Germline Organization Research Articles

Russian Doll Genes and Complex Chromosome Rearrangements in Oxytricha trifallax.

Ciliates have two different types of nuclei per cell, with one acting as a somatic, transcriptionally active nucleus (macronucleus; abbr. MAC) and another serving as a germline nucleus (micronucleus; abbr. MIC). Furthermore, Oxytricha trifallax undergoes extensive genome rearrangements during sexual conjugation and post-zygotic development of daughter cells. These rearrangements are necessary because the precursor MIC loci are often both fragmented and scrambled, with respect to the corresponding MAC loci. Such genome architectures are remarkably tolerant of encrypted MIC loci, because RNA-guided processes during MAC development reorganize the gene fragments in the correct order to resemble the parental MAC sequence. Here, we describe the germline organization of several nested and highly scrambled genes in Oxytricha trifallax. These include cases with multiple layers of nesting, plus highly interleaved or tangled precursor loci that appear to deviate from previously described patterns. We present mathematical methods to measure the degree of nesting between precursor MIC loci, and revisit a method for a mathematical description of scrambling. After applying these methods to the chromosome rearrangement maps of O. trifallax we describe cases of nested arrangements with up to five layers of embedded genes, as well as the most scrambled loci in O. trifallax.

Automated three-dimensional reconstruction of the Caenorhabditis elegans germline

The Caenorhabditis elegans germline is widely used as a model to study stem cell development, chromosome dynamics and apoptosis. Major readouts of germline phenotypes such as cell counting and protein expression profiling are routinely analyzed manually and in a two-dimensional manner. The major disadvantages of the existing approaches are 1) they are time-consuming and laborious and 2) there is an inability to study the effects of genetic mutations in three dimensions. Here, we demonstrate a rapid, automated method for analyzing the three-dimensional distribution of proteins, germline nuclei and cytoskeletal structures in the C. elegans germline. Using this method, we have revealed previously unappreciated germline organization and cytoskeletal structures that will have a major impact on the characterization of germline phenotypes. To conclude, our new method dramatically enhances the efficiency and resolution of C. elegans germline analysis and may be applied to other cellular structures.

Germline organization in Strongyloides nematodes reveals alternative differentiation and regulation mechanisms.

Nematodes of the genus Strongyloides are important parasites of vertebrates including man. Currently, little is known about their germline organization or reproductive biology and how this influences their parasitic life strategies. Here, we analyze the structure of the germline in several Strongyloides and closely related species and uncover striking differences in the development, germline organization, and fluid dynamics compared to the model organism Caenorhabditis elegans. With a focus on Strongyloides ratti, we reveal that the proliferation of germ cells is restricted to early and mid-larval development, thus limiting the number of progeny. In order to understand key germline events (specifically germ cell progression and the transcriptional status of the germline), we monitored conserved histone modifications, in particular H3Pser10 and H3K4me3. The evolutionary significance of these events is subsequently highlighted through comparisons with six other nematode species, revealing underlying complexities and variations in the development of the germline among nematodes.Electronic supplementary materialThe online version of this article (doi:10.1007/s00412-015-0562-5) contains supplementary material, which is available to authorized users.

Evolutionary immunology: Early vertebrates reveal diverse immune recognition strategies

Adaptive immune responses in most vertebrates (the gnathostomes: jawed fishes, amphibians, reptiles, birds and mammals) are mediated by lymphocyte-associated antigen receptors encoded by rearranging genes—immunoglobulin (Ig) on B cells and T-cell receptors on T cells. These hypervariable recognition molecules share commonality in their immunoglobulin superfamily (IgSF) domains and the germ-line organization of their rearranging gene elements.

FLI-1 Flightless-1 and LET-60 Ras control germ line morphogenesis in C. elegans

BackgroundIn the C. elegans germ line, syncytial germ line nuclei are arranged at the cortex of the germ line as they exit mitosis and enter meiosis, forming a nucleus-free core of germ line cytoplasm called the rachis. Molecular mechanisms of rachis formation and germ line organization are not well understood.ResultsMutations in the fli-1 gene disrupt rachis organization without affecting meiotic differentiation, a phenotype in C. elegans referred to here as the germ line morphogenesis (Glm) phenotype. In fli-1 mutants, chains of meiotic germ nuclei spanned the rachis and were partially enveloped by invaginations of germ line plasma membrane, similar to nuclei at the cortex. Extensions of the somatic sheath cells that surround the germ line protruded deep inside the rachis and were associated with displaced nuclei in fli-1 mutants. fli-1 encodes a molecule with leucine-rich repeats and gelsolin repeats similar to Drosophila flightless 1 and human Fliih, which have been shown to act as cytoplasmic actin regulators as well as nuclear transcriptional regulators. Mutations in let-60 Ras, previously implicated in germ line development, were found to cause the Glm phenotype. Constitutively-active LET-60 partially rescued the fli-1 Glm phenotype, suggesting that LET-60 Ras and FLI-1 might act together to control germ line morphogenesis.ConclusionFLI-1 controls germ line morphogenesis and rachis organization, a process about which little is known at the molecular level. The LET-60 Ras GTPase might act with FLI-1 to control germ line morphogenesis.

A Role forCaenorhabditis elegansChromatin-Associated Protein HIM-17 in the Proliferationvs. Meiotic Entry Decision

Chromatin-associated protein HIM-17 was previously shown to function in the chromosomal events of meiotic prophase. Here we report an additional role for HIM-17 in regulating the balance between germ cell proliferation and meiotic development. A cryptic function for HIM-17 in promoting meiotic entry and/or inhibiting proliferation was revealed by defects in germline organization in him-17 mutants grown at high temperature (25 degrees) and by a synthetic tumorous germline phenotype in glp-1(ar202); him-17 mutants at 15 degrees.

Allelic exclusion in anamniotic vertebrates

Clonal selection, a central principle in immunology, is predicated on one lymphocyte making one kind of antibody or T cell receptor. At loci encoding antigen receptors only one allele is used, and this has been shown in normal lymphocytes from frogs to humans. Fish antibody chains, however, are encoded by multiple loci, and at some of these loci the gene segments are already rearranged in the germline. The differences in germline organization and the uncoupling of rearrangement and expression raise questions as to whether some of the early vertebrates might be an exception to the clonal selection theory.

Chicken immunoglobulin μ-chain gene: germline organization and tandem repeats characteristic of class switch recombination

We have isolated the phage clones covering the region spanning from the heavy (H)-chain joining (J) region to the end of the μ-chain gene of the chicken immunoglobulin (Ig). The distance from J H to the first exon of the μ-chain constant (C) region is approximately 13 kb, and introns between the C region exons measure more than 3 kb. These distances are significantly larger than those of known μ-chain genes. We found a region cross-hybridizing to the switch regions of the mouse Cμ and Cα genes just in front of the first exon of Cμ. Partial nucleotide sequencing of this region revealed that this region consists of tandem repeats of pentamers ( C T )( C A ) CAG complementary to the mammalian switch repetitive region. These findings suggest that this region is a good candidate for a class switch region of the μ-chain gene of chicken Ig.

Genomic organization of the human T-cell antigen-receptor alpha/delta locus.

Two clusters of overlapping cosmid clones comprising about 100 kilobases (kb) at the human T-cell antigen-receptor alpha/delta locus were isolated from a genomic library. The structure of the germ-line V delta 1 variable gene segment was determined. V delta 1 is located 8.5 kb downstream of the V alpha 13.1 gene segment, and both V segments are arranged in the same transcriptional orientation. The V alpha 17.1 segment is located between V delta 1 and the D delta, J delta, C delta region (containing the diversity, joining, and constant gene segments). Thus, V delta and V alpha segments are interspersed along the chromosome. The germ-line organization of the D delta 2, J delta 1, and J delta 2 segments was determined. Linkage of C delta to the J alpha region was established by identification of J alpha segments within 20 kb downstream of C delta. The organization of the locus was also analyzed by field-inversion gel electrophoresis. The unrearranged V delta 1 and D delta, J delta, C delta regions are quite distant from each other, apparently separated by a minimum of 175-180 kb.

Germ-line configuration of the T-cell receptor beta-chain gene in B-cell chronic lymphoproliferative disorders which co-express T-cell antigens.

In 7 cases of chronic B-cell lymphoproliferative disorders-6 chronic lymphocytic leukaemias and 1 non-Hodgkin lymphoma in leukaemic phase--which co-expressed T-cell markers (CD3, CD2) the clonal origin was investigated at the DNA level. In accordance with the diagnosis, all cases showed a monoclonally rearranged configuration of the immunoglobulin genes. On the contrary, the T-cell receptor beta chain gene always retained a germ-line organization. These findings demonstrate that B-cell chronic lymphoproliferative disorders which co-express T-cell-related markers are truly composed of monoclonal B-cell elements.

Germline organization of the murine T cell receptor beta-chain genes.

The complete germline organization of the beta-chain genes of the murine T cell receptor was elucidated in order to obtain the structural basis for understanding the mechanisms of somatic DNA rearrangements. Twenty of the 22 known variable (V beta) genes are clustered within 250 kilobases of DNA 5' to the constant region (C beta) genes. These V beta genes share the same transcriptional orientation as the diversity (D beta), joining (J beta), and C beta genes, which implies that chromosomal deletion is the mechanism for most V beta to D beta-J beta rearrangements. Within this V beta cluster, the distance between the most proximal V beta gene and the D beta-J beta-C beta cluster is 320 kilobases, as determined by field-inversion gel electrophoresis. The large distance between V beta and D beta, relative to that between D beta and J beta, may have significant implications for the ordered rearrangement of the T cell receptor beta-chain genes.