An AP-1 binding site in the upstream region of Deb-A is part of a developmentally regulated negative element.

The Deb-A gene from Drosophila melanogaster encodes a small membrane-associated protein, regulated during development, with peak abundance at 12-15 h of embryogenesis. The cis-acting regulatory elements that control expression of Deb-A during embryogenesis were localized using a somatic transformation assay. The Adh gene of D. melanogaster was used as a 'reporter' gene. The promoterless ADH coding sequence was fused to the 5'-upstream control region of Deb-A. Deletions were introduced into the 5'-region using various restriction sites and Bal31 deletion mutagenesis. A negative regulatory element, or silencer, was localized to a segment 47 base pairs long, between -395 and -442. It is responsible for 80% of the repression of gene expression during late development and reduces levels of Deb-A RNA nearly 5-fold. This negative element is temporally functional. It becomes active after 15 h of embryogenesis and it has no effect on gene expression prior to that. Within this negative element of 47 base pairs, two footprint regions were protected from DNase I digestion by embryonic nuclear extracts: one region contains an AP-1 binding site, but the other footprint is due to unknown element(s). High molecular weight DNA-protein complexes on an oligonucleotide probe spanning the AP-1 binding site were identified in gel retardation assays using partially purified bacterially expressed Djun protein or nuclear extracts from Drosophila embryos. These data suggest that the AP-1 site may be partly responsible for decreasing Deb-A expression during the late embryonic developmental stages of D. melanogaster.

Isolation and characterization of cDNA complementary to transient maternal poly(A)+ RNA from the Drosophila oocyte.

cDNA complementary to total oocyte poly(A)+ RNA from Drosophila melanogaster was enriched for sequences complementary to transient maternal sequences; that is, those sequences which disappear from the oocyte during subsequent. A seven- to ten-fold enrichment factor was obtained, from 5.3% to about 50% of the total cDNA. Kinetic analysis of this enriched fraction indicates that the transient maternal sequences include 44 +/- 14 different sequences.

The expression and genomic organization of randomly selected cloned Drosophila melanogaster genes.

A lambda recombinant DNA library containing Drosophila melanogaster nuclear DNA inserts was screened with cDNA made from oocyte and gastrula poly(A)+ RNA. 124 clones were isolated which represented sequences complementary to a distribution of abundancies of their RNAs. The clone set was then used as probes to identify those whose RNA abundancies changed during embryonic development. The vast majority of clones showed little difference during development. Four different clones were identified whose poly(A)+ RNAs were quantitatively regulated; two were oocyte-specific, and two were embryonic-specific. 44 clones were chosen for in situ hybridization to salivary gland polytene chromosomes. The location and distribution of their sites are described. A class of clones, identified by in situ hybridization to the nucleolus, is further described. These clones contain a scrambled array of ribosomal intervening sequences.

Sequence and expression of two regulated transcription units during Drosophila melanogaster development: Deb-A and Deb-B.

Two transcription units, Deb-A and Deb-B (developmental embryonic), are present in a recombinant bacteriophage clone containing a DNA insert from Drosophila melanogaster. The insert maps cytologically to chromosome 2R at 48EF. Sequence analysis of the open reading frames indicates that both are putative membrane proteins with similar hydropathy profiles. The expression of the encoded poly(A)+ RNAs is regulated during oocyte embryonic and larval development. The 600 base Deb-A RNA is present in oocytes and increases in abundance until 15 h of embryogenesis. Its abundance level drops dramatically between 15 and 19 h and is modulated at a low level in the three larval instars and pupae. The 525 base Deb-B RNA is present in oocytes at a 10-fold lower level than the Deb-A transcript. Its abundance increases until a maximum level is reached at 9-12 h. After this time the transcript is undetectable on Northern transfers. Both RNAs are present in greater than 95% polyadenylated form in the cytoplasm. Deb-B RNA is found on polysomes in proportions similar to the bulk of poly(A)+ RNA populations. Deb-A RNA, on the other hand is mostly nonpolysomal during embryogenesis. The RNAs from both Deb-A and Deb-B are found uniformly distributed throughout the embryo during development.

The gene structure of the Drosophila melanogaster homolog of the human proto-oncogene fos.

The Drosophila melanogaster homolog of the human proto-oncogene fos is Dfos. It is the only fos homolog in the Drosophila genome. Fos functions as a subunit of the heterodimeric transcription factor AP-1. There are two models of the Dfos gene. The first comes from a cDNA sequence of Dfos (Perkins et al., Genes Dev. 4 (1990) 822). The second is from the gene sequence published by the Drosophila genome project (Adams et al., Science 287 (2000) 2185), and there are notable contradictions between the two models. The promoter and the 5' end of the transcript sequence were not identified in either model. In this paper, we present the gene structure of Dfos and identify the promoter. This promoter has an initiator and a downstream promoter element sequence, but lacks a TATA box. Through comparison of the mRNA and genomic DNA sequences, three introns varying in length from 66 bp to 17.57 kb were found and verified by RT-PCR. The Dfos gene is 21.2 kb in length, giving a transcript of 3438 bp, coding for a predicted protein of 595 amino acids. The 3' untranslated region is confirmed to be 1092 bp in length.

Cardiopulmonary rehabilitation specialization: survey and suggested curriculum.

Findings exploring further educational commitment and job potential in the carciopulmonary rehabilitation area are presented based on a nationwide survey to 100 physical therapy departments. Results show 1) 99 percent of the sample population believed postgraduate work was necessary; 2) 72 percent indicated that caridac and plumonary rehabilitation techniques should be offered together; 3) 51 percent idinicated willingness to complete a clinical internship; and 4) 79 percent indicated that this specialty training could be utilized effectively in their respective hospital settings, with 20 percent indicating that hospital administrators would willingly pay salary differentials for this clinical specialty. A clinical Master of Arts degree curriculum design is presented with brief course descriptions currently in progess in the Department of Physical Therapy, New York University School of Education, Health, Nursing, and the Arts, Professions.

mRNA usage during Drosophila melanogaster embryonic development. Analysis of nine cloned DNA segments.

A set of nine phage lambda clones containing inserts from Drosophila melanogaster which are complementary to cDNA made from oocyte poly(A)+ RNA were selected from a larger group. These cloned elements code for a range of middle abundant RNA sequences which show no appreciable change in abundance during Drosophila embryogenesis. Seven of the nine clones are complementary to two oocyte RNAs, one to three RNAs and one to four RNAs. This study describes the changes that occur in these RNAs during embryonic development in the polysomal and non-polysomal fraction, and in the poly(A)+ RNA and poly(A)- RNA fraction. In all nine of these clones, greater than 70% of the complementary RNA is found in the polysomal region of a sucrose gradient. This proportion increases somewhat during development. Specific changes have been found during development in the proportion of RNA that is poly(A)+. Depending to the cloned sequence, this proportion may increase, decrease, or remain unchanged. For those clones that show a change, most of this change occurs between 8 and 19 h of development. Our data suggest, furthermore, the presence of a class of non-adenylated RNA being utilized during embryogenesis.

Transcription of Djun from D. melanogaster is positively regulated by DTF-1, AP-1, and CREB binding sites.

Djun is the homolog of the mammalian proto-oncogene jun in D. melanogaster, where it encodes a component of an AP-1-like nuclear DNA binding protein, or transcription factor. Djun, unlike its vertebrate counterparts, contains an intron in its 5' noncoding region. The expression of Djun in cultured Schneider line 2 cells is controlled by multiple cis-acting elements in its promoter region and the 5' noncoding region of the transcription unit. A 43-bp 5' upstream promoter region is necessary for the transcription activity of Djun. Deletion of this fragment decreased transcriptional activity by 67-fold. This region includes a TATA box and a sequence similar to the Drosophila transcription factor 1 (DTF-1) consensus sequence (GCAACAT/GC/C). A large DNase I footprint covering both the DTF-1 binding site and the TATA box was detected in this region when incubated with nuclear extract from Drosophila embryos, suggesting interactions with related transcription factors. This 43-bp sequence alone, containing the DTF-1 binding site and TATA box, however, is not sufficient for transcription activity. An 80-bp sequence including the start of transcription has considerable basal activity. An intragenic region containing an AP-1 site and a CRE site modulates or fine tunes activity of the promoter. Its activity as an enhancer is reduced when moved upstream in either orientation. An extragenic region containing two AP-1 sites similarly affects promoter activity.

Response of Djun and Dfos mRNA abundance to signal transduction pathways in cultured cells of Drosophila melanogaster.

The mammalian proto-oncogenes c-jun and c-fos are situated at the end of multiple signal transduction pathways and activation of their products Jun and Fos, components of the transcription factor AP-1, are able to regulate gene transcription in response to extracellular stimuli. Djun and Dfos, the products of the Drosophila proto-oncongenes Djun and Dfos, are similar in size and sequence to their mammalian counterparts c-Jun and c-Fos and are related to their mammalian counterparts by their antigenic properties. However, very little is known about how they are regulated through signal transduction pathways. This paper has investigated the response of their mRNA abundance levels to three signal transduction pathways in Drosophila cultured cells. Various agonists and antagonists that stimulate and inhibit specific enzymes in the pathways have been tested. The results suggest that Djun and Dfos mRNA are continuously expressed and their abundance levels are transiently regulated by multiple signaling pathways, the peak response coming at 1-2 hours after perturbation. Dfos is more highly regulated than Djun which is only modulated. The receptor tyrosine kinase pathways positively regulate Dfos and Djun. The cAMP-mediated pathway positively regulates Dfos but negatively regulates Djun. The protein kinase C-activated pathway does not affect Djun whereas it negatively regulates Dfos.

Drosophila Jun relays the Jun amino-terminal kinase signal transduction pathway to the Decapentaplegic signal transduction pathway in regulating epithelial cell sheet movement.

We have characterized mutations in the Drosophila homolog of the mammalian proto-oncogene c-Jun gene (Djun). We demonstrate that DJUN in the embryo is a downstream target of the JNK signal transduction pathway during dorsal closure formation, and that the function of the JNK/DJUN pathway is to control the localized expression of decapentalegic (dpp), a member of the TGF-beta growth factor family. In contrast to previous observations, we find that both in the embryo and during photoreceptor cell determination, DJUN is not regulated by a pathway that involves MAPK.

The regulation of protein synthesis in mammalian cells VI. Soluble and polyribosome associated components in controlling in vitro polypeptide initiation in HeLa cells.

The in vitro initiation of polypeptides on endogenous polyribosomes has been studied in extracts from HeLa cells. Regulation of the rate of initiation of polypeptides can be examined. In these experiments an assay using [(35)S]fMet-tRNA(f) (Met) has been developed, and the system further characterized. The system has been separated into a fraction containing polyribosomes with subunits and a fraction containing soluble components. The regulation of initiation has at least two distinct components. There is one factor in the soluble fraction which develops a stimulated response after protein synthesis has been inhibited in intact cells. This stimulation does not require new RNA synthesis during the period of cell "stress."A second component is associated with ribosomes. This factor is necessary for the initiation of polypeptides on endogenous polyribosomes. It disappears gradually when cells are exposed to actinomycin. The disappearance is first manifested by an inability of polyribosomes to respond to stimulated supernatants. This unstable component, which decays in the presence of actinomycin, has no apparent counterpart in systems that measure initiation on exogenous mRNA.

Isolation and characterization of complementary deoxyribonucleic acid complementary to the highly abundant class of poly(adenylic acid)-containing ribonucleic acid from oocytes of Drosophila melanogaster.

The complementary deoxyribonucleic acid (cDNA) complementary to the highly abundant class of poly(adenylic acid)-containing ribonucleic acid [poly(A+) RNA] from Drosophila melanogaster oocytes has been isolated and characterized. Analysis of the kinetics of hybridization of this cDNA (cDNAHA) to total poly(A+) RNA of oocytes indicates this class contains approximately 86 different sequences. Hybridization kinetics of cDNAHA annealed to poly(A+) RNA from 19-old embryos is essentially the same as that of oocyte poly(A+) RNA. This suggests the highly abundant class of poly(A+) RNA persists in approximately the same frequency through early development. Analysis of the hybridization of cDNAHA to genomic DNA suggests that the highly abundant poly(A+) RNA from oocytes is not enriched for transcripts from repetitive sequences of the genome.

DHR3, an ecdysone-inducible early-late gene encoding a Drosophila nuclear receptor, is required for embryogenesis.

Response to the steroid hormone ecdysone in Drosophila is controlled by genetic regulatory hierarchies that include eight members of the nuclear receptor protein family. The DHR3 gene, located within the 46F early-late ecdysone-inducible chromosome puff, encodes an orphan nuclear receptor that recently has been shown to exert both positive and negative regulatory effects in the ecdysone-induced genetic hierarchies at metamorphosis. We used a reverse genetics approach to identify 11 DHR3 mutants from a pool of lethal mutations in the 46F region on the second chromosome. Two DHR3 mutations result in amino acid substitutions within the conserved DNA binding domain. Analysis of DHR3 mutants reveals that DHR3 function is required to complete embryogenesis. All DHR3 alleles examined result in nervous system defects in the embryo.

Drosophila MEF2, a transcription factor that is essential for myogenesis.

mef2 encodes the only apparent Drosophila homolog of the vertebrate myocyte-specific enhancer factor 2 (MEF2). We show herein that the Drosophila MEF2 protein is expressed throughout the mesoderm following gastrulation. Later in embryogenesis, its expression is maintained in precursors and differentiated cells of the somatic and visceral musculature, as well as the heart. We have characterized genetic deficiencies and EMS-induced point mutations that result in complete loss of MEF2 protein in homozygous mutant embryos. These embryos exhibit a dramatic absence of myosin heavy chain (MHC)-expressing myoblasts and lack differentiated muscle fibers. Examination of earlier events of muscle development indicates that the specification and early differentiation of somatic muscle precursors are not affected because even-skipped-, nautilus-, and beta 3-tubulin-expressing myoblasts are present. However, these partially differentiated cells are unable to undergo further differentiation to form muscle fibers in the absence of mef2. The later aspects of differentiation of the visceral mesoderm and the heart are also disrupted in mef2 mutant embryos, although the specification and early development of these tissues appear unaffected. Midgut morphogenesis is disrupted in the mutant embryos, presumably as a consequence of abnormal development of the visceral mesoderm. In the heart, the cardial cells do not express MHC. These results indicate that MEF2 is required for later aspects of differentiation of the three major types of musculature, which include body wall muscles, gut musculature, and the heart, in the Drosophila embryo.

A genetic analysis of the cytological region 46C-F containing the Drosophila melanogaster homolog of the jun proto-oncogene.

The cytogenetic region 46C-F on the right arm of Drosophila chromosome 2, which contains the homolog of the human jun proto-oncogene, has been genetically mapped and characterized. This project led to the identification and characterization of a Jra (jun-related antigen) mutation, which has been described in detail elsewhere. Three mutagens, EMS, DEB and gamma-rays, were used to isolate 126 lethal lines for this interval. Complementation analysis of the 126 lethal lines identified 29 lethal complementation groups in the region; nine of which have now been correlated with known genes or phenotypes. The region has been subdivided into ten intervals using various small deletions, seven intervals in 46C/D and three intervals in 46E/F. Sixteen P-element lines have been mapped to this interval and are allelic to eight of our complementation groups. The remaining unidentified complementation groups have been analyzed for critical phase, which is when the first observable defect arises and/or when death occurs. There are twelve embryonic lethal groups and seven larval lethal groups. Three lines show visible abnormalities in gut and tracheal development prior to death.