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.

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 Calliphora erythrocephala 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.

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.

Genes from two intermoult puffs in Drosophila virilis polytene chromosomes are differentially transcribed during larval development.

Genes from two Drosophila virilis intermoult puffs were isolated by microcloning. From puff 16A on the X-chromosome a 2.9 kb DNA fragment was obtained, which hybridizes with three transcripts. Two of them represent the mRNAs for larval glue proteins. They are found in different abundancies in third larval instar salivary glands, but also in minor amounts in midgut and in fat body. In puff 55E on chromosome III two genes were identified. They are transcribed exclusively in salivary glands during all three larval instars. Therefore, their products must be related to another gland-specific function, which is sustained throughout larval life.

Glue protein genes in Drosophila virilis: their organization, developmental control of transcription and specific mRNA degradation.

The gene Lgp-1, which is localized in the intermoult puff 16A of D. virilis polytene chromosomes, encodes the major larval glue protein Igp-1. The gene consists of two exons interrupted by a short intron. In the 5' flanking region of Lgp-1, we find putative ecdysone receptor binding sites and two proximal conserved sequence motifs which are possibly involved in gene regulation. The amino acid sequence deduced from the DNA sequence reveals a relationship to the 68C glue protein family of D. melanogaster. The size of the Lgp-1 transcripts decreases in late third instar larvae concomitantly with their disappearance. This is caused by deadenylation followed by distinct nucleolytic attacks in the 3' untranslated region. Preliminary data suggest the presence of another glue protein gene in the 16A puff region which is related to the Lgp-1 gene.

Genetic transfer of the pigment bacteriorhodopsin into the eukaryote Schizosaccharomyces pombe.

The gene encoding for bacterio-opsin (bop gene) from Halobacterium halobium has been introduced in a yeast expression vector. After transformation in Schizosaccharomyces pombe, bacterio-opsin (BO) is expressed and was detected by antisera. The precursor protein of BO (pre-BO) is processed by cleavage of amino acids at the N-terminal end as in H. halobium. Addition of the chromophore, retinal, to the culture medium results in a slight purple colour of the yeast cells indicating the in vivo regeneration of BO to bacteriorhodopsin (BR) and its incorporation into membranes. Therefore, in contrast to the expression in E. coli, isolation of the membrane protein and reconstitution in lipid vesicles is not necessary for functional analysis. The kinetics of the ground state signal of the photocycle BR in protoplasts is demonstrated by flash spectroscopy and is comparable to that of the natural system. The present investigation shows for the first time the transfer of an energy converting protein from archaebacteria to eukaryotes by genetic techniques. This is a basis for further studies on membrane biogenesis, genetics, and bioenergetics by analysis of in vivo active mutants.

A non-conserved sequence in the 5'region of the CYH2 intron from Saccharomyces cerevisiae controls splicing efficiency of the pre-mRNA.

The CYH2 gene from Saccharomyces cerevisiae containing one 510 bp intron is spliced inefficiently. We have shown previously that a non-conserved sequence within the intron is responsible for this low splicing efficiency. Using synthetic oligonucleotides comprising the identified region we show in this report that a very short region contains the specificity to act negatively on the splicing efficiency of the CYH2 gene. Furthermore, this sequence influences the splicing efficiency only when it is placed close to the 5' splice site of the gene. Investigations with chimeric CYH2/beta-actin genes show that this sequence acts independent from its natural surroundings. We propose that this sequence might interact with splicing factor(s).

Primary structure of the ribosomal protein gene S6 from Schizosaccharomyces pombe.

We have determined the nucleotide sequence of a ribosomal protein gene which codes for the ribosomal protein S6 (rps6). The sequence analysis revealed that the gene comprises 239 amino acids, giving rise to a basic protein with a molecular weight of 27,502 Da. The product of this gene is the equivalent of the ribosomal protein S10 from Saccharomyces cerevisiae. Northern analyses and S1 mapping of both the 5' and the 3' end of the transcripts of this gene show that it is transcribed into three distinct transcripts with different sizes and heterogeneous termini. In the DNA region flanking the coding sequence, several conserved elements are present that may be involved in the transcription initiation and termination.

Sequence and regulatory responses of a ribosomal protein gene from the fission yeast Schizosaccharomyces pombe.

We have determined the nucleotide sequence and mapped the 5' and 3' termini of a ribosomal protein gene. The gene is transcribed into a RNA molecule of about 770 nt and appears to initiate at multiple sites, as judged by SI nuclease analysis. Gene dosage experiments with a plasmid born gene leads to a proportional increase of the messenger RNA, but not to an overproduction of the protein, suggesting a posttranscriptional control mechanism. However, the heat shock response of this gene indicates that there is also a potential for transcriptional control. Comparison of the 5' flanking region of this gene with the ribosomal protein gene S 6 from Schizosaccharomyces pombe and with ribosomal protein genes from Saccharomyces cerevisiae revealed homologous sequences, which may be involved in the regulation of ribosomal protein genes.

Intron mutations that affect the splicing efficiency of the CYH2 gene of Saccharomyces cerevisiae.

To define the extent of intervening sequences required for efficient splicing of the CYH2 gene in Saccharomyces cerevisiae, we have constructed a series of intron mutations. Artificial intron extensions of more than 300 bp of the natural intron lead to an inhibition of splicing whereas intron deletions lead to a drastic improvement of the splicing efficiency. It is shown that deletion of a 32 bp sequence element within the intron is responsible for this drastic improvement.

The occurrence of two ribosomal ribonucleases depending on growth phase in yeast. Induction of ribonuclease in glucose-starved cells.

There are two latent ribonucleases associated with the 40 S subunits of yeast ribosomes which differ in their digestion products, pH optimum, molecular weight, and in their activity during growth phase. The 3'-nucleotide-producing enzyme is active only in the late logarithmic or stationary growth phase, whereas the ribonuclease which produces 5'-nucleotides is present at all growth phases. The enzymes were separated by affinity chromatography and were partially characterized. By changing growth conditions--i.e. decreasing and increasing the glucose concentration in the medium--the activity of the 3'-ribonuclease could be induced or reduced.

A ribonuclease from yeast associated with the 40 S ribosomal subunit.

1. Autodegradation of yeast ribosomes is due to a 'latent' ribonuclease which is associated with the 40 S ribosomal subunit. 2. The ribonuclease was extracted in the presence of EDTA from ribosomes and purified 118-rold by protamine sulphate precipitation, (NH4)2SO4 fractionation and chromatography on DEAE-cellulose. 3. The optimum pH for this enzyme is 5 to 6.5 while the optimum temperature is 45 to 50 degrees C. Incubation for 10 min at 60 degrees C caused a reduction in enzyme activity of 70%. 4. The ribonuclease has an endonucleolytic activity against rRNA, tRNA, poly(A), poly(U) and poly(C) but does not degrade poly(G) or DNA. It hydrolyzes the homopolymers to nucleoside 3'-phosphates. 5. Zn2+, Mn2+, heparin, glutathione and p-chloromercuribenzoate inhibit the ribonuclease, while Na+, K+, EDTA and sermidine have only little or no effect. 6. It binds tightly to yeast ribosomes but only loosely to ribonuclease-free wheat germ ribosomes. 7. Polyribosomes possess less autodegradation activity than monoribosomes, isolated from the same homogenate.