DNA sequences, gene regulation and modular protein evolution in the Drosophila 68C glue gene cluster

The 68C locus of the Drosophila melanogaster polytene chromosomes contains the structural genes for three glue polypeptides (sgs-3, sgs-7 and sgs-8) synthesized in the larval salivary glands during the third larval instar. When the messenger RNAs for the glue polypeptides are being synthesized, the locus is puffed; the puff regresses in response to the steroid hormone ecdysterone. The three 68C glue mRNAs are coded in a gene cluster of less than 5000 base-pairs, and are expressed co-ordinately. In the experiments described here we show that the coordinate expression of these RNAs does not result from amplification of the puff DNA, nor is it associated with puff DNA rearrangement. We also report the nucleotide sequence of 6751 base-pairs of genomic DNA that includes the entire gene cluster, and describe coding and non-coding sequences with possible regulatory roles. In addition, we deduce the amino acid sequences of the primary translation products of the glue mRNAs, and show that the glue proteins form a diverged gene family. The members of the family all contain an amino-terminal hydrophobic block of amino acids, which is absent in the mature, secreted glue proteins, and a cysteine-rich carboxy-terminal module. sgs-3 differs from sgs-7 and sgs-8 by containing a third module between the other two, comprised largely of tandem repeats of the five amino acids Pro-Thr-Thr-Thr-Lys.     

Adjacent chromosomal regions can evolve at very different rates: Evolution of theDrosophila 68C glue gene cluster

Summary The 68C puff is a highly transcribed region of theDrosophila melanogaster salivary gland polytene chromosomes. Three different classes of messenger RNA originate in a 5000-bp region in the puff; each class is translated to one of the salivary gland glue proteins sgs-3, sgs-7, or sgs-8. These messenger RNA classes are coordinately controlled, with each RNA appearing in the third larval instar and disappearing at the time of puparium formation. Their disappearance is initiated by the action of the steroid hormone ecdysterone. In the work reported here, we studied evolution of this hormone-regulated gene cluster in themelanogaster species subgroup ofDrosophila. Genome blot hybridization experiments showed that five other species of this subgroup have DNA sequences that hybridize toD. melanogaster 68C sequences, and that these sequences are divided into a highly conserved region, which does not contain the glue genes, and an extraordinarily diverged region, which does. Molecular cloning of this DNA fromD. simulans, D. erecta, D. yakuba, andD. teissieri confirmed the division of the region into a slowly and a rapidly evolving protion, and also showed that the rapidly evolving region of each species codes for third instar larval salivary gland RNAs homologous to theD. melanogaster glue mRNAs. The highly conserved region is at least 13,000 bp long, and is not known to code for any RNAs.     

Drosophila: the genetics of two major larval proteins.

A series of irradiation-induced deficiencies covering 62 polytene chromosome bands in chromosome arm 3L of Drosophila melanogaster includes the loci of two abundant developmentally regulated larval proteins. The structural gene for larval serum protein 2 (LSP 2) lies at 68E3 or 4, and that for salivary glue secretion protein 3 between 68A8 and 68C11, coincident with a major intermoult puff active in the salivary gland at the time of glue synthesis. The structural genes for esterase 6 and four visible recessive loci lie within the same region.     

IN SITU DEMONSTRATION OF DNA HYBRIDIZING WITH CHROMOSOMAL AND NUCLEAR SAP RNA IN CHIRONOMUS TENTANS

Cytological hybridization combined with microdissection of Chironomus tentans salivary gland cells was used to locate DNA complementary to newly synthesized RNA from chromosomes and nuclear sap and from a single chromosomal puff, the Balbiani ring 2 (BR 2). Salivary glands were incubated with tritiated nucleosides. The labeled RNA was extracted from microdissected nuclei and hybridized to denatured squash preparations of salivary gland cells under conditions which primarily allow repeated sequences to interact. The bound RNA, resistant to ribonuclease treatment, was detected radioautographically. It was found that BR 2 RNA hybridizes specifically with the BR 2 region of chromosome IV. Nuclear sap RNA was fractionated into high and low molecular-weight RNA; the former hybridizes with the BR 2 region of chromosome IV, the latter in a diffuse distribution over the whole chromosome set. RNA from chromosome I hybridizes diffusely with all chromosomes. Nucleolar RNA hybridizes specifically with the nucleolar organizers, contained in chromosomes II and III. It is concluded that the BR 2 region of chromosome IV contains repeated DNA sequences and that nuclear sap contains BR 2 RNA.     

A trans-acting regulatory product necessary for expression of the Drosophila melanogaster 68C glue gene cluster

The mutation I(1)npr-1 is located at cytological location 2B5 on the X chromosome in Drosophila melanogaster. We have found that this mutation causes absence of the normal product of the 2B5 locus and that it has the following phenotypes: the 68C glue puff on the third chromosome does not regress when mutant salivary glands are cultured in the presence of ecdysterone; the three 68C glue protein mRNAs are not synthesized; and a transformed Drosophila strain carrying both a normal resident 68C Sgs-3 gene and an introduced functional Sgs-3 gene with only a few kb of flanking sequences expresses neither Sgs-3 RNA if the I(1)npr-1 mutation is crossed into the stock. Thus the normal product of the I(1)npr11 gene is required for regression of the 68C puff, and the I(1)npr-1 gene product allows expression of the Sgs-3 gene by interacting, either directly or indirectly, with DNA sequences near this glue protein gene.     

Sequences sufficient for correct regulation of Sgs-3 lie close to or within the gene.

The Drosophila melanogaster 68C chromosomal locus is the site of a prominent polytene chromosome puff that harbors the genes Sgs-3, Sgs-7 and Sgs-8. These genes code for proteins that are part of the salivary glue that Drosophila larvae secrete as a means of fixing themselves to an external substrate for the duration of the pre-pupal and pupal period. The 68C glue genes are regulated by the steroid hormone ecdysterone, with the hormone required for both initiation and cessation of gene expression during the third larval instar. Previous work has defined sequences sufficient for expression of abundant levels of Sgs-3 mRNA at the correct time and in the correct tissue. We show here that sequences sufficient for normal tissue- and stage-specific accumulation of Sgs-3 RNA, but adequate only for low levels of expression, lie within 130 bp of the 5' end of the gene, or within the gene.     

Characterization of a Drosophila repeat mapping at the early-ecdysone puff 63F and present in many eucaryotic genomes

In this report we describe the nucleotide sequence of a 229 bp tandemly repeated sequence that hybridizes in situ to the early-ecdysone puff site 63F on salivary gland polytene chromosome 3 (Izquierdo, M., Arribas, C. and Alonso, C. (1981) Chromosoma 83, 363–366). Restriction analysis of genomic clones from the region indicates the existence of a minimum of 15 copies tandemly arranged at two separated sites, within the 63F puff region. The 229 basic units include conserved and variable segments and have two possible open-reading frames. A slight variation in the length of basic repeats was also observed. Some fly-stocks from Drosophila melanogaster contain particular RNA size classes complementary to the 63F repeat, while other RNAs remain constant in all stocks analyzed. A 5 kb fragment containing the repeat is present in many eucaryotic living beings, including plants and humans.     

An expandable gene that encodes a Drosophila glue protein is not expressed in variants lacking remote upstream sequences

The Drosophila melanogaster gene Sgs4 encodes one of the glue polypeptides, sgs-4, synthesized in the larval salivary gland. We have examined the structure and expression of Sgs4 in five strains that produce abundant amounts of sgs-4 and its mRNA and in four that do not. The nonproducers include three Japanese strains that accumulate trace amounts of mRNA and one strain, BER-1, that contains no detectable Sgs4 RNA. Sgs4 carries a tandem array of repeated 21 bp elements within its coding sequence. The number of elements per array varies, causing considerable differences in the lengths of Sgs4 and its mRNA among the strains. These differences in length are not correlated with differences in mRNA abundance; rather, the low or zero abundance in nonproducers correlates with the loss of DNA upstream from the gene. The Japanese nonproducers carry a 52 bp deletion 305 bp upstream from the 5′ end of Sgs4, and BER-1 carries a 95 bp deletion 392 bp upstream. Curiously, each deletion encompasses one or more of the salivary-gland-specific DNAase I-hypersensitive sites which are known to flank the Sgs4 gene.     

Photoinduced bonding of endogenous ecdysterone to salivary gland chromosomes of Chironomus tentans

Endogenous ecdysterone has been bonded to chromosomal loci by irradiation of Ch. tentans salivary glands. The hormone has been localized on the polytene chromosomes by indirect immunofluorescence microscopy. Hormone binding to chromosomes is stage-specific. Seven chromosomal loci could be identified which specifically bound hormone in larval salivary glands, and 21 chromosomal loci which specifically bound hormone in prepupal salivary glands. All puffs that have been described by Clever (1961) as being inducible by ecdysterone have been found to contain irreversibly bound ecdysterone in prepupal salivary gland chromosomes. A small number of puff sites in larval salivary gland chromosomes exhibited varying amounts of bound ecdysterone, (as judged by fluorescence intensity) most notably 117B and Balbiani rings 1 and 3 on chromosome IV. In addition to stage specific binding sites, there were many others showing equal binding of the hormone in both, larval and prepupal, stages of development. — Fluorescence intensities (reflecting the amount of bonded hormone) at puff sites along the tip section of the prepupal salivary gland chromosome arm IR have been computed indicating that differences between fluorescence intensities of different puffs can be expressed as multiples of a basic fluorescence intensity. Thus, the amount of fluorescence intensity (bonded hormone) in the various puffs may be quantized. — The data indicate that in Ch. tentans salivary glands ecdysterone acts, at the chromosomal level. The development of larvae into prepupae generates more puff sites and more hormone binding. This is discussed in the light of current models of hormone-receptor function.