Overexpression of kermit/dGIPC is associated with lethality in Drosophila melanogaster

Insertional mutagenesis is an important tool for functional genomics in Drosophila melanogaster. The insertion site in the KG00562 mutant fly line has been mapped to the CG8709 (herein named DmLpin) locus and to the 3’ of kermit (also called dGIPC). This mutant line presents a high lethality rate resulting from a gain of function. To obtain some insight into the biological role of the mutated locus, we have characterized the mutation and its relation to the high mortality of the KG00562 fly line. In this mutant, we did not detect one of the DmLpin transcripts, namely DmLpinK, but we did detect an unusual 2.3-kb mRNA (LpinK-w). Further investigation revealed that the LpinK-w transcript results from an aberrant splicing between the untranslated first exon of DmLpinK and the mini-white marker gene. Lack of DmLpinK or LpinK-w expression does not contribute to lethality, since heterozygous KG00562/Def7860 animals presented lethality rates comparable to those of the wild type. In contrast, the overexpression of kermit was associated with lethality of the KG00562 fly line. Significantly higher levels of kermit were detected in the Malpighian tubules of KG00562/+ flies that presented higher lethality rates than wild-type or KG00562/Def7860 animals, in which the lethality was rescued. In agreement with a recently reported study, our data support the hypothesis that misexpression of kermit/dGIPC could interfere with Drosophila development, with further investigations being needed in this direction.

Overexpression of kermit/dGIPC is associated with lethality in Drosophila melanogaster.

Insertional mutagenesis is an important tool for functional genomics in Drosophila melanogaster. The insertion site in the KG00562 mutant fly line has been mapped to the CG8709 (herein named DmLpin) locus and to the 3' of kermit (also called dGIPC). This mutant line presents a high lethality rate resulting from a gain of function. To obtain some insight into the biological role of the mutated locus, we have characterized the mutation and its relation to the high mortality of the KG00562 fly line. In this mutant, we did not detect one of the DmLpin transcripts, namely DmLpinK, but we did detect an unusual 2.3-kb mRNA (LpinK-w). Further investigation revealed that the LpinK-w transcript results from an aberrant splicing between the untranslated first exon of DmLpinK and the mini-white marker gene. Lack of DmLpinK or LpinK-w expression does not contribute to lethality, since heterozygous KG00562/Def7860 animals presented lethality rates comparable to those of the wild type. In contrast, the overexpression of kermit was associated with lethality of the KG00562 fly line. Significantly higher levels of kermit were detected in the Malpighian tubules of KG00562/+ flies that presented higher lethality rates than wild-type or KG00562/Def7860 animals, in which the lethality was rescued. In agreement with a recently reported study, our data support the hypothesis that misexpression of kermit/dGIPC could interfere with Drosophila development, with further investigations being needed in this direction.

Broad-Complex, E74, and E75 early genes control DNA puff BhC4-1 expression in prepupal salivary glands.

The DNA puff BhC4-1 gene of the sciarid Bradysia hygida is induced in salivary glands prior to the pupal molt as a secondary response to the increase in ecdysone titers. Previous studies demonstrated that the BhC4-1 promoter is activated in transgenic Drosophila melanogaster salivary glands as a late response to the ecdysone peak that triggers metamorphosis, revealing that this aspect of BhC4-1 transcriptional regulation is conserved in the Drosophila background. To identify regulators of BhC4-1 expression, we utilized a candidate gene approach and tested the roles of the ecdysone-induced genes BR-C, E74, and E75. Our results reveal that the BR-C Z3 isoform is essential for BhC4-1-lacZ induction in prepupal salivary glands and constitute the first demonstration of the participation of early genes products on DNA puff genes regulation.

Immunocharacterization of the DNA puff BhC4-1 protein of Bradysia hygida (Diptera: Sciaridae).

The DNA puff BhC4-1 gene is amplified and highly expressed in the salivary gland of Bradysia hygida late larvae. Using affinity-purified polyclonal antibodies we have identified the product of the BhC4-1 gene as a 43 kDa polypeptide which is present in extracts of salivary glands from late fourth instar larvae and in the corresponding gland secretion, but not in glands from earlier stages. We also demonstrate that this protein is produced mainly in the S1 and S3 regions of the salivary gland, where BhC4-1 amplification levels are more pronounced and larger amounts of mRNA are produced. By immunoelectron microscopy the BhC4-1 protein was detected in secretory granules of the S1 and S3 regions, and localized in fibrous structures present in the saliva.

Analysis of the amplification and transcription of the C3-22 gene of Rhynchosciara americana (Diptera: Sciaridae) in transgenic lines of Drosophila melanogaster.

Drosophila melanogaster was transformed with an 18 kb fragment of the C3 DNA puff of Rhynchosciara americana, including the C3-22 gene and the origins of replication that direct amplification. Different tissues and developmental stages of five independent transgenic lines were analyzed by quantitative Southern blot hybridization. No indication was found that the transformed fragment was amplified, strongly suggesting that factors involved in DNA puff amplification have not been conserved in Drosophila. Transcription of the C3-22 gene in the transgenic lines was found to be at a low and constitutive level throughout development. These results indicate that, unlike other DNA puff genes, the factors that regulate the C3-22 gene are not conserved in Drosophila.

Identification of regulatory regions in the DNA puff BhC4-1 promoter.

The mechanisms that control DNA puff BhC4-1 expression in the salivary gland of sciarid late larvae have been shown to be conserved in Drosophila. By analysing Drosophila transformed with constructs carrying progressive deletions of the BhC4-1 promoter fragment (-3314/+40) fused to the lacZ reporter gene we show that the elements required for the correct BhC4-1-lacZ developmental regulation in prepupal salivary glands are contained in a 226 bp fragment (-186/+40). Also, interestingly, this study identified a 67 bp fragment (-253/-187) that activates BhC4-1-lacZ expression specifically in the ring gland.

The control of BhB10-1 gene expression in the salivary gland of Bradysia hygida (Diptera, Sciaridae) is disrupted in vivo by a delayed effect of cycloheximide.

BhB10-1 is an amplified gene present in DNA puff B10. This gene is very active in the salivary gland regions S1 and S3 at the end of the larval development. Two transcripts of this gene, 1.3 and 1.1 kb in size, were detected. A secretory protein, SP23, is the product of BhB10-1. In this work, we present evidence supporting the hypothesis that a biphasic process of mRNA degradation is an important component in the control of BhB10-1 gene expression. The 1.3 kb transcript, by a process of poly(A) tail shortening, is converted to the inactive transcript of 1.1 kb which is detected during and after the period of SP23 expression. Cycloheximide in very low concentration, if applied at a proper time, can disrupt this process leading to extended periods of 1.3 kb RNA detection and SP23 synthesis. A tentative model is proposed to explain this phenomenon.

The induction of DNA puff BhC4-1 gene is a late response to the increase in 20-hydroxyecdysone titers in last instar dipteran larvae.

The characterization of DNA puff BhC4-1 expression was extended and its response to 20-hydroxyecdysone investigated in Bradysia hygida and in transgenic Drosophila carrying the BhC4-1 gene. In both organisms the activation of BhC4-1 in salivary glands occurs at the end of the larval stage coinciding with the peak in ecdysone titers which induces metamorphosis. Injections of 20-hydroxyecdysone into mid-fourth instar larvae of B. hygida show that the induction of BhC4-1 expression, as well as amplification and puff C4 expansion, are late events induced by the hormone. This late response of BhC4-1 expression was also observed in transgenic salivary glands cultivated in the presence of 20-hydroxyecdysone. In vitro studies using transgenic Drosophila indicate that both repressor and activator factors regulate the timing of BhC4-1 expression in salivary glands.

The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome.

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription-PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning.

Mapping of intrinsic bent DNA sites in the upstream region of DNA puff BhC4-1 amplified gene.

We have identified bent DNA sites in the distal and proximal DNA puff BhC4-1 amplified gene promoter region of Bradysia hygida. The 2D modeling of the 3D DNA path and the ENDS ratio values calculated in this promoter region resulted in the identification of ten pronounced bent sites named BhC4B - 9 to + 1. The 1847 bp fragment (- 3697 to - 1850) in relation to the transcription start site shows multiple bending sites, BhC4B - 9 to BhC4B - 4, with periodicity approximately 300 bp. The analysis of the other identified bent region, starting at position - 957, reveals that the BhC4B + 1 bent site colocalizes with the putative BhC4-1 minimal promoter. The sequence analysis of bent site BhC4B - 4 shows a distribution of dA*dT at approximately 10 bp intervals between the middle of each tract, but intervals with more than one turn, approximately 20 bp, two helix turns, were detected in the other bent sites described here. The bent sites BhC4B - 6 and BhC4B - 4, contain two consensus sequences, with 60 bp each. The apparent molecular weight of fragments in the BhC4-1 promoter region were estimated in agarose gels and compared with the data obtained in polyacrylamide gels without and with ethidium bromide. The mobility reduction ratios (R-values) were determined, and a high R-value, 1.80, for a 1215 bp fragment in the distal promoter region and a 1.23 significant R-value for a 662 bp fragment in the proximal segment were found. To further analyze the predicted bent DNA sites in these fragments, the 2D trajectories of the 3D DNA path and other parameters, AT percentage, roll angle, ENDS ratio and DeltaG, were determined. The role of these bent sites in the BhC4-1 transcription regulation is discussed.

The DNA puff BhB10-1 gene is differentially expressed in various tissues of Bradysia hygida late larvae and constitutively transcribed in transgenic Drosophila.

We extended the characterization of the DNA puff BhB10-1 gene of Bradysia hygida by showing that, although its mRNA is detected only at the end of the fourth larval instar, BhB10-1 expression is not restricted to the salivary gland, the tissue in which this gene is amplified. Different amounts of BhB10-1 mRNA were detected in other larval tissues such as gut, Malpighian tubules, fat body, brain and cuticle, suggesting that this gene is expressed differentially in the various tissues analyzed. Analysis of transgenic Drosophila carrying the BhB10-1 transcription unit and flanking sequences revealed that the tested fragment promotes transcription in a constitutive manner. We suggest that either cis-regulatory elements are missing in the transgene or factors that temporally regulate the BhB10-1 gene in B. hygida are not conserved in Drosophila.

The DNA puff BhB10-1 gene is differentially expressed in various tissues of Bradysia hygida late larvae and constitutively transcribed in transgenic Drosophila

We extended the characterization of the DNA puff BhB10-1 gene of Bradysia hygida by showing that, although its mRNA is detected only at the end of the fourth larval instar, BhB10-1 expression is not restricted to the salivary gland, the tissue in which this gene is amplified. Different amounts of BhB10-1 mRNA were detected in other larval tissues such as gut, Malpighian tubules, fat body, brain and cuticle, suggesting that this gene is expressed differentially in the various tissues analyzed. Analysis of transgenic Drosophila carrying the BhB10-1 transcription unit and flanking sequences revealed that the tested fragment promotes transcription in a constitutive manner. We suggest that either cis-regulatory elements are missing in the transgene or factors that temporally regulate the BhB10-1 gene in B. hygida are not conserved in Drosophila

PCR template preparation for capillary DNA sequencing.

Fluorescence-based capillary DNA sequencing has facilitated the early completion of several complex sequencing projects. While capillary systems offer great benefits in terms of ease of use and automation, we find that they are sufficiently different from slab gel separation methodologies, demanding re-examination of the protocols used to generate and use DNA sequencing templates. We have recently initiated a large-scale Human Open Reading Frame EST project involving 30 laboratories feeding 11 MegaBace 1000 capillary sequencers. The group has already produced more than 300,000 valid sequences. The most successful template preparation protocol we have found is described here. We have found that a crucial step is the standardization of the quantity and quality of the templates, which have been achieved by overnight bacterial culture followed by PCR using limiting amounts of primers. Using this protocol, there is no need for post-PCR purification, and the final preparation cost is US $0.09/template. After sequencing 10,848 templates using this protocol, 78% of the reads were accepted (after discarding vectors without inserts and inserts smaller than 100 nucleotides), and 85% of the total number of bases had Phred scores of 15 or above.

Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags.

Transcribed sequences in the human genome can be identified with confidence only by alignment with sequences derived from cDNAs synthesized from naturally occurring mRNAs. We constructed a set of 250,000 cDNAs that represent partial expressed gene sequences and that are biased toward the central coding regions of the resulting transcripts. They are termed ORF expressed sequence tags (ORESTES). The 250,000 ORESTES were assembled into 81,429 contigs. Of these, 1, 181 (1.45%) were found to match sequences in chromosome 22 with at least one ORESTES contig for 162 (65.6%) of the 247 known genes, for 67 (44.6%) of the 150 related genes, and for 45 of the 148 (30.4%) EST-predicted genes on this chromosome. Using a set of stringent criteria to validate our sequences, we identified a further 219 previously unannotated transcribed sequences on chromosome 22. Of these, 171 were in fact also defined by EST or full length cDNA sequences available in GenBank but not utilized in the initial annotation of the first human chromosome sequence. Thus despite representing less than 15% of all expressed human sequences in the public databases at the time of the present analysis, ORESTES sequences defined 48 transcribed sequences on chromosome 22 not defined by other sequences. All of the transcribed sequences defined by ORESTES coincided with DNA regions predicted as encoding exons by genscan. (http://genes.mit.edu/GENSCAN.html).

The DNA puff BhB10-1 gene encodes a glycine-rich protein secreted by the late stage larval salivary glands of Bradysia hygida.

We present the molecular characterization of a gene of Bradysia hygida DNA puff B10 whose temporal expression in the salivary gland correlates with the puff expansion. The transcription unit of this gene, named BhB10-1, was mapped in a 2-kb EcoRI genomic fragment that is amplified in the salivary gland of late fourth instar larvae. Its 1.3-kb transcript undergoes poly-A tail shortening during development, indicating that post-transcriptional controls as well as transcription activation are involved in the temporal regulation of the BhB10-1 gene. Analysis of the deduced amino acid sequence from the cDNA indicates that the BhB10-1 protein is a glycine-rich secretory protein. A BhB10-1-fusion protein expressed in bacteria was used to raise polyclonal antibodies. Using an immunopurified antibody, we identified the product of the DNA puff BhB10-1 gene as a 23-kDa polypeptide that is produced mainly by the salivary gland regions S1 and S3 and is present in the saliva of late larvae. This is the first direct identification of a protein encoded by a DNA puff amplified gene.

The DNA puff gene BhC4-1 of Bradysia hygida is specifically transcribed in early prepupal salivary glands of Drosophila melanogaster.

The BhC4-1 gene of the sciarid Bradysia hygida is located at DNA puff C4 and is amplified and actively transcribed in the salivary gland at the end of the last larval instar. We show here that a 3.6 kb fragment from the upstream region of the BhC4-1 gene is able to drive transcription in transgenic Drosophila specifically in prepupal salivary gland in a temporally regulated manner. The mRNA is present in maximal amounts in prepupae +3 h; in prepupae +9 h the levels of BhC4-1 mRNA decline, and it is no longer detected in pupae +24 h. Taken together these results suggest that most, if not all, of the key promoter regulatory elements were included in the DNA fragments employed to transform Drosophila. Moreover, strong expression of the transgenes implies conservation of the regulatory elements involved, since Drosophila transcription factors appear to recognize B. hygida regulatory DNA sequences. Quantitative Southern blot hybridization indicates that the sequences from DNA puff C4 are not amplified at detectable levels in salivary glands of transgenic prepupae when the BhC4-1 gene is transcribed. Transcription of a DNA puff in the absence of amplification indicates that the induction of these processes involves distinct mechanisms.

Molecular characterization of an 18 kb segment of DNA puff C4 of Bradysia hygida (Diptera, sciaridae).

The data presented here are an extension of the molecular characterization of DNA puff C4 of Bradysia hygida. A cDNA related to a gene amplified in this puff and expressed when puff C4 expands was cloned and sequenced. Analysis of the amino acid sequence deduced from the open reading frame present in the cDNA indicate that the encoded protein is secreted and comprises mostly alpha-helical coiled-coil. An 18 kb genomic segment containing the transcription unit of this gene was also cloned and the structure and expression of the 1.4 kb mRNA was determined. Quantitative slot blot hybridization of DNA complementary to the transcription unit shows that this gene is amplified about 21 times in the salivary gland, confirming data previously obtained. Fragments upstream of the 5' end, and beyond the 3' end, of the gene transcription unit were also analysed and shown to be amplified at least eight and five times, respectively. Based on these data we discuss how amplification could occur at DNA puffs.

Sequence of a cDNA encoding bothropstoxin I, a myotoxin from the venom of Bothrops jararacussu.

With the aim of further understanding the structure/function relationships in the membrane-damaging activity of the Lys49 phospholipase A2 (Lys49-PLA2) sub-family, we used PCR (polymerase chain reaction) on total venom gland cDNAs from Bothrops jararacussu with degenerate oligodeoxyribonucleotides encoding the N- and C-termini of myotoxin II, a Lys49-PLA2 from Bothrops asper. A 350-bp cDNA coding for bothropstoxin I (BtxtxI) was amplified. Sequencing of the amplified fragment shows that BtxtxI has a Lys49, and comparison with the known structure of myotoxin II showed that the amino acids involved in the formation of a novel dimeric structure in this protein were also conserved.