Locus 67B of Drosophila melanogaster contains seven, not four, closely related heat shock genes.

The four small hsp genes of Drosophila melanogaster as well as three genes regulated during development (genes 1, 2 and 3) are localized at the chromosomal locus 67B. The four small hsp genes share strong sequence homologies between themselves which were detected here by cross-hybridization. Under the same stringency conditions, each of the genes 1, 2 and 3 hybridize to some of the small hsp genes. By DNA sequencing of gene 1, the homology was localized within the same two regions already conserved between the small hsp genes: a central region of 83 amino acids, homologous with the mammalian alpha crystallin and the first 15 N-terminal amino acids. The transcriptional inducibility of the genes 1, 2 and 3 was also compared with that of the four small hsp genes during various stages of Drosophila development at either the normal growth temperature or after a heat shock. We confirm previous reports on the developmental patterns of all seven genes and find moreover that genes 1, 2 and 3 are heat-shock inducible at any of the stages tested. We conclude that genes 1, 2 and 3 are also heat shock genes. Therefore, the locus 67B contains seven, not four, small heat shock genes.

Nucleotide sequences responsible for the thermal inducibility of the Drosophila small heat-shock protein genes in monkey COS cells.

The promoter regions of the Drosophila melanogaster small heat-shock protein genes have been analysed in order to localize those sequences responsible for their heat-shock transcriptional inducibility. Different lengths of the 5' DNA sequences of these four genes were each fused individually to the Herpes simplex virus thymidine kinase (HSV-tk) transcription unit. These hybrid genes were constructed in a simian virus 40 recombinant vector for transfection in permissive monkey COS cells and tested for their heat-shock inducibility. The hsp22/HSV-tk and hsp26/HSV-tk fusion genes were found to be heat-inducible at 43 degrees C, giving rise to correctly initiated transcripts, but transcriptionally quiescent at 37 degrees C (control temperature). The hsp23 and hsp27 fusion gene constructs are, however, not heat-shock-inducible; no transcripts being detectable from hsp27/HSV-tk constructs at either temperature and all hsp23/HSV-tk clones being faithfully but constitutively expressed at low levels at both temperatures. By testing a series of 5' deletion mutants in hsp22/HSV-tk, a homologous sequence located adjacent to the TATA box in both the hsp22 and hsp26 genes was identified as being responsible for their heat-shock activation. This control element corresponds to the Pelham "consensus sequence", previously described for the Drosophila hsp70 genes. The possible modes of transcriptional induction of all four genes are discussed.

Nucleotide sequence analysis of the Drosophila small heat shock gene cluster at locus 67B.

The four small heat shock protein genes of Drosophila melanogaster clustered at cytological locus 67B have been characterized by DNA sequencing. Over 6250 nucleotides, covering the 5', protein-coding and 3' regions of these genes have been determined together with their predicted amino acid sequences. Each gene possesses characteristic eukaryotic 5' and 3' sequence elements and a single uninterrupted protein-coding region. The four encoded polypeptides of 19,700, 20,600, 23,000 and 23,600 Mr share a homologous stretch of 108 amino acid residues, representing 51 to 62% of their lengths. This region is flanked by sequences of dissimilar length and amino acid composition, located mainly at the amino-terminal end, but also at the extreme carboxyl termini of these proteins. The first 14 amino acids exhibit a small degree of homology, both amongst themselves and with some signal peptides and a transmembrane protein. Investigation of the hydrophilic/hydrophobic characteristics of the four polypeptides revealed, within the conserved 108 amino acid stretch, the presence of an alpha-helical region of very prominent local hydrophilicity, which probably represents a surface structural domain common to each protein. Sequence analysis with respect to transcription initiation and termination and possible regulatory signals is discussed together with some structural predictions for the four proteins.