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M L Paçó-Larson

Publications and source records attributed to M L Paçó-Larson.

15 recordsLinked to original sources
PubMed2006

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.

Animals↗
PubMed2003

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.

Animals↗
PubMed2003

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.

Animals↗
PubMed2002

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.

Animals↗
PubMed2002

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.

Animals↗
PubMed2001

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.

Animals↗
PubMed2001

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.

Polymerase Chain Reaction↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed1998

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.

Animals↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1993

DNA puff C4 of Bradysia hygida (Diptera: Sciaridae) contains genes unequally amplified and differentially expressed during development.

We report here the isolation and characterization of a 2.3 kb genomic EcoRI fragment that co-localizes in the DNA puff C4 of Bradysia hygida with a 4 kb EcoRI fragment previously characterized as containing part of a gene amplified and expressed in the salivary gland at the time when puff C4 expands. Verification of the relative amount of DNA complementary to these two genomic fragments shows that they are unequally amplified in the salivary gland. The fragment containing part of the gene expressed when puff C4 expands amplifies about eight times more than the 2.3 kb fragment. This 2.3 kb fragment also carries sequences complementary to RNA species present in the gland in a period when puff C4 has already receded. Based on these data we discuss the nature of the DNA puff and the possible way in which amplification is occurring at these sites.

Animals↗
PubMed1992

Cloning of a B10 DNA puff sequence developmentally amplified and expressed in the salivary gland of Bradysia hygida.

A recombinant clone carrying a 2-kb fragment was isolated from a mini-library of the B10 DNA puff of Bradysia hygida. This fragment was amplified in the salivary gland during the period of DNA puff formation. Amplification started when DNA puff anlage was formed and continued to increase, reaching a maximum of about 10-fold 28 h later. Northern blot hybridization experiments showed that this 2-kb fragment was complementary to two RNA species of about 1.3 kb and 1.1 kb, which are developmentally regulated in the salivary gland. Maximum amounts of these messages were present when the B10 puff is fully expanded.

Animals↗
PubMed1989

Hemoglobin H disease caused by two gene deletions.

1. A white Brazilian woman not of Asian origin was found to have Hb H disease of moderate severity. 2. Gene mapping demonstrated that the disease was caused by the association of two abnormal alpha-globin gene clusters on chromosome 16: one with a deletion which removed the two functional alpha genes and the other carrying the 3.7-kb rightward deletion, which leaves a single functional alpha gene. 3. These data illustrate the necessity for systematic molecular approaches to the diagnosis of this heterogeneous group of diseases.

Blotting, Southern↗
PubMedDate not supplied

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.

Animals↗