Comparative analysis of zygotic developmental genes in Rhodnius prolixus genome shows conserved features on the tracheal developmental pathway.

Most of the in-depth studies on insect developmental genetic have been carried out in the fruit fly Drosophila melanogaster, an holometabolous insect, so much more still remains to be studied in hemimetabolous insects. Having Rhodnius prolixus sequenced genome available, we search for orthologue genes of zygotic signaling pathways, segmentation, and tracheogenesis in the R. prolixus genome and in three species of Triatoma genus transcriptomes, concluding that there is a high level of gene conservation. We also study the function of two genes required for tracheal system development in D. melanogaster - R. prolixus orthologues: trachealess (Rp-trh) and empty spiracles (Rp-ems). From that we see that Rp-trh is required for early tracheal development since Rp-trh RNAi shows that the primary tracheal branches fail to form. On the other hand, Rp-ems is implied in the proper formation of the posterior tracheal branches, in a similar way to D. melanogaster. These results represent the initial characterization of the genes involved in the tracheal development of an hemimetabolous insect building a bridge between the current genomic era and V. Wigglesworth's classical studies on insects' respiratory system physiology.

Neuropeptide precursor gene discovery in the Chagas disease vector Rhodnius prolixus.

We show a straightforward workflow combining homology search in Rhodnius prolixus genome sequence with cloning by rapid amplification of cDNA ends and mass spectrometry. We have identified 32 genes and their transcripts that encode a number of neuropeptide precursors leading to 194 putative peptides. We validated by mass spectrometry 82 of those predicted neuropeptides in the brain of R. prolixus to achieve the first comprehensive genomic, transcriptomic and neuropeptidomic analysis of an insect disease vector. Comparisons of available insect neuropeptide sequences revealed that the R. prolixus genome contains most of the conserved neuropeptides in insects, many of them displaying specific features at the sequence level. Some gene families reported here are identified for the first time in the order Hemiptera, a highly biodiverse group of insects that includes many human, animal and plant disease agents.

Different modes of translation for hid, grim and sickle mRNAs in Drosophila.

Protein synthesis is inhibited during apoptosis. However, the translation of many mRNAs still proceeds driven by internal ribosome entry sites (IRESs). Here we show that the 5'UTR of hid and grim mRNAs promote translation of uncapped-mRNA reporters in cell-free embryonic extracts and that hid and grim mRNA 5'UTRs drive IRES-mediated translation. The translation of capped-reporters proceeds in the presence of cap competitor and in extracts where cap-dependent translation is impaired. We show that the endogenous hid and grim mRNAs are present in polysomes of heat-shocked embryos, indicating that cap recognition is not required for translation. In contrast, sickle mRNA is translated in a cap-dependent manner in all these assays. Our results show that IRES-dependent initiation may play a role in the translation of Drosophila proapoptotic genes and suggest a variety of regulatory pathways.

Aromatic amino acids are critical for stability of the bicoid homeodomain.

The Drosophila Bicoid (Bcd) protein plays a dual role as a transcription and translation factor dependent on the unique DNA and RNA binding properties of the homeodomain (HD). We have used circular dichroism and fluorescence spectroscopy to probe the structure and stability of the Bcd-HD, for which a high resolution structure is not yet available. The fluorescence from the single tryptophan residue in the HD (Trp-48) is strongly quenched in the native state but is dramatically enhanced ( approximately 20-fold) upon denaturation. Similar results were obtained with the Ultrabithorax HD (Ubx-HD), suggesting that the unusual tryptophan fluorescence may be a general phenomenon of HD proteins. We have used site-directed mutagenesis to explore the role of aromatic acids in the structure of the Bcd-HD and to evaluate the proposal that interactions between the strictly conserved Trp residue in HDs and nearby aromatic residues are responsible for the fluorescence quenching in the native state. We determined that both Trp-48 and Phe-8 in the N-terminal region of the HD are individually necessary for structural stability of the Bcd-HD, the latter most likely as a factor coordinating the orientation of the N-terminal helix I and the recognition helix for efficient binding to a DNA target.

Arenavirus nucleocapsid protein displays a transcriptional antitermination activity in vivo.

RNA polymerase pausing and transcriptional antitermination regulates gene activity in several systems. In arenavirus infected cells the switch from transcription to replication is subjected to a hairpin-dependent termination and requires protein synthesis to bypass this signal. The transcriptional antitermination control by Junín virus nucleocapsid protein N, has been demonstrated in vivo by infecting BHK-21 cells expressing this viral protein in the presence of translation inhibitors. This is the first demonstration in vivo of a transcriptional antitermination control in arenavirus-infected cells.

Homeodomain position 54 specifies transcriptional versus translational control by Bicoid.

Bicoid (BCD), the anterior determinant of Drosophila, controls embryonic gene expression by transcriptional activation and translational repression. Both functions require the homeodomain (HD), which recognizes DNA motifs at target gene enhancers and a specific sequence interval in the 3' untranslated region of caudal (cad) mRNA. Here we show that the BCD HD is a nucleic acid-binding unit. Its helix III contains an arginine-rich motif (ARM), similar to the RNA-binding domain of the HIV-1 protein REV, needed for both RNA and DNA recognition. Replacement of arginine 54, within this motif, alters the RNA but not the DNA binding properties of the HD. Corresponding BCD mutants fail to repress cad mRNA translation, whereas the transcriptional target genes are still activated.

Sequence interval within the PEST motif of Bicoid is important for translational repression of caudal mRNA in the anterior region of the Drosophila embryo.

The Drosophila body organizer Bicoid (Bcd) is a maternal homeodomain protein. It forms a concentration gradient along the longitudinal axis of the preblastoderm embryo and activates early zygotic segmentation genes in a threshold-dependent fashion. In addition, Bcd acts as a translational repressor of maternal caudal (cad) mRNA in the anterior region of the embryo. This process involves a distinct Bcd-binding region (BBR) in the 3' untranslated region (UTR) of cad mRNA. Using cotransfection assays, we found that Bcd represses translation in a cap-dependent manner. Bcd-dependent translational repression involves a portion of the PEST motif of Bcd, a conserved protein motif best known for its function in protein degradation. Rescue experiments with Bcd-deficient embryos expressing transgene-derived Bcd mutants indicate that amino acid replacements within the C-terminal portion of the PEST motif prevent translational repression of cad mRNA but allow for Bcd-dependent transcriptional activation. Thus, Bcd contains separable protein domains for transcriptional and translational regulation of target genes. Maternally-derived cad protein in the anterior region of embryos interferes with head morphogenesis, showing that cad mRNA suppression by Bcd is an important control event during early Drosophila embryogenesis.

Cooperative DNA-binding by Bicoid provides a mechanism for threshold-dependent gene activation in the Drosophila embryo.

The Bicoid morphogen directs pattern formation along the anterior-posterior (A-P) axis of the Drosophila embryo. Bicoid is distributed in a concentration gradient that decreases exponentially from the anterior pole, however, it transcribes target genes such as hunchback in a step-function-like pattern; the expression domain is uniform and has a sharply defined posterior boundary. A 'gradient-affinity' model proposed to explain Bicoid action states that (i) cooperative gene activation by Bicoid generates the sharp on/off switch for target gene transcription and (ii) target genes with different affinities for Bicoid are expressed at different positions along the A-P axis. Using an in vivo yeast assay and in vitro methods, we show that Bicoid binds DNA with pairwise cooperativity; Bicoid bound to a strong site helps Bicoid bind to a weak site. These results support the first aspect of the model, providing a mechanism by which Bicoid generates sharp boundaries of gene expression. However, contrary to the second aspect of the model, we find no significant difference between the affinity of Bicoid for the anterior gene hunchback and the posterior gene knirps. We propose, instead, that the arrangement of Bicoids bound to the target gene presents a unique signature to the transcription machinery that, in combination with overall affinity, regulates the extent of gene transcription along the A-P axis.

Activation of posterior pair-rule stripe expression in response to maternal caudal and zygotic knirps activities.

Drosophila pair-rule gene expression, in an array of seven evenly spaced stripes along the anterior-posterior axis of the blastoderm embryo, is controlled by distinct cis-acting stripe elements. In the anterior region, such elements mediate transcriptional activation in response to the maternal concentration gradient of the anterior determinant BICOID and repression by spatially distinct activities of zygotic gap genes. In the posterior region, activation of hairy stripe 6 has been shown to depend on the activity of the gap gene knirps, suggesting that posterior stripe expression is exclusively controlled by zygotic regulators. Here we show that the zygotic activation of hairy stripe 6 expression is preceded by activation in response to maternal caudal activity. Thus, transcriptional activation of posterior stripe expression is likely to be controlled by maternal and zygotic factors as has been observed for anterior stripes. The results suggest that activation and the expression level mediated by the hairy stripe 6-element depend on the number of activator binding sites, likely to involve additive rather than synergistic interactions. We found an identical transacting factor requirement for hairy stripe 6 and 7 expression. The arrangement of the corresponding binding sites for the common factors involved in the control of the two stripes share a high degree of similarity, but some of the factors exert opposite regulatory functions within the two enhancer elements.

Mechanism and Bicoid-dependent control of hairy stripe 7 expression in the posterior region of the Drosophila embryo.

Pair-rule gene hairy (h) expression in seven evenly spaced stripes, along the longitudinal axis of the Drosophila blastoderm embryo, is mediated by a modular array of separate stripe enhancer elements. The minimal enhancer element, which generates reporter gene expression in place of the most posterior h stripe 7 (h7-element), contains a dense array of binding sites for factors providing the trans-acting control of h stripe 7 expression as revealed by genetic analyses. The h7-element mediates position-dependent gene expression by sensing region-specific combinations and concentrations of both the maternal homeodomain transcriptional activators, Caudal and Bicoid, and of transcriptional repressors encoded by locally expressed zygotic gap genes. Caudal and Bicoid, which form complementing concentration gradients along the longitudinal axis of the embryo, function as redundant activators, indicating that the anterior determinant Bicoid is able to activate gene expression in the most posterior region of the embryo. The spatial limits of the h stripe-7 domain are brought about by the local activities of repressors which prevent activation. The results suggest that the gradients of Bicoid and Caudal combine their activities to activate segmentation genes along the entire axis of the embryo.

From gradients to stripes in Drosophila embryogenesis: filling in the gaps.

Pattern formation along the anterior-posterior axis of the Drosophila embryo is organized by asymmetrically distributed maternal transcription factors. They initiate a cascade of spatially restricted and interacting zygotic gene activities that provide a molecular blueprint of the larval body at blastoderm stage. The key players in the pattern forming process have been identified. Recent progress has begun to reveal the mechanisms by which coherent positional information of maternal origin becomes transferred into serially repeated zygotic gene expression domains reflecting the metameric body plan of the larva.

Gene regulation in the Drosophila embryo.

Pattern formation in Drosophila depends on hierarchical interactions between the maternal and zygotic gene activities which subdivide the embryo into increasingly smaller metameric units along the anterior posterior axis. Here we describe those genes that encode the transcription factors which control precisely the expression of subordinate transcription factors in time and space. This regulation operates through the protein-protein interactions between transcription factors bound to the cis-acting enhancers, which eventually determine the frequency of transcription initiation by polymerase II. Our data show that taking into account the multiple transcriptional activators and repressors that bind to a typical enhancer element, it is likely that the regulation of gene expression in a given cell is defined by their concentration-dependent interplay which directs target gene expression in a position-dependent fashion.

RNA binding and translational suppression by bicoid.

The anterior determinant bicoid (bcd) of Drosophila is a homeodomain protein. It forms an anterior-to-posterior gradient in the embryo and activates, in a concentration-dependent manner, several zygotic segmentation genes during blastoderm formation. Its posterior counterpart, the homeodomain transcription factor caudal (cad), forms a concentration gradient in the opposite direction, emanating from evenly distributed messenger RNA in the egg. In embryos lacking bcd activity as a result of mutation, the cad gradient fails to form and cad becomes evenly distributed throughout the embryo. This suggests that bcd may act in the region-specific control of cad mRNA translation. Here we report that bcd binds through its homeodomain to cad mRNA in vitro, and exerts translational control through a bcd-binding region of cad mRNA.

Activation of posterior gap gene expression in the Drosophila blastoderm.

The process of body prepatterning during Drosophila blastoderm formation relies on the localized activities of zygotic segmentation genes, which are controlled by asymmetrically distributed maternal determinants. The anterior determinant bicoid, a homeodomain transcription factor, forms an anterior-to-posterior concentration gradient. It interacts with the maternal transcription factor hunchback to activate the anterior zygotic patterning genes, including the central gap gene Krüppel (Kr). In contrast, the posterior maternal system does not provide such a decisive transcription factor, but rather prevents the repressor hunchback from acting in the posterior half so that the gap genes giant (gt) and knirps (kni) are activated by an as yet unknown transcription factor. Here we show that caudal, a conserved homeodomain protein that forms a posterior-to-anterior concentration gradient, and the anterior determinant bicoid cooperate to form a partly redundant activator system in the posterior region of the embryo.

Molecular organization of Junin virus S RNA: complete nucleotide sequence, relationship with other members of the Arenaviridae and unusual secondary structures.

In this study, overlapping cDNA clones covering the entire S RNA molecule of Junin virus, an arenavirus that causes Argentine haemorrhagic fever, were generated. The complete sequence of this 3400 nucleotide RNA was determined using the dideoxynucleotide chain termination method. The nucleocapsid protein (N) and the glycoprotein precursor (GPC) genes were identified as two non-overlapping open reading frames of opposite polarity, encoding primary translation products of 564 and 481 amino acids, respectively. Intracellular processing of the latter yields the glycoproteins found in the viral envelope. Comparison of the Junin virus N protein with the homologous proteins of other arenaviruses indicated that amino acid sequences are conserved, the identity ranging from 46 to 76%. The N-terminal half of GPC exhibits an even higher degree of conservation (54 to 82%), whereas the C-terminal half is less conserved (21 to 50%). In all comparisons the highest level of amino acid sequence identity was seen when Junin virus and Tacaribe virus sequences were aligned. The nucleotide sequence at the 5' end of Junin virus S RNA is not identical to that determined of the other sequenced arenaviruses. However, it is complementary to the 3'-terminal sequences and may form a very stable panhandle structure (delta G-242.7 kJ/mol) involving the complete non-coding regions upstream from both the N and GPC genes. In addition, a distinct secondary structure was identified in the intergenic region, downstream from the coding sequences; Junin virus S RNA shows a potential secondary structure consisting of two hairpin loops (delta G -163.2 and -239.3 kJ/mol) instead of the single hairpin loop that is usually found in other arenaviruses. The analysis of the arenavirus S RNA nucleotide sequences and their encoded products is discussed in relation to structure and function.

Patterns of transient expression of the arenavirus nucleocapsid protein gene in transfected cells.

The cloned genes for the nucleocapsid proteins N of Junín and LCM (lymphocytic choriomeningitis) arenaviruses were inserted into the SV40-derived expression vector designated pKG4. When BHK-21 (baby hamster kidney fibroblasts) and CV-1 (African green monkey kidney fibroblasts) cell lines were transfected using these constructions, the transient expression yielded a polypeptide that could not be distinguished either by size nor by immunoreactivity from the N protein synthesized during the viral infection. The immunofluorescence analysis showed a pattern of intracellular localization similar to that observed in virus infected cells, i.e. varying from a diffuse cytoplasmic staining to granules, either distributed throughout the cytoplasm or concentrated in the perinuclear region. The association of the N protein with basophilic granules is similar to that observed in the cytopathic effect caused by arenaviruses, and could be related to the physicochemical properties of this polypeptide containing numerous basic amino acid sequences, that would allow for the interaction with cellular RNAs.

Patterns of transient expression of the arenavirus nucleocapsid protein gene in transfected cells.

The cloned genes for the nucleocapsid proteins N of Junín and LCM (lymphocytic choriomeningitis) arenaviruses were inserted into the SV40-derived expression vector designated pKG4. When BHK-21 (baby hamster kidney fibroblasts) and CV-1 (African green monkey kidney fibroblasts) cell lines were transfected using these constructions, the transient expression yielded a polypeptide that could not be distinguished either by size nor by immunoreactivity from the N protein synthesized during the viral infection. The immunofluorescence analysis showed a pattern of intracellular localization similar to that observed in virus infected cells, i.e. varying from a diffuse cytoplasmic staining to granules, either distributed throughout the cytoplasm or concentrated in the perinuclear region. The association of the N protein with basophilic granules is similar to that observed in the cytopathic effect caused by arenaviruses, and could be related to the physicochemical properties of this polypeptide containing numerous basic amino acid sequences, that would allow for the interaction with cellular RNAs.