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

Abstract

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.

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

“…A Physical map of an 18 kb segment from DNA puff C4. The thick line represents a restriction map of the segment and the arrow above indicates the direction of BhC4-1 transcription (Monesi et al 1995). B Diagram of the A3 construct.…”

“…Initially a 3.6 kb XbaI-EcoRI fragment, containing 293 bp of the 5© end of the BhC4-1 transcription unit and 3.3 kb of upstream sequences, was excised from clone pBC4 ) and inserted into the P-element vector pCaSpeR 4 (Pirrotta 1988), generating the construct pCaSpeR 4±3.6-X/E. In a second cloning step a 7.5 kb EcoRI-EcoRI genomic fragment, containing the 3© end of the BhC4-1 transcription unit in addition to 6 kb of downstream sequences, was excised from phage ldBC4±2C (Monesi et al 1995) and cloned into the construct pCaSpeR 4±3.6-X/E, which had been previously digested with EcoRI and dephosphorylated. The recombinant plasmids from the second cloning step were digested with different restriction enzymes to ensure that the orientation of the 7.5 kb fragment, in relation to the 3.6 kb fragment, corresponded to the organization of these fragments in the B. hygida genome.…”

“…), following the instructions of the manufacturer. The BhC4-1 probe consisted of a 706 bp EcoRI-HindIII fragment from the BhC4-1 coding sequence (Monesi et al 1995) cloned into the plasmid pBluescript II KS+. After digestion with EcoRI, in vitro transcription was performed in the presence of [a-32 P]UTP, using the MAXIscript T7/T3 kit (Ambion, Austin, Tex.).…”

“…To date, the highest amplification levels in the analyzed genomic segments have been detected in the regions containing the transcription unit of those genes whose transcription accompanies the formation of the puff (Coelho et al 1993;Monesi et al1995;Stocker et al 1996). On the basis of these observations, and the results of early autoradiographic and cytophotometric studies, it has been suggested that amplification in DNA puffs is a consequence of repeated reinitiation of replication at the puff loci Monesi et al 1995;Stocker et al 1996). This hypothesis was further supported by the demonstration of active bidirectional replication origins in the vicinity of the promoter region of the II/9±1 gene of DNA puff II/9A of S. coprophila (Liang et al1993) and of the gene mapped to DNA puff C3 of R. americana (Yokosawa 1995).…”

“…The amplification levels estimated for these cloned regions are 16-and 32-fold in DNA puffs C8 and C3 of R. americana (Stocker et al 1996), 18-and 17-fold in DNA puffs II/2B and II/9A of S. coprophila , and 21-and 10-fold in DNA puffs C4 and B10 of B. hygida (Fontes et al 1992;Paçó-Larson et al 1992). The characterization of amplified regions has been extended to 60 kb and 50 kb in DNA puffs C8 and C3 of R. americana (Stocker et al 1996;Penalva et al 1997), 35 kb in DNA puff II/9A of S. coprophila and 18 kb in DNA puff C4 of B. hygida (Monesi et al 1995). To date, the highest amplification levels in the analyzed genomic segments have been detected in the regions containing the transcription unit of those genes whose transcription accompanies the formation of the puff (Coelho et al 1993;Monesi et al1995;Stocker et al 1996).…”