P element transformation vectors for studying Drosophila melanogaster oogenesis and early embryogenesis

Serano, Thomas L.; Cheung, Hung-Kam; Frank, Lawrence; Cohen, Robert S.

doi:10.1016/0378-1119(94)90804-4

Cited by 31 publications

(20 citation statements)

References 26 publications

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“…Our starting point for these studies was a K10-lacZ-K10 (KZK) reporter construct that lacks the TLS due to the deletion of a 308 base pair sequence from the trailing (K10 3 0 UTR) portion of the construct (see Materials and Methods). This construct, called D, and all other constructs described in this paper contain the endogenous K10 promoter, which is active exclusively in nurse cells (Cheung et al 1992;Serano et al 1994). In situ hybridization experiments show that transcripts produced from D transgenes are completely defective for transport/ anterior localization ( Fig.…”

Section: Resultsmentioning

confidence: 91%

The positional, structural, and sequence requirements of theDrosophilaTLS RNA localization element

Cohen

Zhang²,

Dollar³

2005

RNA

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The subcellular localization of mRNAs is a key step in the polarization of cells in organisms from yeast to man. Here, we use a transgenic fly/in situ hybridization assay system to define the positional, structural, and sequence requirements of the TLS, a stem loop RNA sequence element that mediates the subcellular localization of K10 and Orb transcripts in Drosophila oocytes. We find that the TLS is a highly robust and modular element. It mediates efficient RNA localization regardless of sequence context or position within the transcript. Site-specific mutagenesis experiments indicate that the size and shape of the stem and loop regions are critical determinants of TLS activity. Such experiments also identify specific base residues that are important for TLS activity. All such residues map to the stem portion of the structure. Significantly, mutations at these residues interfere with TLS activity only when they alter the stereochemistry of the stem's minor groove. For example, mutation of the A:U base pair at position 3 of the TLS stem to G:C severely reduces TLS activity, while mutation of the same base pair to U:A has no effect. Extensive searches for TLS-like elements in other Drosophila mRNAs using sequence and structural parameters defined by our experiments indicate that the TLS is unique to K10 and Orb mRNAs. This unexpected finding raises important questions as to how the many hundreds of other mRNAs that are known or thought to exhibit K10 and Orb-like localization are localized.

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Section: Resultsmentioning

confidence: 91%

The positional, structural, and sequence requirements of theDrosophilaTLS RNA localization element

Cohen

Zhang²,

Dollar³

2005

RNA

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“…The critical feature is the use of promoters that are strongly expressed during oogenesis but silent in early embryogenesis. The two promoters used in this study are rpA1 (14) and hsp26/sgs3 (24,25). The latter promoter contains the nurse cell-specific enhancer from the hsp26 gene linked to the basal sgs3 promoter.…”

Section: Resultsmentioning

confidence: 99%

Functional Mapping of Destabilizing Elements in the Protein-coding Region of the Drosophila fushi tarazumRNA

Ito

Jacobs-Lorena

2001

Journal of Biological Chemistry

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The instability of the fushi tarazu (ftz) mRNA is essential for the proper development of the Drosophila embryo. Previously, we identified a 201-nucleotide instability element (FIE3) in the 3 untranslated region (UTR) of the ftz mRNA. Here we report on the identification of two additional elements in the protein-coding region of the message: the 63-nucleotide-long FIE5-1 and the 69-nucleotide-long FIE5-2. The function of both elements was position-dependent; the same elements destabilized RNAs when present within the coding region but did not when embedded in the 3 UTR of the hybrid mRNAs. We conclude that ftz mRNA has three redundant instability elements, two in the protein-coding region and one in the 3 UTR. Although each instability element is sufficient to destabilize a heterologous mRNA, the destabilizing activity of the two 5-elements depended on their position within the message.Drosophila embryonic development depends on the precise temporal and spatial expression of maternal and zygotic pattern-forming genes (1). Maternal pattern-forming genes are transcribed during oogenesis, and their mRNA abundance decreases rapidly in the early embryo. Moreover, many mRNAs encoded by zygotic pattern-forming genes undergo dramatic changes in abundance and spatial distribution during early embryogenesis. To achieve these rapid changes, especially for rapid down-regulation, transcriptional control alone is insufficient, and regulation at the level of mRNA stability is essential. For instance, the maternal bicoid mRNA is completely stable during the first 2 h of embryogenesis but is rapidly destabilized at cellularization of the blastoderm (2). As discussed in the following text, the zygotic fushi tarazu (ftz) mRNA is one of the most unstable eukaryotic mRNAs known. Given that most mRNAs in the Drosophila embryo are constitutively stable (30), the question arises how selected mRNAs in the same embryo cytoplasm are targeted for degradation. Recognition of the targeted RNAs by the RNA degrading machinery must involve cis-acting sequences. These sequences are the focus of the present study.ftz is a member of the pair-rule class of segmentation genes and one of the best characterized early zygotic genes. In early embryos ftz mRNA is detected only from about 1.5 to 4.5 h after fertilization. When first expressed, ftz mRNA is uniformly distributed through the embryo (4). As development progresses, its distribution first becomes restricted to a region comprising from 15 to 65% of egg length, then to four broad bands, and finally to seven narrow stripes that encircle the embryo (4 -6). The seven stripes are short-lived, and no ftz mRNA is detected by 5 h after fertilization. This rapid change of expression pattern and formation of stripes in a short time span can be attributed to the termination of transcription in interstripe regions coupled with rapid mRNA turnover. The need for rapid mRNA turnover is emphasized by the fact that the FTZ protein activates its own transcription in a positive feedback loop (7). Thus, it is impor...

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“…cDNA containing the complete 7548-nucleotide tud coding sequence (CDS) (from the Berkeley Drosophila Genome Project) and mini-tud constructs were cloned into pP{CaSpeR-2} with the nos promoter and 5ЈUTR for germline expression (Wang and Lehmann, 1991), an HA tag at the N terminus and a K10 3ЈUTR for mRNA stability (Serano et al, 1994). For the generation of a mini-tud ⌬1 construct, a NruI-Bsu36I fragment of tud CDS was excised and the Bsu36I-protruding end of the CDS remainder was filled in and blunt-ligated with the NruI end.…”

Section: Transgene Constructionmentioning

confidence: 99%

The role of Tudor domains in germline development and polar granule architecture

Arkov

Wang

Ramos³

et al. 2006

Development

125

180

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Tudor domains are found in many organisms and have been implicated in protein-protein interactions in which methylated protein substrates bind to these domains. Here, we present evidence for the involvement of specific Tudor domains in germline development. Drosophila Tudor, the founder of the Tudor domain family, contains 11 Tudor domains and is a component of polar granules and nuage, electron-dense organelles characteristic of the germline in many organisms, including mammals. In this study, we investigated whether the 11 Tudor domains fulfil specific functions for polar granule assembly, germ cell formation and abdomen formation. We find that even a small number of non-overlapping Tudor domains or a substantial reduction in overall Tudor protein is sufficient for abdomen development. In stark contrast, we find a requirement for specific Tudor domains in germ cell formation, Tudor localization and polar granule architecture. Combining genetic analysis with structural modeling of specific Tudor domains, we propose that these domains serve as 'docking platforms' for polar granule assembly.

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P element transformation vectors for studying Drosophila melanogaster oogenesis and early embryogenesis

Cited by 31 publications

References 26 publications

The positional, structural, and sequence requirements of theDrosophilaTLS RNA localization element

The positional, structural, and sequence requirements of theDrosophilaTLS RNA localization element

Functional Mapping of Destabilizing Elements in the Protein-coding Region of the Drosophila fushi tarazumRNA

The role of Tudor domains in germline development and polar granule architecture

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