Somatic Effects of P Element Activity in Drosophila melanogaster: Pupal Lethality

Nonautonomous P elements normally excise and transpose only when a source of transposase is supplied, and only in the germline. The germline specificity depends on one of the introns of the transposase gene which is not spliced in somatic cells. To study the effects of somatic P activity, a modified P element (delta 2-3) lacking this intron was used as a source of transposase. Nonautonomous P elements from a strain called Birmingham, when mobilized in somatic cells by delta 2-3, were found to cause lethality, although neither component was lethal by itself. The three major Birmingham chromosomes acted approximately independently in producing the lethal effect. This lethality showed a strong dependence on temperature. Although temperature sensitivity was limited to larval stages, the actual deaths occurred at the pupal stage. Survivors, which could be recovered by decreasing the temperature or by reducing the proportion of the Birmingham genome present, often showed multiple developmental anomalies and reduced longevity reminiscent of the effects of cell death from radiation damage. Although the genetic damage occurred in dividing imaginal disc cells, the phenotypic manifestations--death and abnormalities--are not observed until later. The survivors also showed gonadal dysgenic (GD) sterility, a well-known characteristic of P-M hybrid dysgenesis. To explain these findings, we suggest that pupal lethality and GD sterility are both caused by massive chromosome breakage in larval cells, resulting from excision and transposition of genomic P elements acting as substrate for the transposase.     

An Organizational Hub of Developmentally Regulated Chromatin Loops in the Drosophila Antennapedia Complex

Chromatin boundary elements (CBEs) are widely distributed in the genome and mediate formation of chromatin loops, but their roles in gene regulation remain poorly understood. The complex expression pattern of the Drosophila homeotic gene Sex combs reduced (Scr) is directed by an unusually long regulatory sequence harboring diverse cis elements and an intervening neighbor gene fushi tarazu (ftz). Here we report the presence of a multitude of CBEs in the Scr regulatory region. Selective and dynamic pairing among these CBEs mediates developmentally regulated chromatin loops. In particular, the SF1 boundary plays a central role in organizing two subsets of chromatin loops: one subset encloses ftz, limiting its access by the surrounding Scr enhancers and compartmentalizing distinct histone modifications, and the other subset subdivides the Scr regulatory sequences into independent enhancer access domains. We show that these CBEs exhibit diverse enhancer-blocking activities that vary in strength and tissue distribution. Tandem pairing of SF1 and SF2, two strong CBEs that flank the ftz domain, allows the distal enhancers to bypass their block in transgenic Drosophila, providing a mechanism for the endogenous Scr enhancer to circumvent the ftz domain. Our study demonstrates how an endogenous CBE network, centrally orchestrated by SF1, could remodel the genomic environment to facilitate gene regulation during development.     

The Peculiarities of Large Intron Splicing in Animals

In mammals a considerable 92% of genes contain introns, with hundreds and hundreds of these introns reaching the incredible size of over 50,000 nucleotides. These “large introns” must be spliced out of the pre-mRNA in a timely fashion, which involves bringing together distant 5′ and 3′ acceptor and donor splice sites. In invertebrates, especially Drosophila, it has been shown that larger introns can be spliced efficiently through a process known as recursive splicing—a consecutive splicing from the 5′-end at a series of combined donor-acceptor splice sites called RP-sites. Using a computational analysis of the genomic sequences, we show that vertebrates lack the proper enrichment of RP-sites in their large introns, and, therefore, require some other method to aid splicing. We analyzed over 15,000 non-redundant, large introns from six mammals, 1,600 from chicken and zebrafish, and 560 non-redundant large introns from five invertebrates. Our bioinformatic investigation demonstrates that, unlike the studied invertebrates, the studied vertebrate genomes contain consistently abundant amounts of direct and complementary strand interspersed repetitive elements (mainly SINEs and LINEs) that may form stems with each other in large introns. This examination showed that predicted stems are indeed abundant and stable in the large introns of mammals. We hypothesize that such stems with long loops within large introns allow intron splice sites to find each other more quickly by folding the intronic RNA upon itself at smaller intervals and, thus, reducing the distance between donor and acceptor sites.     

Functional Dissection of YA, an Essential, Developmentally Regulated Nuclear Lamina Protein in Drosophila melanogaster

The Drosophila YA protein is a nuclear lamina component whose function is essential to initiate embryonic development. To identify regions of YA required for its action in its normal cellular context, we made targeted mutations in the YA protein and tested their consequences in flies and embryos in vivo. We found that critical amino acids are distributed along the length of the YA molecule, with functionally important regions including the N- and the C-terminal ends, the cysteine residues in YA’s two potential zinc fingers, a serine/threonine-rich region, and a potential maturation-promoting factor or mitogen-activated protein kinase phosphorylation target site, ITPIR. In addition, several Ya mutations showed intragenic complementation, with N-terminal mutations complementing C-terminal mutations, suggesting that YA proteins interact with one another. In support of this interaction, we demonstrated by immunoprecipitation that YA molecules are present in complexes with each other. Finally, we showed that the C-terminal 179 amino acids of YA are necessary to target, or retain, YA in the nuclear envelope.     

Palmdelphin在出生后发育的大鼠小脑中的表达模式研究

Palmdelphin是参与质膜的动态变化与细胞形态的调控的paralemmin家族新成员,与神经发育的相关性尚不明确.前期工作提示,它与调控小脑发育的一种肌动蛋白结合蛋白Mtss1(metastasis suppressor1)具有一定相关性.为了探索该基因与小脑出生后发育的相关性,利用原位杂交技术研究Palmdelphin在小脑中的时空表达,结果表明,Palmdelphin在出生后第7d大鼠小脑中有明显的表达,且分布主要集中在浦肯野神经元.半定量RT-PCR的结果进一步表明Palmdelphin的转录水平在小脑发育过程中受到调控,在出生后7d有表达高峰.这些结果显示Palmdelphin与小脑出生后神经元发育存在一定相关性.     

Developmentally programmed remodeling of the Drosophila olfactory circuit

Neural circuits are often remodeled after initial connections are established. The mechanisms by which remodeling occurs, in particular whether and how synaptically connected neurons coordinate their reorganization, are poorly understood. In Drosophila, olfactory projection neurons (PNs) receive input by synapsing with olfactory receptor neurons in the antennal lobe and relay information to the mushroom body (MB) calyx and lateral horn. Here we show that embryonic-born PNs participate in both the larval and adult olfactory circuits. In the larva, these neurons generally innervate a single glomerulus in the antennal lobe and one or two glomerulus-like substructures in the MB calyx. They persist in the adult olfactory circuit and are prespecified by birth order to innervate a subset of glomeruli distinct from larval-born PNs. Developmental studies indicate that these neurons undergo stereotyped pruning of their dendrites and axon terminal branches locally during early metamorphosis. Electron microscopy analysis reveals that these PNs synapse with MB gamma neurons in the larval calyx and that these synaptic profiles are engulfed by glia during early metamorphosis. As with MB gamma neurons, PN pruning requires cell-autonomous reception of the nuclear hormone ecdysone. Thus, these synaptic partners are independently programmed to prune their dendrites and axons.     

华山松胚性愈伤组织诱导与幼胚离体培养

探索华山松（Pinus armandii）体细胞胚胎发生技术对其实施规模化无性繁殖和开展遗传转化具有重要意义。本文以1／2LM为基本培养基,通过激素调节等措施对华山松的胚性愈伤组织诱导和幼胚的离体培养技术进行了初步研究。研究结果：胚性愈伤组织诱导率最高可达52．71％,但愈伤组织继代培养后没有体细胞胚胎的分化;首次从其子叶期的幼胚中直接诱导出具有根和茎的完整植株,诱导率达92％以上。文章确认了采集的幼胚发育状态对胚性愈伤组织的诱导有重要影响,并对诱导的培养条件等进行了探讨。     

Precipitated Proteins Improve the Production of Carrot Somatic Embryos

Extracellular compounds isolated from embryogenic carrot cell suspension cultures increase, by 1.5 to 6-fold, end-stage embryo production when added back to carrot cultures initiating embryogenesis. The causative factors related to the enhancement of embryo production are most likely to be extracellular, high molecular weight proteins found in the embryo-free medium (EFM) after somatic embryos have been formed. The addition of heat-treated EFM to fresh cultures did not result in enhancing effects on the production of end-stage embryos. However, the addition of compounds precipitated from EFM, by high concentrations of salt, accelerated by four days the formation of comparable amounts of end-stage embryos and surpassed total end-stage embryo levels by a factor of 4-6, dependent on the precipitate dose. These results suggest that heat-labile polypeptide molecules may be responsible for growth factor-like effects during somatic embryogenesis.     

Effects of P Element Insertions on Quantitative Traits in Drosophila Melanogaster

P element mutagenesis was used to construct 94 third chromosome lines of Drosophila melanogaster which contained on average 3.1 stable P element inserts, in an inbred host strain background previously free of P elements. The homozygous and heterozygous effects of the inserts on viability and abdominal and sternopleural bristle number were ascertained by comparing the chromosome lines with inserts to insert-free control lines of the inbred host strain. P elements reduced average homozygous viability by 12.2% per insert and average heterozygous viability by 5.5% per insert, and induced recessive lethal mutations at a rate of 3.8% per insert. Mutational variation for the bristle traits averaged over both sexes was 0.03Ve per homozygous P insert and 0.003Ve per heterozygous P insert, where Ve is the environmental variance. Mutational variation was greater for the sexes considered separately because inserts had large pleiotropic effects on sex dimorphism of bristle characters. The distributions of homozygous effects of inserts on the bristle traits were asymmetrical, with the largest effects in the direction of reducing bristle number; and highly leptokurtic, with most of the increase in variance contributed by a few lines with large effects. The inserts had partially recessive effects on the bristle traits. Insert lines with extreme bristle effects had on average greatly reduced viability.     

Distinct, Developmentally Regulated Brain mRNAs Direct the Synthesis of Neurotransmitter Transporters

The Xenopus laevis oocyte expression system was utilized to define developmental and structural properties of neurotransmitter transporter mRNAs and the pharmacological characteristics of encoded carriers independent of the complexities of brain tissue preparations. Poly(A)+ RNA from dissected brain regions of neonatal and adult rats was microinjected into Xenopus oocytes and the expression of Na(+)-dependent neurotransmitter transporters determined 48 h later. Transport studies conducted with oocytes injected with RNAs derived from juvenile rat tissues indicate a region- and transporter-specific, postnatal increase in mRNA abundance as a major factor in the developmental changes observed for brain high-affinity amino acid uptake systems. Both L-glutamic acid (Glu) and gamma-aminobutyric acid (GABA) uptake systems were detectable by day 3 in postnatal forebrain mRNA and became progressively enriched during the next 2 weeks of forebrain development. In contrast, brainstem Glu and GABA transporter enrichment was 60-70% of adult values by day 3 and exceeded adult levels by day 10. Parallel determinations of L-glutamic acid decarboxylase mRNA abundance during development argue for distinct regulatory influences on mRNAs directing transmitter synthesis and reuptake. Glycine uptake could not be detected at any point of forebrain development and exhibited a gradual postnatal rise to adult levels over the first 3 postnatal weeks of brainstem development. Uptake studies conducted with well-characterized inhibitors of Glu, GABA, dopamine, and choline transport (D-aspartate, nipecotic acid, nomifensine, and hemicholinium-3, respectively) revealed that oocyte transporters encoded by adult rat brain mRNAs retained antagonist sensitivities exhibited by in vitro brain preparations. In addition, a differential regional sensitivity to the Glu transport antagonist dihydrokainate (1 mM) was observed, lending support to previous reports of region-specific Glu transporter subtypes. To determine the structural diversity present among brain transporter mRNAs, poly(A)+ RNA was size-fractionated on linear (10-31%) sucrose density gradients prior to oocyte injection. These experiments revealed two mRNA size classes (2.4-3.0 kb, 4.0-4.5 kb) independently capable of directing the synthesis of Glu, GABA, and glycine transporters. In regions other than the cerebellum, Glu and GABA transporter activities migrated as single, yet distinct, peaks of 4.0-4.5 kb. In contrast, both Glu and GABA transporters exhibited major peaks of activity at 2.5-3.0 kb with size-fractionated cerebellar mRNA. Brainstem glycine uptake exhibited a broad sedimentation profile, with peaks apparent at 2.4 and 4.0 kb. Taken together, these findings indicate previously unappreciated complexity in mRNA structure and regulation which underlies the expression of amino acid neurotransmitter uptake systems in the rodent CNS.     

A Stable Genomic Source of P Element Transposase in Drosophila Melanogaster

A single P element insert in Drosophila melanogaster, called P[ry+ delta 2-3](99B), is described that caused mobilization of other elements at unusually high frequencies, yet is itself remarkably stable. Its transposase activity is higher than that of an entire P strain, but it rarely undergoes internal deletion, excision or transposition. This element was constructed by F. Laski, D. Rio and G. Rubin for other purposes, but we have found it to be useful for experiments involving P elements. We demonstrate that together with a chromosome bearing numerous nonautonomous elements it can be used for P element mutagenesis. It can also substitute efficiently for "helper" plasmids in P element mediated transformation, and can be used to move transformed elements around the genome.     

猪H-FABP基因第三内含子的遗传变异

以马身猪、大白猪、长白猪和杜洛克猪四个品种共190头猪为实验动物,成功扩增出猪H-FABP基因intron 3的全序列,全长1 350 bp,已向GenBank提交,检索号为DQ 002993。根据序列测定结果设计引物P2,扩增其中255bp的片段,应用PCR-SSCP分析发现P2-SSCP多态位点,该位点上具有两个等位基因A和B。在4个猪种中,AA基因型频率最高,A等位基因频率明显高于B等位基因频率。序列测定的结果表明,P2-SSCP的变异是由碱基A→G的替换造成的。