Horst Kress

Biochemical and ontogenetic aspects of glycoprotein synthesis in Drosophila virilis salivary glands.

Abstract After SDS-polyacrylamide gel electrophoresis two glycosylated glue proteins are found in the salivary glands of Drosophila virilis late third instar larvae. Synthesis of larval glue protein 1 occurs in three successive steps: at first a precursor protein with a molecular weight of about 138,000 daltons is formed. This is modified by two subsequent steps of glycosylation, the first one involving hexosamine, the second one hexoses. Studies with tunicamycin and β-hydroxynorvaline suggest that glycosylation occurs at threonine residues. Larval glue protein 2 has a molecular weight of approximately 15,000 daltons and is weakly glycosylated. The synthesis of glue proteins is stage specific. It starts at about 120 hr after oviposition and attains its maximal rate about 20 hr later. At this time the larvae leave the food. Between ecdysone release and puparium formation (146–151 hr) larval glue protein synthesis is terminated. Throughout the prepupal stage a different set of glycoproteins is synthesized. Thus, the larval-prepupal transition is accompanied by the reprogramming of glycoprotein synthesis in salivary glands. The secretion products formed during the two developmental stages seem to possess different biological functions.

Glucosamine metabolism in Drosophila salivary glands. Separation of metabolites and some characteristics of three enzymes involved.

The conversion of fructose 6-phosphate to mucopolysaccharide precursors was studied in extracts of Drosophila virilis salivary glands. 1. Methods for chromatography of sugar phosphates were adapted and modified to allow routine separation and quantitation of radioactivity of the metabolites from milligram amounts of tissue. Anion exchange chromatography was performed on Dowex 1-X8 employing steps of increasing ammonium formate. Final isolation of each compound was achieved by various thin-layer chromatographic systems. 2. Data obtained by isotope incorporation into glucosamine 6-phosphate compare well with results of the Morgan-Elson colorimetric assay for amino-sugars. 3. Glucosaminephosphate isomerase (glutamine-forming) (EC 5.3.1.19) in gland extracts has a Km of 0.35 mM for fructose 6-phosphate, and of 0.25 mM for glutamine. The enzyme is inhibited at glutamine concentrations exceeding 1 mM and by UDP-N-acetylglucosamine (50% at 0.6 mM). Feedback inhibition by UDP-N-acetylglucosamine is enhanced by AMP and by glucose 6-phosphate. 4. Glucosaminephosphate isomerase (EC 5.3.1.10) has a twenty fold lower affinity towards fructose 6-phosphate (Km = 6.0 mM) compared to the glutamine-forming isomerase. Km (NH+4) is 7.4 mM. In the presence of 20 mM glucose 6-phosphate, the pH optimum is shifted from 6.6 to 7.4, and V increased by a factor of 2.5. Furthermore, the affinity is approximately doubled for both substrates. 5. Glucosamine acetyltransferase (EC 2.3.1.3) has a Km of 2 mM for glucoseamine 6-phosphate. Its activity is not rate-limiting in salivary glands. Since N-acetylglucosamine 6-phosphate and 1-phosphate were found near equilibrium concentrations, acetylglucosamine phosphomutase (EC 2.7.5.2) must also be present in the extracts.

Evolution of 5S rRNA gene families in Drosophila.

In Drosophila virilis, the three clusters of 5S rRNA genes on chromosome 5 comprise two different gene families (B and C), which differ profoundly in the organization of their spacer sequences. While C-type genes, which are found in two of the clusters, exhibit a true repetitive character, the B-type genes of the third cluster are each embedded in completely different genomic environments. Southern blots of genomic DNA of different D. virilis subspecies, D. hydei and D. melanogaster probed with 5S rRNA gene spacer and coding sequences demonstrate the specificity of C-type sequences for the D. virilis species group. The comparative analysis of flanking sequences of 5S rRNA genes of D. virilis, members of the D. melanogaster species subgroup and of the blowfly Calliphoraerythrocephala reveals the existence of conserved sequence motifs both in the 5′ upstream and 3′ downstream flanking regions. Their possible roles in the control of expression and processing of the 5S rRNA precursor molecule are discussed.

Molecular cloning of the Drosophila virilis larval glue protein gene Lgp-3 and its comparative analysis with other Drosophila glue protein genes

DNA comprising the larval glue protein gene Lgp-3 of Drosophila virilis was isolated from a lambda genomic and a cDNA library. The transcription start site, two polyadenylation sites and the boundaries of the single intron were determined. An open reading frame encoding 379 amino acids was found. At the DNA level the presence of similar introns and three conserved sequence motifs in the proximal promoters suggest that the gene is related to those of the D. virilis lgp-1 and the D. melanogaster sgs-3, -7 and -8 glue proteins. Their common ancestry is also substantiated by the comparisons of the deduced amino acid sequences and the profiles of hydropathic indices, which reveal striking similarities of the N- and C-termini and of the central repeat domains, although the lengths and the primary structures of the proteins diverged considerably during 60 million years of separate evolution of the two Drosophila species.

Glucosamine metabolism in Drosophila virilis salivary glands: Ontogenetic changes of enzyme activities and metabolite synthesis☆

Abstract In Drosophila virilis salivary glands the in vitro activities of enzymes involved in the glucosamine pathway were examined during the third larval instar and in the prepupa. While glutamine-fructose-6-phosphate aminotransferase (EC 5.3.1.19) becomes inactive at the time of puparium formation, glucosamine-6-phosphate isomerase (EC 5.3.1.10) and glucosamine-6-phosphate N-acetyltransferase (EC 2.3.1.3) show maximal activities in the prepupal gland. The activity of UDP-N-acetylglucosamine pyrophosphorylase (EC 2.7.7.23) may also decrease prior to puparium formation. Incubation of larval and prepupal glands in medium containing [3H]glucose + [14C]-uridine or [14C]glucosamine and subsequent separation of intermediates of the glucosamine pathway by chromatographic procedures reveal that the capacity of the glands to incorporate the isotopes into these intermediates decreases significantly at the time of puparium formation. The results suggest that in D. virilis salivary glands the formation of aminosugars is mainly controlled by the activities of the two enzymes glutamine-fructose-6-phosphate aminotransferase and UDP-N-acetylglucosamine pyrophosphorylase.

Sequence, molecular organization and products of the Drosophila virilis homologs of the D. melanogaster nested genes lethal(2)tumorous imaginal discs [l(2)tid] and lethal(2)neighbour of tid [l(2)not]

In this study, we describe the isolation of the Drosophila virilis (Dvir) 6201-bp genomic fragment homologous to a 7047-bp genomic region of D. melanogaster (Dmel) that harbors the nested genes lethal(2) tumorous imaginal discs (l(2)tid), lethal(2) neighbour of tid (l(2)not) and lethal(2) relative of tid (l(2)rot). The isolated fragment, which maps at the cytogenetic position 50A5 on chromosome 5, carries the Dvir homologs of the Dmel genes l(2)tid and l(2)not. In both cases, the interspecific comparison of the determined sequences reveals a high homology regarding the protein coding regions and a high degree of evolutionary divergence concerning the intronic parts of the genes. In the two distantly related species, the particular gene within gene arrangement of the two genes is conserved, namely, Dvir tid is located in the intron of Dvir not, on the non-coding DNA strand. Interestingly, the Dvir homolog of the Dmel l(2)rot gene residing in the l(2)not intron on its coding strand, opposite l(2)tid, is not present in the 6201-bp genomic fragment. The protein predicted from the Dvir tid sequence, Dvir Tid58, exhibits 76.5% identity with the putative Tid56 protein of Dmel. The putative Dvir Not58 protein shows 71% identity with its Dmel homolog Not56. The developmental transcript and protein patterns, as well as the characteristics of the protein products encoded by the genes Dvir tid and Dvir not are similar to those identified for their Dmel homologs.

Drosophila salivary glands exhibit a regional reprogramming of gene expression during the third larval instar

In D. virilis salivary glands transcripts of two early gland protein genes, Egp-1 and Egp-2, which encode putative secretory proteins, accumulate in all cells from the first to mid third larval instar. Subsequently the transcripts disappear from the cytoplasm of the corpus cells, but not from their nuclei, where they accumulate at the chromosomal site of their synthesis. In the collum cells, however, Egp-transcripts continue to be detectable in the cytoplasm until the end of larval life. In the salivary glands of transgenic D. melanogaster the presence of a Egp-1/lacZ fusion protein shows the same regional shift as the cytoplasmic Egp-transcripts in D. virilis. We predict that the expression of Egp-genes is related to an early secretory function of the larval salivary glands which is executed by all cells during earlier larval stages but becomes restricted exclusively to the collum cells during the third larval instar.

Drosophila glue protein gene expression: a proposal for its ecdysone-dependent developmental control

The primary targets of steroid hormones are genes. For the ecdysone-controlled genes of Drosophila larval glue proteins proximal and distal control elements were identified by mutagenesis and sequence comparison. Their presence is required for the correct stage- and tissue-specific expression of these genes. The supposed function of these elements is described in a working model.

Polytene chromosomes: A general model for the eucaryotic interphase state

Abstract The euchromatic structures of insect polytene chromosomes represent an amplified version of the chromomeric organization in diploid chromatin. As such, they are an excellent model system for studying structure/function relationships of eucaryotic interphase chromosomes. Polytenization is accomplished by replication patterns that are different for euchromatic and heterochromatic chromatin and also seem to depend on the functional state of chromatin. In Diptera, polytene chromosomes are characterized by genetically determined discontinuities of DNA packing in bands and interbands that are modified by functional demands. The cytological visualization of proteins that are associated with the compaction and decondensation of chromatin, allows the analysis of the temporal and spatial dynamics of DNA/protein interactions in the context of structure, transcription, and the processing of RNA. Ribonucleoprotein particles may be followed on their way via the nuclear matrix through the pores of the nuclear envelope to the cytoplasmic compartment. Thus, polytene nuclei provide unique opportunities for studying the flow of genetic information from the site of storage to the site of action, i.e. from gene to phenotype.

Ecdysone-controlled mRNA stability in Drosophila salivary glands: deadenylation-independent degradation of larval glue protein gene message during the larval/prepupal transition

20-Hydroxyecdysone induces poly(A) shortening and the subsequent degradation of transcripts encoding the larval glue protein LGP-1 in Drosophila virilis late third larval instar salivary glands. Degradation concurs with the transient increase of ribonucleolytic activities in the gland cells. In vitro nuclease assays using crude cytoplasmic extracts of ecdysone-treated salivary glands demonstrate degradation to be deadenylation-independent and that the induced ribonucleolytic activities initiate the degradation of the Lgp-1 transcripts in putative single-stranded loop regions. The independence of degradation from deadenylation is also found in vivo in transformed D. melanogaster carrying a modified Lgp-1 gene.