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BackgroundPrimary analysis of the double-blind, phase III Efficacy of XL184 (Cabozantinib) in Advanced Medullary Thyroid Cancer (EXAM) trial demonstrated significant improvement in progression-free survival with cabozantinib versus placebo in patients with progressive medullary thyroid cancer (MTC). Final analysis of overall survival (OS), a key secondary endpoint, was carried out after long-term follow-up.Patients and methodsEXAM compared cabozantinib with placebo in 330 patients with documented radiographic progression of metastatic MTC. Patients were randomized (2:1) to cabozantinib (140 mg/day) or placebo. Final OS and updated safety data are reported.ResultsMinimum follow-up was 42 months. Kaplan-Meier analysis showed a 5.5-month increase in median OS with cabozantinib versus placebo (26.6 versus 21.1 months) although the difference did not reach statistical significance [stratified hazard ratio (HR), 0.85; 95% confidence interval (CI), 0.64-1.12; P = 0.24]. In an exploratory assessment of OS, progression-free survival, and objective response rate, cabozantinib appeared to have a larger treatment effect in patients with RET M918T mutation-positive tumors compared with patients not harboring this mutation. For patients with RET M918T-positive disease, median OS was 44.3 months for cabozantinib versus 18.9 months for placebo [HR, 0.60; 95% CI, 0.38-0.94; P = 0.03 (not adjusted for multiple subgroup analyses)], with corresponding values of 20.2 versus 21.5 months (HR, 1.12; 95% CI, 0.70-1.82; P = 0.63) in the RET M918T-negative subgroup. Median treatment duration was 10.8 months with cabozantinib and 3.4 months with placebo. The safety profile for cabozantinib remained consistent with that of the primary analysis.ConclusionThe secondary end point was not met in this final OS analysis from the trial of cabozantinib in patients with metastatic, radiographically progressive MTC. A statistically nonsignificant increase in OS was observed for cabozantinib compared with placebo. Exploratory analyses suggest that patients with RET M918T-positive tumors may experience a greater treatment benefit with cabozantinib.Trial registration numberNCT00704730.

Introduction: METEOR was a phase 3 trial (NCT01865747) of cabozantinib versus everolimus in adults with advanced or metastatic clear cell RCC previously treated with VEGF receptor (VEGFR) tyrosine kinase inhibitors (TKIs). This post hoc analysis of METEOR compared outcomes for patients recruited from European and non-European countries., Material and Methods: Adults with advanced/metastatic clear cell RCC who had received ≥ 1 prior VEGFR-TKI treatment were randomized 1:1 to receive cabozantinib or everolimus. Patients were categorized by recruitment region: Europe or outside of Europe (rest of world [RoW]). Progression-free survival (PFS), overall survival (OS), objective response rate (ORR), and adverse events (AEs) were compared between regional subgroups., Results: In total, there were 320 eligible patients from Europe (cabozantinib, 167; everolimus, 153) and 338 from RoW (North America, 240 patients; Asia-Pacific, 86; Latin America, 12; randomized as cabozantinib, 163; everolimus, 175). PFS and OS were longer with cabozantinib than with everolimus and similar for the Europe and RoW subgroups. For PFS, the hazard ratio (HR) for cabozantinib versus everolimus was 0.54 for the Europe subgroup ( p < .001) and 0.50 for the RoW subgroup ( p < .001). For OS, the HR was 0.75 for the Europe subgroup ( p = .034) and 0.69 for the RoW subgroup ( p = .006). ORR in the Europe subgroup was 15% for cabozantinib and 3.9% for everolimus ( p < .001). For the RoW subgroup, ORR was 20% for cabozantinib and 2.9% for everolimus ( p < .001). Incidence of grade 3/4 AEs were similar for the Europe (cabozantinib, 74%; everolimus, 58%) and RoW subgroups (cabozantinib, 69%; everolimus, 64%)., Conclusion: In the METEOR trial, efficacy outcomes for patients recruited from European and non-European countries favored cabozantinib over everolimus. The efficacy and safety results for the regional subgroups were consistent with those of the overall METEOR population.

Cabozantinib is an oral tyrosine kinase inhibitor (TKI) approved for the treatment of patients with advanced renal cell carcinoma (RCC) at a dose of 60 mg/day. As with other TKIs, cabozantinib is associated with high interpatient variability in drug clearance and exposure that can significantly impact safety and tolerability across a patient population. To optimize cabozantinib exposure (maintaining efficacy and tolerability) for the individual, patients may require treatment interruption with dose reduction (40 mg/day and then 20 mg/day). In the pivotal Phase 3 METEOR trial, cabozantinib significantly improved overall survival, progression-free survival and the objective response rate compared with everolimus in patients with advanced RCC who had received previous treatment with a VEGFR TKI. Dose reductions were common for patients receiving cabozantinib (60%) but effective as only 9% discontinued treatment due to adverse events (AEs). In this review, we discuss pharmacometric analyses that evaluated the impact of cabozantinib dose on efficacy and safety outcomes during the METEOR study. Exposure-response models demonstrate that the risk of experiencing adverse events and dose reduction is increased in patients with low cabozantinib clearance versus typical clearance and decreased in patients with high clearance. Dose reduction of cabozantinib to manage AEs is predicted to have minimal impact on efficacy as AEs are more likely to occur in patients with low clearance and higher exposure to cabozantinib. These analyses further support a dose modification strategy to optimize cabozantinib exposure for individual patients., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Cabozantinib significantly prolonged progression-free survival (PFS) versus a placebo in patients with progressive, metastatic medullary thyroid cancer (MTC; P < .001). An exploratory analysis of phase 3 trial data evaluated the influence of rearranged during transfection (RET) and RAS (HRAS, KRAS, and NRAS) mutations on cabozantinib clinical activity., Methods: Patients (n = 330) were randomized to cabozantinib (140 mg/day) or a placebo. The primary endpoint was PFS. Additional outcome measures included PFS, objective response rates (ORRs), and adverse events in RET and RAS mutation subgroups., Results: Among all study patients, 51.2% were RET mutation-positive (38.2% with RET M918T), 34.8% were RET mutation-unknown, and 13.9% were RET mutation-negative. Sixteen patients were RAS mutation-positive. Cabozantinib appeared to prolong PFS versus the placebo in the RET mutation-positive subgroup (hazard ratio [HR], 0.23; 95% confidence interval [CI], 0.14-0.38; P < .0001), the RET mutation-unknown subgroup (HR, 0.30; 95% CI, 0.16-0.57; P = .0001), and the RAS mutation-positive subgroup (HR, 0.15; 95% CI, 0.02-1.10; P = .0317). The RET M918T subgroup achieved the greatest observed PFS benefit from cabozantinib versus the placebo (HR, 0.15; 95% CI, 0.08-0.28; P < .0001). The ORRs for RET mutation-positive, RET mutation-negative, and RAS mutation-positive patients were 32%, 22%, and 31%, respectively. No PFS benefit was observed in patients lacking both RET and RAS mutations, although the ORR was 21%. The safety profile for all subgroups was similar to that for the overall cabozantinib arm., Conclusions: These data suggest that cabozantinib provides the greatest clinical benefit to patients with MTC who have RET M918T or RAS mutations. However, a prospective trial is needed to confirm the relation between genetic variation and the response to cabozantinib. Cancer 2016;122:3856-3864. © 2016 American Cancer Society., (© 2016 American Cancer Society.)

Background XL281 is a potent and selective inhibitor of wild-type and mutant RAF kinases with anti-tumor activity in multiple xenograft models. Mutations in KRAS or BRAF can activate the RAF/MEK/ERK pathway in human tumors and may confer sensitivity to RAF kinase inhibitors. Methods We performed a phase I study of XL281 in patients with advanced solid tumors. Patients were enrolled in successive cohorts of XL281 orally once daily in 28-day cycles. Twice daily dosing, different formulations, and the effect of food and famotidine were also studied. At the MTD expanded cohorts with defined mutations were treated. Results In total, 160 patients were treated. The MTD on the QD schedule was 150 mg. The most common toxicities were diarrhea, nausea, and fatigue. Plasma Cmax and AUC increased with dose. Famotidine resulted in lower AUC while food had no effect. Two patients had partial responses by RECIST: One with papillary thyroid cancer with NRAS mutation and one with uveal melanoma. Another nine patients had tumor decrease of >10% but did not meet RECIST criteria for PR. Matched tumors pairs from 33 patients showed evidence of RAF inhibition with significant decreases in pERK, pMEK and pAKT. Conclusions XL281 was generally well tolerated and the MTD was established at 150 mg/day. Partial responses and clinical benefit were observed in several patients. Tumor biopsies demonstrated effective target inhibition.

This phase I study evaluated selective JAK2 inhibitor XL019 in 30 patients with myelofibrosis. The initial dose cohorts were 100, 200, and 300 mg orally on days 1-21 of a 28-day cycle. Central and/or peripheral neurotoxicity developed in all patients. Subsequently, patients were treated on lower doses; neurotoxicity was again observed, leading to study termination. Peripheral neuropathy resolved in 50%, and central neurotoxicity in all patients within months after therapy cessation. Myelosuppression was minimal. The terminal half-life of XL019 was approximately 21 h, with steady state reached by Day 8. International Working Group defined responses were seen in three (10%) patients., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

About 10% of patients with essential thrombocythemia (ET) or myelofibrosis (MF) that lack mutations in JAK2 harbor an activating mutation in the thrombopoietin receptor, MPLW515L. Distinct from the JAK2V617F retroviral transplant model, the MPLW515L model recapitulates many features of ET and MF, including severe fibrosis and thrombocytosis. We have tested EXEL-8232, an experimental potent JAK2 inhibitor, for efficacy in suppression of thrombocytosis in vivo and for its ability to attenuate MPLW515L myeloproliferative disease. EXEL-8232 was administered for 28 days q12 h by oral gavage at doses of 30 or 100 mg/kg, prospectively. Animals treated with EXEL-8232 at 100 mg/kg had normalized high platelet counts, eliminated extramedullary hematopoiesis in the spleen and eliminated bone marrow fibrosis, whereas the wild-type controls did not develop thrombocytopenia. Consistent with a clinical response in this model, we validated surrogate end points for response to treatment, including a reduction of endogenous colony growth and signaling inhibition in immature erythroid and myeloid primary cells both in vitro and upon treatment in vivo. We conclude that EXEL-8232 has efficacy in treatment of thrombocytosis in vivo in a murine model of ET and MF, and may be of therapeutic benefit for patients with MPL-mutant MPN.

Chk1 and Chk2 kinases are critically involved in modulating DNA damage checkpoints. In particular, Chk1, a key activator of the S-phase DNA damage response, may be involved in resistance to genotoxic therapies that target DNA synthesis. We studied the in vitro and in vivo effects of EXEL-9844 (XL844), a potent, orally available, and specific inhibitor of Chk1 and Chk2, in combination with gemcitabine. In clonogenic assays using multiple cell lines in vitro, EXEL-9844 had only minor effects as a single agent but substantially enhanced gemcitabine-induced cell killing. Correspondingly, in PANC-1 cells, EXEL-9844 increased gemcitabine-induced H2AX phosphorylation, blocked Cdc25A phosphorylation, and induced premature mitotic entry. In a PANC-1 xenograft model, EXEL-9844 significantly enhanced gemcitabine antitumor activity but had limited effect as a single agent. Together, these data show that cell cycle checkpoint inhibitors may have significant clinical utility in potentiating the activity of gemcitabine.

During embryonic development, complex events, such as cellular proliferation, differentiation, survival, and guidance of axons, are orchestrated and regulated by a variety of extracellular signals. Receptor tyrosine kinases mediate many of these events, with several playing critical roles in neuronal survival and axonal guidance. It is evident that not all the receptor tyrosine kinases that play key roles in regulating neuronal development have been identified. In this study, we have characterized the spatial-temporal expression profile of a recently identified receptor tyrosine kinase, anaplastic lymphoma kinase (ALK), in embryonic chick by means of whole-mount in situ hybridization in conjunction with immunohistochemistry. Our findings reveal that Alk is expressed in sympathetic and dorsal root ganglia as early as stage 19. In addition, mRNA is expressed from stage 23/24 (E4) to stage 39 (E13) in discrete motor neuron subsets of chick spinal cord along with a select group of muscles that are innervated by one of these particular motor neuron clusters. Expression within the spinal cord is coincident with the onset and duration of motor neuron programmed cell death and during the period of musculature innervation and synapse formation. Hence, the data presented here identify ALK as a novel candidate receptor for regulating critical events in the development of neurons in both the central and the peripheral nervous systems., (Copyright 2006 Wiley-Liss, Inc.)

With the sequence of the human genome at hand, target discovery strategies are needed that can rapidly identify novel gene products involved in human disease pathways. In this article, the authors describe a cell-based, high-throughput assay that can identify gene products capable of modulating the vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha (TNFalpha) signaling pathways in human endothelial cells. The assay uses real-time PCR technology to measure downstream reporter mRNA transcripts induced upon cytokine stimulation in a 96-well plate format and has been adapted for use with recombinant adenoviruses. The authors specifically demonstrate modulation of cytokine-driven reporter transcripts using drug inhibitors and through adenoviral-mediated expression of known signaling intermediates of the respective pathways. In addition, they have used an arrayed library of 350 recombinant adenoviruses to screen for novel modulators of the VEGF and TNFalpha pathways. The high-throughput screening capacity and sensitivity of this system make it a useful tool for new drug target identification.

To facilitate the characterization of proteins that negatively regulate tumor cell proliferation in vitro, the authors have implemented a high-throughput functional assay that measures S-phase progression of tumor cell lines. For 2 tumor cell lines-human melanoma A375 and human lung carcinoma A549-conditions were established using the cyclin-dependent kinase inhibitor, p27kip; the tumor suppressor p53, a kinase-inactive allele of the cell cycle-regulated serine/threonine kinase Aurora2; and the G1/S drug block, aphidicolin. For screening purposes, gene libraries were delivered by adenoviral infection. Cells were fixed and labeled by immunocytochemistry, and an automated image acquisition and analysis package on a Cellomics ArrayScanII was used to quantify the effects of these treatments on cell proliferation. The assay can be used to identify novel proteins involved in proliferation and serves as a more robust, reproducible, and sensitive alternative to enzyme-linked immunosorbent assay (ELISA)-based technologies.

Nerve growth factor is known to stimulate neurite outgrowth and support neuronal survival during embryonic development. We have studied the expression of the nerve growth factor receptor, TrkA, at both mRNA and protein levels during the course of chicken retinal development. Furthermore, we have compared the expression of trkA mRNA with that of the 75-kD low-affinity neurotrophin receptor (p75NTR). RNase protection assay identified peak-levels of trkA mRNA in the late embryonic retina. Using in situ hybridization and immunohistochemistry, we found cells expressing TrkA in both the internal and the external part of the inner nuclear layer, corresponding to amacrine and horizontal cells, respectively. The TrkA-expressing amacrine cell has a unistratified dendritic arborization in the second sublamina of the inner plexiform layer, and may represent the stellate amacrine cell described by Cajal. The horizontal cells, possessing arciform dendrite processes in the outer plexiform layer, showed strong TrkA immunoreactivity in both dendrites and cell bodies. During the course of retinal development, the TrkA-expressing amacrine cells decreased in number, whereas the TrkA-expressing horizontal cells persisted. Because nerve growth factor was expressed where the horizontal cells, but not where the amacrine cells were located, these findings raise the question of whether nerve growth factor could locally support the survival of TrkA-expressing interneurons during retinal development.

We report the presence in rat spinal cord of a novel neuronal system expressing tyrosine kinase receptor (trkA), the high affinity receptor for nerve growth factor (NGF). TrkA immunoreactive cell bodies were observed in the intermediate grey matter of the spinal cord and were classified into three main groups: central canal cells located dorsolateral to the aqueduct, partition cells located between lamina X, and the lateral border of the intermediate grey, and a morphologically heterogeneous group which included large cells located near the lateral border. In situ hybridization confirmed that cells in all these areas express trkA mRNA. Combined immunofluorescence and retrograde Fluoro-Gold labelling was used to further characterise the projections and neurotransmitter profile of the trkA cells. Although often located in the vicinity of preganglionic cell groups, trkA immunoreactive cells are not themselves preganglionic. Rather, the central canal and partition cells belong to a neurochemically complex cholinergic propriospinal system. Many partition cells coexpress trkA, choline acetyltransferase (ChAT), the low affinity neurotrophin receptor, p75, and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d). In contrast, trkA immunoreactive central canal cells express ChAT, but do not express p75 and only a subpopulation express NADPH-d. The large trkA immunoreactive cells located on the lateral border do not express ChAT. TrkA immunoreactive fibres were also present and were located in the dorsal horn, in the dorsal columns, and in a bundle ventral to the aqueduct. However, double labelling revealed that the trkA immunoreactive fibres are not intrinsic but are primary afferent in origin and coexpress p75. The location of this novel trkA neuronal system is consistent with it having a role in the segmental integration of autonomic outflow. NGF could affect this system by modulating neuronal phenotype and/or synaptic efficacy.

The neurons of the dorsal root ganglia (DRG) that supply muscle spindles require target-derived factors for survival. One necessary factor for these neurons is neurotrophin-3 (NT3). To determine whether NT3 can promote the survival of these neurons in the absence of other target-derived factors, we analyzed the effects of exogenous NT3 after early limb bud deletion in the chick. In control embryos, limb bud deletion eliminated approximately 90% of the trkC-positive (trkC+) neurons in lumbar DRG on the deleted side. In addition, the deletion led to a dramatic loss of collateral sensory projections to motoneurons. Exogenous NT3 restored a normal population of trkC+ neurons in lumbar DRG on the deleted side and increased the number of trkC+ neurons in DRG with normal targets (contralateral lumbar and thoracic). The effect was highly selective; NT3 increased the number of trkC+ neurons without significantly changing the number of either trkA+ or trkB+ neurons. The effect of NT3 was attributable to the rescue of DRG neurons from cell death, because exogenous NT3 reduced the number of pyknotic nuclei without significantly altering proliferation. Analysis of spinal projections showed further that many of the trkC+ neurons rescued by NT3 projected to the ventral spinal cord. These neurons thus had central projections characteristic of muscle spindle afferents. Together, our results indicate that NT3 signaling is both necessary and sufficient for the development of the proprioceptive phenotype, even in the absence of other signals from limb muscle.

In development approximately 70-80% of dorsal root ganglion (DRG) cells are dependent on nerve growth factor (NGF) for their survival, while in the adult only some 40% of DRG cells express the high-affinity NGF receptor, trkA. This discrepancy suggests that trkA expression, and therefore neurotrophin sensitivity, may alter as the animal matures. We have tested this possibility by counting the number of L4/5 DRG neurons showing immunoreactivity for trkA in rats from the day of birth to postnatal day 14. We also examined changes in p75 and IB4 labelling. On the day of birth, 71% of DRG cells were found to express trkA. However, this percentage gradually fell with age and reached adult levels at postnatal day 14. The expression of p75 did not parallel that of trkA, remaining relatively constant at between 45 and 50% of cells from birth to postnatal day 14. Over the same period there was a marked increase in the proportion of cells which bind the lectin IB4 from 9 (day of birth) to 40% (day 14). Since in the adult the IB4 population consists of small cells which mostly do not express trkA, this finding suggests that the postnatal down-regulation of trkA occurs in this population. Consistent with this suggestion are the results of double labelling for trkA and IB4, which confirmed that at times intermediate between birth and postnatal day 14 there was a high degree of coexpression between these markers (which is absent in the adult). This result also suggests that the down-regulation of trkA is unlikely to be directly responsible for the emerging IB4 binding.

To assess functions of neurotrophins at defined times in development, we have prepared antibodies of the extracellular domains of each of the trk receptors. Here, antibodies to trkC, the major receptor for NT-3, are used to examine trkC expression and function during the formation and maturation of the chick dorsal root ganglion (DRG). Our results show that in the immature DRG, the majority of cells express trkC, and inhibition of trkC activation results in reductions in neuronal numbers before the period of target-mediated cell death, the time when neurotrophins previously have been shown to regulate survival. Furthermore, blockade of trkC in ovo induced reductions in subpopulations of DRG neurons known to be dependent on NGF, in addition to those dependent on NT-3 during the target-regulated cell death period. An early function for NT-3 on immature DRG neurons is supported further by data presented here that demonstrate that whereas BDNF and NGF can support a subset of immature DRG neurons in vitro, activation of the trkC receptor either by NT-3 binding or via antibody-mediated cross-linking induces the most robust survival response. When all three neurotrophins are combined, the number of surviving neurons does not exceed that supported by NT-3 alone. Together, these data are consistent with coexpression of more than one trk receptor family member on immature sensory neurons, and they demonstrate that inhibition of trkC activation has surprisingly early and pleiotrophic effects on the development of spinal sensory ganglia.

The receptors involved in retrograde transport of neurotrophins from the retina to the isthmo-optic nucleus (ION) of chick embryos were characterized using antibodies to the p75 neurotrophin receptor and trkB receptors. Survival of neurons in the ION has been shown previously to be regulated by target-derived trophic factors with survival promoted or inhibited by ocular injection of brain-derived neurotrophic factor (BDNF) or nerve growth factor (NGF), respectively. In the present paper, we show that during the period of target dependence, these neurons express trkB and p75 neurotrophin receptor but not trkA or trkC mRNAs. We also show that BDNF and NT-3 were transported efficiently at low doses, whereas NGF was transported significantly only at higher doses. The transport of BDNF and NT-3 was reduced by high concentrations of NGF or by antibodies to either trkB or the p75 neurotrophin receptor. Thus both receptors help mediate retrograde transport of these neurotrophins. Ocular injection of the comparatively specific trk inhibitor K252a did not reduce transport of exogenous BDNF, but did induce significant neuronal death in the ION, which could not be prevented by co-injection of BDNF. Thus, transport of BDNF alone does not generate a trophic signal at the cell body when axonal trkB is inactivated. In summary, our results indicate that both p75 neurotrophin and trkB receptors can mediate internalization and retrograde transport of BDNF, but activation of trkB seems to be essential for the survival-promoting actions of this neurotrophin.

To test the molecular nature of the NGF receptor responsible for the ability of NGF to rescue septal cholinergic neurons following axotomy, we infused polyclonal antibodies that act as specific agonists of trkA (RTA) into the lateral ventricle of fimbria-fornix lesioned animals. Rats receiving chronic intraventricular infusions of RTA showed significantly more low affinity NGF receptor immunoreactive (p75NGFR-IR) neurons on the lesioned side than did control animals 2 weeks following unilateral fimbria-fornix lesion. RTA also initiated cholinergic sprouting. Infusions of RTA in combination with an antibody that blocks p75NGFR (REX) did not reduce the cell savings effect observed with RTA alone. However, animals infused with RTA plus REX demonstrated significantly less sprouting. These findings suggest that antibody-induced trkA activation is sufficient to mediate NGF-promoted survival of axotomized cholinergic neurons in vivo.

Although central neurons do not naturally recover following injury, damaged adult septal neurons can regenerate when nerve growth factor (NGF) is provided along with a suitable cellular substrate. This study investigates the outgrowth of axotomized septal neurons grafted with primary fibroblasts genetically modified to produce NGF. Confocal microscope images of double staining for neuritic markers (neurofilament or low-affinity NGF receptor) and the astrocytic marker glial fibrillary acidic protein (GFAP) demonstrated that regenerating neurites crossed dense buildups of astrocytic processes at the edges of NGF-producing grafts and were in apposition with astrocytic processes within NGF-producing grafts. Immunoreactivity for acetylcholinesterase and low-(p75) and high-affinity (TrkA) NGF receptors was dense in NGF-producing grafts but absent in control grafts. NGF-grafted rats exhibited significantly increased hippocampal density of p75-immunoreactive fibers and significantly decreased ectopic hippocampal sympathetic ingrowth as compared to control-grafted rats. Rats with unilateral fimbria-fornix lesions and NGF-producing grafts exhibited ameliorated performance on a simple memory task. These findings demonstrate that implantation of NGF-producing grafts to the lesion cavity allows axotomized septal cholinergic neurons to reinnervate the hippocampus, and that rats receiving these grafts show a partial recovery of function.

Receptor-type protein tyrosine phosphatase beta (RPTP beta) is expressed in the developing nervous system and contains a carbonic anhydrase (CAH) domain as well as a fibronectin type III repeat in its extracellular domain. Fusion proteins containing these domains were used to search for ligands of RPTP beta. The CAH domain bound specifically to a 140 kDa protein expressed on the surface of neuronal cells. Expression cloning in COS7 cells revealed that this protein is contactin, a GPI membrane-anchored neuronal cell recognition molecule. The CAH domain of RPTP beta induced cell adhesion and neurite growth of primary tectal neurons, and differentiation of neuroblastoma cells. These responses were blocked by antibodies against contactin, demonstrating that contactin is a neuronal receptor for RPTP beta. These experiments show that an individual domain of RPTP beta acts as a functional ligand for the neuronal receptor contactin. The interaction between contactin and RPTP beta may generate unidirectional or bidirectional signals during neural development.

Immunocytochemistry has been used to examine the location of trkA, the high-affinity receptor for nerve growth factor, in adult rat dorsal root ganglia, trigeminal ganglia and spinal cord. TrkA immunoreactivity was observed in small and medium sized ganglion cells and in the dorsal horn of the spinal cord. In lumbar L4 and L5 ganglia trkA-immunoreactive cells constitute 40% of dorsal root ganglion cells and range in size from 15 to 45 microns in diameter. Double labelling using markers for various dorsal root ganglion subpopulations revealed that virtually all (92%) trkA-immunoreactive cells express calcitonin gene-related peptide (CGRP) immunoreactivity. In contrast only 4 and 13% of trkA-immunoreactive cells are labelled by the monoclonal antibody LA4 or the lectin Griffonia simplicifolia IB4, markers for small non-peptide-containing cells. Eighteen percent of trkA-immunoreactive cells belong to the 'large light' subpopulation, identified by their strong immunostaining by the neurofilament antibody RT97. TrkA immunoreactivity in the dorsal horn is heaviest in laminae I and II outer, has a similar distribution to CGRP, and is depleted by dorsal rhizotomy. Our results show that trkA-expressing cells in dorsal root ganglia correspond almost exactly with the CGRP, peptide-producing population. The receptor is present not only on cell bodies but also on central terminals. Non-peptide-containing small cells, which constitute 30% of dorsal root ganglion cells, are not trkA-immunoreactive and therefore most probably are functionally independent of nerve growth factor.

The present investigation used an antibody directed against the extracellular domain of the signal transducing nerve growth factor receptor, trkA, to reveal immunoreactive perikarya or fibers within the olfactory bulb and tubercle, cingulate cortex, nucleus accumbens, striatum, endopiriform nucleus, septal/diagonal band complex, nucleus basalis, hippocampal complex, thalamic paraventricular and reuniens nuclei, periventricular hypothalamus, interpeduncular nucleus, mesencephalic nucleus of the fifth nerve, dorsal nucleus of the lateral lemniscus, prepositus hypoglossal nucleus, ventral cochlear nucleus, ventral lateral tegmentum, medial vestibular nucleus, spinal trigeminal nucleus oralis, nucleus of the solitary tract, raphe nuclei, and spinal cord. Colocalization experiments revealed that virtually all striatal trkA-immunoreactive neurons (> 99%) coexpressed choline acetyltransferase (ChAT) but not p75 nerve growth factor receptor (NGFR). Within the septal/diagonal band complex virtually all trkA neurons (> 95%) coexpressed both ChAT and p75 NGFR. More caudally, dual stained sections revealed numerous trkA/ChAT (> 80%) and trkA/p75 NGFR (> 95%) immunoreactive neurons within the nucleus basalis. In the brainstem, raphe serotonergic neurons (45%) coexpressed trkA. Sections stained with a pan-trk antibody that recognizes primarily trkA, as well as trkB and trkC, labeled neurons within all of these regions as well as within the hypothalamic arcuate, supramammilary, and supraoptic nuclei, hippocampus, inferior and superior colliculus, substantia nigra, ventral tegmental area of T'sai, and cerebellular Purkinje cells. Virtually all of these other regions with the exception of the cerebellum also expressed pan-trk immunoreactivity in the monkey. The widespread expression of trkA throughout the central neural axis suggests that this receptor may play a role in signal transduction mechanisms linked to NGF-related substances in cholinergic basal forebrain and noncholinergic systems. These findings suggest that pharmacological use of ligands for trkA could have beneficial effects on the multiple neuronal systems that are affected in such disorders as Alzheimer's disease.

TrkA, a member of the receptor tyrosine kinase family, binds nerve growth factor (NGF) and subsequently activates intracellular signaling pathways. Previous studies have found variable and weak interaction of the TrkA receptor with neurotrophin 3 (NT-3), another member of the NGF family. TrkA is expressed in two splice forms, differing in the presence of an 18-bp exon in the extracellular domain. The biological responses of each isoform of the TrkA receptor were tested after transfection into the cell line PC12nnr5, a variant of PC12 cells lacking functional TrkA protein. NGF was found to activate each form of the receptor comparably. However, the TrkA isoform containing the variable exon showed significantly higher activation by NT-3, which was detected by stimulation of TrkA autophosphorylation, induction of ZIF268 transcription, and cellular differentiation. Function-perturbing antibodies to the p75 low-affinity NGF receptor potentiated the NT-3 responses of both forms of TrkA in the transfected PC12nnr5 cell lines, suggesting that the low-affinity NGF receptor suppresses the ability of TrkA to respond to NT-3.

Nerve growth factor (NGF) acts through trkA receptors to serve as a trophic factor for cholinergic neurones in the medial septal nucleus (MSN) and vertical limb of the diagonal band (VDB). Herein, we show that brain nitric oxide synthase (NOS), which synthesizes the neuromodulator nitric oxide, is selectively expressed in a large fraction of trkA-containing neurones in the MSN and VDB. Axotomy of these neurones gave evidence that NOS expressing cholinergic neurones innervate the hippocampus. NGF infusion induced a robust, specific increase in NOS expression in basal forebrain cholinergic neurones. These results indicate that brain NOS can be regulated by a neurotrophic factor and suggest that NGF influences forebrain function by regulating production of nitric oxide as well as acetylcholine.

TrkA, a tyrosine kinase receptor, is an essential component of the nerve growth factor (NGF) response pathway. The binding of NGF to the receptor induces receptor autophosphorylation and activation of intracellular signaling pathways, resulting in diverse biological effects. We prepared polyclonal antibodies against the entire extracellular domain of rat trkA produced using a baculovirus expression system. These antibodies specifically recognize rat trkA on antigen blots and in immunoprecipitations. Both IgG and Fab fragments block binding of NGF to trkA expressed by the PC12 cell line. In NGF binding studies using anti-trkA and anti-low-affinity NGF receptor (LNGFR) immunoglobulin (Ig) G, essentially all binding of NGF can be inhibited. The results imply that > or = 97% of the NGF binding sites on PC12 cells are accounted for by trkA and the LNGFR. The binding data also argue that all low-affinity NGF binding sites on PC12 cells reflect interactions with the LNGFR, while all high-affinity sites are trkA dependent. A fraction of the high-affinity (or slow) binding sites seem to require both trkA and the LNGFR. Although the monovalent anti-trkA Fab fragments inhibited the biological effects of NGF, such as induction of tyrosine phosphorylation, and survival and neurite outgrowth of sympathetic neurons, the IgG preparation was not effective as an inhibitor. Instead, the IgG fraction by itself was almost as effective as NGF at stimulating receptor activation, cell survival, and neurite outgrowth. Thus, it appears oligomerization of trkA by antibody-induced cross-linking is sufficient to produce the known cellular effects of NGF.

The soluble NSF attachment proteins (SNAPs) enable N-ethyl-maleimide-sensitive fusion protein (NSF) to bind to target membranes. Here we report the cloning and sequencing of complementary DNAs encoding alpha-, beta- and gamma-SNAPs. Two of these proteins, alpha and gamma, are found in a wide range of tissues, and act synergistically in intra-Golgi transport. The third, beta, is a brain-specific isoform of alpha-SNAP. Thus, NSF and SNAPs appear to be general components of the intracellular membrane fusion apparatus, and their action at specific sites of fusion must be controlled by SNAP receptors particular to the membranes being fused, as described in the accompanying article.

We have used an in vitro Golgi protein transport assay dependent on high molecular weight (greater than 100 kD) cytosolic and/or peripheral membrane proteins to study the requirements for transport from the cis- to the medial-compartment. Fractionation of this system indicates that, besides the NEM-sensitive fusion protein (NSF) and the soluble NSF attachment protein (SNAP), at least three high molecular weight protein fractions from bovine liver cytosol are required. The activity from one of these fractions was purified using an assay that included the second and third fractions in a crude state. The result is a protein of 115-kD subunit molecular mass, which we term p115. Immunodepletion of the 115-kD protein from a purified preparation with mAbs removes activity. Peptide sequence analysis of tryptic peptides indicates that p115 is a "novel" protein that has not been described previously. Gel filtration and sedimentation analysis indicate that, in its native state, p115 is a nonglobular homo-oligomer. p115 is present on purified Golgi membranes and can be extracted with high salt concentration or alkaline pH, indicating that it is peripherally associated with the membrane. Indirect immunofluorescence indicates that p115 is associated with the Golgi apparatus in situ.

The nucleotide sequences of the mitochondrial DNA (mtDNA) molecules of two nematodes, Caenorhabditis elegans [13,794 nucleotide pairs (ntp)], and Ascaris suum (14,284 ntp) are presented and compared. Each molecule contains the genes for two ribosomal RNAs (s-rRNA and l-rRNA), 22 transfer RNAs (tRNAs) and 12 proteins, all of which are transcribed in the same direction. The protein genes are the same as 12 of the 13 protein genes found in other metazoan mtDNAs: Cyt b, cytochrome b; COI-III, cytochrome c oxidase subunits I-III; ATPase6, Fo ATPase subunit 6; ND1-6 and 4L, NADH dehydrogenase subunits 1-6 and 4L: a gene for ATPase subunit 8, common to other metazoan mtDNAs, has not been identified in nematode mtDNAs. The C. elegans and A. suum mtDNA molecules both include an apparently noncoding sequence that contains runs of AT dinucleotides, and direct and inverted repeats (the AT region: 466 and 886 ntp, respectively). A second, apparently noncoding sequence in the C. elegans and A. suum mtDNA molecules (109 and 117 ntp, respectively) includes a single, hairpin-forming structure. There are only 38 and 89 other intergenic nucleotides in the C. elegans and A. suum mtDNAs, and no introns. Gene arrangements are identical in the C. elegans and A. suum mtDNA molecules except that the AT regions have different relative locations. However, the arrangement of genes in the two nematode mtDNAs differs extensively from gene arrangements in all other sequenced metazoan mtDNAs. Unusual features regarding nematode mitochondrial tRNA genes and mitochondrial protein gene initiation codons, previously described by us, are reviewed. In the C. elegans and A. suum mt-genetic codes, AGA and AGG specify serine, TGA specifies tryptophan and ATA specifies methionine. From considerations of amino acid and nucleotide sequence similarities it appears likely that the C. elegans and A. suum ancestral lines diverged close to the time of divergence of the cow and human ancestral lines, about 80 million years ago.

We report conditions under which Golgi membranes depleted of peripheral membrane proteins can be reconstituted for intra-cisternal vesicular transport. Analysis of the reconstitution reveals requirements for N-ethylmaleimide-sensitive fusion protein, a purified peripheral protein involved in the fusion stage of vesicular transport, as well as other peripheral protein activities which can be provided by mammalian cytosol but not yeast cytosol. The restorative activity in bovine brain cytosol is found in two broad and complementing fractions, of average native molecular masses of about 500 and 40 kDa, termed Fr1 and Fr2, respectively. This resolved transport system was used to develop a purification scheme for Fr2. Three proteins of apparent molecular masses of 35, 36, and 39 kDa (Fr2-alpha, -beta, and -gamma, respectively) were found to be responsible for Fr2 activity and were purified to homogeneity. Each Fr2 protein has activity by itself in the reconstituted in vitro Golgi transport assay, although each exhibits a different specific activity and plateau value. No synergy of the three Fr2 proteins was observed during mixing experiments. The three Fr2 proteins seem to be closely related based on size, in vitro activities, chromatographic properties, and peptide maps and may comprise a new family of proteins involved in vesicular transport.

Three new and likely related components of the cellular fusion machinery have been purified from bovine brain cytosol, termed alpha-SNAP (35 kd), beta-SNAP (36 kd), and gamma-SNAP (39 kd). Transport between cisternae of the Golgi complex measured in vitro requires SNAP activity during the membrane fusion stage, and each SNAP is capable of binding the general cellular fusion protein NSF to Golgi membranes. The SNAP-NSF-membrane complex may be an early stage in the assembly of a proposed multisubunit "fusion machine" on the target membrane. SNAP transport factor activity is also found in yeast. Yeast cytosol prepared from a secretion mutant defective in export from the endoplasmic reticulum (sec17) lacks SNAP activity, which can be restored in vitro by the addition of pure alpha-SNAP, but not beta- or gamma-SNAPs. These data suggest that the mechanism of action of SNAPs in membrane fusion is conserved in evolution.

Genes for URF2, tRNAtrp, tRNAcys, tRNAtyr and cytochrome c oxidase subunit I (COI) have been identified within a sequenced segment of the Drosophila yakuba mtDNA molecule. The five genes are arranged in the order given. Transcription of the tRNAcys and tRNAtyr genes is in the same direction as replication, while transcription of the URF2, tRNAtrp and COI genes is in the opposite direction. A similar arrangement of these genes is found in mammalian mtDNA except that in the latter, the tRNAala and tRNAasn genes are located between the tRNAtrp and tRNAcys genes. Also, a sequence found between the tRNAasn and tRNAcys genes in mammalian mtDNA, which is associated with the initiation of second strand DNA synthesis, is not found in this region of the D. yakuba mtDNA molecule. As the D. yakuba COI gene lacks a standard translation initiation codon, we consider the possibility that the quadruplet ATAA may serve this function. As in other D. yakuba mitochondrial polypeptide genes, AGA codons in the URF2 and COI genes do not correspond in position to arginine-specifying codons in the equivalent genes of mouse and yeast mtDNAs, but do most frequently correspond to serine-specifying codons.

Genes for tRNAgly and tRNAserUCN have been identified within sequences of mtDNA of Drosophila yakuba. The tRNAgly gene lies between the genes for cytochrome c oxidase subunit III and URF3, and all three of these genes are contained in the same strand of the mtDNA molecule. The tRNAserUCN gene is adjacent to the URF1 gene. These genes are contained in opposite strands of the mtDNA molecule and their 3' ends overlap. The structures of the tRNAgly and tRNAserUCN genes, and of the four tRNA genes of D. yakuba mtDNA reported earlier (tRNAile, tRNAgln, tRNAf-met and tRNAval) are compared to each other, to non-organelle tRNAs, and to corresponding mammalian mitochondrial tRNA genes. Within 19 nucleotides upstream from the 5' terminal nucleotide of each of the Drosophila mitochondrial tRNAgly, tRNAserUCN, tRNAile, tRNAgln and tRNAf-met genes occurs the sequence 5'TTTATTAT, or a sequence differing from it by one nucleotide substitution. Upstream from this octanucleotide sequence, and separated from it by 3, 4 and 11 nucleotides, respectively, in the 5' flanking regions of the tRNAile, tRNAserUCN and tRNAgly genes occurs the sequence 5'GATGAG.

Part of the replication origin-containing A+T-rich region of the Drosophila yakuba mtDNA molecule and segments on either side of this region have been sequenced, and the genes within them identified. The data confirm that the small and large rRNA genes lie in tandem adjacent to that side of the A+T-rich region which is replicated first, and establish that a tRNAval gene lies between the two rRNA genes and that URF1 follows the large rRNA gene. The data further establish that the genes for tRNAile, tRNAgln, tRNAf-met and URF2 lie in the order given, on the opposite side of the A+T-rich region to the rRNA genes and, except for tRNAgln, are contained in the opposite strand to the rRNA, tRNAval and URF1 genes. This is in contrast to mammalian mtDNAs where all of these genes are located on the side of the replication origin which is replicated last, within the order tRNAphe, small (12S) rRNA, tRNAval, large (16S) rRNA, tRNAleu, URF1, tRNAile, tRNAgln, tRNAf-met and URF2, and, except tRNAgln, are all contained in the same (H) strand. In D. yakuba URF1 and URF2, the triplet AGA appears to specify an amino acid, which is again different from the situation found in mammalian mtDNAs, where AGA is used only as a rare termination codon.

Genes for URF3, tRNAala, tRNAarg, tRNAasn, tRNAserAGY, tRNAglu, tRNAphe, and the carboxyl terminal segment of the URF5 gene have been identified within a sequenced segment of the mtDNA molecule of Drosophila yakuba. The genes occur in the order given. The URF5 and tRNAphe genes are transcribed in the same direction as replication while the URF3 and remaining five tRNA genes are transcribed in the opposite direction. Considerable differences exist in the relative arrangement of these genes in D. yakuba and mammalian mtDNA molecules. In the tRNAserAGY gene an eleven nucleotide loop, within which secondary structure formation seems unlikely, replaces the dihydrouridine arm, and both the variable loop (six nucleotides) and the T phi C loop (nine nucleotides) are larger than in any other D. yakuba tRNA gene. As available evidence is consistent with AGA codons specifying serine rather than arginine in the Drosophila mitochondrial genetic code, the possibility is considered that the 5'GCU anticodon of the D. yakuba tRNAserAGY gene can recognize AGA as well as AGY codons.

Vesicular transport between successive compartments of the mammalian Golgi apparatus has recently been reconstituted in a cell-free system. In addition to ATP, transport requires both membrane-bound and cytosolic proteins. Here we report that the cytosol fraction from yeast will efficiently substitute for mammalian cytosol. Mammalian cytosol contains several distinct transport factors, which we have distinguished on the basis of gel filtration and ion-exchange chromatography. Yeast cytosol appears to contain the same collection of transport factors. Resolved cytosol factors from yeast and mammals complement each other in a synergistic manner. These findings suggest that the molecular mechanisms of intracellular protein transport have been conserved throughout evolution. Moreover, this hybrid cell-free system will enable the application of yeast genetics to the identification and isolation of cytosolic proteins that sustain intracellular protein transport.

We have compared nucleotide sequences of corresponding segments of the mitochondrial DNA (mtDNA) molecules of Drosophila yakuba and Drosophila melanogaster, which contain the genes for six proteins and seven tRNAs. The overall frequency of substitution between the nucleotide sequences of these protein genes is 7.2%. As was found for mtDNAs from closely related mammals, most substitutions (86%) in Drosophila mitochondrial protein genes do not result in an amino acid replacement. However, the frequencies of transitions and transversions are approximately equal in Drosophila mtDNAs, which is in contrast to the vast excess of transitions over transversions in mammalian mtDNAs. In Drosophila mtDNAs the frequency of C----T substitutions per codon in the third position is 2.5 times greater among codons of two-codon families than among codons of four-codon families; this is contrary to the hypothesis that third position silent substitutions are neutral in regard to selection. In the third position of codons of four-codon families transversions are 4.6 times more frequent than transitions and A----T substitutions account for 86% of all transversions. Ninety-four percent of all codons in the Drosophila mtDNA segments analyzed end in A or T. However, as this alone cannot account for the observed high frequency of A----T substitutions there must be either a disproportionately high rate of A----T mutation in Drosophila mtDNA or selection bias for the products of A----T mutation. --Consideration of the frequencies of interchange of AGA and AGT codons in the corresponding D. yakuba and D. melanogaster mitochondrial protein genes provides strong support for the view that AGA specifies serine in the Drosophila mitochondrial genetic code.

The nucleotide sequence of a segment of the mtDNA molecule of Drosophila yakuba has been determined, within which have been identified the 3' end of the large rRNA gene and the entire genes for tRNAleuCUN , URF1 , tRNAserUCN , cytochrome b, URF6 , tRNApro, tRNAthr , URF4L , URF4 , tRNAhis and URF5 . The genes are arranged in the order given, with the large rRNA gene being closest to the A+T-rich region which contains the origin of replication. Transcription of all of these genes except those for cytochrome b, URF6 , tRNAserUCN and tRNAthr proceeds in the same direction as replication. Differences occur in the relative arrangement and in the direction of transcription of these twelve genes between D. yakuba and mammalian mtDNA molecules. Internal AGA codons occur in all of the polypeptide genes except URF6 . Comparisons of the positions of these AGA codons to codons in corresponding mouse genes is consistent with the view that in the D. yakuba mitochondrial genetic code AGA specifies serine. Genes equivalent to all of the polypeptide, tRNA and rRNA genes found in mammalian mtDNA have now been identified in D. yakuba mtDNA.

The nucleotide sequence of a segment of the mtDNA molecule of Drosophila yakuba which contains the A+T-rich region and the small and large rRNA genes separated by the tRNAval gene has been determined. The 5' end of the small rRNA gene was located by S1 protection analysis. In contrast to mammalian mtDNA, a tRNA gene was not found at the 5' end of the D. yakuba small rRNA gene. The small and large rRNA genes are 20.7% and 16.7% G+C and contain only 789 and 1326 nucleotides. The 5' regions of the small rRNA gene (371 nucleotides) and of the large rRNA gene (643 nucleotides) are extremely low in G+C (14.6% and 9.5%, respectively) and convincing sequence homologies between these regions and the corresponding regions of mouse mt-rRNA genes were found only for a few short segments. Nevertheless, the entire lengths of both of the D. yakuba mt-rRNA genes can be folded into secondary structures which are remarkably similar to secondary structures proposed for the rRNAs of mouse mtDNA. The replication origin-containing, A+T-rich region (1077 nucleotides; 92.8% A+T), which lies between the tRNAile gene and the small rRNA gene, lacks open reading frames greater than 123 nucleotides.

The nucleotide sequence of a segment of the mtDNA molecule of Drosophila yakuba has been determined, within which have been identified the genes for tRNAleuUUR, cytochrome c oxidase subunit II (COII), tRNAlys, tRNAasp, URFA6L, ATPase subunit 6 (ATPase6), cytochrome c oxidase subunit III (COIII) and tRNAgly. The genes are arranged in the order given and all are transcribed from the same strand of the molecule in a direction opposite to that in which replication proceeds around the molecule. The tRNAlys gene is unusual among mitochondrial tRNAlys genes in that it contains a CTT anticodon. The triplet AGA is used to specify an amino acid in all of the COII, COIII, ATPase6, and URFA6L genes. However, the AGA codons found in these four polypeptide genes correspond in position to codons which specify nine different amino acids, but never arginine, in the equivalent polypeptide gene which have been sequenced from mtDNAs of mouse, yeast and Zea mays.

The complete nucleotide sequence of the mitochondrial DNA (mtDNA) molecule of the parasitic nematode worm Ascaris suum has been determined. This molecule lacks genes for tRNAs of the standard form. Instead, 21 sequences are found that can be folded into structures that resemble tRNAs in which the T psi C arm and variable loop are missing and replaced with a single loop of between 4 and 12 nucleotides. Considerations of various properties of these sequences, including the number, predicted anticodons, conserved nucleotides, direction of transcription, base composition, and relative gene arrangements are consistent with the interpretation that they are genes for a different sort of tRNA. Transfer RNA genes with a similar potential secondary structure are found in mtDNA of the free-living nematode Caenorhabditis elegans, suggesting that this unusual form of tRNA is used by all nematode mitochondria.

The sequence of a segment of the Drosophila virilis mitochondrial DNA (mtDNA) molecule that contains the A + T-rich region, the small rRNA gene, the tRNA(f-met), tRNA(gln), and tRNA(ile) genes, and portions of the ND2 and tRNA(val) genes is presented and compared with the corresponding segment of the D. yakuba mtDNA molecule. The A + T-rich regions of D. virilis and D. yakuba contain two correspondingly located sequences of 49 and 276/274 nucleotides that appear to have been conserved during evolution. In each species the replication origin of the mtDNA molecule is calculated to lie within a region that overlaps the larger conserved sequence, and within this overlap is found a potential hairpin structure. Substitutions between the larger conserved sequences of the A + T-rich regions, the small mt-rRNA genes, and the ND2 genes are biased in favor of transversions, 71-97% of which are A----T changes. There is a 13.8 times higher frequency of nucleotide differences between the 5' halves than between the 3' halves of the D. virilis and D. yakuba small mt-rRNA genes. Considerations of the effects of observed substitutions and deletion/insertions on possible nucleotide pairing within the small mt-rRNA genes of D. virilis and D. yakuba strongly support the secondary structure model for the Drosophila small mt-rRNA that we previously proposed.

The mitochondrial genome of Drosophila yakuba is a circular DNA molecule of 16019 nucleotide pairs. The sequence contains the genes for two rRNA molecules, 22 tRNA molecules, five known polypeptides (cytochrome b, cytochrome c oxidase subunits I, II, III and ATPase subunit 6) and eight unidentified polypeptides (URF1, 2, 3, 4L, 4, 5, 6 and A6L). Between the tRNA(ile) and small rRNA genes there occurs a sequence of 1077 nucleotides that is 92.8 per cent A + T and lacks reading frames greater than 123 nucleotides. Replication of the molecule originates in this A + T-rich region and proceeds toward the small rRNA gene. Non-coding nucleotides between genes are either absent or occur in low numbers (1 to 31). A sequence equivalent in size and secondary structure potential to the sequence associated with the initiation of second strand synthesis in mammalian mtDNA is missing in Drosophila mtDNA. While the genes found in D. yakuba and mammalian mtDNAs are the same, the relative arrangement of many of these genes differs considerably in the two molecules. The proportions of the two strands of the D. yakuba molecule which serve as template for transcription of genes are approximately equal. This contrasts with the situation in mammalian mtDNAs where all genes except those for URF6 and eight tRNAs are transcribed from one strand. The dihydrouridine and T psi C loops of D. yakuba mt-tRNA genes are highly variable in size, and among these genes there is a general deficiency of nucleotides which are highly conserved in prokaryotic and eukaryotic nuclear-coded tRNAs. The D. yakuba tRNA(AGYser) gene is unusual in that an eleven nucleotide loop replaces the dihydrouridine arm. D. yakuba mitochondrial polypeptide genes utilize 59 sense codons. However, 93.8 per cent of all codons used end in A or T. Unique variations occur in the Drosophila mitochondrial genetic code. AGA appears to specify serine rather than arginine as in the standard code, or termination as in the mammalian mitochondrial code. The Drosophila COI gene lacks a standard translation initiation codon, and may utilize a four nucleotide codon ATAA for that purpose. As in other metazoan mitochondria, TGA and ATA specify tryptophan and methionine, respectively. As a tRNA with an anticodon (TCT) specific for AGA codons does not appear to be encoded in D. yakuba mtDNA, it seems likely that the GCU anticodon of the D. yakuba tRNA which recognizes AGY (serine) codons can also recognize AGA.(ABSTRACT TRUNCATED AT 400 WORDS)

The sequence of the 16,019 nucleotide-pair mitochondrial DNA (mtDNA) molecule of Drosophila yakuba is presented. This molecule contains the genes for two rRNAs, 22 tRNAs, six identified proteins [cytochrome b, cytochrome c oxidase subunits I, II, and III (COI-III), and ATPase subunits 6 and 8] and seven presumptive proteins (URF1-6 and URF4L). Replication originates within a region of 1077 nucleotides that is 92.8% A + T and lacks any open reading frame larger than 123 nucleotides. An equivalent to the sequence found in all mammalian mtCDNAs that is associated with initiation of second-strand DNA synthesis is not present in D. yakuba mtDNA. Introns are absent from D. yakuba mitochondrial genes and there are few (0-31) intergenic nucleotides. The genes found in D. yakuba and mammalian mtDNAs are the same, but there are differences in their arrangement and in the relative proportions of the complementary strands of the molecule that serve as templates for transcription. Although the D. yakuba small and large mitochondrial rRNA genes are exceptionally low in G and C and are shorter than any other metazoan rRNA genes reported, they can be folded into secondary structures remarkably similar to the secondary structures proposed for mammalian mitochondrial rRNAs. D. yakuba mitochondrial tRNA genes, like their mammalian counterparts, are more variable in sequence than nonorganelle tRNAs. In mitochondrial protein genes ATG, ATT, ATA, and in one case (COI) ATAA appear to be used as translation initiation codons. The only termination codon found in these genes is TAA. In the D. yakuba mitochondrial genetic code, AGA, ATA, and TGA specify serine, isoleucine, and tryptophan, respectively. Fifty-nine types of sense condon are used in the D. yakuba mitochondrial protein genes, but 93.8% of all codons end in A or T. Codon-anticodon interactions may include both G-A and C-A pairing in the wobble position. Evidence is summarized that supports the hypothesis that A and T nucleotides are favored at all locations in the D. yakuba mtDNA molecule where these nucleotides are compatible with function.