A transient expression assay for tissue-specific gene expression of alcohol dehydrogenase in Drosophila

The regulation of expression of the alcohol dehydrogenase gene of Drosophila was examined by injecting plasmids containing the gene directly into preblastoderm embryos and subsequently staining for alcohol dehydrogenase activity in somatic cells of larvae and adults. The alcohol dehydrogenase genes introduced in this manner were expressed normally in both adults and larvae; i.e., alcohol dehydrogenase activity was found exclusively in tissues where it would normally be expressed. Activity was found in some cells in more than 90% of all surviving third instar larvae, but not all cells which would normally express the enzyme were positive, presumably due to the random distribution of the injected DNA to the cells of the embryo. Regulated expression was not dependent on the vector used: tissue-specific expression was obtained from alcohol dehydrogenase genes inserted in the P-element vector, Carnegie-4; in pBR322; in pUC18; or in bacteriophage lambda. The bulk of the injected DNA was not integrated into the chromosome and appeared to persist throughout development as supercoiled and nicked circles. Using the procedure and in vitro mutagenesis, we were able to show that the alcohol dehydrogenase gene was expressed in a normal tissue-specific manner in larvae if there were 777 nucleotides of upstream information present.     

The expression of a tissue-specific self-peptide is required for allo-recognition

We have compared the functional properties of I-Ad expressed on different cell types. Specifically, we have transfected I-A alpha d and I-A beta d cDNA into a panel of T cell thymomas of various phenotypes. Excellent class II surface expression was achieved in all T cell tumors, equivalent in level to that found on the B cell lymphoma A20. Interestingly, however, two allo-I-Ad-specific Thy differed in their recognition of the transfected tumor cells: whereas the 42H11 T cell hybridoma (THy) was stimulated very efficiently by all transfectants, the RK38.2.2 Thy did not react to any of them. Both THy responded equally well to I-Ad on A20 B lymphoma cells. Purified macrophages isolated from various sources were also differentially recognized by the two THy, although there was only a quantitative difference in stimulation. Taken together, these results are best interpreted to show that the TCR of the RK38.2.2 THy is specific for I-Ad in the context of a B cell-specific determinant, possibly a self-peptide that is naturally associated with Ia. A cross-reactive molecule could be expressed by macrophages and COS-1 cells, but not by T cells.     

Multimerization of the Drosophila zeste protein is required for efficient DNA binding.

The Drosophila zeste protein forms multimeric species in vitro through its C-terminal domain. Multimerization is required for efficient binding to DNA containing multiple recognition sequences and increasing the number of binding sites stimulates binding in a cooperative manner. Mutants that can only form dimers still bind to a dimeric site, but with lower affinity. Mutations or progressive deletions from the C-terminal show that when even dimer formation is prevented, DNA-binding activity is lost. Surprisingly, binding activity is regained with larger deletions that leave only the DNA-binding domain. Additional protein sequences apparently inhibit DNA binding unless they permit multimerization. The DNA-binding domain peptides bind strongly even to isolated recognition sequences and they bind as monomers. The ability of various zeste peptides to stimulate white gene expression in vivo shows that multimeric forms are the functional species of the zeste product in vivo. The DNA-binding domain peptide binds well to DNA in vitro, but it cannot stimulate white gene expression in vivo. This failure may reflect the need for an activation domain or it may be caused by indiscriminate binding of this peptide to non-functional isolated sites. Multimerization increases binding specificity, selecting only sites with multiple recognition sequences.     

The cyclophilin homolog ninaA is a tissue-specific integral membrane protein required for the proper synthesis of a subset of Drosophila rhodopsins.

Mutations in the Drosophila ninaA gene cause dramatic reductions in rhodopsin levels, leading to impaired visual function. The ninaA protein is a homolog of peptidyl-prolyl cis-trans isomerases. We find that ninaA is unique among this family of proteins in that it is an integral membrane protein, and it is expressed in a cell type-specific manner. We have used transgenic animals misexpressing different rhodopsins in the major class of photoreceptor cells to demonstrate that ninaA is required for normal function by two homologous rhodopsins, but not by a less conserved member of the Drosophila rhodopsin gene family. This demonstrates in vivo substrate specificity in a cyclophilin-like molecule. We also show that vertebrate retina contains a ninaA-related protein and that ninaA is a member of a gene family in Drosophila. These data offer insights into the in vivo role of this important family of proteins.     

Isolation of silencer-containing sequences causing a tissue-specific position effect on alcohol dehydrogenase expression in Drosophila melanogaster

A transient expression assay has been used to investigate the cause of a tissuespecific position effect on Adh expression from a transgene insertion in Drosophila. A 15.4-kb genomic clone containing the 3.2-kb Adh insert along with flanking regions of genomic DNA is expressed in this assay in a tissue-specific pattern resembling the abnormal expression pattern of the position effect. The 3.2-kb Adh insert is expressed normally without the flanking sequences. A silencer element is located upstream of the Adh gene within a 2-kb fragment that acts in both orientations and at a distance of at least 6.5 kb from the larval Adh promoter to suppress ADH expression in a nontissue specific fashion. The DNA sequence of the 2-kb fragment indicates that it is a noncoding region. A 17-bp sequence is repeated within this region and may be associated with the silencer activity, since subclones from the 2-kb fragment, each containing one of the repeated regions, both retain full silencer activity. This silencer fails to suppress expression from an α₁-tubulin promoter-LacZ fusion construct or an hsp70 promoter-Ach fusion construct. In addition to the silencer, another element is located downstream of the Adh gene that produces a higher level of anterior than posterior midgut expression. These results suggest that the 5′ silencer and the 3′ element act together to create the tissue specific pcsition effect characteristic of the GC-1 line. © 1994 Wiley-Liss, Inc.     

Similar tissue-specific expression of the Adh genes from different Drosophila species is mediated by distinct arrangements of cis-acting sequences

In Drosophila melanogaster transformants, the alcohol dehydrogenase (Adh) genes from D. affinidisjuncta and D. grimshawi show similar levels of expression except in the adult midgut where the D. affinidisjuncta gene is expressed about 10- to 20-fold more strongly. To study the arrangement of cis-acting sequences responsible for this regulatory difference, homologous restriction sites were used to create a series of chimeric genes that switched fragments from the 5′ and 3′ flanking regions of these two genes. Chimeric genes were introduced into the germ-line of D. melanogaster, and Adh gene expression was analyzed by measuring RNA levels. Various gene fragments in the promoter region and elsewhere influence expression in the adult midgut and in whole larvae and adults. Comparison of these results with earlier studies involving chimeras between the D. affinidisjuncta and D. hawaiiensis genes indicates that expression in the adult midgut is influenced by multiple regulatory sequences and that distinct arrangements of regulatory sequences can result in similar levels of expression both in the adult midgut and in the whole organism.     

The protein CTCF is required for the enhancer blocking activity of vertebrate insulators.

An insulator is a DNA sequence that can act as a barrier to the influences of neighboring cis-acting elements, preventing gene activation, for example, when located between an enhancer and a promoter. We have identified a 42 bp fragment of the chicken beta-globin insulator that is both necessary and sufficient for enhancer blocking activity in human cells. We show that this sequence is the binding site for CTCF, a previously identified eleven-zinc finger DNA-binding protein that is highly conserved in vertebrates. CTCF sites are present in all of the vertebrate enhancer-blocking elements we have examined. We suggest that directional enhancer blocking by CTCF is a conserved component of gene regulation in vertebrates.     

The primary structure of alcohol dehydrogenase from Drosophila lebanonensis. Extensive variation within insect 'short-chain' alcohol dehydrogenase lacking zinc

Insect alcohol dehydrogenase is highly different from the well-known yeast and mammalian alcohol dehydrogenases. The enzyme from Drosophila lebanonensis has now been characterized by protein analysis and was found to have a 254-residue protein chain with an acetyl-blocked N-terminal Met. Comparisons with the structures of the enzyme from other species allows judgement of the extent of variability within the insect alcohol dehydrogenases. They have diverged to a considerable extent; two forms analyzed at the protein level differ at 18% of all residues, and all the known Drosophila alcohol dehydrogenase structures reveal differences at 72 positions. Some deviations, against a background similarity, in the extent of changes are noted among the parts corresponding to different exons. The structural variation within Drosophila is about as large as the one for the mammalian zinc-containing alcohol dehydrogenase. Consequently, the results illustrate Drosophila relationships and establish great variations also for group of alcohol dehydrogenases lacking zinc.     

Drosophila stem-loop binding protein intracellular localization is mediated by phosphorylation and is required for cell cycle-regulated histone mRNA expression

Stem-loop binding protein (SLBP) is an essential component of the histone pre-mRNA processing machinery. SLBP protein expression was examined during Drosophila development by using transgenes expressing hemagglutinin (HA) epitope-tagged proteins expressed from the endogenous Slbp promoter. Full-length HA-dSLBP complemented a Slbp null mutation, demonstrating that it was fully functional. dSLBP protein accumulates throughout the cell cycle, in contrast to the observed restriction of mammalian SLBP to S phase. dSLBP is located in both nucleus and cytoplasm in replicating cells, but it becomes predominantly nuclear during G2. dSLBP is present in mitotic cells and is down-regulated in G1 when cells exit the cell cycle. We determined whether mutation at previously identified phosphorylation sites, T120 and T230, affected the ability of the protein to restore viability and histone mRNA processing to dSLBP null mutants. The T120A SLBP restored viability and histone pre-mRNA processing. However, the T230A mutant, located in a conserved TPNK sequence in the RNA binding domain, did not restore viability and histone mRNA processing in vivo, although it had full activity in histone mRNA processing in vitro. The T230A protein is concentrated in the cytoplasm, suggesting that it is defective in nuclear targeting, and accounting for its failure to function in histone pre-mRNA processing in vivo.     

A conserved inositol phospholipid binding site within the pleckstrin homology domain of the Gab1 docking protein is required for epithelial morphogenesis.

Stimulation of the hepatocyte growth factor receptor tyrosine kinase, Met, induces the inherent morphogenic program of epithelial cells. The multisubstrate binding protein Gab1 (Grb2-associated binder-1) is the major phosphorylated protein in epithelial cells following activation of Met. Gab1 contains a pleckstrin homology domain and multiple tyrosine residues that act to couple Met with multiple signaling proteins. Met receptor mutants that are impaired in their association with Gab1 fail to induce a morphogenic program in epithelial cells, which is rescued by overexpression of Gab1. The Gab1 pleckstrin homology domain binds to phosphatidylinositol 3,4, 5-trisphosphate and contains conserved residues, shown from studies of other pleckstrin homology domains to be crucial for phospholipid binding. Mutation of conserved phospholipid binding residues tryptophan 26 and arginine 29, generates Gab1 proteins with decreased phosphatidylinositol 3,4,5-trisphosphate binding, decreased localization at sites of cell-cell contact, and reduced ability to rescue Met-dependent morphogenesis. We conclude that the ability of the Gab1 pleckstrin homology domain to bind phosphatidylinositol 3,4,5-trisphosphate is critical for subcellular localization of Gab1 and for efficient morphogenesis downstream from the Met receptor.     

The quinone binding site in Escherichia coli succinate dehydrogenase is required for electron transfer to the heme b

We have examined the role of the quinone-binding (Q(P)) site of Escherichia coli succinate:ubiquinone oxidoreductase (succinate dehydrogenase) in heme reduction and reoxidation during enzyme turnover. The SdhCDAB electron transfer pathway leads from a cytosolically localized flavin adenine dinucleotide cofactor to a Q(P) site located within the membrane-intrinsic domain of the enzyme. The Q(P) site is sandwiched between the [3Fe-4S] cluster of the SdhB subunit and the heme b(556) that is coordinated by His residues from the SdhC and SdhD subunits. The intercenter distances between the cluster, heme, and Q(P) site are all within the theoretical 14 A limit proposed for kinetically competent intercenter electron transfer. Using EPR spectroscopy, we have demonstrated that the Q(P) site of SdhCDAB stabilized a ubisemiquinone radical intermediate during enzyme turnover. Potentiometric titrations indicate that this species has an E(m,8) of approximately 60 mV and a stability constant (K(STAB)) of approximately 1.0. Mutants of the following conserved Q(P) site residues, SdhC-S27, SdhC-R31, and SdhD-D82, have severe consequences on enzyme function. Mutation of the conserved SdhD-Y83 suggested to hydrogen bond to the ubiquinone cofactor had a less severe but still significant effect on function. In addition to loss of overall catalysis, these mutants also affect the rate of succinate-dependent heme reduction, indicating that the Q(P) site is an essential stepping stone on the electron transfer pathway from the [3Fe-4S] cluster to the heme. Furthermore, the mutations result in the elimination of EPR-visible ubisemiquinone during potentiometric titrations. Overall, these results demonstrate the importance of a functional, semiquinone-stabilizing Q(P) site for the observation of rapid succinate-dependent heme reduction.     

The Arabidopsis CLAVATA2 gene encodes a receptor-like protein required for the stability of the CLAVATA1 receptor-like kinase.

The CLAVATA2 (CLV2) gene regulates both meristem and organ development in Arabidopsis. We isolated the CLV2 gene and found that it encodes a receptor-like protein (RLP), with a presumed extracellular domain composed of leucine-rich repeats similar to those found in plant and animal receptors, but with a very short predicted cytoplasmic tail. RLPs lacking cytoplasmic signaling domains have not been previously shown to regulate development in plants. Our prior work has demonstrated that the CLV1 receptor-like kinase (RLK) is present as a disulfide-linked multimer in vivo. We report that CLV2 is required for the normal accumulation of CLV1 protein and its assembly into protein complexes, indicating that CLV2 may form a heterodimer with CLV1 to transduce extracellular signals. Sequence analysis suggests that the charged residue in the predicted transmembrane domain of CLV2 may be a common feature of plant RLPs and RLKs. In addition, the chromosomal region in which CLV2 is located contains an extremely high rate of polymorphism, with 50 nucleotide and 15 amino acid differences between Landsberg erecta and Columbia ecotypes within the CLV2 coding sequence.     

LUMP is a putative double-stranded RNA binding protein required for male fertility in Drosophila melanogaster

In animals, male fertility requires the successful development of motile sperm. During Drosophila melanogaster spermatogenesis, 64 interconnected spermatids descended from a single germline stem cell are resolved into motile sperm in a process termed individualization. Here we identify a putative double-stranded RNA binding protein LUMP that is required for male fertility. lump(1) mutants are male-sterile and lack motile sperm due to defects in sperm individualization. We show that one dsRNA binding domains (dsRBD) is essential for LUMP function in male fertility. These findings reveal LUMP is a novel factor required for late stages of male germline differentiation.