ChromosomeResearch 1993, 1, 121-126 DNA puff C4 of Bradysiahygida (Diptera- Sciaridae) contains genes unequally amplified and differentially expressed during development P. S. R. Coelho, N. Monesi, J. C. DeAImeida, F. Toledo, G. Buttin & M. L. Pa96-Larson Received 7 April 1993; received in revised form 18 May 1993: Accepted for publication by Herbert Macgregor 20 May 1993 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 frag- ment 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. Key words: Developmental gene amplification, DNA puff, molecular organization. Introduction DNA puffs formed in the polytene chromosomes of Scia- ridae salivary glands at the end of the larval stage have been characterized as sites of differential DNA replication in addition to transcription. Cytological studies on these puffs led to the first indications of gene amplification in somatic cells (Breuer & Pavan 1955). Cloning of cDNAs (Glover et al. 1982) and genomic fragments (Pa~6-Larson et al. 1992) from DNA puffs directly demonstrated the occurrence of genes developmentally amplified and expressed in these chromosomal regions. Besides the chorion genes of Drosophila, DNA puffs of Sciaridae are the only other known case of developmentally amplified genes coding for proteins. However, unlike the case of Drosophila, very little is known about the molecular organ- ization, the products of the amplified genes and the mechanism that underlies amplification in DNA puffs (for review see Lara et al. 1991). Considering the peculiarity of this system that couples replication and transcription in a defined chromosomal site, our laboratory is working on the molecular definition of the DNA puff C4 of Bradysia hygida. In this context, we report here the isolation of a 2.3 kb genomic EcoRI fragment in DNA puff C4 that co- localizes with a 4 kb fragment previously characterized as containing part of a gene amplified and expressed in the salivary gland at the time when this puff expands (Pa~6- Larson et al. 1992). 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 this data we discuss the nature of the DNA puff and the possible way in which amplification is occurring at these sites. Material and methods Salivary glands were obtained from female larvae, pupae and adult flies from a laboratory culture kept at 20°C (Sauaia et al. 1971). The salivary glands comprise three distinct morphological regions, namely $i, $2, and $3, and there are four chromosomes, A, B, C and X, in the salivary gland cells. Figure 1 shows the chromosome segments that in $1 form the DNA puffs C4 and C5, late in the last larval instar, in a time sequence from E3 throughout E7 + 20h (Laicine et al. 1984). The DNA puff C4 mini- library used here was described elsewhere (Pa~6-Larson et al. 1992). P. S. R. Coelho, N. Monesi, J. C. De Almeida and M. L. Paqd-Larson (corresponding author) are at Departamento de Morfologia, Faculdade de Medicina de Ribeirdo Preto, Universidade de S4o Paulo, Ribeirdo Preto, SP 14049-900, Brasil. Fax: (+55) 16 6331586; E-maih MLPLARSO@BRUSP. F. Toledo and G. Buttin are at Unitd de Gdndtique Somatique (URA CNRS 361), Institute Pasteur, 25 rue du Dr Roux, 75724 Paris Cddex 15, France. © 1993 Rapid Communicationsof Oxford Chromosome Research Vol 1 1993 121