Mutations in SUPPRESSOR OF VARIEGATION1, a factor required for normal chloroplast translation, suppress var2-mediated leaf variegation in Arabidopsis

The Arabidopsis thaliana yellow variegated2 (var2) mutant is variegated due to lack of a chloroplast FtsH-like metalloprotease (FtsH2/VAR2). We have generated suppressors of var2 variegation to gain insight into factors and pathways that interact with VAR2 during chloroplast biogenesis. Here, we describe two such suppressors. Suppression of variegation in the first line, TAG-FN, was caused by disruption of the nuclear gene (SUPPRESSOR OF VARIEGATION1 [SVR1]) for a chloroplast-localized homolog of pseudouridine (Psi) synthase, which isomerizes uridine to Psi in noncoding RNAs. svr1 single mutants were epistatic to var2, and they displayed a phenotypic syndrome that included defects in chloroplast rRNA processing, reduced chloroplast translation, reduced chloroplast protein accumulation, and elevated chloroplast mRNA levels. In the second line (TAG-IE), suppression of variegation was caused by a lesion in SVR2, the gene for the ClpR1 subunit of the chloroplast ClpP/R protease. Like svr1, svr2 was epistatic to var2, and clpR1 mutants had a phenotype that resembled svr1. We propose that an impairment of chloroplast translation in TAG-FN and TAG-IE decreased the demand for VAR2 activity during chloroplast biogenesis and that this resulted in the suppression of var2 variegation. Consistent with this hypothesis, var2 variegation was repressed by chemical inhibitors of chloroplast translation. In planta mutagenesis revealed that SVR1 not only played a role in uridine isomerization but that its physical presence was necessary for proper chloroplast rRNA processing. Our data indicate that defects in chloroplast rRNA processing are a common, but not universal, molecular phenotype associated with suppression of var2 variegation.     

Molecular characterization of Pegarn: a <Emphasis Type="Italic">Drosophila</Emphasis> homolog of UNC-51 kinase

We have isolated and characterized the gene encoding a Drosophila melanogaster homolog of Caenorhabditis elegans UNC-51 (uncoordinated movement-51): Pegarn. Developmental Northern blot shows the Pegarn gene is expressed at all stages of development. The protein is detected throughout the Drosophila third instar larval central nervous system (CNS) in axons projecting out from the ventral ganglion and in the optic anlagen of the optic lobe. Heterozygous Pegarn mutant embryos show defects in larval axonal neuronal patterning, but survive to adulthood. Homozygous mutants have an even more deformed pattern of neuronal development and do not survive through the larval stages. The data from this research suggest the critical roles of Pegarn in CNS and PNS axonal formation in Drosophila melanogaster and indicates its similar role in other multicellular species.     

Interaction of molecular forms of ceruloplasmin with specific membrane receptors from healthy persons and patients with hepatolenticular degeneration]

The ceruloplasmin (CP) interaction with the specific receptor localized on the erythrocyte membrane of healthy donors and of patients with hepatolenticular degeneration (HLD) was studied by using Scatchard analysis. It was found that in patients with the Wilson's disease the CP receptor is unaffected by the pathological process. The molecular mass of the erythrocyte receptor of CP is about 130 kDa. The CP binding to the specific receptor is followed by limited proteolysis of the latter. No CP binding to the receptor occurs in the presence of serine protease inhibitors. The CP-like protein previously detected in the sera of Wilson gene carriers can specifically interact with the CP receptor.     

The Ketel(D) dominant-negative mutations identify maternal function of the Drosophila importin-beta gene required for cleavage nuclei formation

The Ketel(D) dominant female-sterile mutations and their ketel(r) revertant alleles identify the Ketel gene, which encodes the importin-beta (karyopherin-beta) homologue of Drosophila melanogaster. Embryogenesis does not commence in the Ketel(D) eggs deposited by the Ketel(D)/+ females due to failure of cleavage nuclei formation. When injected into wild-type cleavage embryos, cytoplasm of the Ketel(D) eggs does not inhibit nuclear protein import but prevents cleavage nuclei formation following mitosis. The Ketel(+) transgenes slightly reduce effects of the Ketel(D) mutations. The paternally derived Ketel(D) alleles act as recessive zygotic lethal mutations: the Ketel(D)/- hemizygotes, like the ketel(r)/ketel(r) and the ketel(r)/- zygotes, perish during second larval instar. The Ketel maternal dowry supports their short life. The Ketel(D)-related defects originate most likely following association of the Ketel(D)-encoded mutant molecules with a maternally provided partner. As in the Ketel(D) eggs, embryogenesis does not commence in eggs of germline chimeras with ketel(r)/- germline cells and normal soma, underlining the dominant-negative nature of the Ketel(D) mutations. The ketel(r) homozygous clones are fully viable in the follicle epithelium in wings and tergites. The Ketel gene is not expressed in most larval tissues, as revealed by the expression pattern of a Ketel promoter-lacZ reporter gene.     

Plasmodium falciparum: structural and functional domains of the mature-parasite-infected erythrocyte surface antigen

The mature parasite-infected erythrocyte surface antigen (MESA) is a protein exported to the membrane skeleton of the infected red cell, where it forms a strong noncovalent interaction with the host red cell protein, protein 4.1. The complete gene structure of MESA from the Ugandan isolate Palo Alto is described. Comparison to the previously reported MESA sequence from the Papua New Guinean cloned line D10 reveals strong conservation of the general gene structure of a short first exon and a long second exon. The exact exon/intron boundaries were determined by the generation and sequencing of a cDNA from this region. The MESA gene from both isolates consists of seven blocks of repeats that are identical in order. Repeat blocks are conserved to a high degree; however, differences are noted in most blocks in the form of scattered mutations or differences in repeat numbers. Previous work had shown that synthetic peptides spanning a 19-residue region could inhibit the binding of MESA to protein 4.1. Removal of this region from MESA almost completely abolished the binding of MESA to IOVs. Sequencing of this region from a number of laboratory and field isolates demonstrates complete conservation of the cytoskeletal binding domain and flanking sequences.     

Characterization of the enzymatic properties of the first and second domains of metallocarboxypeptidase D.

Carboxypeptidase D (CPD) contains three domains with homology to other metallocarboxypeptidases. To further characterize the various domains, we constructed a series of point mutants with a critical active site Glu of duck CPD converted to Gln. The proteins were expressed in the baculovirus system, purified to homogeneity, and characterized. Point mutations within both the first and second domains eliminated enzyme activity, indicating that the third domain is inactive toward dansyl-Phe-Ala-Arg. CPD removed only the C-terminal Lys or Arg from peptides, with the first domain more efficient toward Arg and the second domain more efficient toward Lys. Peptides containing Pro in the penultimate position were poorly cleaved by either domain. Cleavage of a peptide with Ala in the penultimate position was most efficient, with the relative order Ala >/= Met > Ser, Phe > Tyr > Trp > Thr >/= Gln, Asp, Leu, Gly > Pro for CPD with both domains active. There were only minor differences between the first and the second domains regarding the influence of the penultimate amino acid. The first domain was optimally active at pH 6.3-7.5, whereas the second domain was optimally active at pH 5. 0-6.5. Thus, the first and second carboxypeptidase domains have complementary enzyme activities. Furthermore, the finding that CPD with both domains active shows a broad activity to a wide range of substrates is consistent with a role for this enzyme in the processing of many proteins that transit the secretory pathway.     

利用 CRISPR/Cas9 系统定向编辑黑腹果蝇Osiris24基因

Osiris基因在几丁质沉积过程中表达,可能参与昆虫表皮的发育。本研究利用CRISPR/Cas9 基因编辑系统对Osiris24基因进行编辑,进而观察Osiris24突变体果蝇的性状并且检测Osiris24的表达特征。在Osiris24第1外显子设计2个sgRNA靶位点,插入到pCFD4敲除载体骨架中,同时构建酵母Gal4蛋白序列的供体(donor)载体,将2个载体同时注射到nos-Cas9胚胎中获得G0代转基因果蝇。结果显示,G0代基因编辑阳性率为92.8%,Osiris24纯合突变体在胚胎或1龄幼虫期致死,杂合突变体未观察到可见表型。将阳性G0代雄虫与UAS-GFP雌虫杂交,检测不同龄期和不同组织GFP信号表达情况。结果发现,Osiris24在不同龄期幼虫中均有表达,幼虫期主要在体壁、气管、前肠和后肠高表达,蛹期主要在体壁和翅上表达,推测其在果蝇发育中发挥重要作用,本研究为深入探究Osiris基因功能提供了研究模型。     

The PIKE Homolog Centaurin gamma Regulates Developmental Timing in Drosophila

Phosphoinositide-3-kinase enhancer (PIKE) proteins encoded by the PIKE/CENTG1 gene are members of the gamma subgroup of the Centaurin superfamily of small GTPases. They are characterized by their chimeric protein domain architecture consisting of a pleckstrin homology (PH) domain, a GTPase-activating (GAP) domain, Ankyrin repeats as well as an intrinsic GTPase domain. In mammals, three PIKE isoforms with variations in protein structure and subcellular localization are encoded by the PIKE locus. PIKE inactivation in mice results in a broad range of defects, including neuronal cell death during brain development and misregulation of mammary gland development. PIKE -/- mutant mice are smaller, contain less white adipose tissue, and show insulin resistance due to misregulation of AMP-activated protein kinase (AMPK) and insulin receptor/Akt signaling. here, we have studied the role of PIKE proteins in metabolic regulation in the fly. We show that the Drosophila PIKE homolog, ceng1A, encodes functional GTPases whose internal GAP domains catalyze their GTPase activity. To elucidate the biological function of ceng1A in flies, we introduced a deletion in the ceng1A gene by homologous recombination that removes all predicted functional PIKE domains. We found that homozygous ceng1A mutant animals survive to adulthood. In contrast to PIKE -/- mouse mutants, genetic ablation of Drosophila ceng1A does not result in growth defects or weight reduction. Although metabolic pathways such as insulin signaling, sensitivity towards starvation and mobilization of lipids under high fed conditions are not perturbed in ceng1A mutants, homozygous ceng1A mutants show a prolonged development in second instar larval stage, leading to a late onset of pupariation. In line with these results we found that expression of ecdysone inducible genes is reduced in ceng1A mutants. Together, we propose a novel role for Drosophila Ceng1A in regulating ecdysone signaling-dependent second to third instar larval transition.     

Intron-dependent evolution of the nucleotide-binding domains within alcohol dehydrogenase and related enzymes.

It has been suggested that the intron/exon structure of a gene corresponds to its evolutionary history. Accordingly, early in evolution DNA segments encoding short functional polypeptides may have been rearranged (exon-shuffling) to create full-length genes and RNA splicing may have been developed to remove intervening sequences (introns) in order to preserve translational reading frames. A conflicting viewpoint would be that introns were randomly inserted into previously uninterrupted genes after their initial evolutionary development. If so, the sites of introns would be unlikely to consistently reflect the domain structure of the protein. To address this question, the intron/exon structure of the gene encoding human alcohol dehydrogenase (ADH) was determined and compared to the gene structures for other ADHs and related proteins, all of which possess nucleotide-binding domains. Our results indicate that the introns in the nucleotide-binding domains of all the genes examined do indeed fall at positions which separate the short functional polypeptides (i.e. beta strands) which are believed to comprise this domain. We argue that our data is most easily explained by the hypothesis that introns were present in an ancestral nucleotide-binding domain which was later rearranged by exon-shuffling to form the various dehydrogenases and kinases which utilize such a domain.     

Identification of mouse CPX-1, a novel member of the metallocarboxypeptidase gene family with highest similarity to CPX-2

The recent finding that Cpe(fat)/Cpe(fat) mice, which lack carboxypeptidase E (CPE) activity because of a point mutation, are still capable of a reduced amount of neuroendocrine peptide processing suggested that additional carboxypeptidases (CPs) participate in this processing reaction. Searches for novel members of the CPE gene family led to the discovery of CPD, CPZ, AEBP1, and CPX-2. In the present report, we describe mouse CPX-1, another novel member of this gene family. Like AEBP1 and CPX-2, CPX-1 contains an N-terminal region of 160 amino acids with sequence similarity to the discoidin domain of a variety of proteins. The 410-residue CP-like domain of CPX-1 has 54% to 62% amino acid sequence identity with AEBP1 and CPX-2 and 33% to 49% amino acid identity with other members of the CPE subfamily. However, several active-site residues that are important for catalytic activity of other CPs are not conserved in CPX-1. Furthermore, CPX-1 expressed in either the baculovirus system or the mouse AtT-20 cell line does not cleave standard CP substrates. Northern blot analysis showed the highest levels of CPX-1 mRNA in testis and spleen and lower levels in salivary gland, brain, heart, lung, and kidney. In situ hybridization of CPX-1 mRNA in embryonic and fetal mouse tissue showed expression throughout the head and thorax, with abundance in primordial cartilage and skeletal structures. In the head, high levels of CPX-1 mRNA were associated with the nasal mesenchyme, primordial cartilage structures in the ear, and the meninges. In the thorax, CPX-1 mRNA was expressed in multiple developing skeletal structures, including chondrocytes and perichondrial cells of the rib, vertebral, and long-bone primordia. Taken together, these findings suggest that it is unlikely that CPX-1 functions in the processing of neuroendocrine peptides. Instead, CPX-1 may have a role in development, possibly mediating cell interactions via its discoidin domain.     

Structural Changes in the Cytoplasmic Domain of the Mechanosensitive Channel MscS During Opening

The bacterial mechanosensitive channel MscS forms a homoheptamer of subunits composed of a transmembrane (TM) domain and a large cytoplasmic (CP) domain. Recent studies suggest that a lateral expansion of the TM domain, structural change in the CP domain, and TM-CP interactions are essential to open the channel. However, it has not been examined whether the CP domain undergoes structural changes during channel opening. The aim of this study was to estimate structural changes in the CP domain during channel opening using fluorescence resonance energy transfer (FRET) spectroscopy. To monitor changes in the horizontal diameter of the CP domain, four point mutants (A132C, F178C, L246C, and R259C), all of which had channel activity, were created and labeled with Alexa488 and Alexa568 for FRET analysis. The FRET efficiency of these mutants decreased when lysophosphatidylcholine was applied to open the channel, suggesting that the CP domain swells up when the channel opens. The degree of the decease in FRET efficiency after lysophosphatidylcholine treatment was smaller in the D62N/F178C mutant, which was deficient in the TM-CP interactions, than in the F178C mutant. These findings provide the first, to our knowledge, experimental evidence that the CP domain swells up during channel opening, and the swelling is mediated by the TM-CP interactions.     

TnaA,an SP-RING Protein,Interacts with Osa,a Subunit of the Chromatin Remodeling Complex BRAHMA and with the SUMOylation Pathway in Drosophila melanogaster

Tonalli A (TnaA) is a Drosophila melanogaster protein with an XSPRING domain. The XSPRING domain harbors an SP-RING zinc-finger, which is characteristic of proteins with SUMO E3 ligase activity. TnaA is required for homeotic gene expression and is presumably involved in the SUMOylation pathway. Here we analyzed some aspects of the TnaA location in embryo and larval stages and its genetic and biochemical interaction with SUMOylation pathway proteins. We describe that there are at least two TnaA proteins (TnaA₁₃₀ and TnaA₁₂₃) differentially expressed throughout development. We show that TnaA is chromatin-associated at discrete sites on polytene salivary gland chromosomes of third instar larvae and that tna mutant individuals do not survive to adulthood, with most dying as third instar larvae or pupae. The tna mutants that ultimately die as third instar larvae have an extended life span of at least 4 to 15 days as other SUMOylation pathway mutants. We show that TnaA physically interacts with the SUMO E2 conjugating enzyme Ubc9, and with the BRM complex subunit Osa. Furthermore, we show that tna and osa interact genetically with SUMOylation pathway components and individuals carrying mutations for these genes show a phenotype that can be the consequence of misexpression of developmental-related genes.     

amontillado,the Drosophila homolog of the prohormone processing protease PC2, is required during embryogenesis and early larval development

Biosynthesis of most peptide hormones and neuropeptides requires proteolytic excision of the active peptide from inactive proprotein precursors, an activity carried out by subtilisin-like proprotein convertases (SPCs) in constitutive or regulated secretory pathways. The Drosophila amontillado (amon) gene encodes a homolog of the mammalian PC2 protein, an SPC that functions in the regulated secretory pathway in neuroendocrine tissues. We have identified amon mutants by isolating ethylmethanesulfonate (EMS)-induced lethal and visible mutations that define two complementation groups in the amon interval at 97D1 of the third chromosome. DNA sequencing identified the amon complementation group and the DNA sequence change for each of the nine amon alleles isolated. amon mutants display partial embryonic lethality, are defective in larval growth, and arrest during the first to second instar larval molt. Mutant larvae can be rescued by heat-shock-induced expression of the amon protein. Rescued larvae arrest at the subsequent larval molt, suggesting that amon is also required for the second to third instar larval molt. Our data indicate that the amon proprotein convertase is required during embryogenesis and larval development in Drosophila and support the hypothesis that AMON acts to proteolytically process peptide hormones that regulate hatching, larval growth, and larval ecdysis.