Evaluation of Multiple Immunoassay Technology Platforms to Select the Anti-Drug Antibody Assay Exhibiting the Most Appropriate Drug and Target Tolerance.

The aim of this study was, at the assay development stage and thus with an appropriate degree of rigor, to select the most appropriate technology platform and sample pretreatment procedure for a clinical ADA assay. Thus, ELISA, MSD, Gyrolab, and AlphaLISA immunoassay platforms were evaluated in association with target depletion and acid dissociation sample pretreatment steps. An acid dissociation step successfully improved the drug tolerance for all 4 technology platforms and the required drug tolerance was achieved with the Gyrolab and MSD platforms. The target tolerance was shown to be better for the ELISA format, where an acid dissociation treatment step alone was sufficient to achieve the desired target tolerance. However, inclusion of a target depletion step in conjunction with the acid treatment raised the target tolerance to the desired level for all of the technologies. A higher sensitivity was observed for the MSD and Gyrolab assays and the ELISA, MSD, and Gyrolab all displayed acceptable interdonor variability. This study highlights the usefulness of evaluating the performance of different assay platforms at an early stage in the assay development process to aid in the selection of the best fit-for-purpose technology platform and sample pretreatment steps.

The Spartan ortholog maternal haploid is required for paternal chromosome integrity in the Drosophila zygote.

The animal sperm nucleus is characterized by an extremely compacted organization of its DNA after the global replacement of histones with sperm-specific nuclear basic proteins, such as protamines. In the absence of DNA repair activity in the mature gamete, the integrity of the paternal genome is potentially challenged by the unique topological constraints exerted on sperm DNA. In addition, the maintenance of paternal DNA integrity during the rapid remodeling of sperm chromatin at fertilization has long been regarded as a maternal trait. However, little is known about the nature of the egg proteins involved in this essential aspect of zygote formation. We had previously characterized the unique phenotype of the classical Drosophila maternal effect mutant maternal haploid (mh), which specifically affects the integration of paternal chromosomes in the zygote. Here we show that MH is the fly ortholog of the recently identified human DVC1/Spartan protein, a conserved regulator of DNA damage tolerance. Like Spartan, MH protein is involved in the resistance to UV radiation and recruits the p97/TER94 segregase to stalled DNA replication forks in somatic cells. In the zygote, we found that the mh phenotype is consistent with perturbed or incomplete paternal DNA replication. Remarkably, however, the specific accumulation of MH in the male pronucleus before the first S phase suggests that this maternal protein is required to maintain paternal DNA integrity during nuclear decondensation or to set the paternal chromatin landscape in preparation of the first zygotic cycle.

Drosophila Yemanuclein and HIRA cooperate for de novo assembly of H3.3-containing nucleosomes in the male pronucleus.

The differentiation of post-meiotic spermatids in animals is characterized by a unique reorganization of their nuclear architecture and chromatin composition. In many species, the formation of sperm nuclei involves the massive replacement of nucleosomes with protamines, followed by a phase of extreme nuclear compaction. At fertilization, the reconstitution of a nucleosome-based paternal chromatin after the removal of protamines requires the deposition of maternally provided histones before the first round of DNA replication. This process exclusively uses the histone H3 variant H3.3 and constitutes a unique case of genome-wide replication-independent (RI) de novo chromatin assembly. We had previously shown that the histone H3.3 chaperone HIRA plays a central role for paternal chromatin assembly in Drosophila. Although several conserved HIRA-interacting proteins have been identified from yeast to human, their conservation in Drosophila, as well as their actual implication in this highly peculiar RI nucleosome assembly process, is an open question. Here, we show that Yemanuclein (YEM), the Drosophila member of the Hpc2/Ubinuclein family, is essential for histone deposition in the male pronucleus. yem loss of function alleles affect male pronucleus formation in a way remarkably similar to Hira mutants and abolish RI paternal chromatin assembly. In addition, we demonstrate that HIRA and YEM proteins interact and are mutually dependent for their targeting to the decondensing male pronucleus. Finally, we show that the alternative ATRX/XNP-dependent H3.3 deposition pathway is not involved in paternal chromatin assembly, thus underlining the specific implication of the HIRA/YEM complex for this essential step of zygote formation.

Engineering silkworms for resistance to baculovirus through multigene RNA interference.

Bombyx mori nucleopolyhedrovirus (BmNPV) that infects the silkworm, B. mori, accounts for >50% of silk cocoon crop losses globally. We speculated that simultaneous targeting of several BmNPV essential genes in transgenic silkworm would elicit a stable defense against the virus. We introduced into the silkworm germline the vectors carrying short sequences of four essential BmNPV genes in tandem, either in sense or antisense or in inverted-repeat arrangement. The transgenic silkworms carrying the inverted repeat-containing transgene showed stable protection against high doses of baculovirus infection. Further, the antiviral trait was incorporated to a commercially productive silkworm strain highly susceptible to BmNPV. This led to combining the high-yielding cocoon and silk traits of the parental commercial strain and a very high level of refractoriness (>75% survival rate as compared to <15% in nontransgenic lines) to baculovirus infection conferred by the transgene. We also observed impaired infectivity of the occlusion bodies derived from the transgenic lines as compared to the wild-type ones. Currently, large-scale exploitation of these transgenic lines is underway to bring about economic transformation of sericulture.

Novel genes differentially expressed between posterior and median silk gland identified by SAGE-aided transcriptome analysis.

Serial analysis of gene expression (SAGE) profiles, from posterior and median cells of the silk gland of Bombyx mori, were analyzed and compared, so as to identify their respective distinguishing functions. The annotation of the SAGE libraries was performed with a B. mori reference tag collection, which was extracted from a novel set of Bombyx ESTs, sequenced from the 3' side. Most of the tags appeared at similar relative concentration within the two libraries, and corresponded with region-specific and highly abundant silk proteins. Strikingly, in addition to tags from silk protein mRNAs, 19 abundant tags were found (≥ 0.1%), in the median cell library, which were absent in the posterior cell tag collection. With the exception of tags from SP1 mRNA, no PSG specific tags were found in this subset class. The analysis of some of the MSG-specific transcripts, suggested that middle silk gland cells have diversified functions, in addition to their well characterized role in silk sericins synthesis and secretion.

Specialization of a Drosophila capping protein essential for the protection of sperm telomeres.

BACKGROUND: A critical function of telomeres is to prevent fusion of chromosome ends by the DNA repair machinery. In Drosophila somatic cells, assembly of the protecting capping complex at telomeres notably involves the recruitment of HOAP, HP1, and their recently identified partner, HipHop. We previously showed that the hiphop gene was duplicated before the radiation of the melanogaster subgroup of species, giving birth to K81, a unique paternal effect gene specifically expressed in the male germline. RESULTS: Here we show that K81 specifically associates with telomeres during spermiogenesis, along with HOAP and HP1, and is retained on paternal chromosomes until zygote formation. In K81 mutant testes, capping proteins are not maintained at telomeres in differentiating spermatids, resulting in the transmission of uncapped paternal chromosomes that fail to properly divide during the first zygotic mitosis. Despite the apparent similar capping roles of K81 and HipHop in their respective domain of expression, we demonstrate by in vivo reciprocal complementation analyses that they are not interchangeable. Strikingly, HipHop appeared to be unable to maintain capping proteins at telomeres during the global chromatin remodeling of spermatid nuclei. CONCLUSIONS: Our data demonstrate that K81 is essential for the maintenance of capping proteins at telomeres in postmeiotic male germ cells. In species of the melanogaster subgroup, HipHop and K81 have not only acquired complementary expression domains, they have also functionally diverged following the gene duplication event. We propose that K81 specialized in the maintenance of telomere protection in the highly peculiar chromatin environment of differentiating male gametes.

Drosophila I-R hybrid dysgenesis is associated with catastrophic meiosis and abnormal zygote formation.

The Drosophila I-R type of hybrid dysgenesis is a sterility syndrome (SF sterility) associated with the mobilization of the I retrotransposon in female germ cells. SF sterility results from a maternal-effect embryonic lethality whose origin has remained unclear since its discovery about 40 years ago. Here, we show that meiotic divisions in SF oocytes are catastrophic and systematically fail to produce a functional female pronucleus at fertilization. As a consequence, most embryos from SF females rapidly arrest their development with aneuploid or damaged nuclei, whereas others develop as non-viable, androgenetic haploid embryos. Finally, we show that, in contrast to mutants affecting the biogenesis of piRNAs, SF egg chambers do not accumulate persistent DNA double-strand breaks, suggesting that I-element activity might perturb the functional organization of meiotic chromosomes without triggering an early DNA damage response.

Epigenetic and replacement roles of histone variant H3.3 in reproduction and development.

The nucleosomal organization of eukaryotic chromatin is generally established during DNA replication by the deposition of canonical histones synthesized in S phase. However, cells also use a Replication Independent (RI) nucleosome assembly pathway that allows the incorporation of non-canonical histone variants in the chromatin. H3.3 is a conserved histone variant that is structurally very close to its canonical counterpart but nevertheless possesses specific properties. In this review, we discuss the dual role of H3.3 which functions as a neutral replacement histone, but also participates in the epigenetic transmission of active chromatin states. These properties of H3.3 are also explored in the light of recent studies that implicate this histone and its associated chromatin assembly factors in large scale, replication-independent chromatin remodeling events. In particular, H3.3 appears as a critical player in the transmission of the paternal genome, from sperm to zygote.

Serial Analysis of Gene Expression in Plasmodium berghei salivary gland sporozoites.

BACKGROUND: The invasion of Anopheles salivary glands by Plasmodium sporozoites is an essential step for transmission of the parasite to the vertebrate host. Salivary gland sporozoites undergo a developmental programme to express genes required for their journey from the site of the mosquito bite to the liver and subsequent invasion of, and development within, hepatocytes. A Serial Analysis of Gene Expression was performed on Anopheles gambiae salivary glands infected or not with Plasmodium berghei and we report here the analysis of the Plasmodium sporozoite transcriptome. RESULTS: Annotation of 530 tag sequences homologous to Plasmodium berghei genomic sequences identified 123 genes expressed in salivary gland sporozoites and these genes were classified according to their transcript abundance. A subset of these genes was further studied by quantitative PCR to determine their expression profiles. This revealed that sporozoites modulate their RNA amounts not only between the midgut and salivary glands, but also during their storage within the latter. Among the 123 genes, the expression of 66 is described for the first time in sporozoites of rodent Plasmodium species. CONCLUSION: These novel sporozoite expressed genes, especially those expressed at high levels in salivary gland sporozoites, are likely to play a role in Plasmodium infectivity in the mammalian host.

The essential role of Drosophila HIRA for de novo assembly of paternal chromatin at fertilization.

In many animal species, the sperm DNA is packaged with male germ line--specific chromosomal proteins, including protamines. At fertilization, these non-histone proteins are removed from the decondensing sperm nucleus and replaced with maternally provided histones to form the DNA replication competent male pronucleus. By studying a point mutant allele of the Drosophila Hira gene, we previously showed that HIRA, a conserved replication-independent chromatin assembly factor, was essential for the assembly of paternal chromatin at fertilization. HIRA permits the specific assembly of nucleosomes containing the histone H3.3 variant on the decondensing male pronucleus. We report here the analysis of a new mutant allele of Drosophila Hira that was generated by homologous recombination. Surprisingly, phenotypic analysis of this loss of function allele revealed that the only essential function of HIRA is the assembly of paternal chromatin during male pronucleus formation. This HIRA-dependent assembly of H3.3 nucleosomes on paternal DNA does not require the histone chaperone ASF1. Moreover, analysis of this mutant established that protamines are correctly removed at fertilization in the absence of HIRA, thus demonstrating that protamine removal and histone deposition are two functionally distinct processes. Finally, we showed that H3.3 deposition is apparently not affected in Hira mutant embryos and adults, suggesting that different chromatin assembly machineries could deposit this histone variant.

RNA interference-mediated silencing of the bursicon gene induces defects in wing expansion of silkworm.

We studied the role of the bursicon gene in wing expansion. First, we investigated its expression at different developmental stages in the silkworm, Bombyx mori. Bursicon gene was expressed at low levels in larvae, high levels in pupae, and low levels again in adults. Then, we injected the double-stranded bursicon RNA into B. mori pupae to test RNA interference. The level of bursicon mRNA was reduced significantly in pupae, and a deficit in wing expansion was observed in adults. In addition, the differential display reverse transcription polymerase chain reaction (DD-RT-PCR) was used to reveal differences in the expression of transcripts in response to the inhibition of bursicon. In conclusion, bursicon plays a key role in the stereotyped behavioral program involved in wing expansion.

SAGE analysis of mosquito salivary gland transcriptomes during Plasmodium invasion.

Invasion of the vector salivary glands by Plasmodium is a critical step for malaria transmission. To describe salivary gland cellular responses to sporozoite invasion, we have undertaken the analysis of Anopheles gambiae salivary gland transcriptome using Serial Analysis of Gene Expression (SAGE). Statistical analysis of the more than 160000 sequenced tags generated from four libraries, two from glands infected by Plasmodium berghei, two from glands of controls, revealed that at least 57 Anopheles genes are differentially expressed in infected salivary glands. Among the 37 immune-related genes identified by SAGE tags, four (Defensin1, GNBP, Serpin6 and Cecropin2) were found to be upregulated during salivary gland invasion, while five genes encoding small secreted proteins display induction patterns strongly reminiscent of that of Cecropin2. Invasion by Plasmodium has also an impact on the expression of genes involved in transport, lipid and energy metabolism, suggesting that the sporozoite may exploit the metabolism of its host. In contrast, protein composition of saliva is predicted to be only slightly modified after infection. This study, which is the first transcriptome analysis of the salivary gland response to Plasmodium infection, provides a basis for a better understanding of Plasmodium/Anopheles salivary gland interactions.

The histone H3.3 chaperone HIRA is essential for chromatin assembly in the male pronucleus.

In sexually reproducing animals, a crucial step in zygote formation is the decondensation of the fertilizing sperm nucleus into a DNA replication-competent male pronucleus. Genome-wide nucleosome assembly on paternal DNA implies the replacement of sperm chromosomal proteins, such as protamines, by maternally provided histones. This fundamental process is specifically impaired in sésame (ssm), a unique Drosophila maternal effect mutant that prevents male pronucleus formation. Here we show that ssm is a point mutation in the Hira gene, thus demonstrating that the histone chaperone protein HIRA is required for nucleosome assembly during sperm nucleus decondensation. In vertebrates, HIRA has recently been shown to be critical for a nucleosome assembly pathway independent of DNA synthesis that specifically involves the H3.3 histone variant. We also show that nucleosomes containing H3.3, and not H3, are specifically assembled in paternal Drosophila chromatin before the first round of DNA replication. The exclusive marking of paternal chromosomes with H3.3 represents a primary epigenetic distinction between parental genomes in the zygote, and underlines an important consequence of the critical and highly specialized function of HIRA at fertilization.

Biosynthesis and cocoon-export of a recombinant globular protein in transgenic silkworms.

A gene construct was made by fusing the coding sequence of the red fluorescent protein (DsRed) to the exon 2 of the fibrohexamerin gene (fhx), that encodes a subunit of fibroin, the major silk protein of the silkworm Bombyx mori. The fusion gene was inserted into a piggyBac vector to establish a series of transgenic lines. The expression of the transgene was monitored during the course of larval life and was found restricted to the posterior silk gland cells as the endogenous fhx gene, in all the selected transgenic lines. The exogenous polypeptide was secreted into the lumen of the posterior silk gland together with fibroin, and further exported with the silk proteins as a foreign constituent of the cocoon fiber. The capacity of DsRed to emit fluorescence in the air-dried silk thread led to show that the recombinant protein was distributed over the whole length of the fiber. A remarkable property of the system lies in the localization of the globular protein at the periphery of the silk thread, allowing its rapid and easy recovery in aqueous solutions, without dissolving fibroin. The procedure represents a novel and promising strategy for the production of massive recombinant proteins of biomedical and pharmaceutical interest, with reduced cost.

Serial analysis of gene expression in the silkworm, Bombyx mori.

The silkworm Bombyx mori is one of the most economically important insects and serves as a model for Lepidoptera insects. We used serial analysis of gene expression (SAGE) to derive profiles of expressed genes during the developmental life cycle of the silkworm and to create a reference for understanding silkworm metamorphosis. We generated four SAGE libraries, one from each of the four developmental stages of the silkworm. In total we obtained 257,964 SAGE tags, of which 39,485 were unique tags. Sorted by copy number, 14.1% of the unique tags were detected at a median to high level (five or more copies), 24.2% at lower levels (two to four copies), and 61.7% as single copies. Using a basic local alignment search tool on the EST database, 35% of the tags matched known silkworm expressed sequence tags. SAGE demonstrated that a number of the genes were up- or down-regulated during the four developmental phases of the egg, larva, pupa, and adult. Furthermore, we found that the generation of longer cDNA fragments from SAGE tags constituted the most efficient method of gene identification, which facilitated the analysis of a large number of unknown genes.

Origin and neofunctionalization of a Drosophila paternal effect gene essential for zygote viability.

BACKGROUND: Although evolutionary novelty by gene duplication is well established, the origin and maintenance of essential genes that provide entirely new functions (neofunctionalization) is still largely unknown. Drosophila is a good model for the search of genes that are young enough to allow deciphering the molecular details of their evolutionary history. Recent years have seen increased interest in genes specifically required for male fertility because they often evolve rapidly. A special class of genes affecting male fertility, the paternal effect genes, have also become a focus of study to geneticists and reproductive biologists interested in fertilization and sperm-egg interactions. RESULTS: Using molecular genetics and the annotated Drosophila melanogaster genome, we identified CG14251 as the Drosophila paternal effect gene, ms(3)K81 (K81). This assignment was subsequently confirmed by P-element rescue of K81. A search for orthologous K81 sequences revealed that the distribution of K81 is surprisingly restricted to the 9 species comprising the melanogaster subgroup. Phylogenetic analyses indicate that K81 arose through duplication, most likely retroposition, of a ubiquitously expressed gene before the radiation of the melanogaster subgroup, followed by a period of rapid divergence and acquisition of a critical male germline-specific function. Interestingly, K81 has adopted the expression profile of a flanking gene suggesting that transcriptional coregulation may have been important in the neofunctionalization of K81. CONCLUSION: We present a detailed case history of the origin and evolution of a new essential gene and, in so doing, provide the first molecular identification of a Drosophila paternal effect gene, ms(3)K81 (K81).

Junonia coenia densovirus-based vectors for stable transgene expression in Sf9 cells: influence of the densovirus sequences on genomic integration.

The invertebrate parvovirus Junonia coenia densovirus (JcDNV) shares similarities with terminal hairpins and nonstructural (NS) protein activities of adeno-associated virus (AAV) despite their evolutionary divergence (B. Dumas, M. Jourdan, A. M. Pascaud, and M. Bergoin, Virology, 191:202-222, 1992, and C. Ding, M. Urabe, M. Bergoin, and R. M. Kotin, J. Virol. 76:338-345, 2002). We demonstrate here that persistent transgene expression in insect cells results from stable integration of transfected JcDNV-derived vectors into the host genome. To assess the integrative properties of JcDNV vectors, the green fluorescent protein (GFP) gfp marker gene was fused in frame into the major open reading frame (ORF1) of the viral sequence under the control of the P9 capsid protein promoter. In addition, the influence of the nonstructural proteins on the posttransfection maintenance of the vectors was examined by interruption of one or all three NS ORFs. Following transfection of Sf9 cells with each of the JcDNV constructs, clones showing persistent GFP expression were isolated. Structural analyses revealed that the majority of the JcDNV plasmid sequence was integrated into the genome of the fluorescent clones. Integration was observed whether or not NS proteins were expressed. However, the presence of NS genes in the constructs greatly influenced the number of integrated copies and their distribution in the host genome. Disruption of NS genes expression resulted in integration of head-to-tail concatemers at multiple sites within the genome. Further analyses demonstrated that the cis JcDNV 5' inverted terminal repeat region was the primary site of recombination. Sequence analyses of integration junctions showed rearrangements of both flanking and internal sequences for most integrations. These findings demonstrate that JcDNV vectors integrate into insect cells in a manner similar to AAV plasmids in mammalian cells.