Non-random chromosome segregation and chromosome eliminations in the fly Bradysia (Sciara).

Mendelian inheritance is based upon random segregation of homologous chromosomes during meiosis and perfect duplication and division of chromosomes in mitosis so that the entire genomic content is passed down to the daughter cells. The unusual chromosome mechanics of the fly Bradysia (previously called Sciara) presents many exceptions to the canonical processes. In male meiosis I, there is a monopolar spindle and non-random segregation such that all the paternal homologs move away from the single pole and are eliminated. In male meiosis II, there is a bipolar spindle and segregation of the sister chromatids except for the X dyad that undergoes non-disjunction. The daughter cell that is nullo-X degenerates, whereas the sperm has two copies of the X. Fertilization restores the diploid state, but there are three copies of the X chromosome, of which one or two of the paternally derived X chromosomes will be eliminated in an early cleavage division. Bradysia (Sciara) coprophila also has germ line limited L chromosomes that are eliminated from the soma. Current information and the molecular mechanisms for chromosome imprinting and eliminations, which are just beginning to be studied, will be reviewed here.

Anatomy and evolution of a DNA replication origin.

DNA amplification occurs at the DNA puff II/9A locus in the fungus fly Sciara coprophila. As a foundation to study the molecular mechanism for the initiating events of II/9A DNA re-replication, we have sequenced 14 kb spanning a DNase hypersensitive site (DHS) upstream of the 1 kb amplification origin and through transcription units II/9-1 and II/9-2 downstream of the origin. These elements are annotated as well as the ORC binding site at the origin and the transition point (TP) between continuous and discontinuous DNA syntheses that marks the origin of bidirectional replication at the nucleotide level. A 9 bp motif found at the TP is repeated near the other end of the 1 kb ORI and may identify a putative second TP. The steroid hormone ecdysone induces DNA amplification as well as transcription and puffing at locus II/9A. Within the 14 kb, several matches to the ecdysone response element (EcRE) consensus sequence were identified, including some in the amplification origin region. EcRE O-P is at a central axis of a remarkable symmetry, equidistant to the TPs that are themselves equidistant to EcRE O-1 and EcRE O-2. DNA sequence alterations have occurred throughout the II/9A region in a newly discovered polymorphism (#2). Polymorphism #2 is not specific to developmental stage, sex, or tissue, and it does not impair DNA amplification. The DHS, both 9 bp TP sequences, and EcREs O-1, O-P, and O-2 are conserved between the polymorphism #1 and #2 sequences, suggesting their functional importance and retention during evolutionary selection. Moreover, a 72 bp sequence in the Sciara DHS at DNA puff II/9A is conserved in DNA puff C-3 of Rhynchosciara americana. Comparisons are discussed between the Sciara II/9A amplicon and the chorion locus amplicon on the third chromosome of Drosophila.

High contiguity de novo genome assembly and DNA modification analyses for the fungus fly, Sciara coprophila, using single-molecule sequencing.

BACKGROUND: The lower Dipteran fungus fly, Sciara coprophila, has many unique biological features that challenge the rule of genome DNA constancy. For example, Sciara undergoes paternal chromosome elimination and maternal X chromosome nondisjunction during spermatogenesis, paternal X elimination during embryogenesis, intrachromosomal DNA amplification of DNA puff loci during larval development, and germline-limited chromosome elimination from all somatic cells. Paternal chromosome elimination in Sciara was the first observation of imprinting, though the mechanism remains a mystery. Here, we present the first draft genome sequence for Sciara coprophila to take a large step forward in addressing these features. RESULTS: We assembled the Sciara genome using PacBio, Nanopore, and Illumina sequencing. To find an optimal assembly using these datasets, we generated 44 short-read and 50 long-read assemblies. We ranked assemblies using 27 metrics assessing contiguity, gene content, and dataset concordance. The highest-ranking assemblies were scaffolded using BioNano optical maps. RNA-seq datasets from multiple life stages and both sexes facilitated genome annotation. A set of 66 metrics was used to select the first draft assembly for Sciara. Nearly half of the Sciara genome sequence was anchored into chromosomes, and all scaffolds were classified as X-linked or autosomal by coverage. CONCLUSIONS: We determined that X-linked genes in Sciara males undergo dosage compensation. An entire bacterial genome from the Rickettsia genus, a group known to be endosymbionts in insects, was co-assembled with the Sciara genome, opening the possibility that Rickettsia may function in sex determination in Sciara. Finally, the signal level of the PacBio and Nanopore data support the presence of cytosine and adenine modifications in the Sciara genome, consistent with a possible role in imprinting.

Making ends meet: targeted integration of DNA fragments by genome editing.

Targeted insertion of large pieces of DNA is an important goal of genetic engineering. However, this goal has been elusive since classical methods for homology-directed repair are inefficient and often not feasible in many systems. Recent advances are described here that enable site-specific genomic insertion of relatively large DNA with much improved efficiency. Using the preferred repair pathway in the cell of nonhomologous end-joining, DNA of up to several kb could be introduced with remarkably good precision by the methods of HITI and ObLiGaRe with an efficiency up to 30-40%. Recent advances utilizing homology-directed repair (methods of PITCh; short homology arms including ssODN; 2H2OP) have significantly increased the efficiency for DNA insertion, often to 40-50% or even more depending on the method and length of DNA. The remaining challenges of integration precision and off-target site insertions are summarized. Overall, current advances provide major steps forward for site-specific insertion of large DNA into genomes from a broad range of cells and organisms.

Characterizing and controlling intrinsic biases of lambda exonuclease in nascent strand sequencing reveals phasing between nucleosomes and G-quadruplex motifs around a subset of human replication origins.

Nascent strand sequencing (NS-seq) is used to discover DNA replication origins genome-wide, allowing identification of features for their specification. NS-seq depends on the ability of lambda exonuclease (λ-exo) to efficiently digest parental DNA while leaving RNA-primer protected nascent strands intact. We used genomics and biochemical approaches to determine if λ-exo digests all parental DNA sequences equally. We report that λ-exo does not efficiently digest G-quadruplex (G4) structures in a plasmid. Moreover, λ-exo digestion of nonreplicating genomic DNA (LexoG0) enriches GC-rich DNA and G4 motifs genome-wide. We used LexoG0 data to control for nascent strand-independent λ-exo biases in NS-seq and validated this approach at the rDNA locus. The λ-exo-controlled NS-seq peaks are not GC-rich, and only 35.5% overlap with 6.8% of all G4s, suggesting that G4s are not general determinants for origin specification but may play a role for a subset. Interestingly, we observed a periodic spacing of G4 motifs and nucleosomes around the peak summits, suggesting that G4s may position nucleosomes at this subset of origins. Finally, we demonstrate that use of Na(+) instead of K(+) in the λ-exo digestion buffer reduced the effect of G4s on λ-exo digestion and discuss ways to increase both the sensitivity and specificity of NS-seq.

Universal and domain-specific sequences in 23S-28S ribosomal RNA identified by computational phylogenetics.

Comparative analysis of ribosomal RNA (rRNA) sequences has elucidated phylogenetic relationships. However, this powerful approach has not been fully exploited to address ribosome function. Here we identify stretches of evolutionarily conserved sequences, which correspond with regions of high functional importance. For this, we developed a structurally aligned database, FLORA (full-length organismal rRNA alignment) to identify highly conserved nucleotide elements (CNEs) in 23S-28S rRNA from each phylogenetic domain (Eukarya, Bacteria, and Archaea). Universal CNEs (uCNEs) are conserved in sequence and structural position in all three domains. Those in regions known to be essential for translation validate our approach. Importantly, some uCNEs reside in areas of unknown function, thus identifying novel sequences of likely great importance. In contrast to uCNEs, domain-specific CNEs (dsCNEs) are conserved in just one phylogenetic domain. This is the first report of conserved sequence elements in rRNA that are domain-specific; they are largely a eukaryotic phenomenon. The locations of the eukaryotic dsCNEs within the structure of the ribosome suggest they may function in nascent polypeptide transit through the ribosome tunnel and in tRNA exit from the ribosome. Our findings provide insights and a resource for ribosome function studies.

Biomedical science postdocs: an end to the era of expansion.

After >3 decades of steady growth, the number of biological and medical science postdoctorates at doctoral degree-granting institutions recently began to decline. From 2010 through 2013, the most recent survey years, the postdoctoral population decreased from 40,970 to 38,719, a loss of 5.5%. This decline represents a notable departure from the previous long-standing increases in the number of postdoctorates in the biomedical workforce. The rate of contraction appears to be accelerating in the most recent survey years, and this has important implications for the biomedical workforce.

Isolation and characterization of the ecdysone receptor and its heterodimeric partner ultraspiracle through development in Sciara coprophila.

Regulation of DNA replication is critical, and loss of control can lead to DNA amplification. Naturally occurring, developmentally regulated DNA amplification occurs in the DNA puffs of the late larval salivary gland giant polytene chromosomes in the fungus fly, Sciara coprophila. The steroid hormone ecdysone induces DNA amplification in Sciara, and the amplification origin of DNA puff II/9A contains a putative binding site for the ecdysone receptor (EcR). We report here the isolation, cloning, and characterizing of two ecdysone receptor isoforms in Sciara (ScEcR-A and ScEcR-B) and the heterodimeric partner, ultraspiracle (ScUSP). ScEcR-A is the predominant isoform in larval tissues and ScEcR-B in adult tissues, contrary to the pattern in Drosophila. Moreover, ScEcR-A is produced at amplification but is absent just prior. We discuss these results in relation to the model of ecdysone regulation of DNA amplification.

A remarkable career in science-Joseph G. Gall.

A festive group of ∼150 current and former students, postdoctoral and other associates, and colleagues gathered during the weekend of April 12-14, 2013 to celebrate Joe Gall's 85th birthday. The gathering, hosted by the Carnegie Institution for Science, Department of Embryology (Allan Spradling, Director) and organized by a group of Joe's current and former students (Zehra Nizami, Alison Singer, Ji-Long Liu, Virginia Zakian, Susan Gerbi), was held in Baltimore, MD. Dinners and symposia extending over 3 days celebrated Joe's scientific findings over the years, together with those of his former students, postdoctoral fellows, and other associates (see program at https://sites.google.com/site/gallsymposium2013/ ).

The ecdysone receptor (ScEcR-A) binds DNA puffs at the start of DNA amplification in Sciara coprophila.

The steroid hormone ecdysone induces DNA amplification and subsequent DNA puff formation in late fourth larval instar salivary gland polytene chromosomes of the fungus fly, Sciara coprophila. Previous in vitro studies on DNA puff II/9A in Sciara demonstrated that the ecdysone receptor (ScEcR-A) efficiently binds an ecdysone response element adjacent to the origin recognition complex binding site within the II/9A amplification origin, implying a role for ScEcR-A in amplification. Here, we extrapolate the molecular details from locus II/9A to the rest of the genome using immunofluorescence with a ScEcR-A-specific antibody. ScEcR-A binds all DNA puff sites just as amplification begins and persists throughout the processes of amplification, transcription, and puffing. Ecdysone injections into pre-amplification stage larvae prematurely induce both DNA amplification and ScEcR-A binding to DNA puff sites. These data are consistent with a direct role for ScEcR-A in DNA amplification.

Laboratory Maintenance of the Lower Dipteran Fly Bradysia (Sciara) coprophila: A New/Old Emerging Model Organism.

Laboratory stocks of the lower dipteran fly, Bradysia (Sciara) coprophila, have been maintained for over a century. Protocols for laboratory upkeep of B. coprophila are presented here. These protocols will be useful for the rapidly increasing number of laboratories studying B. coprophila to take advantage of its unique biological features, which include (1) a monopolar spindle in male meiosis I; (2) non-disjunction of the X dyad in male meiosis II; (3) chromosome imprinting to distinguish maternal from paternal homologs; (4) germ line-limited (L) chromosomes; (5) chromosome elimination (paternal chromosomes in male meiosis I; one to two X chromosomes in early embryos; L chromosomes from the soma in early embryos); (6) sex determination by the mother (there is no Y chromosome); and (7) developmentally regulated DNA amplification at the DNA puff loci in larval salivary gland polytene chromosomes. It is now possible to explore these many unique features of chromosome mechanics by using the recent advances in sequencing and assembly of the B. coprophila genome and the development of transformation methodology for genomic engineering. The growing scientific community that uses B. coprophila for research will benefit from the protocols described here for mating the flies (phenotypic markers for mothers that will have only sons or only daughters; details of mass mating for biochemical experiments), checking embryo hatch, feeding larvae, and other comments on its rearing.

Bradysia (Sciara) coprophila larvae up-regulate DNA repair pathways and down-regulate developmental regulators in response to ionizing radiation.

The level of resistance to radiation and the developmental and molecular responses can vary between species, and even between developmental stages of one species. For flies (order: Diptera), prior studies concluded that the fungus gnat Bradysia (Sciara) coprophila (sub-order: Nematocera) is more resistant to irradiation-induced mutations that cause visible phenotypes than the fruit fly Drosophila melanogaster (sub-order: Brachycera). Therefore, we characterized the effects of and level of resistance to ionizing radiation on B. coprophila throughout its life cycle. Our data show that B. coprophila embryos are highly sensitive to even low doses of gamma-irradiation, whereas late-stage larvae can tolerate up to 80 Gy (compared to 40 Gy for D. melanogaster) and still retain their ability to develop to adulthood, though with a developmental delay. To survey the genes involved in the early transcriptional response to irradiation of B. coprophila larvae, we compared larval RNA-seq profiles with and without radiation treatment. The up-regulated genes were enriched for DNA damage response genes, including those involved in DNA repair, cell cycle arrest, and apoptosis, whereas the down-regulated genes were enriched for developmental regulators, consistent with the developmental delay of irradiated larvae. Interestingly, members of the PARP and AGO families were highly up-regulated in the B. coprophila radiation response. We compared the transcriptome responses in B. coprophila to the transcriptome responses in D. melanogaster from 3 previous studies: whereas pathway responses are highly conserved, specific gene responses are less so. Our study lays the groundwork for future work on the radiation responses in Diptera.

Correction: Yamamoto, Y.; Gerbi, S.A. Development of Transformation for Genome Editing of an Emerging Model Organism. Genes 2022, 13, 1108.

There was a typographical error in the original publication [...].

Development of Transformation for Genome Editing of an Emerging Model Organism.

With the advances in genomic sequencing, many organisms with novel biological properties are ripe for use as emerging model organisms. However, to make full use of them, transformation methods need to be developed to permit genome editing. Here, we present the development of transformation for the fungus fly Bradysia (Sciara) coprophila; this may serve as a paradigm for the development of transformation for other emerging systems, especially insects. Bradysia (Sciara) has a variety of unique biological features, including locus-specific developmentally regulated DNA amplification, chromosome imprinting, a monopolar spindle in male meiosis I, non-disjunction of the X chromosome in male meiosis II, X chromosome elimination in early embryogenesis, germ-line-limited (L) chromosomes and high resistance to radiation. Mining the unique biology of Bradysia (Sciara) requires a transformation system to test mutations of DNA sequences that may play roles for these features. We describe a Bradysia (Sciara) transformation system using a modified piggyBac transformation vector and detailed protocols we have developed to accommodate Bradysia (Sciara) specific requirements. This advance will provide a platform for us and others in the growing Bradysia (Sciara) community to take advantage of this unique biological system. In addition, the versatile piggyBac vectors described here and transformation methods will be useful for other emerging model systems.

The nucleolus: a site of ribonucleoprotein maturation.

The nucleolus is the site of ribosomal RNA synthesis, processing and ribosome maturation. Various small ribonucleoproteins also undergo maturation in the nucleolus, involving RNA modification and RNA-protein assembly. Such steps and other activities of small ribonucleoproteins also take place in Cajal (coiled) bodies. Events of ribosome biogenesis are found solely in the nucleolus, which is the final destination of small nucleolar RNAs after their traffic through Cajal bodies. However, nucleoli are just a stopping point in the intricate cellular traffic for small nuclear RNAs and other ribonucleoproteins.

U4 snRNA nucleolar localization requires the NHPX/15.5-kD protein binding site but not Sm protein or U6 snRNA association.

All small nuclear RNAs (snRNAs) of the [U4/U6.U5] tri-snRNP localize transiently to nucleoli, as visualized by microscopy after injection of fluorescein-labeled transcripts into Xenopus laevis oocyte nuclei. Here, we demonstrate that these RNAs traffic to nucleoli independently of one another, because U4 snRNA deleted in the U6 base-pairing region still localizes to nucleoli. Furthermore, depletion of endogenous U6 snRNA does not affect nucleolar localization of injected U4 or U5. The wild-type U4 transcripts used here are functional: they exhibit normal nucleocytoplasmic traffic, associate with Sm proteins, form the [U4/U6] di-snRNP, and localize to nucleoli and Cajal bodies. The nucleolar localization element (NoLE) of U4 snRNA was mapped by mutagenesis. Neither the 5'-cap nor the 3'-region of U4, which includes the Sm protein binding site, are essential for nucleolar localization. The only region in U4 snRNA required for nucleolar localization is the 5'-proximal stem loop, which contains the binding site for the NHPX/15.5-kD protein. Even mutation of just five nucleotides, essential for binding this protein, impaired U4 nucleolar localization. Intriguingly, the NHPX/15.5-kD protein also binds the nucleolar localization element of box C/D small nucleolar RNAs, suggesting that this protein might mediate nucleolar localization of several small RNAs.

DNA replication and chromatin.

The study of DNA replication in eukaryotic chromosomes has revealed a multitude of different regulatory levels. Nuclear and chromosomal location as well as chromatin structure may affect the activity of replication origins and their modulation during development.

Bundling up DNA.

Structures known as chromocenters, comprising satellite DNA and proteins such as D1 or HMGA1, help to contain DNA inside the nucleus between cell divisions.

Developmental changes in the Sciara II/9A initiation zone for DNA replication.

Developmentally regulated initiation of DNA synthesis was studied in the fly Sciara at locus II/9A. PCR analysis of nascent strands revealed an initiation zone that spans approximately 8 kb in mitotic embryonic cells and endoreplicating salivary glands but contracts to 1.2 to 2.0 kb during DNA amplification of DNA puff II/9A. Thus, the amplification origin occurs within the initiation zone used for normal replication. The initiation zone left-hand border is constant, but the right-hand border changes during development. Also, there is a shift in the preferred site for initiation of DNA synthesis during DNA amplification compared to that in preamplification stages. This is the first demonstration that once an initiation zone is defined in embryos, its borders and preferred replication start sites can change during development. Chromatin immunoprecipitation showed that the RNA polymerase II 140-kDa subunit occupies the promoter of gene II/9-1 during DNA amplification, even though intense transcription will not start until the next developmental stage. RNA polymerase II is adjacent to the right-hand border of the initiation zone at DNA amplification but not at preamplification, suggesting that it may influence the position of this border. These findings support a relationship between the transcriptional machinery and establishment of the replication initiation zone.