Molecular characterization of Pegarn: a Drosophila homolog of UNC-51 kinase.

We have isolated and characterized the gene encoding a Drosophila melanogaster homolog of Caenorhabditis elegans UNC-51 (uncoordinated movement-51): Pegarn. Developmental Northern blot shows the Pegarn gene is expressed at all stages of development. The protein is detected throughout the Drosophila third instar larval central nervous system (CNS) in axons projecting out from the ventral ganglion and in the optic anlagen of the optic lobe. Heterozygous Pegarn mutant embryos show defects in larval axonal neuronal patterning, but survive to adulthood. Homozygous mutants have an even more deformed pattern of neuronal development and do not survive through the larval stages. The data from this research suggest the critical roles of Pegarn in CNS and PNS axonal formation in Drosophila melanogaster and indicates its similar role in other multicellular species.

pcdr, a novel gene with sexually dimorphic expression in the pigment cells of the Drosophila eye.

We report the molecular cloning and characterization of pcdr (pigment cell dehydrogenase reductase), a Drosophila visual system-specific gene with novel properties of spatial, temporal and sexual regulation. Short chain dehydrogenase/reductases are a family of proteins that catalyze mechanistically conserved dehydrogenase/reductase reactions in a wide range of cells and tissues. These enzymes are required in a variety of reactions ranging from steroid metabolism and prostaglandin synthesis to alcohol detoxification. The Drosophila pcdr gene encodes a new member of this family, displaying 42% amino acid sequence identity to the mammalian prostaglandin dehydrogenase. pcdr expression is restricted to the visual system with very high levels found in the pigment cells. Interestingly, expression of pcdr mRNA is sexually dimorphic with males showing higher levels of expression than females. This sexual dimorphism is under the control of the sex differentiation cascade as transformer and transformer 2 mutations shift females to a male-like level of expression. Finally, we demonstrate that a region of 335 nucleotides including sequences upstream and just downstream of the transcription start is sufficient to reproduce the normal expression pattern.

Defective intracellular transport is the molecular basis of rhodopsin-dependent dominant retinal degeneration.

Retinitis pigmentosa (RP) is a group of hereditary human diseases that cause retinal degeneration and lead to eventual blindness. More than 25% of all RP cases in humans appear to be caused by dominant mutations in the gene encoding the visual pigment rhodopsin. The mechanism by which the mutant rhodopsin proteins cause dominant retinal degeneration is still unclear. Interestingly, the great majority of these mutants appear to produce misfolded rhodopsin. We now report the isolation and characterization of 13 rhodopsin mutations that act dominantly to cause retinal degeneration in Drosophila; four of these correspond to identical substitutions in human autosomal dominant RP patients. We demonstrate that retinal degeneration results from interference in the maturation of wild-type rhodopsin by the mutant proteins.

Identification of two bone morphogenetic protein type I receptors in Drosophila and evidence that Brk25D is a decapentaplegic receptor.

Drosophila sequences at chromosomal positions 25D (Brk25D) and 43E (Brk43E) are similar to the TGF beta type I receptor serine/threonine kinases and are expressed broadly during embryogenesis. Brk25D binds dpp protein and bone morphogenetic protein 2 with high affinity. Mutations affecting Brk25D map to the gene thick veins and block the expression of two decapentaplegic-responsive (dpp-responsive) genes, dpp and labial, in the embryonic midgut. Defects in Brk25D receptor function combined with reduced expression of dpp ligand produce mutant phenotypes in the embryo and adult. Brk43E is the product of the gene saxophone, which also interacts with dpp. We conclude that dpp signaling in vivo is mediated by at least two receptors, Brk25D and Brk43E.

Isolation of Drosophila genes encoding G protein-coupled receptor kinases.

G protein-coupled receptors are regulated via phosphorylation by a variety of protein kinases. Recently, termination of the active state of two such receptors, the beta-adrenergic receptor and rhodopsin, has been shown to be mediated by agonist- or light-dependent phosphorylation of the receptor by members of a family of protein-serine/threonine kinases (here referred to as G protein-coupled receptor kinases). We now report the isolation of a family of genes encoding a set of Drosophila protein kinases that appear to code for G protein-coupled receptor kinases. These proteins share a high degree of sequence homology with the bovine beta-adrenergic receptor kinase. The presence of a conserved family of G protein-coupled receptor kinases in vertebrates and invertebrates points to the central role of these kinases in signal transduction cascades.

Resolution by diagonal gel mobility shift assays of multisubunit complexes binding to a functionally important element of the rat growth hormone gene promoter.

DNase I footprinting identifies a tissue-general factor, GHF3, binding to the rat growth hormone promoter between nucleotides -239 and -219. Mutation of the GHF3-binding site reduces promoter activity to 30% of that of the wild-type promoter after transfection into GC cells. Southwestern blotting and protein/DNA cross-linking experiments demonstrate that the GHF3-binding factor migrates as a 43-kDa protein. However, multiple GHF3 factor/DNA complexes with different electrophoretic mobilities are detected by gel retardation analysis. A novel technique, the diagonal gel mobility shift assay, is used to demonstrate that five of the different complexes represent multisubunit structures containing a common DNA-binding subunit. In this method, the multisubunit complexes resolved by one-dimensional gel mobility shift assays are observed to partially dissociate during electrophoresis in a second dimension with the DNA-binding subunit detected as a common signal directly below those signals representing the undissociated complexes which lie on a diagonal line. Two of the five complexes also contain an additional subunit in common whereas two other complexes appear to contain completely different subunits interacting with the common DNA-binding subunit. All five complexes copurify during GHF3-binding site-specific DNA affinity chromatography, and this fraction stimulates in vitro transcription in a GHF3-binding site-dependent fashion. Thus, a functionally important region of the rat growth hormone gene promoter interacts with a DNA-binding transcription factor which in turn acts as a docking site for other proteins.

A naturally occurring deletion in the enhancer repeats of the human papovavirus BK optimizes early enhancer function at the expense of late promoter activity.

The late promoter of the human papovavirus BK (prototype) is contained within the same region of DNA as the early promoter enhancer. This region, consisting of a 68-50-68-bp repeat and the flanking region to the late side, contains elements important for both late promoter activity and enhancer function for the early transcription unit. We have studied the importance of the naturally occurring 18-bp deletion in the middle repeat by constructing three repeats containing every possible combination of the 50- and 68-bp segments and testing their abilities to promote late transcription and activate an enhancerless SV40 early promoter. We find that the wild-type configuration of repeats gives optimal enhancer function, but is less active than other combinations for late transcription. This suggests that different interactions between the transcription elements contained within the same region of DNA are responsible for modulation of early and late transcription.

Unidirectional deletion and linker scan analysis of the late promoter of the human papovavirus BK.

We have previously shown that the late promoter of the human papovavirus BK (prototype) is contained within the three 68-bp repeats and a 66-bp region to the late side of the repeats which together constitute the early promoter enhancer. We have now carried out unidirectional deletion and linker scan analyses of these sequences to identify the major elements of the late promoter in human and monkey cells. Several important sequence motifs involved in late promoter function are found throughout this region. The most active ones correspond to previously defined binding sites for the transcription factors NF1 and Sp1 and a GC-rich region known to be important for early promoter function. The NF1 sequences may also be involved in negative regulation in some situations.

The late promoter of the human papovavirus BK is contained within the early promoter enhancer region.

We have analyzed the sequences necessary for late promoter function and mapped the late mRNA start sites of the human papovavirus BK (prototype). Our results show that, under both replicating and nonreplicating conditions, the BKV late promoter is contained within the same region defined as the enhancer for the early promoter. This region consists of three 68-bp repeats (the middle one of which has an 18-bp deletion) and a 66-bp region containing an enhancer element denoted as c, located to the late side of the 68-bp repeats. Deletions within the early enhancer domain indicate that elements of the late promoter are found throughout the entire region.

Analysis of the early regulatory region of the human papovavirus BK.

BKV is a human papovavirus which latently infects a majority of the world population and whose DNA has been found in human tumor tissue. Along with simian virus 40 (SV40) and JCV, it is one of several highly homologous polyomaviruses which display distinct host ranges, tissue tropisms, and transformation potentials. Determination of these properties is thought to reside, in part, in the noncoding regulatory region of these viruses. We have studied the regulation of gene expression by the early promoter and enhancer of BKV. Our results show that the early promoter of BKV consists of elements found both to the early side of and within the proximal 18 bp of the first enhancer element itself. At least one BKV regulatory element appears to be downstream of the mRNA start sites. The BKV enhancer consists of two different types of elements, three direct repeats, and an element (denoted "c") found in the 30 bp to the late side of the distal repeat. When used with the BKV promoter the enhancer repeat elements were found to be redundant, optimal promoter activity requiring only two of the three repeats plus the c element. Using the heterologous SV40 promoter the optimal BKV enhancer consisted of either three repeats or two repeats plus the c element. Subfragments of the enhancer region were capable of partial activation of homologous and heterologous promoters. We conclude that the regulation of BKV early gene expression involves novel elements arranged in ways not previously described in other papovaviruses.