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1.
Curr Protoc Plant Biol ; 2(1): 89-107, 2017 Mar.
Article in English | MEDLINE | ID: mdl-31725974

ABSTRACT

This article presents protocols for fluorescence in situ hybridization (FISH) in the cultivated soybean, Glycine max. The protocols represent soybean-optimized versions developed for maize. We describe the use of two different probes types: genomic-repeat-based fluorescently-tagged oligonucleotides and bacterial artificial chromosomes (BACs). The two probe types can be used either individually or together, depending on the experimental questions. The article also includes starting points for executing FISH in additional legume species. © 2017 by John Wiley & Sons, Inc.

2.
Curr Protoc Plant Biol ; 2(1): 78-88, 2017 Mar.
Article in English | MEDLINE | ID: mdl-31725978

ABSTRACT

This unit presents a highly reliable protocol to produce and screen metaphase chromosome spreads from root tip cell suspensions of soybean (Glycine max), or other legumes. The procedures represent soybean-optimized versions of protocols developed for maize. The use of pressurized nitrous oxide to reliably generate metaphase-arrested chromosomes is crucial to overcoming one of the challenges of working with tiny and numerous soybean chromosomes. © 2017 by John Wiley & Sons, Inc.

3.
G3 (Bethesda) ; 6(4): 1013-22, 2016 04 07.
Article in English | MEDLINE | ID: mdl-26865698

ABSTRACT

Fluorescence in situ hybridization (FISH)-based karyotyping is a powerful cytogenetics tool to study chromosome organization, behavior, and chromosome evolution. Here, we developed a FISH-based karyotyping system using a probe mixture comprised of centromeric and subtelomeric satellite repeats, 5S rDNA, and chromosome-specific BAC clones in common bean, which enables one to unambiguously distinguish all 11 chromosome pairs. Furthermore, we applied the karyotyping system to several wild relatives and landraces of common bean from two distinct gene pools, as well as other related Phaseolus species, to investigate repeat evolution in the genus Phaseolus Comparison of karyotype maps within common bean indicates that chromosomal distribution of the centromeric and subtelomeric satellite repeats is stable, whereas the copy number of the repeats was variable, indicating rapid amplification/reduction of the repeats in specific genomic regions. In Phaseolus species that diverged approximately 2-4 million yr ago, copy numbers of centromeric repeats were largely reduced or diverged, and chromosomal distributions have changed, suggesting rapid evolution of centromeric repeats. We also detected variation in the distribution pattern of subtelomeric repeats in Phaseolus species. The FISH-based karyotyping system revealed that satellite repeats are actively and rapidly evolving, forming genomic features unique to individual common bean accessions and Phaseolus species.


Subject(s)
Centromere/genetics , In Situ Hybridization, Fluorescence , Karyotyping , Phaseolus/genetics , Repetitive Sequences, Nucleic Acid , Base Sequence , DNA, Ribosomal/genetics , Evolution, Molecular , Genes, Plant , Genetic Variation , Karyotype , Phaseolus/classification , Phylogeny , Species Specificity
4.
Plant Biotechnol J ; 14(1): 332-41, 2016 Jan.
Article in English | MEDLINE | ID: mdl-25973713

ABSTRACT

Three soybean [Glycine max (L) Merr.] small RNA libraries were generated and sequenced using the Illumina platform to examine the role of miRNAs during soybean nodulation. The small RNA libraries were derived from root hairs inoculated with Bradyrhizobium japonicum (In_RH) or mock-inoculated with water (Un_RH), as well as from the comparable inoculated stripped root samples (i.e. inoculated roots with the root hairs removed). Sequencing of these libraries identified a total of 114 miRNAs, including 22 novel miRNAs. A comparison of miRNA abundance among the 114 miRNAs identified 66 miRNAs that were differentially expressed between root hairs and stripped roots, and 48 miRNAs that were differentially regulated in infected root hairs in response to B. japonicum when compared to uninfected root hairs (P ≤ 0.05). A parallel analysis of RNA ends (PARE) library was constructed and sequenced to reveal a total of 405 soybean miRNA targets, with most predicted to encode transcription factors or proteins involved in protein modification, protein degradation and hormone pathways. The roles of gma-miR4416 and gma-miR2606b during nodulation were further analysed. Ectopic expression of these two miRNAs in soybean roots resulted in significant changes in nodule numbers. miRNA target information suggested that gma-miR2606b regulates a Mannosyl-oligosaccharide 1, 2-alpha-mannosidase gene, while gma-miR4416 regulates the expression of a rhizobium-induced peroxidase 1 (RIP1)-like peroxidase gene, GmRIP1, during nodulation.


Subject(s)
Bradyrhizobium/physiology , Gene Expression Regulation, Plant , Glycine max/genetics , Glycine max/microbiology , MicroRNAs/genetics , Plant Diseases/microbiology , Plant Roots/genetics , Plant Roots/microbiology , Gene Expression Profiling , Gene Library , MicroRNAs/metabolism , Plant Diseases/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Root Nodulation/genetics , RNA, Plant/genetics , RNA, Plant/metabolism , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , RNA, Transfer/genetics , RNA, Transfer/metabolism , Reproducibility of Results , Sequence Analysis, RNA
5.
Genome Biol ; 14(6): r53, 2013 Jun 03.
Article in English | MEDLINE | ID: mdl-23731509

ABSTRACT

BACKGROUND: Theobroma cacao L. cultivar Matina 1-6 belongs to the most cultivated cacao type. The availability of its genome sequence and methods for identifying genes responsible for important cacao traits will aid cacao researchers and breeders. RESULTS: We describe the sequencing and assembly of the genome of Theobroma cacao L. cultivar Matina 1-6. The genome of the Matina 1-6 cultivar is 445 Mbp, which is significantly larger than a sequenced Criollo cultivar, and more typical of other cultivars. The chromosome-scale assembly, version 1.1, contains 711 scaffolds covering 346.0 Mbp, with a contig N50 of 84.4 kbp, a scaffold N50 of 34.4 Mbp, and an evidence-based gene set of 29,408 loci. Version 1.1 has 10x the scaffold N50 and 4x the contig N50 as Criollo, and includes 111 Mb more anchored sequence. The version 1.1 assembly has 4.4% gap sequence, while Criollo has 10.9%. Through a combination of haplotype, association mapping and gene expression analyses, we leverage this robust reference genome to identify a promising candidate gene responsible for pod color variation. We demonstrate that green/red pod color in cacao is likely regulated by the R2R3 MYB transcription factor TcMYB113, homologs of which determine pigmentation in Rosaceae, Solanaceae, and Brassicaceae. One SNP within the target site for a highly conserved trans-acting siRNA in dicots, found within TcMYB113, seems to affect transcript levels of this gene and therefore pod color variation. CONCLUSIONS: We report a high-quality sequence and annotation of Theobroma cacao L. and demonstrate its utility in identifying candidate genes regulating traits.


Subject(s)
Fruit/genetics , Gene Expression Regulation, Plant , Genes, Plant , Genome, Plant , Quantitative Trait, Heritable , Cacao/genetics , Cacao/metabolism , Chromosome Mapping , Chromosomes, Plant , Color , Fruit/metabolism , Genome Size , High-Throughput Nucleotide Sequencing , Quantitative Trait Loci , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic
6.
Plant Physiol ; 161(1): 36-47, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23124322

ABSTRACT

Insertional mutagenesis is a powerful tool for determining gene function in both model and crop plant species. Tnt1, the transposable element of tobacco (Nicotiana tabacum) cell type 1, is a retrotransposon that replicates via an RNA copy that is reverse transcribed and integrated elsewhere in the plant genome. Based on studies in a variety of plants, Tnt1 appears to be inactive in normal plant tissue but can be reactivated by tissue culture. Our goal was to evaluate the utility of the Tnt1 retrotransposon as a mutagenesis strategy in soybean (Glycine max). Experiments showed that the Tnt1 element was stably transformed into soybean plants by Agrobacterium tumefaciens-mediated transformation. Twenty-seven independent transgenic lines carrying Tnt1 insertions were generated. Southern-blot analysis revealed that the copy number of transposed Tnt1 elements ranged from four to 19 insertions, with an average of approximately eight copies per line. These insertions showed Mendelian segregation and did not transpose under normal growth conditions. Analysis of 99 Tnt1 flanking sequences revealed insertions into 62 (62%) annotated genes, indicating that the element preferentially inserts into protein-coding regions. Tnt1 insertions were found in all 20 soybean chromosomes, indicating that Tnt1 transposed throughout the soybean genome. Furthermore, fluorescence in situ hybridization experiments validated that Tnt1 inserted into multiple chromosomes. Passage of transgenic lines through two different tissue culture treatments resulted in Tnt1 transposition, significantly increasing the number of insertions per line. Thus, our data demonstrate the Tnt1 retrotransposon to be a powerful system that can be used for effective large-scale insertional mutagenesis in soybean.


Subject(s)
Chromosomes, Plant , Genomics/methods , Glycine max/genetics , Mutagenesis, Insertional/methods , Retroelements , Agrobacterium tumefaciens/genetics , Agrobacterium tumefaciens/metabolism , Blotting, Southern , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Dosage , In Situ Hybridization, Fluorescence , Plant Somatic Embryogenesis Techniques , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Glycine max/metabolism , Nicotiana/genetics , Nicotiana/metabolism , Transformation, Genetic
7.
Appl Environ Microbiol ; 77(22): 8106-13, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21948825

ABSTRACT

The rumen, the foregut of herbivorous ruminant animals such as cattle, functions as a bioreactor to process complex plant material. Among the numerous and diverse microbes involved in ruminal digestion are the ruminal protozoans, which are single-celled, ciliated eukaryotic organisms. An activity-based screen was executed to identify genes encoding fibrolytic enzymes present in the metatranscriptome of a bovine ruminal protozoan-enriched cDNA expression library. Of the four novel genes identified, two were characterized in biochemical assays. Our results provide evidence for the effective use of functional metagenomics to retrieve novel enzymes from microbial populations that cannot be maintained in axenic cultures.


Subject(s)
Glycoside Hydrolases/genetics , Glycoside Hydrolases/metabolism , Metagenome , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Rumen/parasitology , Animals , Cattle , Cluster Analysis , DNA, Protozoan/chemistry , DNA, Protozoan/genetics , Gene Library , Molecular Sequence Data , Phylogeny , Sequence Analysis, DNA , Sequence Homology
8.
G3 (Bethesda) ; 1(2): 117-29, 2011 Jul.
Article in English | MEDLINE | ID: mdl-22384324

ABSTRACT

Soybean (Glycine max [L.] Merr.) is a major crop species and, therefore, a major target of genomic and genetic research. However, in contrast to other plant species, relatively few chromosomal aberrations have been identified and characterized in soybean. This is due in part to the difficulty of cytogenetic analysis of its small, morphologically homogeneous chromosomes. The recent development of a fluorescence in situ hybridization -based karyotyping system for soybean has enabled our characterization of most of the chromosomal translocation lines identified to date. Utilizing genetic data from existing translocation studies in soybean, we identified the chromosomes and approximate breakpoints involved in five translocation lines.

9.
Methods Mol Biol ; 701: 67-96, 2011.
Article in English | MEDLINE | ID: mdl-21181525

ABSTRACT

Fluorescence in situ hybridization (FISH) is an invaluable tool for chromosome analysis and engineering. The ability to visually localize endogenous genes, transposable elements, transgenes, naturally occurring organellar DNA insertions - essentially any unique sequence larger than 2 kb - greatly facilitates progress. This chapter details the labeling procedures and chromosome preparation techniques used to produce high-quality FISH signals on somatic metaphase and meiotic pachytene spreads.


Subject(s)
Chromosome Painting/methods , Chromosomes, Plant , DNA, Plant/genetics , In Situ Hybridization, Fluorescence/methods , Plants/genetics , Biotechnology/methods , Metaphase
10.
Plant Physiol ; 153(4): 1808-22, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20534735

ABSTRACT

Nodulation of soybean (Glycine max) root hairs by the nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum is a complex process coordinated by the mutual exchange of diffusible signal molecules. A metabolomic study was performed to identify small molecules produced in roots and root hairs during the rhizobial infection process. Metabolites extracted from roots and root hairs mock inoculated or inoculated with B. japonicum were analyzed by gas chromatography-mass spectrometry and ultraperformance liquid chromatography-quadrupole time of flight-mass spectrometry. These combined approaches identified 2,610 metabolites in root hairs. Of these, 166 were significantly regulated in response to B. japonicum inoculation, including various (iso)flavonoids, amino acids, fatty acids, carboxylic acids, and various carbohydrates. Trehalose was among the most strongly induced metabolites produced following inoculation. Subsequent metabolomic analyses of root hairs inoculated with a B. japonicum mutant defective in the trehalose synthase, trehalose 6-phosphate synthase, and maltooligosyltrehalose synthase genes showed that the trehalose detected in the inoculated root hairs was primarily of bacterial origin. Since trehalose is generally considered an osmoprotectant, these data suggest that B. japonicum likely experiences osmotic stress during the infection process, either on the root hair surface or within the infection thread.


Subject(s)
Bradyrhizobium/metabolism , Glycine max/microbiology , Plant Roots/metabolism , Symbiosis , Bradyrhizobium/physiology , Gas Chromatography-Mass Spectrometry , Metabolome , Plant Roots/microbiology , RNA, Plant/genetics , Glycine max/genetics , Glycine max/metabolism , Trehalose/biosynthesis
11.
Genetics ; 185(3): 727-44, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20421607

ABSTRACT

The development of a universal soybean (Glycine max [L.] Merr.) cytogenetic map that associates classical genetic linkage groups, molecular linkage groups, and a sequence-based physical map with the karyotype has been impeded due to the soybean chromosomes themselves, which are small and morphologically homogeneous. To overcome this obstacle, we screened soybean repetitive DNA to develop a cocktail of fluorescent in situ hybridization (FISH) probes that could differentially label mitotic chromosomes in root tip preparations. We used genetically anchored BAC clones both to identify individual chromosomes in metaphase spreads and to complete a FISH-based karyotyping cocktail that permitted simultaneous identification of all 20 chromosome pairs. We applied these karyotyping tools to wild soybean, G. soja Sieb. and Zucc., which represents a large gene pool of potentially agronomically valuable traits. These studies led to the identification and characterization of a reciprocal chromosome translocation between chromosomes 11 and 13 in two accessions of wild soybean. The data confirm that this translocation is widespread in G. soja accessions and likely accounts for the semi-sterility found in some G. soja by G. max crosses.


Subject(s)
Chromosomes, Plant/genetics , Glycine max/genetics , In Situ Hybridization, Fluorescence , Karyotyping/methods , Base Sequence , Chromosomes, Artificial, Bacterial , DNA, Plant , Molecular Sequence Data , Nucleic Acid Hybridization
12.
Plant Physiol ; 151(3): 1167-74, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19605552

ABSTRACT

Recent studies have documented that the soybean (Glycine max) genome has undergone two rounds of large-scale genome and/or segmental duplication. To shed light on the timing and nature of these duplication events, we characterized and analyzed two subfamilies of high-copy centromeric satellite repeats, CentGm-1 and CentGm-2, using a combination of computational and molecular cytogenetic approaches. These two subfamilies of satellite repeats mark distinct subsets of soybean centromeres and, in at least one case, a pair of homologs, suggesting their origins from an allopolyploid event. The satellite monomers of each subfamily are arranged in large tandem arrays, and intermingled monomers of the two subfamilies were not detected by fluorescence in situ hybridization on extended DNA fibers nor at the sequence level. This indicates that there has been little recombination and homogenization of satellite DNA between these two sets of centromeres. These satellite repeats are also present in Glycine soja, the proposed wild progenitor of soybean, but could not be detected in any other relatives of soybean examined in this study, suggesting the rapid divergence of the centromeric satellite DNA within the Glycine genus. Together, these observations provide direct evidence, at molecular and chromosomal levels, in support of the hypothesis that the soybean genome has experienced a recent allopolyploidization event.


Subject(s)
DNA, Satellite/genetics , Evolution, Molecular , Glycine max/genetics , Polyploidy , Centromere/genetics , Chromosomes, Plant , Computational Biology , DNA, Plant/genetics , Genome, Plant , In Situ Hybridization, Fluorescence , Molecular Sequence Data , Phylogeny
13.
BMC Genomics ; 9: 28, 2008 Jan 22.
Article in English | MEDLINE | ID: mdl-18211698

ABSTRACT

BACKGROUND: Integrated genetic and physical maps are extremely valuable for genomic studies and as important references for assembling whole genome shotgun sequences. Screening of a BAC library using molecular markers is an indispensable procedure for integration of both physical and genetic maps of a genome. Molecular markers provide anchor points for integration of genetic and physical maps and also validate BAC contigs assembled based solely on BAC fingerprints. We employed a six-dimensional BAC pooling strategy and an in silico approach to anchor molecular markers onto the soybean physical map. RESULTS: A total of 1,470 markers (580 SSRs and 890 STSs) were anchored by PCR on a subset of a Williams 82 BstY I BAC library pooled into 208 pools in six dimensions. This resulted in 7,463 clones (approximately 1x genome equivalent) associated with 1470 markers, of which the majority of clones (6,157, 82.5%) were anchored by one marker and 1106 (17.5%) individual clones contained two or more markers. This contributed to 1184 contigs having anchor points through this 6-D pool screening effort. In parallel, the 21,700 soybean Unigene set from NCBI was used to perform in silico mapping on 80,700 Williams 82 BAC end sequences (BES). This in silico analysis yielded 9,835 positive results anchored by 4152 unigenes that contributed to 1305 contigs and 1624 singletons. Among the 1305 contigs, 305 have not been previously anchored by PCR. Therefore, 1489 (78.8%) of 1893 contigs are anchored with molecular markers. These results are being integrated with BAC fingerprints to assemble the BAC contigs. Ultimately, these efforts will lead to an integrated physical and genetic map resource. CONCLUSION: We demonstrated that the six-dimensional soybean BAC pools can be efficiently used to anchor markers to soybean BACs despite the complexity of the soybean genome. In addition to anchoring markers, the 6-D pooling method was also effective for targeting BAC clones for investigating gene families and duplicated regions in the genome, as well as for extending physical map contigs.


Subject(s)
Chromosomes, Artificial, Bacterial/genetics , Genetic Markers/physiology , Genomic Library , Glycine max/genetics , Physical Chromosome Mapping/methods , Polymerase Chain Reaction , Base Sequence , Expressed Sequence Tags , Genome, Plant , Models, Biological , Sequence Homology, Nucleic Acid
14.
Genes Dev ; 21(18): 2300-11, 2007 Sep 15.
Article in English | MEDLINE | ID: mdl-17875665

ABSTRACT

The interface between cellular systems involving small noncoding RNAs and epigenetic change remains largely unexplored in metazoans. RNA-induced silencing systems have the potential to target particular regions of the genome for epigenetic change by locating specific sequences and recruiting chromatin modifiers. Noting that several genes encoding RNA silencing components have been implicated in epigenetic regulation in Drosophila, we sought a direct link between the RNA silencing system and heterochromatin components. Here we show that PIWI, an ARGONAUTE/PIWI protein family member that binds to Piwi-interacting RNAs (piRNAs), strongly and specifically interacts with heterochromatin protein 1a (HP1a), a central player in heterochromatic gene silencing. The HP1a dimer binds a PxVxL-type motif in the N-terminal domain of PIWI. This motif is required in fruit flies for normal silencing of transgenes embedded in heterochromatin. We also demonstrate that PIWI, like HP1a, is itself a chromatin-associated protein whose distribution in polytene chromosomes overlaps with HP1a and appears to be RNA dependent. These findings implicate a direct interaction between the PIWI-mediated small RNA mechanism and heterochromatin-forming pathways in determining the epigenetic state of the fly genome.


Subject(s)
Chromatin/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Proteins/metabolism , Amino Acid Motifs , Animals , Argonaute Proteins , Binding Sites , Chromobox Protein Homolog 5 , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Embryo, Nonmammalian , Female , Gene Silencing , Male , Models, Molecular , Nuclear Proteins/metabolism , Protein Binding , Proteins/chemistry , Proteins/physiology , RNA-Induced Silencing Complex
15.
Plant Physiol ; 144(2): 623-36, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17449649

ABSTRACT

The lysin motif (LysM) domain is an ancient and ubiquitous protein module that binds peptidoglycan and structurally related molecules. A genomic survey in a large number of species spanning all kingdoms reveals that the combination of LysM and receptor kinase domains is present exclusively in plants. However, the particular biological functions and molecular evolution of this gene family remain largely unknown. We show that LysM domains in plant LysM proteins are highly diversified and that a minimum of six distinct types of LysM motifs exist in plant LysM kinase proteins and five additional types of LysM motifs exist in nonkinase plant LysM proteins. Further, motif similarities suggest that plant LysM motifs are ancient. Although phylogenetic signals are not sufficient to resolve the earliest relationships, plant LysM motifs may have arisen through common ancestry with LysM motifs in other kingdoms. Within plants, the gene family has evolved through local and segmental duplications. The family has undergone further duplication and diversification in legumes, where some LysM kinase genes function as receptors for bacterial nodulation factor. Two pairs of homeologous regions were identified in soybean (Glycine max) based on microsynteny and fluorescence in situ hybridization. Expression data show that most plant LysM kinase genes are expressed predominantly in the root and that orthologous LysM kinase genes share similar tissue expression patterns. We also examined synteny around plant LysM kinase genes to help reconstruct scenarios for the evolution of this important gene family.


Subject(s)
Amino Acid Motifs/genetics , Evolution, Molecular , Magnoliopsida/enzymology , Plant Proteins/genetics , Protein Kinases/genetics , Gene Expression Regulation, Plant , Genome, Plant , Genomics , In Situ Hybridization, Fluorescence , Magnoliopsida/genetics , Molecular Sequence Data , Multigene Family , Synteny
16.
Development ; 131(13): 3169-81, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15175253

ABSTRACT

The Notch signaling pathway controls the follicle cell mitotic-to-endocycle transition in Drosophila oogenesis by stopping the mitotic cycle and promoting the endocycle. To understand how the Notch pathway coordinates this process, we have identified and performed a functional analysis of genes whose transcription is responsive to the Notch pathway at this transition. These genes include the G2/M regulator Cdc25 phosphatase, String; a regulator of the APC ubiquitination complex Hec/CdhFzr and an inhibitor of the CyclinE/CDK complex, Dacapo. Notch activity leads to downregulation of String and Dacapo, and activation of Fzr. All three genes are independently responsive to Notch. In addition, CdhFzr, an essential gene for endocycles, is sufficient to stop mitotic cycle and promote precocious endocycles when expressed prematurely during mitotic stages. In contrast, overexpression of the growth controller Myc does not induce premature endocycles but accelerates the kinetics of normal endocycles. We also show that Archipelago (Ago), a SCF-regulator is dispensable for mitosis, but crucial for endocycle progression in follicle epithelium. The results support a model in which Notch activity executes the mitotic-to-endocycle switch by regulating all three major cell cycle transitions. Repression of String blocks the M-phase, activation of Fzr allows G1 progression and repression of Dacapo assures entry into the S-phase. This study provides a comprehensive picture of the logic that external signaling pathways may use to control cell cycle transitions by the coordinated regulation of the cell cycle.


Subject(s)
Drosophila melanogaster/metabolism , Membrane Proteins/metabolism , Ovarian Follicle/metabolism , Animals , Cdh1 Proteins , Cell Cycle , Cell Cycle Proteins/metabolism , Cell Nucleus/metabolism , Cyclin D , Cyclin E/metabolism , Cyclins/metabolism , Cytoskeletal Proteins , Down-Regulation , Drosophila Proteins/metabolism , Female , Flow Cytometry , G1 Phase , G2 Phase , Mitosis , Models, Biological , Nuclear Proteins/metabolism , Protein Tyrosine Phosphatases/metabolism , Receptors, Notch , S Phase , Signal Transduction , Temperature , Time Factors , cdc25 Phosphatases/metabolism
17.
Development ; 130(5): 859-71, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12538514

ABSTRACT

A hallmark of germline cells across the animal kingdom is the presence of perinuclear, electron-dense granules called nuage. In many species examined, Vasa, a DEAD-box RNA helicase, is found in these morphologically distinct particles. Despite its evolutionary conservation, the function of nuage remains obscure. We have characterized a null allele of maelstrom (mael) and shown that Maelstrom protein is localized to nuage in a Vasa-dependent manner. By phenotypic characterization, we have defined maelstrom as a spindle-class gene that affects Vasa modification. In a nuclear transport assay, we have determined that Maelstrom shuttles between the nucleus and cytoplasm, which may indicate a nuclear origin for nuage components. Interestingly, Maelstrom, but not Vasa, depends on two genes involved in RNAi phenomena, aubergine and spindle-E (spn-E), for its nuage localization. Furthermore, maelstrom mutant ovaries show mislocalization of two proteins involved in the microRNA and/or RNAi pathways, Dicer and Argonaute2, suggesting a potential connection between nuage and the microRNA-pathway.


Subject(s)
Adenosine Triphosphatases , Cytoplasmic Granules/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Insect Proteins/metabolism , Peptide Initiation Factors/metabolism , RNA Helicases/metabolism , Active Transport, Cell Nucleus/physiology , Alleles , Animals , Cytoplasmic Granules/chemistry , DEAD-box RNA Helicases , Drosophila Proteins/genetics , Drosophila melanogaster/physiology , Fatty Acids, Unsaturated/metabolism , Female , Germ Cells/cytology , Germ Cells/physiology , Humans , Immunohistochemistry , In Situ Hybridization , Insect Proteins/genetics , Oogenesis/physiology , Ovary/cytology , Ovary/metabolism , RNA Helicases/genetics , RNA Interference
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