Expression during embryogenesis of a mouse gene with sequence homology to the Drosophila engrailed gene

Regions of the mouse and human genomes with strong homology to the Drosophila engrailed gene have been identified by Southern blot analysis. One mouse engrailed-like region, Mo-en.1, has been cloned and partially sequenced; homology with the engrailed gene is localized to a 180 bp engrailed-like homeo box and 63 nucleotides immediately 3' to it. The protein sequence this region can encode includes 81 amino acids, of which 60 (75%) are identical with those of the putative translation product of the corresponding engrailed sequence. These data suggest that Mo-en.1 represents a mouse homolog of a gene of the Drosophila engrailed gene complex. Mo-en.1 has been mapped to chromosome 1, indicating it is not linked to other homeo box sequences that have been mapped in the mouse genome. Analysis of poly(A)+ RNA extracted from teratocarcinoma cells and whole mouse embryos demonstrates that the conserved homeo box region of Mo-en.1 is expressed differentially during mouse embryogenesis.     

The engrailed locus of Drosophila: structural analysis of an embryonic transcript

cDNA clones originating from the engrailed gene of Drosophila have been isolated from recombinant phage libraries that were made using poly(A)+ RNA extracted from early embryos. The DNA sequence of one of these clones includes a homeo box, a 180 bp sequence present in several other Drosophila genes important in formation of body pattern during development. The homeo boxes found in the other Drosophila genes, as well as in cognate sequences from a wide range of segmented animals, including higher vertebrates, are highly conserved. By contrast, the homeo box within the engrailed gene diverges substantially and, unlike the other homeo boxes, is interrupted by an intervening sequence. The engrailed homeo box is located near the 3' end of a 1700 bp open reading frame. If translated, this sequence would produce a protein of unusual composition. We also show that a neighboring gene has a large region with strong homology to engrailed, and that it also contains a homeo box.     

Nanos is the localized posterior determinant in Drosophila

Segmental pattern in the Drosophila embryo is established by two maternal factors localized to the anterior and posterior poles of the egg cell. Here we provide molecular evidence that the localized posterior factor is the RNA of the nanos (nos) gene. nos RNA is localized to the posterior pole of early embryos, and nos protein acts at a distance to direct abdomen formation. Synthetic nos RNA has biological activity identical to that of the posterior pole plasm. Injection of nos RNA rescues the segmentation defect of embryos derived from females mutant for all nine known posterior group genes. Injection of nos RNA into the anterior is able to direct formation of ectopic posterior structures. Our results demonstrate that a localized source of nos RNA is sufficient to specify abdominal segmentation and imply that other posterior group genes are required for localization, stabilization, or distribution of the nos gene product.     

Cloning,expression and immunolocalization pattern of a cinnamyl alcohol dehydrogenase gene from strawberry (Fragaria x ananassa cv. Chandler)

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) catalyses the conversion of p-hydroxy-cinnamaldehydes to the corresponding alcohols and is considered a key enzyme in lignin biosynthesis. By a differential screening of a strawberry (Fragariax ananassa cv. Chandler) fruit specific subtractive cDNA library, a full-length clone corresponding to a cad gene was isolated (Fxacad1). Northern blot and quantitative real time PCR studies indicated that the strawberry Fxacad1 gene is expressed in fruits, runners, leaves, and flowers but not in roots. In addition, the gene presented a differential expression in fruits along the ripening process. Moreover, by screening of a strawberry genomic library a cad gene was isolated (Fxacad2). Similar to that found in other cad genes from higher plants, this strawberry cad gene is structured in five exons and four introns. Southern blot analyses suggest that, probably, a small cad gene family exists in strawberry. RT-PCR studies indicated that only the Fxacad1 gene was expressed in all the fruit ripening stages and vegetative tissues analysed. The Fxacad1 cDNA was expressed in E. coli cells and the corresponding protein was used to raise antibodies against the strawberry CAD polypeptide. The antibodies obtained were used for immunolocalization studies. The results showed that the CAD polypeptide was localized in lignifying cells of all the tissues examined (achenes, fruit receptacles, runners, leaves, pedicels, and flowers). Additionally, the cDNA was also expressed in yeast (Pichia pastoris) as an extracellular protein. The recombinant protein showed activity with the characteristic substrates of CAD enzymes from angiosperms, indicating that the gene cloned corresponds to a CAD protein.     

The Drosophila fragile X protein functions as a negative regulator in the orb autoregulatory pathway

Translational regulation of maternal mRNAs in distinct temporal and spatial patterns underlies many key decisions in developing eggs and embryos. In Drosophila, Orb is responsible for mediating the translational activation of mRNAs localized within the developing oocyte. Orb is a germline-specific RNA binding protein and is one of the founding members of the CPEB family of translational regulators. Here we show that Orb associates with the Drosophila Fragile X Mental Retardation (dFMR1) protein as part of a ribonucleoprotein complex that controls the localized translation of mRNAs in developing egg chambers. One of the key orb regulatory targets is orb mRNA, and this autoregulatory activity is critical for ensuring that Orb protein is expressed at high levels in the oocyte. We show that dFMR1 functions as a negative regulator in the orb autoregulatory circuit, downregulating orb mRNA translation.     

Localization of nanos RNA controls embryonic polarity.

Anterior-posterior polarity of the Drosophila embryo is initiated during oogenesis through differential maternal RNA localization. The RNA of the anterior morphogen bicoid is localized to the anterior pole of the embryo, where bicoid protein controls head and thorax development. The RNA of the posterior morphogen nanos is localized to the posterior pole, where nanos protein is required for abdomen formation. Here we show that the nanos 3' untranslated region, like that of the bicoid RNA, is sufficient for RNA localization. We have used the bicoid RNA localization signal to mislocalize nanos, producing embryos with two sources of nanos protein. Such embryos form two abdomens with mirror image symmetry. Embryos with nanos RNA localized only to the anterior have greater nanos gene activity than embryos with nanos RNA localized posteriorly. We propose a role for RNA localization in regulating nanos activity.     

Structure of the segmentation gene paired and the Drosophila PRD gene set as part of a gene network

The sequence of paired, a pair-rule gene required for segmentation in Drosophila, is presented. A search for genes with domains homologous to the paired gene was initiated and three homologues from a set of 12 were characterized with respect to temporal or spatial expression and sequence homologies. All four are transcribed in early development, one in the oocyte and during cleavage stages in the form of a gradient. In addition to the prd-specific his-pro repeat, some of the 12 genes contain M-repeats and two new types of homeo boxes not detectable by hybridization with the two known classes of homeo boxes. The observed linking of gene sets through combinations of homologies coding for protein domains is consistent with a general network concept of gene action.     

Germ line specific expression of a protein phosphatase Y interacting protein (PPYR1) in Drosophila

PPYR1, the product of the CG15031 gene, was identified as a protein phosphatase Y (PPY) interacting protein in Drosophila melanogaster using a yeast two-hybrid screen. PPYR1 displays a biphasic expression pattern: the maternal protein is abundant in the developing egg chambers and in the early embryos, while the zygotic protein appears later in development and is localized specifically in the testes of the males. The maternal and zygotic gene products differ from each other in their size having apparent molecular masses of 47 and 66 kDa, respectively. The maternal PPYR1 is localized in the cytoplasm of the follicular and nurse cells and is deposited as a ribonucleoprotein complex in the oocyte. In the early embryos, the PPYR1 is distributed evenly, and it gradually diminishes during embryonic development. Zygotic PPYR1 is expressed exclusively in the testes, predominantly in the cytoplasm of the spermatocytes. PPY is localized in the nuclei of the same cells. Our results suggest that PPYR1 has two distinct developmental isoforms: a maternal protein the expression of which is independent of PPY and a zygotic protein which is co-expressed with PPY.     

Two novel LEM-domain proteins are splice products of the annotated Drosophila melanogaster gene CG9424 (Bocksbeutel)

The LEM motif is a sequence of 40-50 amino acids that has been identified in a number of non-related proteins of the inner nuclear membrane including the lamina-associated polypeptides 2 (LAP2), emerin, MAN1 and the Drosophila protein otefin. This evolutionary conserved sequence motif can mediate via the interaction with the small protein BAF the binding of LEM-domain proteins to DNA. Taking advantage of its sequenced genome we analyzed whether Drosophila possesses beside otefin additional genes coding for proteins with a LEM motif. A putative candidate gene was the annotated gene CG9424 which we named Bocksbeutel. Of all putative Drosophila LEM-domain proteins, otefin and Bocksbeutel exhibited the highest similarity in the LEM motif (53% identical amino acids). The Bocksbeutel gene can code for two isoforms of 399 and 351 amino acids that are produced by alternative splicing. In the alpha-isoform a transmembrane domain is localized close to the carboxyterminus. This segment is absent in the shorter beta-isoform. By RT-PCR we could show that in the embryo the mRNA coding for the alpha-isoform and in significantly lower amounts the mRNA coding for the beta-isoform are expressed. When expressed in transfected cells as GFP fusion proteins, the beta-isoform is localized predominantly in the nucleoplasm and the alpha-isoform is targeted to the nuclear envelope, indicating that Bocksbeutel-alpha is localized in the inner nuclear membrane. Bocksbeutel-alpha is the predominant isoform expressed in cells, larvae, and flies. Indirect immunofluorescence with Bocksbeutel-specific antibodies on tissues and cultured cells revealed that Bocksbeutel proteins are localized in the nuclear envelope and in the cytoplasm. By RNA interference we have down-regulated the expression of Bocksbeutel, BAF, otefin, and lamin DmO in Drosophila Kc167 cells. The down-regulation of Bocksbeutel and otefin had no influence on the viability of Kc167 cells and the intracellular localization of all other nuclear and nuclear envelope proteins analyzed. In contrast, when lamin DmO was reduced by RNAi the distribution of Bocksbeutel and otefin in the nuclear envelope of Kc167 cells was significantly altered. We conclude that the two LEM-domain proteins Bocksbeutel and otefin are no limiting components for the maintenance of the nuclear architecture in cultured Drosophila cells at interphase.