The homeotic gene spalt (sal) evolved during Drosophila speciation.

The region-specific homeotic gene spalt (sal) acts in two separate domains in the head and tail region of the Drosophila melanogaster embryo. Based on comparative morphology, sal is likely to be involved in the establishment of the head during the evolution of invertebrates and thus, it should be conserved. We have analyzed the conservation of the segmentation genes Krüppel (Kr) and even-skipped (eve) in parallel with sal coding sequences in several Drosophila species that are evolutionarily separated by up to 60 million years. To our surprise, sal sequences appear to be conserved in the Sophophora subgenus of the Drosophila genus but not in the Drosophila subgenus. On the other hand, the segmentation and other homeotic genes are conserved in the Drosophila subgroup as well. Our data suggest that sal encodes an accessory function that evolved relatively late during Drosophila speciation rather than playing a fundamental evolutionary role similar to that of other homeotic genes.     

Essential role of Drosophila Hdac1 in homeotic gene silencing

Deacetylation of the N-terminal tails of core histones plays a crucial role in gene silencing. Rpd3 and Hda1 represent two major types of genes encoding trichostatin A-sensitive histone deacetylases. Although they have been widely found, their cellular and developmental roles remain to be elucidated in metazoa. We show that Drosophila Hdac1, an Rpd3-type gene, interacts cooperatively with Polycomb group repressors in silencing the homeotic genes that are essential for axial patterning of body segments. The biochemical copurification and cytological colocalization of HDAC1 and Polycomb group repressors strongly suggest that HDAC1 is a component of the silencing complex for chromatin modification on specific regulatory regions of homeotic genes.     

Segmental determination in Drosophila conferred by hunchback (hb), a repressor of the homeotic gene Ultrabithorax (Ubx).

The activity of homeotic genes in Drosophila cells determines segment-specific morphogenesis. Here, we provide evidence that the product of hunchback (hb), a segmentation gene, acts as a direct repressor or "silencer" of the homeotic gene Ultrabithorax (Ubx) and thus prevents ectopic activity of this gene: we show, by stable integration of reporter gene constructs, that hb protein binding sites are capable of repressing at a distance the activity of an embryonic Ubx enhancer outside the Ubx expression domain. This silencing activity is observed at advanced embryonic stages, at a time when the hb gene product is no longer detectable or required, and is dependent on the function of Polycomb (Pc). We propose a working hypothesis as to how hb protein in a "hit-and-run" fashion may effect stable and heritable silencing of the Ubx gene throughout advanced stages of development, thus mediating repression of this homeotic gene outside its realm of function.     

Intrachromosomal rearrangements mediated by hobo transposons in Drosophila melanogaster.

The recurring intrachromosomal rearrangements observed in an unstable X chromosome, designated Uc, of Drosophila melanogaster are shown to be mediated by hobo transposable elements. Each of 29 chromosome rearrangement breakpoints in 16 gross aberrations detected in the Uc-derived X chromosomes had a hobo element. In one particular unstable X chromosome line selected for detailed studies, a hobo element was found in each of the five hot spots for rearrangements. Furthermore, hobo elements at deletion hot spots were found to lie in the same orientation, whereas those hobo elements at inversion hot spots were in the opposite orientation. The restriction maps of two phage lambda clones containing rearrangement breakpoints indicated that a hobo element was inserted exactly at the breakpoints. Pairing of hobo elements in the same chromosome followed by recombination between the paired hobo elements is suggested as the explanation for the intrachromosomal aberrations observed in the Uc X chromosomes. A clear qualitative difference among the hobo elements in their ability to participate in rearrangement formation was noted. It was also found that each of the 11 recessive lethal mutations mapped in the 6F1-2 doublet had a hobo element in the doublet, whereas none of the 16 independent revertants of the mutation had a hobo element in the site. This observation indicates that hobo movement is responsible for production and subsequent instability of recessive lethal mutations in the 6F region of the Uc X chromosomes.     

Regulation of Drosophila lifespan by JNK signaling

Cellular responses to extrinsic and intrinsic insults have to be carefully regulated to properly coordinate cytoprotection, repair processes, cell proliferation and apoptosis. Stress signaling pathways, most prominently the Jun-N-terminal Kinase (JNK) pathway, are critical regulators of such cellular responses and have accordingly been implicated in the regulation of lifespan in various organisms. JNK signaling promotes cytoprotective gene expression, but also interacts with the insulin signaling pathway to influence growth, metabolism, stress tolerance and regeneration. Here, we review recent studies in Drosophila that elucidate the tissue-specific and systemic consequences of JNK activation that ultimately impact lifespan of the organism.     

Myocardial infarction mediated by endothelin receptor signaling in hypercholesterolemic mice.

Myocardial infarction is linked to atherosclerosis, yet the sequence leading from silent coronary atherosclerosis to acute myocardial infarction has remained unclear. Here we show that hypercholesterolemic apolipoprotein E-/- low density lipoprotein receptor-/- mice develop not only coronary atherosclerosis but also myocardial infarction. Exposure of mice to mental stress or hypoxia led to acute ischemia, which, in a large proportion of the mice, was followed by electrocardiographic changes, leakage of troponin T, and loss of dehydrogenase from the myocardium, all indicative of acute myocardial infarction. Apoptotic death of cardiomyocytes was followed by inflammation and fibrosis in the heart. All these pathological changes could be prevented by a blocker of the endothelin type A receptor. Thus, stress elicits myocardial infarction through endothelin receptor signaling in coronary atherosclerosis caused by hypercholesterolemia.     

Genetic analysis of insulin signaling in Drosophila.

Studies in the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans have revealed that components of the insulin signaling pathway have been highly conserved during evolution. Genetic analysis in Drosophila suggests that structural conservation also extends to the functional level. Flies carrying mutations that reduce insulin signaling have a growth deficiency phenotype similar to that seen in mice with disruptions of genes encoding insulin-like growth factors (IGFs) or the IGF-I receptor. Recent studies in flies have demonstrated a role for the insulin signaling pathway in the regulation of metabolism, reproduction and lifespan via modulation of central neuroendocrine pathways. Similarly, mice with loss of brain insulin receptors or insulin receptor substrate 2 deficiency exhibit neuroendocrine defects and female infertility. These parallels suggest that the insulin system has multiple conserved roles, acting directly to modulate growth and indirectly, via the neuroendocrine system, to modulate peripheral physiology in response to changes in nutrient availability.     

Targeted gene mutations in Drosophila.

A cloned gene can be of interest because of its expression in a particular tissue or at a certain developmental stage, or because of homology to an interesting gene from another organism. In Drosophila its location in the genome is readily determined by in situ hybridization to the banded larval salivary gland polytene chromosomes, but it is more difficult to isolate mutations that may reveal its function. This paper describes a general method for detecting transposable element insertions into the gene in question. This "reverse genetics" then offers the possibility of observing a consequent mutant phenotype, providing a key to the normal function of the gene. The sensitivity of the polymerase chain reaction makes it possible to detect the occurrence of a single appropriate P-element transposon insertion among a population of mutagenized flies. This is accomplished by the use of oligonucleotide primers--one a sequence from within the cloned gene and the other homologous to the terminal sequence of the P-element DNA--to prime synthesis into the DNA flanking an insertion site. A segment of DNA, bounded by the two primers, will be a target for amplification only in a fly in which a P-element has inserted within about 2 kilobases of the gene primer. This technique has been used to detect P-element insertions near a gene expressed in the Drosophila compound eye. Potential problems with the technique and possible refinements in the screen are discussed. In principle, it could be utilized to detect insertion of a foreign element into any gene for which at least a partial sequence is known and could be extended to other organisms.     

Control of seed mass and seed yield by the floral homeotic gene APETALA2

APETALA2 (AP2) is best known for its role in the regulation of flower meristem and flower organ identity and development in Arabidopsis. We show here that AP2 also plays an important role in determining seed size, seed weight, and the accumulation of seed oil and protein. We demonstrate genetically that AP2 acts through the maternal sporophyte and endosperm genomes to control seed weight and seed yield. Thus, AP2 functions outside the boundaries of flower meristem and flower organ development to affect agronomically relevant traits in Arabidopsis.     

Expression and novel structure of a collagen gene in Drosophila.

We report the structure and developmental expression of collagen gene sequences in Drosophila melanogaster. Collagen-like genomic clones were isolated by screening a Drosophila genomic library with a chicken pro alpha 2(I) cDNA clone as a hybridization probe. A 1.5-kilobase (kb) DNA sequence from a 9.2-kb DNA clone (pDCg1) is presented. Unlike the highly fragmented genes for vertebrate type I collagen, there is no evidence of a 54-base-pair primordial unit within this gene segment. Instead, the fragment is composed of two large coding sequences. Together they specify a sequence of 469 amino acids. This collagen product is composed almost entirely of the Gly-X-Y repeat characteristic of peptides involved in triple helix formation. Within the polypeptide there are four minor discontinuities in the Gly-X-Y pattern. Similar interruptions have been observed in a mouse basement membrane collagen protein sequence. Therefore, the Drosophila collagen gene may encode a nonfibrous collagen such as a basement membrane or cuticle collagen or a novel collagenous protein. By using the DNA segment of known sequence as a hybridization probe, a developmental sequence of polyadenylylated RNA samples was screened for the presence of homologous sequences. A RNA species 6.4 kb in length was detected as a prominent band only in the first- and second-instar larval stages. This pattern of developmental hybridization correlates with the production of the cuticle and basement membranes, and the large size of the RNA is consistent with its identification as a collagen-encoding RNA.     

Long-range signaling within growing neurites mediated by neurotrophin-3

In addition to well established trophic functions, neurotrophins acutely affect neurotransmitter secretion from the presynaptic nerve terminal, influence synaptic development, and may serve as selective retrograde messengers that regulate synaptic efficacy. The crucial question related to the mechanisms of neurotrophin-mediated signaling is whether acute effects of neurotrophins are spatially restricted to the activated synapses. Here we have used a local perfusion technique for local delivery of neurotrophin-3 (NT-3) to various regions of developing Xenopus embryo neurons in culture. Within minutes after a focal exposure of a soma or a small ( approximately 30 micrometer in length) axonal segment to NT-3, we observed an increase in the spontaneous neurotransmitter secretion from the presynaptic nerve terminals located approximately 300-400 micrometer away from the site of NT-3 application. Secretory activity along the axonal shaft was not affected. Our findings suggest that the NT-3-mediated signal may rapidly travel through neuronal cytoplasm over unexpectedly long distances and modulate neurotransmitter release specifically at the presynaptic nerve terminals.     

Intercellular calcium signaling mediated by point-source burst release of ATP

Calcium signaling, manifested as intercellular waves of rising cytosolic calcium, is, in many cell types, the result of calcium-induced secretion of ATP and activation of purinergic receptors. The mechanism by which ATP is released has hitherto not been established. Here, we show by real-time bioluminescence imaging that ATP efflux is not uniform across a field of cells but is restricted to brief, abrupt point-source bursts. The ATP bursts emanate from single cells and manifest the transient opening of nonselective membrane channels, which admits fluorescent indicators of < or = 1.5 kDa. These observations challenge the existence of regenerative ATP release, because ATP efflux is finite and restricted to a point source. Transient efflux of cytosolic nucleotides from a subset of cells may represent a conserved pathway for coordinating local activity of electrically nonexcitable cells, because identical patterns of ATP release were identified in human astrocytes, endothelial cells, and bronchial epithelial cells.     

Targeted gene correction of episomal DNA in mammalian cells mediated by a chimeric RNA.DNA oligonucleotide.

An experimental strategy to facilitate correction of single-base mutations of episomal targets in mammalian cells has been developed. The method utilizes a chimeric oligonucleotide composed of a contiguous stretch of RNA and DNA residues in a duplex conformation with double hairpin caps on the ends. The RNA/DNA sequence is designed to align with the sequence of the mutant locus and to contain the desired nucleotide change. Activity of the chimeric molecule in targeted correction was tested in a model system in which the aim was to correct a point mutation in the gene encoding the human liver/bone/kidney alkaline phosphatase. When the chimeric molecule was introduced into cells containing the mutant gene on an extrachromosomal plasmid, correction of the point mutation was accomplished with a frequency approaching 30%. These results extend the usefulness of the oligonucleotide-based gene targeting approaches by increasing specific targeting frequency. This strategy should enable the design of antiviral agents.