be explained within the framework of the genome imprinting model but are not predicted by preferential germ-line mutation.