Gametic specialization of centromeric histone paralogs in Drosophila virilis

In most eukaryotes, centromeric histone (CenH3) proteins mediate mitosis and meiosis and ensure epigenetic inheritance of centromere identity. We hypothesized that disparate chromatin environments in soma versus germline might impose divergent functional requirements on single CenH3 genes, which could be ameliorated by gene duplications and subsequent specialization. Here, we analyzed the cytological localization of two recently identified CenH3 paralogs, Cid1 and Cid5, in Drosophila virilis using specific antibodies and epitope-tagged transgenic strains. We find that only ancestral Cid1 is present in somatic cells, whereas both Cid1 and Cid5 are expressed in testes and ovaries. However, Cid1 is lost in male meiosis but retained throughout oogenesis, whereas Cid5 is lost during female meiosis but retained in mature sperm. Following fertilization, only Cid1 is detectable in the early embryo, suggesting that maternally deposited Cid1 is rapidly loaded onto paternal centromeres during the protamine-to-histone transition. Our studies reveal mutually exclusive gametic specialization of divergent CenH3 paralogs. Duplication and divergence might allow essential centromeric genes to resolve an intralocus conflict between maternal and paternal centromeric requirements in many animal species.

Gametic specialization of centromeric histone paralogs in Drosophila virilis

In most eukaryotes, centromeric histone (CenH3) proteins mediate the highly conserved process of chromosome segregation as the foundational kinetochore assembly factor. However, in multicellular organisms, CenH3 proteins have to perform their essential functions in different chromatin environments. CenH3 proteins not only mediate mitosis and meiosis but also ensure epigenetic inheritance of centromere identity on sperm chromatin, which is highly compact and almost completely stripped of histones during spermiogenesis. We hypothesized that such disparate chromatin environments might impose different functional constraints on CenH3. If so, gene duplications could ameliorate the difficulty of encoding divergent and even potentially incompatible centromeric functions in the same gene. Here, we analyzed the cytological localization of two recently identified CenH3 paralogs, Cid1 and Cid5, in D. virilis using specific antibodies and epitope-tagged transgenic strains. We find that only ancestral Cid1 is present in somatic cells, whereas both Cid1 and Cid5 are expressed in testes and ovaries. However, Cid1 and Cid5 are alternately retained in male and female gametes; Cid1 is lost in male meiosis but retained throughout oogenesis, whereas Cid5 is lost during female meiosis but retained in mature sperm. Following fertilization, maternally deposited Cid1 rapidly replaces paternal Cid5 during the protamine-to-histone transition. Our studies reveal mutually exclusive gametic specialization of two divergent CenH3 paralogs. We suggest that centromeric histone duplication and divergence may allow essential genes involved in chromosome segregation to specialize and thereby resolve an intralocus conflict between maternal and paternal centromeric histone requirements in many animal species.

Web-based hybrid-dimensional Visualization and Exploration of Cytological Localization Scenarios

Summary The CELLmicrocosmos 4.2 PathwayIntegration (CmPI) is a tool which provides hybriddimensional visualization and analysis of intracellular protein and gene localizations in the context of a virtual 3D environment. This tool is developed based on Java/Java3D/JOGL and provides a standalone application compatible to all relevant operating systems. However, it requires Java and the local installation of the software. Here we present the prototype of an alternative web-based visualization approach, using Three.js and D3.js. In this way it is possible to visualize and explore CmPI-generated localization scenarios including networks mapped to 3D cell components by just providing a URL to a collaboration partner. This publication describes the integration of the different technologies - Three.js, D3.js and PHP - as well as an application case: a localization scenario of the citrate cycle. The CmPI web viewer is available at: http://CmPIweb.CELLmicrocosmos.org.

Changes in the expression and cytological localization of betacellulin and its receptors (ErbB-1 and ErbB-4) in the trophoblasts in human placenta over the course of pregnancy

OBJECTIVE: Betacellulin (BTC), purified and cloned from mouse beta cell tumor (BTC-JC10), is regarded as a new member of the epidermal growth factor family. The present study was conducted to clarify the expression of BTC and its receptors, ErbB-1 and ErbB-4, in the trophoblasts in the human placenta over the course of pregnancy. DESIGN AND METHODS: Human placental tissues were obtained from 4 pregnant women at the 4th to 5th week of pregnancy (very early placentas), 10 women at the 6th to 12th week (early placentas), 5 women at the 18th to 21st week (mid placentas) and 8 women at the 38th and 40th week (term placentas). The mRNA expressions of BTC, erbB-1 and erbB-4 were evaluated by quantitative RT-PCR with Southern blotting and the expression of the soluble form of BTC was determined by western immunoblot with a specific antibody to BTC protein. Immunohistochemical staining of BTC, ErbB-1 and ErbB-4 was also performed. RESULTS: The levels of BTC mRNA expression in early and mid placentas were significantly higher than those in term placentas. The soluble form of BTC protein with an estimated molecular mass of 9.5 kDa was expressed in early and mid placentas, whereas the soluble form was not detected in term placentas. BTC from very early placentas until mid placentas was immunolocalized in syncytiotrophoblasts (S-cells), and was most abundant in early placentas. In contrast, BTC was immunolocalized in extravillous trophoblasts (EVTs), but not in villous trophoblasts in term placentas. The levels of erbB-1 mRNA in the early and mid placentas were significantly higher than those in term placentas, whereas the levels of erbB-4 mRNA in early placentas were significantly lower than those in mid and term placentas. ErbB-1 was immunolocalized in cytotrophoblasts in very early placentas, whereas it was immunolocalized in S-cells from early until term placentas. ErbB-4 from very early placentas until mid placentas was immunolocalized in S-cells, whereas ErbB-4 in the term placentas was detected in EVTs, but not in villous trophoblasts. CONCLUSIONS: These findings provide evidence for changes in expression and cytological localization of BTC and its receptors in the trophoblasts in human placenta over the course of pregnancy. BTC may play a pivotal role as a local growth factor in promoting the differentiated villous trophoblastic function via ErbB-1 in early placentas and in contributing to placental growth through the maintenance of EVT cell function via ErbB-4 in term placentas.

Polymorphism Patterns in Two Tightly Linked Developmental Genes, Idgf1 and Idgf3, of Drosophila melanogaster

A new developmental gene family, recently identified in D. melanogaster, has been called imaginal disc growth factors (IDGF) because the proteins promote growth of cell lineages derived from imaginal discs. These are the first genes reported that encode polypeptide factors with mitotic activity in invertebrates. Characteristics such as similar arrangement of introns and exons, small size, and different cytological localization make this family an excellent candidate for evolutionary studies. We focus on the loci Idgf1 and Idgf3, two genes that possess the most distinctive features. We examine the pattern of intra- and interspecific nucleotide variation in the sequences from 20 isogenic lines of D. melanogaster and sequences from D. simulans and D. yakuba. While MK, HKA, and Tajima’s tests of neutrality fail to reject a neutral model of molecular evolution, Fu and Li’s test with outgroup and McDonald’s test suggest that balancing selection is modulating the evolution of the Idgf1 locus. The rate of recombination between the two loci is high enough to uncouple any linkage disequilibrium arising between Idgf1 and Idgf3, despite their close physical proximity.

Rolling Circle Amplification of Cyclizable Dna Probes by ∅29 Dna Polymerase: A Tool for in Situ Single-Copy Gene Detection.

Oligonucleotide probes that can be cyclized by ligation (“padlock probes”) provide a very high degree of recognition specificity. Nilsson et al. have demonstrated the used of padlock probes for the cytological localization of alphoid repeats in chromosome 12. We have been extending the use of padlock probes to the detection of single copy sequences, and with this in mind have explored the amplification of DNA circles.We designed a primer complementary to the arbitrary backbone (non-probing) sequence of a 92-base closed circular probe oligonucleotide and investigated the kinetics of rolling circle replication. Using the highly processive, strand-displacing DNA polymerase of phage ∅29 (kindly provided by Dr. Margarita Salas, CSIC, Madrid, Spain) we demonstrated that several hundred tandem copies of the circular oligonucleotide are generated in a few minutes of incubation at 32°C. Because the amplified DNA remains hybridized to the circle in a rolling circle reaction, this method of amplification offers unique advantages for in situ gene detection since the amplified DNA can not diffuse away from the site of synthesis.