Cytoplasmic dynein intermediate-chain isoforms with different targeting properties created by tissue-specific alternative splicing.

The intermediate chains (ICs) are the subunits of the cytoplasmic dynein that provide binding of the complex to cargo organelles through interaction of their N termini with dynactin. We present evidence that in Drosophila, the IC subunits are represented by at least 10 structural isoforms, created by the alternative splicing of transcripts from a unique Cdic gene. The splicing pattern is tissue specific. A constitutive set of four IC isoforms is expressed in all tissues tested; in addition, tissue-specific isoforms are found in the ovaries and nervous tissue. The structural variations between isoforms are limited to the N terminus of the IC molecule, where the interaction with dynactin takes place. This suggests differences in the dynactin-mediated organelle binding by IC isoforms. Accordingly, when transiently expressed in Drosophila Schneider-3 cells, the IC isoforms differ in their intracellular targeting properties from each other. A mechanism is proposed for the regulation of dynein binding to organelles through the changes in the content of the IC isoform pool.

Molecular analysis of a novel Drosophila diacylglycerol kinase, DGKepsilon.

Diacylglycerol kinase plays a central role in the metabolism of diacylglycerol by converting diacylglycerol into phosphatidic acid thus initiating resynthesis of phosphatidylinositols. Diacylglycerol is a known second messenger reversibly activating protein kinase C. In addition, diacylglycerol is a potential precursor for polyunsaturated fatty acids. We describe the identification and molecular analysis of a novel type III Drosophila diacylglycerol kinase isoform, DGKepsilon. Drosophila DGKepsilon is mapped to the cytological position 49C1-3. DGKepsilon mRNA is 1.9 kb in length and is broadly distributed throughout development in different cells, primordia and organs, including testes. In embryogenesis, the transcripts are enriched in the cells, which are in S-phase or undergoing endoreplication. Comparison of the Drosophila DGKepsilon with the human homologue revealed that the first zinc finger-like motif is specific for the type III isoform. Although the testis-specific diacylglycerol kinase activity is dependent upon the dose of DGKepsilon gene, the deletion of DGKepsilon does not modulate the total cellular diacylglycerol level. In spite of a proposed key role of diacylglycerol kinase in termination of the diacylglycerol signal, overexpression of a DGKepsilon transgene in flies under the control of a yeast upstream activating sequence promoter does not disrupt normal development in Drosophila.

Regena (Rga), a Drosophila homolog of the global negative transcriptional regulator CDC36 (NOT2) from yeast, modifies gene expression and suppresses position effect variegation.

A mutation in Regena (Rga) was isolated in screens for modifiers of white eye color gene expression. The reduction in the level of the Rga product results in a complex modulation of white mRNA both positively and negatively, depending on the developmental stage. In addition to white, Rga also affects the expression of several other tested genes, with one of them, Vinculin, being regulated in a strong sex-specific manner. Rga was cloned by transposon tagging. Its predicted product lacks any recognized nucleic acid-binding motif but is homologous to a global negative transcriptional regulator, CDC36 (NOT2), from yeast. Rga also acts as a suppressor of position effect variegation, suggesting that a possible function of Rga could be mediation of an interaction between chromatin proteins and the transcriptional complex.

The oxen gene of Drosophila encodes a homolog of subunit 9 of yeast ubiquinol-cytochrome c oxidoreductase complex: evidence for modulation of gene expression in response to mitochondrial activity.

A P-element insertion in the oxen gene, ox(1), has been isolated in a search for modifiers of white gene expression. The mutation preferentially exerts a negative dosage effect upon the expression of three genes encoding ABC transporters involved in pigment precursor transport, white, brown, and scarlet. A precise excision of the P element reverts the mutant phenotype. Five different transcription units were identified around the insertion site. To distinguish a transcript responsible for the mutant phenotype, a set of deletions within the oxen region was generated. Analysis of gene expression within the oxen region in the case of deletions as well as generation of transgenic flies allowed us to identify the transcript responsible for oxen function. It encodes a 6.6-kD homolog of mitochondrial ubiquinol cytochrome c oxidoreductase (QCR9), subunit 9 of the bc(1) complex in yeast. In addition to white, brown, and scarlet, oxen regulates the expression of three of seven tested genes. Thus, our data provide additional evidence for a cellular response to changes in mitochondrial function. The oxen mutation provides a model for the genetic analysis in multicellular organisms of the effect of mitochondrial activity on nuclear gene expression.

Structure of the Drosophila melanogaster annexin X gene.

The annexin X gene was cloned in the P1 recombinant phage carrying a genomic sequence of approximately 70 kb long. This DNA fragment encompasses at least two annexin X copies and several 7.8-kb tandem units represented by an anonymous sequence fused to the 3' truncated part of the annexin X gene. The proteins of annexin family contain a variable amino-terminal domain and a core domain; the latter includes four structurally conserved repeats that presumably arose as a result of duplications. The annexin X gene of Drosophila is about 2 kb long and contains four exons. Exon 1 encodes four amino-terminal amino acids, exon 2 encodes the remaining part of the amino-terminal domain and the three conserved repeats, and exon 3 and exon 4 encode the fourth repeat. The positions of introns 2 and 3 are strictly conserved with respect to both the amino acid position and codon phase as compared to introns 10 and 12 of the fourth repeat in vertebrate annexin genes. We propose the existence of a primordial annexin coding structure comprising at least two introns whose duplications during evolution have been followed by the loss of ancient introns in the first three repeats of Drosophila and vertebrates. Acquisition of new introns in vertebrates is supposed taking into account that exon borders are not found at homologous locations in four repeats of a given vertebrate annexin. Transcription of the annexin gene was detected in embryonic cell cultures. No profound effects of ecdysterone on the annexin X message content in cell cultures were observed.

Krüppel homolog (Kr h) is a dosage-dependent modifier of gene expression in Drosophila.

A lethal mutation in the Krüppel homolog (Kr h) was isolated in screens of P-element insertion mutations for modifiers of white gene expression. The mutation occurs in the 5' untranslated region of the Kr h gene and causes a lightening of the eye colour for several alleles of white due to a decrease in white steady-state mRNA levels at pupal stages. Two related genes, scarlet and brown, were significantly affected as well in early pupae. Genetic analysis of different white alleles suggests that enhancer sequences are necessary for interaction with KR H. Thus, the Kr h gene is a member of the dosage-dependent hierarchy effective upon white.

The sugarless mutation affects the expression of the white eye color gene in Drosophila melanogaster.

Investigation of a modifier locus displaying a darker eye phenotype in white-apricot flies led to the isolation of the gene encoding UDP-glucose dehydrogenase (UDPGDH). The P-element insertion l(3)05007 occurs upstream of the transcription start site of the sugarless (sgl) gene and greatly reduces its transcription at various developmental stages. A single abundant sgl transcript shows a ubiquitous distribution and encodes a 53-kDa protein which is 64% identical in sequence to bovine UDP-glucose dehydrogenase. Overexpression of sgl in E. coli resulted in synthesis of a protein with high levels of UDPGDH activity. Expression of three genes that participate in pigment deposition, white, scarlet and brown, was significantly affected in populations segregating for sgl, suggesting that it is the decrease in UDPGDH level that produces the modifying effect observed. In addition, genetic effects on white-apricot were observed in sgl-wingless and sgl-hedgehog double mutants. Recent data have indicated an effect of UDPGDH on cell surface glycosaminoglycans (GAGs) which modulate the activity of growth factors, and in particular wingless signaling. Our results suggest that the levels of GAGs are rate limiting for cell-cell signaling pathways which mediate changes in gene expression.

Segmented gene conversion as a mechanism of correction of 18S rRNA pseudogene located outside of rDNA cluster in D. melanogaster.

The peculiarities of the sequences of 18S rDNA included in a 90-kb DNA segment cloned in YAC vector are described. This heterochromatic segment is situated on the X chromosome distal to the main rDNA cluster. The pseudo 18S rDNA sequence comprised undamaged stretches of rDNA interspersed with segments characterized by high density of nucleotide substitutions and insertions/deletions. The observed patchwork arrangement of unaltered rDNA sequences was considered as evidence of segmented gene conversion events between the normal and damaged genes which are thought to constitute one of the mechanisms of rDNA array homogenization. The 18S rDNA fragment (510 bp) located nearby, homologous to the internal, undamaged part of pseudo 18S rDNA, carries comparable density of randomly distributed nucleotide substitutions with no evidence of correction.