A novel Drosophila Minute locus encodes ribosomal protein S13.

Minutes comprise > 50 phenotypically similar Drosophila mutations believed to affect ribosomal protein genes. Common traits of the Minute phenotype are short and thin bristles, slow development, and recessive lethality. To further investigate the proposed Minute to ribosomal protein correspondence, loss-of-function Minute mutations were induced by P-element mutagenesis. Here, we report a previously undescribed Minute locus that maps to 32A on chromosome 2L; this Minute allele is named P{lacW}M(2)32A1 and the gene M(2)32A. Flies heterozygous for P{lacW}M(2)32A1 have a medium Minute phenotype. The gene interrupted by the P-element insertion was cloned. Sequence analyses revealed that it encodes the Drosophila homologue of eukaryotic ribosomal protein S13. It is a single-copy gene and the level of RPS13 transcript is reduced to approximately 50% in P(lacW) M(2)32A1 heterozygotes. Both transcript level and phenotype are restored to wild type by remobilizing the P-element, demonstrating that the mutation is caused by insertion of the P-element construct. These results further strengthen the notion that Minutes encode ribosomal proteins and demonstrate that P-element mutagenesis is a fruitful approach to use in these studies.

A Drosophila third chromosome Minute locus encodes a ribosomal protein.

Minutes (M) are a group of over 50 phenotypically similar Drosophila mutations widely believed to affect ribosomal protein genes. This report describes the characterization of the P element-induced M(3)95A(Plac92) mutation [allelic to M(3)95A]. This mutation can be reversed by the mobilization of the P element, demonstrating that the mutation is caused by insertion of this transposable element. The gene interrupted by insertion of the P element was cloned by use of inverse polymerase chain reaction. Nucleotide sequence analysis revealed a 70-75% identity to the human and rat ribosomal protein S3 genes, and to the Xenopus ribosomal protein S1a gene. At the amino acid level, the overall identity is approximately 78% for all three species. This is only the second time that a Minute has been demonstrated to encode a ribosomal protein.

Ribosomal protein insufficiency and the minute syndrome in Drosophila: a dose-response relationship.

Minutes comprise > 50 phenotypically similar mutations scattered throughout the genome of Drosophila, many of which are identified as mutations in ribosomal protein (rp) genes. Common traits of the Minute phenotype are short and thin bristles, slow development, and recessive lethality. By mobilizing a P element inserted in the 5' UTR of M(3)95A, the gene encoding ribosomal protein S3 (RPS3), we have generated two homozygous viable heteroalleles that are partial revertants with respect to the Minute phenotype. Molecular characterization revealed both alleles to be imprecise excisions, leaving 40 and 110 bp, respectively, at the P-element insertion site. The weaker allele (40 bp insert) is associated with a approximately 15% decrease in RPS3 mRNA abundance and displays a moderate Minute phenotype. In the stronger allele (110 bp insert) RPS3 mRNA levels are reduced by approximately 60%, resulting in an extreme Minute phenotype that includes many morphological abnormalities as well as sterility in both males and females due to disruption of early gametogenesis. The results show that there is a correlation between reduced RPS3 mRNA levels and the severity of the Minute phenotype, in which faulty differentiation of somatic tissues and arrest of gametogenesis represent the extreme case. That heteroalleles in M(3)95A can mimic the phenotypic variations that exist between different Minute/rp-gene mutations strongly suggests that all phenotypes primarily are caused by reductions in maximum protein synthesis rates, but that the sensitivity for reduced levels of the individual rp-gene products is different.

Differentiation of the myofibrils and the intermediate filament system during postnatal development of the rat heart.

The differentiation of the myofibrils and the intermediate filament system during postnatal development of the rat heart has been investigated. Several aspects of some of the structural proteins, that means the intermediate filament subunit skeletin, myosin, and the myofibrillar M-line proteins MM-creatine kinase and myomesin have been studied by using gel electrophoresis as well as enzyme and immunohistochemical techniques in combination with electron microscopy of both plastic and cryosectional material. We show that marked changes take place in the organization of the intermediate filament system and in the contractile apparatus, both in atria and in ventricles of the rat heart during postnatal development. In the newborn rats no dense myofibrillar M-bands were present in the M-region and the sarcomeric bands were irregular while in the four-week-old rats dense M-bands composed of a set of five crossbridges interconnecting the thick filaments were present. The sarcomeric bands were now regular. These observations are related to the presence of different isomyosins in the atria and in the ventricles of the newborn and the four-week-old rats, to the observation that MM-creatine kinase was only present in the M-region in the four-week old rats and to the physiological maturation of the heart.

The ribosomal proteins of drosophila melanogaster. IV. Characterization by two-dimensional gel electrophoresis of the ribosomal proteins from nine postembryonic developmental stages.

The ribosomal proteins from nine postembryonic developmental stages of Drosophila melanogaster were analyzed by two-dimensional gel electrophoresis. The ribosomal protein patterns thus obtained constitute reliable control patterns against which any potential mutant can be compared. Furthermore, the results provide increased evidence for a qualitative and quantitative change in the protein composition of the total population of ribosomes which occurs mainly during the third larval instar. Two acidic proteins were found, which may be homologous with rat liver ribosomal proteins L40/L41 and E. coli proteins L7/L12.

The ribosomal proteins of Drosophila melanogaster. V. Analysis by two-dimensional gel electrophoresis of the ribosomal proteins of the temperature-sensitive lethal allele of suppressor of forked, l(1) su(f)ts67g: a putative ribosomal protein mutant.

The possibility that the ribosomes of a temperature-sensitive lethal allele of suppressor of forked, l(1)su(f)ts67g, contain a mutated protein was studied by two-dimensional gel electrophoresis. The results from these analyses revealed no differences in the ribosomal proteins between the mutant and the wild-type strain Ks. It was found, however, that the transition from larval to adult ribosomal protein complement, which occurs mainly during the third instar in Käs, takes place during puparium formation in the mutant at 25 degrees C while it appears to be fatally delayed at 30 degrees C. Thus mutant larvae shifted up to 30 degrees C at 70 hours after oviposition failed to pupate, but reached the third instar and showed an adult ribosomal protein pattern after 4 days. Also, the ribosome content in these larvae was found to be significantly lower compared with late third instar larvae. It was furthermore found that larvae collected within 50 hours of transfer back to 25 degrees C, after a 5 day, 30 degrees C treatment, showed a reversion to a ribosomal protein pattern identical with that of Käs late third instar larvae. The results suggest that the biosynthesis of imaginal ribosomes in l(1)su(f)ts67g is seriously impaired at 30 degrees C and less so at 25 degrees C. Further studies are necessary, however, in order to understand the exact nature of this mutant, which may turn out to be a useful tool in studies on the biosynthesis of ribosomes in D. melanogaster.

Evolution of the dec-1 eggshell locus in Drosophila. II. Intraspecific DNA sequence analysis reveals length mutations in a repetitive region in D. melanogaster.

The dec-1 eggshell gene in Drosophila melanogaster encodes follicle cell proteins required for proper eggshell assembly. As shown by Southern and Northern analyses the dec-1 gene occurs in four alleles (Fc1-4) among wild-type strains. Its second exon has a distinct feature in the form of 12 repeats with 78-91 nucleotides; the first five show nearly 100% homology. DNA sequence comparison of the repeated region of the alleles revealed that the length polymorphisms are caused by changes in the numbers of the first five repeats. The results suggest that the alleles have been generated by unequal intragenic crossing-over and/or slippage during DNA replication and that the allelic length variants have arisen independently. The possibility that the most common allele, FC1, has a selective advantage over the other alleles is discussed.

Evolution of the dec-1 eggshell locus in Drosophila. I. Restriction site mapping and limited sequence comparison in the melanogaster species subgroup.

We have analyzed approximately 18 kb of DNA in and upstream of the defective chorion-1 (dec-1) locus of the eight known species of the melanogaster species subgroup of Drosophila. The restriction maps of D. simulans, D. mauritiana, D. sechellia, D. erecta, and D. orena are shown to have basically the restriction map of D. melanogaster, whereas the maps of D. teissieri and D. yakuba were more difficult to align. However, the basic amount of DNA and sequence arrangement appear to have been conserved in these species. A small deletion of varying length (65-200 bp) is found in a repeated sequence of the central transcribed region of D. melanogaster, D. simulans, and D. erecta. Restriction site mapping indicated that the dec-1 gene is highly conserved in the melanogaster species subgroup. However, sequence comparison revealed that the amount of nucleotide and amino acid substitution in the repeated region is much larger than in the 5' translated region. The 5' flanking region showed noticeable restriction site polymorphisms between species. Based on calculations from the restriction maps a dendrogram was derived that supports earlier published phylogenetic relationships within the melanogaster species subgroup except that the erecta-orena pair is placed closer to the melanogaster complex than to D. teissieri and D. yakuba.

Characterization of a novel Minute-locus in Drosophila melanogaster: a putative ribosomal protein gene.

We describe a novel Minute locus, M(1)7C, on the X-chromosome of Drosophila melanogaster. Heterozygous deficient females have most, if not all, of the Minute features (short and fine bristles, rough and somewhat larger eyes, thin-textured wings, missing aristae, affected antennae, delayed development, reduced fertility, and decreased viability). Both Minute and non-Minute adult progeny from Minute mothers suffer from Minute maternal effects such as abdominal segmentation defects, fused tergites, and missing or defective legs and halteres. Using a plasmid clone from region 7C5-9, which harbours the D. melanogaster ribosomal protein gene RPS14, we have found that the accumulation of a single transcript of approximately 650 b is extremely reduced in Minute larvae in comparison with wild-type. We have localized the RPS14 gene to approximately 28 kbp distal from the singed locus. The results suggest that M(1)7C and RPS14 may be the same gene.

Steroid regulation of glue protein genes in Drosophila melanogaster.

Transcript accumulation of the 3C glue protein gene Sgs-4 was induced in cultured salivary glands of Drosophila third instar larvae by supplementing the culture medium with 20-OH-ecdysone. The salivary glands were isolated from hormone-deficient larvae of the temperature-sensitive mutant l(1)su(f)ts67g, which were shifted from permissive (25 degrees C) to restrictive temperature (30 degrees C) at 60 h after oviposition. At the permissive temperature the glue protein are expressed during the latter half of the third instar. At the restrictive temperature there is no detectable or an extremely reduced accumulation of the 3C glue protein gene transcripts in these larvae. Induction of transcript accumulation was demonstrated by increased amounts of glue gene RNAs in the 20-OH-ecdysone supplemented salivary glands. Maximum accumulation was reached within 1 h after supplementation. The induction of accumulation was inhibited by a concentration of cycloheximide that repressed total protein synthesis, suggesting that 20-OH-ecdysone acts indirectly on the 3C glue gene by inducing synthesis of a protein(s) required transcript accumulation. We also show that there is a more rapid disappearance of 3C transcripts from salivary glands cultured in the presence of 20-OH-ecdysone than from glands cultured in its absence. This hormone-induced disappearance is, in contrast to the 68C transcripts, not inhibited by cycloheximide.

Evolution of the dec-1 eggshell locus in Drosophila. III. Sequence comparisons of the simulans complex repeated domain reveal non-concerted evolution.

The X-linked female sterile locus dec-1 (defective chorion-1) was examined in the closely related species D. simulans, D. mauritiana, and D. sechellia (the simulans complex). This locus encodes important eggshell proteins produced in the follicle cells during stages 9 and 12 of oogenesis. In D. melanogaster four variant protein forms have been found, differing in 2-3 kDa each. The variation is due to deletions of 1, 2, or 3 units of a 5-times repeated sequence (78 bp long) of the central coding region. The same type of deletions were found in two variants of D. simulans; in this species, however, the maximum number of repeats observed so far is four. The island species D. mauritiana and D. sechellia both have the repeat sequence repeated three times. Sequence comparisons revealed that the repeats in the simulans complex have been less homogenised by the forces of concerted evolution than the repeats in D. melanogaster. Two domains of the repetitive region that evolve at different rates and are subject to different mechanisms of DNA turnover were also defined.

Cloning and characterization of variable-sized gypsy mobile elements in Drosophila melanogaster.

A cosmid genomic library from a known gypsy-induced forked mutation, f1, was screened by 32P-labeled gypsy transposable element. Of more than 250 positive clones we randomly selected 21 for in situ hybridization to wild-type polytene chromosomes. Two clones hybridized to region 15F on the X-chromosome, the cytological position of forked. A third clone hybridized to at least 17 sites on the chromosomes indicating the presence of repetitive sequences in the gypsy flanking DNA. All clones labeled the centromeric regions heavily. Ten clones, including the two hybridizing at 15F, were chosen for further analysis, and restriction mapping allowed us to place them into three groups: (1) full-length, (2) slightly diverging, and (3) highly diverging gypsy elements. Group (2) is missing the XbaI site in both their long terminal repeats (LTRs) as well as the middle HindIII site; four of these gypsy elements also have a approximately 100-bp deletion at the 5' LTR. The group (3) gypsy transposons are missing one LTR and also have highly diverging DNA sequences. The restriction analyses further imply that most of these different gypsy elements are present in more than one copy in the genome of the f1 stock used in this study. The results raise intriguing questions regarding the significance of transposable elements in evolution and biological functions.

Content of histone H1 and histone phosphorylation in relation to the higher order structures of chromatin in Drosophila.

The amount of histone H1 relative to core histones has been determined in three Drosophila species (D. melanogaster, D. texana and D. virilis) in chromatin from several tissues differing in chromatin structure and genetic activity. Low levels of H1 were found in relatively undifferentiated, early embryos as well as in a line of cultured cells. In late embryos the content of H1 was highest in D. virilis which possesses larger amounts of and a partially more compacted constitutive heterochromatin than the two other species. Polytene chromatin from larval salivary glands showed increased levels of H1 compared with diploid chromatin and the degree of phosphorylation of this histone was relatively low. The degree of phosphorylation of H2A was found to be drastically reduced in polytene as compared with diploid embryonic chromatin, which parallels the extensive underreplication of constitutive heterochromatin. Also, in diploid chromatin a qualitative correlation was observed between the relative amounts of heterochromatin and the levels of H2A phosphorylation. These findings suggest a connection between H2A phosphorylation and heavy compaction of interphase chromatin.

The Drosophila ribosomal protein L14-encoding gene, identified by a novel Minute mutation in a dense cluster of previously undescribed genes in cytogenetic region 66D.

The Minute phenotype results from mutations at > 50 loci scattered throughout the genome of Drosophila. Common traits of the Minute phenotype are short and thin bristles, slow development, and recessive lethality. Here, we report a novel P-element induced Minute mutation, P¿lacW¿ M(3)66D1, that maps to region 66D on chromosome 3L. Flies heterozygous for P¿lacW¿ M(3)66D1 have a strong Minute phenotype. Molecular characterisation of the chromosomal region revealed three previously undescribed Drosophila genes clustered within a 5-kb genomic fragment. Two of the genes have significant sequence homology to genes for the mammalian ribosomal proteins L14 and RD, respectively, and share a joint 240-bp promoter region harbouring the P-element insert. Quantitative Northern blot analyses showed the mutation to affect RPL14 mRNA levels only. Interestingly, the reduction in abundance of RPL14 mRNA is not constitutive, indicating that the promoter function abolished by the inserted P-element is utilised with different efficiencies in different developmental situations. Remobilisation of the P element produced wild-type flies with normal levels of RPL14 mRNA, demonstrating that the mutant phenotype is caused by the insertion. P¿lacW¿ M(3)66D1 joins a growing list of Minute mutations associated with ribosomal protein-haploinsufficiency.

Evolutionary conservation of synaptosome-associated protein 25 kDa (SNAP-25) shown by Drosophila and Torpedo cDNA clones.

The neuron-specific proteins SNAP-25 (synaptosome-associated protein 25 kDa), synaptobrevin and syntaxin, are localized to presynaptic terminals in mammals and have been found to associate with proteins involved in vesicle docking and membrane fusion. We describe here SNAP-25 cDNA clones from the fruit fly Drosophila melanogaster and the ray Torpedo marmorata. In situ hybridization showed that SNAP-25 mRNA is exclusively found in brain and ganglia in Drosophila with a pattern suggesting expression in most neurons. The Drosophila and Torpedo proteins show 61 and 81% amino acid identity to mouse SNAP-25, a degree of conservation similar to that previously reported for synaptobrevin. None of the SNAP-25 sequences has a membrane-spanning region, but all contain a cluster of cysteine residues that can be palmitoylated for membrane attachment. SNAP-25 displays sequence similarity to syntaxin A and B. These data show that SNAP-25 and synaptobrevin, which are both implicated in vesicle docking and/or membrane fusion, have both been highly conserved during evolution. This supports the existence of a basic molecular machinery for synaptic vesicle docking in vertebrate and invertebrate synapses.