Evolution of the Germline Actin Gene in Hypotrichous Ciliates: Multiple Nonscrambled IESs at Extremely Conserved Locations in Two Urostylids

In hypotrichous ciliates, macronuclear chromosomes are gene‐sized, and micronuclear genes contain short, noncoding internal eliminated segments (IESs) as well as macronuclear‐destined segments (MDSs). In the present study, we characterized the complete macronuclear gene and two to three types of micronuclear actin genes of two urostylid species, i.e. Pseudokeronopsis rubra and Uroleptopsis citrina. Our results show that (1) the gain/loss of IES happens frequently in the subclass Hypotrichia (formerly Stichotrichia), and high fragmentation of germline genes does not imply for gene scrambling; and (2) the micronuclear actin gene is scrambled in the order Sporadotrichida but nonscrambled in the orders Urostylida and Stichotrichida, indicating the independent evolution of MIC‐actin gene patterns in different orders of hypotrichs; (3) locations of MDS–IES junctions of micronuclear actin gene in coding regions are conserved among closely related species.     

Two two-gene macronuclear chromosomes of the hypotrichous ciliates Oxytricha fallax and O. trifallax generated by alternative processing of the 81 locus

We describe the first known macronuclear chromosomes that carry more than one gene in hypotrichous ciliated protozoa. These 4.9- and 2.8-kbp chromosomes each consist almost exclusively of two protein-coding genes, which are conserved and transcribed. The two chromosomes share a common region that consists of a gene that is a member of the family of mitochondrial solute carrier genes (CR-MSC; [Williams and Herrick (1991): Nucleic Acids Res 19:4717–4724]. Each chromosome also carries another gene appended to its common region: The 4.9-kbp chromosome also carries a gene that encodes a protein that is rich in glutamine and charged amino acids and bears regions of heptad repeats characteristic of coiled-coils. Its function is unknown. The second gene of the 2.8 kbp chromosome is a mitochondrial solute carrier gene (LA-MSC); thus, the 2.8-kbp chromosome consists of two mitochondrial solute carrier paralogs. Phylogenetic analysis indicates that the two genes were duplicated before ciliates diverged from the main eukaryotic lineage and were subsequently juxtaposed. The CR- and LA-MSC genes are each interrupted by three introns. The introns are not in homologous positions, suggesting that they may have originated from multiple group II intron transpositions. These chromosomes and their genes are encoded in the Oxytricha germline by the 81 locus. This locus is alternatively processed to generate a nested set of three macronuclear chromosomes, the 4.9- and 2.8-kbp chromosomes and a third (1.6 kbp) which consists almost exclusively of the shared common gene, CR-MSC. Such alternative processing is common in macronuclear development of O. fallax [Cartinhour and Herrick (1984): Mol Cell Biol 4:931–938]. Possible functions for alternative processing are considered; e.g., it may serve to physically link genes to allow co-regulation or co-replication by a common cis-acting sequence. Dev. Genet. 20:348–357, 1997. © 1997 Wiley-Liss, Inc.     

The organization of macronuclear DNA sequences associated with C4A4 repeats in the polytene chromosomes of Stylonychia lemnae

Gene libraries of the micronucleus and the macronuclear anlagen of the polytene chromosome stage of Stylonychia lemnae were screened for internal C₄A₄ repeats. The number of these internal repeats was shown to be identical in both kinds of nuclei. Analysis of macronuclear sequences associated with C₄A₄ in the polytene chromosomes showed that several macronuclear DNA sequences are clustered. However, interspersed between short exons of one gene are located exons of several other genes, i.e. the exon of one gene is an intron for several other genes.by M. Trendelenburg     

The completed macronuclear genome of a model ciliate Tetrahymena thermophila and its application in genome scrambling and copy number analyses

The ciliate Tetrahymena thermophila has been a powerful model system for molecular and cellular biology. However, some investigations have been limited due to the incomplete closure and sequencing of the macronuclear genome assembly, which for many years has been stalled at 1,158 scaffolds, with large sections of unknown sequences(available in Tetrahymena Genome Database, TGD, http://ciliate.org/). Here we completed the first chromosome-level Tetrahymena macronuclear genome assembly, with approximately 300× long Single Molecule, Real-Time reads of the wild-type SB210 cells—the reference strain for the initial macronuclear genome sequencing project. All 181 chromosomes were capped with two telomeres and gaps were entirely closed. The completed genome shows significant improvements over the current assembly(TGD 2014) in both chromosome structure and sequence integrity. The majority of previously identified gene models shown in TGD were retained,with the addition of 36 new genes and 883 genes with modified gene models. The new genome and annotation were incorporated into TGD. This new genome allows for pursuit in some underexplored areas that were far more challenging previously; two of them, genome scrambling and chromosomal copy number, were investigated in this study. We expect that the completed macronuclear genome will facilitate many studies in Tetrahymena biology, as well as multiple lines of research in other eukaryotes.     

The complete karyotype of Aspergilius niger: the use of introduced electrophoretic mobility variation of chromosomes for gene assignment studies

A method is described for unambiguous assignment of cloned genes to Aspergillus niger chromosomes by CHEF gel electrophoresis and Southern analysis. All of the eight linkage groups (LGs), with the exception of LG VII, have previously been assigned to specific chromosomal bands in the electrophoretic karyotype of A. niger. Using a LG VII-specific probe (nicB gene of A. niger) we have shown that LG VII corresponds to a chromosome of about 4.1 Mb. Furthermore, genetic localization of three unassigned genes (glaA, agIA and pepA) in strains in which these genes had been replaced by a selectable marker gene led to a revised karyotype for the chromosomes corresponding to LGs VIII and VI. The revised electrophoretic karyotype reveals only 5 distinct bands. The presence of three pairs of equally sized chromosomes precluded assignment of genes to one specific chromosome in the wild-type strain. However, unambiguous chromosome assignment of cloned genes using CHEF-Southern analysis was demonstrated using a set of A. niger strains with introduced chromosomal size variation. The availability of these tester strains obviates the need to isolate or construct mutant. strains for the purpose of chromosome assignment.     

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.     

Two genes encoding gas vacuole proteins in Halobacterium halobium

Summary The archaebacterium Halobacterium halobium contains two related gas vacuole protein-encoding genes (vac). One of these genes encodes a protein of 76 amino acids and resides on the major plasmid. The second gene is located on the chromosome in a (G+C)-rich DNA fraction and encodes a slightly larger but highly homologous protein consisting of 79 amino acids. The plasmid encoded vac gene is transcribed constitutively throughout the growth cycle while the chromosomal vac gene is expressed during the stationary phase of growth. Comparison of the nucleotide sequences of the two genes indicates differences in the putative promoter regions as well as 35 single base-pair exchanges within the coding regions of the two genes. The majority of the nucleotide exchanges in the coding region occur in the third position of a codon triplet generating the codon synonym. The only differences between the two encoded proteins are the exchange of 2 amino acids (positions 8 and 29) and a deletion of 3 amino acids near the carboxy-terminus of the plasmid encoded vac protein. The genomic DNAs from other halobacterial isolates (Halobacterium sp. SB3, GN101 and YC819-9) were found to contain only a chromosomal vac gene copy. There is a high conservation of the chromosomal vac gene and the genomic region surrounding it among the halobacterial strains investigated.     

Organization of the Euplotes crassus Micronuclear Genome1

Euplotes crassus, like other hypotrichous ciliated protozoa, eliminates most of its micronuclear chromosomal DNA in the process of forming the small linear DNA molecules that comprise the macronuclear genome. By characterizing randomly selected lambda phage clones of E. crassus micronuclear DNA, we have determined the distribution of repetitive and unique sequences and the arrangement of macronuclear genes relative to eliminated DNA. This allows us to compare the E. crassus micronuclear genome organization to that of another distantly related hypotrichous ciliate, Oxytricha nova. The clones from E. crassus segregate into three prevalent classes: those containing primarily eliminated repetitive DNA (Class I); those containing macronuclear genes in addition to repetitive sequences (Class II); and those containing only eliminated unique sequence DNA (Class III). All of the repetitive sequences in these clones belong to the same highly abundant repetitive element family. Our results demonstrate that the sequence organization of the E. crassus and O. nova micronuclear genomes is related in that the macronuclear genes are clustered together in the micronuclear genome and the eliminated unique sequences occur in long stretches that are uninterrupted by repetitive sequences. In both organisms a single repetitive element family comprises the majority of the eliminated interspersed middle repetitive DNA and appears to be preferentially associated with the macronuclear sequence clusters. The similarities in the sequence organization in these two organisms suggest that clustering of macronuclear genes plays a role in the chromosome fragmentation process.     

Validation of housekeeping genes for gene expression studies in an ice alga <Emphasis Type="Italic">Chlamydomonas</Emphasis> during freezing acclimation

Antarctic ice alga Chlamydomonas sp. ICE-L can endure extreme low temperature and high salinity stress under freezing conditions. To elucidate the molecular acclimation mechanisms using gene expression analysis, the expression stabilities of ten housekeeping genes of Chlamydomonas sp. ICE-L during freezing stress were analyzed. Some discrepancies were detected in the ranking of the candidate reference genes between geNorm and NormFinder programs, but there was substantial agreement between the groups of genes with the most and the least stable expression. RPL19 was ranked as the best candidate reference genes. Pairwise variation (V) analysis indicated the combination of two reference genes was sufficient for qRT-PCR data normalization under the experimental conditions. Considering the co-regulation between RPL19 and RPL32 (the most stable gene pairs given by geNorm program), we propose that the mean data rendered by RPL19 and GAPDH (the most stable gene pairs given by NormFinder program) be used to normalize gene expression values in Chlamydomonas sp. ICE-L more accurately. The example of FAD3 gene expression calculation demonstrated the importance of selecting an appropriate category and number of reference genes to achieve an accurate and reliable normalization of gene expression during freeze acclimation in Chlamydomonas sp. ICE-L.     

Tetrahymena Telomerase RNA levels increase during macronuclear development

Telomeres, the G-rich sequences found at the ends of eukaryotic chromosomes, ensure chromosome stability and prevent sequence loss from chromosome ends during DNA replication. During macronuclear development in Tetrahymena, the chromosomes fragment into pieces ranging from 20 kb to 1,500 kb. Tetrahymena telomerase, a ribonucleoprotein, adds telomeric (TTGGGG)_n repeats onto telomeres and onto the newly generated macronuclear DNA ends. We have investigated whether telomerase RNA levels increase during macronuclear development, since such an increase might be expected during chromosomal fragmentation. The steady-state level of the telomerase RNA component was used to estimate the abundance of telomerase present in mating and nonmating Tetrahymena. Northern blot analysis revealed that in vegetatively growing Tetrahymena, there were 18,000–40,000 copies of telomerase RNA per cell. In mating cultures, the levels of RNA increased 2-to 5-fold at 9–15 h, and 1.5- to 3.5-fold in starved nonmating cultures. This increase in telomerase RNA paralleled telomerase activity, which also increased slightly in mating and starved nonmating cells. © 1992 Wiley-Liss, Inc.     

The origin of an unusual sex chromosome constitution in <Emphasis Type="Italic">Acomys</Emphasis> sp. (Rodentia,Muridae) from Tanzania

This paper describes a case which presents an evident variation from the “standard” XX/XY sex chromosomal constitution in a rodent, Acomys sp. This species known to be found in three localities of East Africa has only recently been separated from A. spinosissimus, its closest relative. In our study, five specimens of Acomys sp. and eight specimens of A. spinosissimus were live-trapped in five localities. Comparisons between the two taxa assed by G- banding show a complete homology in the chromosomal shape and banding pattern for 29 pairs of chromosomes corresponding to the complete autosomal set of A. spinosissimus. However, while all the A. spinosissimus analysed have 2n = 60 and a XY-XX system, in Acomys sp. males and females constitute mosaics for sex chromosomes in the bone marrow cells. Females (2n = 59, 60) have an excess (97%) of aneuploid cells with one single giant X chromosome, and males (2n = 60, 61) show X0/XY cells occurring in somatic tissues and XY cells in the germinal lineage. In addition, an odd heterochromatic submetacentric chromosome was identified in all the cells examined in two males and a female of Acomys sp. Since this chromosome was not related to sex determination and it is not present in all the analysed specimens, it can be considered as a B chromosome. Finally, the in situ fluorescence hybridisation (FISH) with telomeric probes showed a very intense interstitial telomeric signal (ITS) at the medial part on the long heterochromatic arm of the X chromosome. This could be due to recent chromosomal rearrangement.     

Nonrandom Representation of Sex-Biased Genes on Chicken Z Chromosome

Several lines of evidence suggest that the X chromosome of various animal species has an unusual complement of genes with sex-biased or sex-specific expression. However, the study of the X chromosome gene content in different organisms provided conflicting results. The most striking contrast concerns the male-biased genes, which were reported to be almost depleted from the X chromosome in Drosophila but overrepresented on the X chromosome in mammals. To elucidate the reason for these discrepancies, we analysed the gene content of the Z chromosome in chicken. Our analysis of the publicly available expressed sequence tags (EST) data and genome draft sequence revealed a significant underrepresentation of ovary-specific genes on the chicken Z chromosome. For the brain-expressed genes, we found a significant enrichment of male-biased genes but an indication of underrepresentation of female-biased genes on the Z chromosome. This is the first report on the nonrandom gene content in a homogametic sex chromosome of a species with heterogametic female individuals. Further comparison of gene contents of the independently evolved X and Z sex chromosomes may offer new insight into the evolutionary processes leading to the nonrandom genomic distribution of sex-biased and sex-specific genes. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Manyuan Long]     

The karyotype of Alligator mississippiensis,and chromosomal mapping of the ZFY/X homologue,Zfc

Comparative mapping studies of X-linked genes in mammals have provided insights into the evolution of the X chromosome. Many reptiles including the American alligator, Alligator mississippiensis, do not appear to possess heteromorphic sex chromosomes, and sex is determined by the incubation temperature of the egg during embryonic development. Mapping of homologues of mammalian X-linked genes in reptiles could lead to a greater understanding of the evolution of vertebrate sex chromosomes. One of the genes used in the mammalian mapping studies was ZFX, an X-linked copy of the human ZFY gene which was originally isolated as a candidate for the mammalian testis-determining factor (TDF). ZFX is X-linked in eutherians, but maps to two autosomal locations in marsupials and monotremes, close to other genes associated with the eutherian X. The alligator homologue of the ZFY/ZFX genes, Zfc, has been isolated and described previously. A detailed karyotype of A. mississippiensis is presented, together with chromosomal in situ hybridisation data localising the Zfc gene to chromosome 3. Further chromosomal mapping studies using eutherian X-linked genes may reveal conserved chromosomal regions in the alligator that have become part of the eutherian X chromosome during evolution.     

Mitosis-like macronuclear division in a ciliate

The macronucleus of a small marine ciliate of the genus Protocrucia consists of a cluster of ten vesicles which give rise to 20 distinct chromosomal elements in the course of prophase-like condensation stages. Size differences of vesicles and chromosomes are cytological indications of their genetic individuality. In an anaphase-like stage, the chromosomal elements are separated in two daughter groups which re-form 10 vesicles each. The micronucleus divides simultaneously. The existence of a precisely functioning mode of chromosome distribution is also indicated by DNA measurements. Since the macronucleus contains much more DNA than the micronucleus, the macronuclear chromosomes are thought to be oligotenic. This hypothesis is supported by the much larger size of the macronuclear chromosomes. In contrast to other modes of macronuclear division known so far, this ciliate has retained some essential features of mitosis.     

A model for random genetic damage directing selection of diploid or aneuploid tumours

Objectives: To test whether genetic instability may determine whether tumours become aneuploid or diploid. Materials and methods: We have identified genes needed for cell survival or replication by combining Affymetrix gene expression array data from 12 experimental cell lines with in silico GEO+GNF and expO databases. Specific loss of heterozygosis (LOHs), chromosomal abnormalities (called derivative chromosomes) and numbers of normal homologues were identified by SNP and SKY analyses. Random gene losses were calculated under the assumption that bi‐allelic MMR gene inactivation causes a 20‐fold increase in rate of gene loss. Results: There were ～1.23 × 10⁴ genes widely dispersed throughout the genome and possibly expressed by all cells for survival or proliferation, many of these genes performed housekeeping functions. Conservation of the genes may explain the complete haploid genomes found for 15 different cell types and derivative chromosomes selectively retained in aneuploid cancer cell lines after LOH formations, and normal homologue losses. Loss of cell survival/replication genes was calculated to be higher in colon stem cells of carriers of MMR gene mutations than carriers of APC gene mutations. Conclusion: Random loss of cell survival/replication genes was calculated to be low enough for colon stem cells with APC gene mutations to ‘select’ LOH and derivative chromosome combinations favouring tumour cell proliferation. However, cell survival/replication gene loss was calculated to be too high for colonic stem cells lacking MMR genes to survive chromosomal instability, explaining why MMR mutations only produce tumours with diploid chromosome cells.     

Genetic and Chromosomal Control of Development

The last decade has been characterized by extraordinary progress in studies of the molecular mechanisms underlying the gene control of development: complexes of genes with a hierarchic principle of functioning have been identified, evolutionary-conservative systems of genes have been studied that ensure the transmembrane regulatory signaling between cells and play a key role in morphogenesis, and a concept of gene networks that coordinate gene interaction was introduced. Note that temporal and tissue-specific parameters of gene expression are correctly realized only in terms of chromosomes and are largely determined by the gene position on a chromosome and in the interphase nucleus. The epigenetic inheritance of gene status in cell generations is realized at the chromosomal level alone due to the cellular or chromosomal memory. This ontogenetic memory is an immanent property of a chromosome and cis-regulation plays a key role in its maintenance.     

The scattered distribution of actin genes in the mouse and human genomes.

A hamster actin cDNA probe was used to localize actin genes on the major components of mouse and human DNAs, namely on the four families of fragments forming the bulk of these genomes. Over 20 EcoRI fragments hybridizing the probe could be detected; a different subset of these fragments was found in each component. Since the fragment families forming the major components of the mouse and human DNAs derive from very long chromosomal segments, the isochores , the presence of actin genes on all components provides evidence for their dispersion in both genomes. In situ hybridization of 125I-labeled probe to metaphase chromosomes in the presence of dextran sulfate confirmed this dispersion by showing that the 29-30 actin gene sites so identified are distributed on almost all chromosomes. Moreover, some human actin genes could be mapped on specific chromosomal segments; in particular, one gene was localized on the long arm of the X chromosome. Finally, three different mouse actin genes were isolated from a recombinant DNA library and previously investigated interspersed repeated sequences were identified in the vicinity of these genes.     

Krb1, a Suppressor of Mak7-1 (a Mutant Rpl4a), Is Rpl4b, a Second Ribosomal Protein L4 Gene, on a Fragment of Saccharomyces Chromosome Xii

The mak7-1 mutant loses the killer toxin-encoding M(1) dsRNA. MAK7 is RPL4A, one of two genes encoding ribosomal protein L4. KRB1 is a dominant suppressor of mak7-1 that is tightly centromere-linked, but not linked to centromere markers of chromosomes I-XVI. Our orthogonal field agarose gel electrophoresis analysis of chromosomal DNA from strains with KRB1 shows a novel band of ~250 kb. This band hybridizes with an RPL4B-specific probe, but not an RPL4A (MAK7)-specific probe. The RPL4B-specific probe also hybridizes to chromosome XII where the original RPL4B is located. KRB1 is meiotically linked to this extra chromosome. Disruption of either the RPL4B gene on chromosome XII or that on the extra chromosome results in loss of the killer phenotype and a decreased concentration of free 60S subunits. Thus, the RPL4B on the extra chromosome is KRB1 and is active. The extra chromosome contains chromosome XII sequence between Lambda 5345 clone (ATCC70558) and Lambda 6639 clone (ATCC71085) of Olson's Lambda library, indicating that KRB1 represents a chromosomal rearrangement involving chromosome XII and explaining the earlier genetic data.