Biomedical subjects

J Yao

Publications and source records attributed to J Yao.

At least 199 records · Page 11Linked to original sources

PubMed1997

Chemical shift dispersion and secondary structure prediction in unfolded and partly folded proteins.

The intrinsic chemical shift dispersion for 15N, 1HN, 13C(alpha), 1H(alpha), 13C(beta) and 13CO resonances has been evaluated utilizing complete resonance assignment data for unfolded apomyoglobin, together with two other unfolded and five folded proteins, obtained from the literature. The dispersion of 13C(alpha), 1H(alpha), and 13C(beta) resonances for the unfolded proteins is poor, whereas the dispersion of 15N, 1HN and 13CO is much greater, reflecting the sensitivity of these nuclei to the nature of the neighboring amino acid in the primary sequence. By contrast, the dispersion of the 13C(alpha), 1H(alpha), and 13C(beta) nuclei are much greater in the folded proteins, reflecting the well-known dependence of the environments of these nuclei on secondary and tertiary structure. These differences in chemical shift dispersion dictate differences in strategies for resonance assignment in unfolded proteins compared with those most commonly used for folded proteins. Strategies utilizing the superior chemical shift dispersion of the 15N, 1HN and, in particular, the 13CO nuclei, are indicated for use with unfolded or partially folded proteins.

Animals↗

PubMed1997

Thermodynamic and kinetic basis of interfacial activation: resolution of binding and allosteric effects on pancreatic phospholipase A2 at zwitterionic interfaces.

A general kinetic model for catalysis by interfacial enzymes is developed. It couples the Michaelis-Menten catalytic turnover cycle at the interface with that in the aqueous phase through the distribution equilibria between the interface and the surrounding aqueous phase. Analysis under two limiting conditions fully describes the steady-state kinetics of hydrolysis and resolves the allosteric effects from apparent modes of interfacial activation in terms of the primary rate and equilibrium parameters for pig pancreatic phospholipase A2 (PLA2). One limit is observed in dispersions of anionic phospholipid vesicles, in which intervesicle exchange of enzyme, substrate, and hydrolysis products is absent and reaction occurs only on vesicles containing enzyme. A complete analysis at this highly processive limit, called kinetics in the scooting mode, has been published [Berg et al. (1991) Biochemistry 30, 7283]. Here is reported the analysis in the other limit, PLA2-catalyzed hydrolysis of zwitterionic micelles of short-chain phosphatidylcholines, at which substrate and products are in rapid exchange. Hydrolysis occurs either in bulk aqueous solution with phospholipid monomers or at the micellar interface. Above the critical micelle concentration (cmc), the hydrolysis rate shows a hyperbolic dependence on the bulk substrate concentration present as micelles. This dependence, characterized by the fitting parameters KMapp and VMapp, is analyzed in terms of the primary rate and equilibrium constants. The kinetic analysis is based on the assumption that the microscopic steady-state condition is satisfied because substrate replenishment in the micro-environment of the enzyme is fast relative to the catalytic turnover time. Added NaCl and anionic interface increase the hydrolysis rate in zwitterionic micelles dramatically. The overall interfacial rate enhancement is attributed to three factors: (a) promotion of PLA2 binding by net anionic charge of the interface, (b) enhancement of substrate affinity of PLA2 at the interface (Ks* allostery), and (c) stimulation of the rate-limiting chemical step (kcat* allostery).

Allosteric Regulation↗

PubMed1997

Spectroscopic and enzymatic characterization of the active site dinuclear metal center of calcineurin: implications for a mechanistic role.

The active site of bovine brain calcineurin contains an Fe3+-Zn2+ dinuclear metal center. Replacement of Zn2+ with Fe2+ yields a mixed valence Fe3+-Fe2+ center that exhibits a characteristic EPR signal that can be used as a convenient spectroscopic probe of the active site. Addition of product phosphate to both the Fe3+-Fe2+ and Fe3+-Zn2+ forms of calcineurin led to perturbations of the respective EPR signals, indicating that phosphate affects the environment of the paramagnetic centers. Anaerobic titrations of the iron-substituted Fe3+-Fe2+ enzyme with dithionite resulted in a gradual loss of activity toward pNPP that paralleled the loss of intensity of the EPR signal of the mixed valence diiron center. During dithionite reduction, an EPR resonance with g approximately 12 appeared. The intensity of this resonance increased when the spectrum was recorded in a parallel mode cavity and was therefore attributed to a paramagnetic center with integer spin. Oxidation of the Fe3+-Fe2+ cluster to the diferric state by hydrogen peroxide also led to a loss of activity. These results indicate that the mixed valence oxidation state represents the catalytically competent form of the cluster. The dependence of the enzyme activity on the redox state of the cluster has implications for a mechanistic role.

Aniline Compounds↗

PubMed1997

Three-dimensional echocardiographic estimation of infarct mass based on quantification of dysfunctional left ventricular mass.

BACKGROUND: Two-dimensional echocardiography is useful for estimating the extent of infarct-related wall motion abnormalities. Such estimation, however, is based on a few selected views and extrapolated for the whole left ventricle (LV). This approach does not provide us with the actual amount of dysfunctional myocardium. Volume-rendered three-dimensional echocardiography (3DE) might overcome these limitations. In this study we explored (1) how well volume-rendered 3DE delineates regional dysfunction of the infarcted LV and (2) how well dysfunctional myocardial mass quantified by 3DE reflects the actual anatomic infarct mass. METHODS AND RESULTS: 3DE was performed before and 3 hours after coronary occlusion in 16 dogs. With the LV viewed in equidistant short-axis slices, the region of dysfunction was demarcated, and the dysfunctional myocardial mass was derived from this. With triphenyltetrazolium chloride staining, anatomic infarct regions were delineated, dissected, and weighed. The anatomic infarct mass was 16.3+/-7.7 g (mean+/-SD) (range, 6.4 to 31.4 g); the dysfunctional mass estimated by 3DE was 17.4+/-9.1 g (range, 5.2 to 39.0 g). The mean difference was 1.0 g. The correlation between dysfunctional mass (y) and infarct mass (x) was y=l.lx-0.6, r=.93 (P<.0001). The percentage of LV involved in infarction was 18.2+/-5.8% (range, 9.1% to 26.1%); the percentage of LV involved in regional dysfunction was 18.3+/-6.9% (range, 7.9% to 31.2%). The mean difference was 0.1%. The correlation between percentage of LV involved in infarction (x) and percentage of LV involved in dysfunction (y) was y=1.0x-1.1, r=.92 (P<.0001). CONCLUSIONS: Volume-rendered 3DE crisply displays regional dysfunction of infarcted LV. 3DE-measured dysfunctional mass accurately reflects the anatomic infarct mass.

Animals↗

PubMed1997

Lipopolysaccharide induction of the tumor necrosis factor-alpha promoter in human monocytic cells. Regulation by Egr-1, c-Jun, and NF-kappaB transcription factors.

Biosynthesis of tumor necrosis factor-alpha (TNF-alpha) is predominantly by cells of the monocytic lineage. This study examined the role of various cis-acting regulatory elements in the lipopolysaccharide (LPS) induction of the human TNF-alpha promoter in cells of monocytic lineage. Functional analysis of monocytic THP-1 cells transfected with plasmids containing various lengths of TNF-alpha promoter localized enhancer elements in a region (-182 to -37 base pairs (bp)) that were required for optimal transcription of the TNF-alpha gene in response to LPS. Two regions were identified: region I (-182 to -162 bp) contained an overlapping Sp1/Egr-1 site, and region II (-119 to -88) contained CRE and NF-kappaB (designated kappaB3) sites. In unstimulated THP-1, CRE-binding protein and, to a lesser extent, c-Jun complexes were found to bind to the CRE site. LPS stimulation increased the binding of c-Jun-containing complexes. In addition, LPS stimulation induced the binding of cognate nuclear factors to the Egr-1 and kappaB3 sites, which were identified as Egr-1 and p50/p65, respectively. The CRE and kappaB3 sites in region II together conferred strong LPS responsiveness to a heterologous promoter, whereas individually they failed to provide transcriptional activation. Furthermore, increasing the spacing between the CRE and the kappaB3 sites completely abolished LPS induction, suggesting a cooperative interaction between c-Jun complexes and p50/p65. These studies indicate that maximal LPS induction of the TNF-alpha promoter is mediated by concerted participation of at least two separate cis-acting regulatory elements.

Activating Transcription Factor 2↗

PubMed1997

The direct cause of photodamage-induced lysosomal destabilization.

Whether membrane lipid photoperoxidation is the immediate cause for lysosomal lysis is still unclear. In this study, we investigated the direct causal factor of photoinduced lysosomal destabilization in a K+-containing solution. Methylene blue (MB)-mediated photodamage caused lysosomal membrane lipid peroxidation and loss of membrane fluidity. Compared with unirradiated lysosomes, the photodamaged lysosomes significantly lost enzyme latency in an isotonic K+-containing solution during a 20-min period of incubation. It indicates an increase in lysosomal K+ permeability. The inward K+ permeation of photodamaged lysosomes was further proved by a K+-induced elevation of internal membrane potential. In addition, the photodamaged lysosomes displayed an increased osmotic sensitivity, showing that MB-mediated photodamage promotes lysosomal osmotic fragility. Although these photoinduced alterations occurred, the lysosomes were relatively stable in an isotonic sucrose medium. In contrast, the organelle destabilized in a photodamage-dependent fashion in an isotonic K+-containing solution. The results indicate that membrane lipid peroxidation does not definitely destabilize lysosomes. The direct cause for the lysosomal destabilization is photoinduced osmotic imbalance across its membrane via an increased K+ uptake, while the increase in osmotic sensitivity favors the destabilization of photodamaged lysosomes.

Animals↗

PubMed1997

Human tau becomes phosphorylated and forms filamentous deposits when overexpressed in lamprey central neurons in situ.

Microinjection of plasmids encoding human tau (htau) protein into identified lamprey reticulospinal neurons (anterior bulbar cells, or ABCs) in situ induces chronic htau expression. htau protein is transported to both the axon and dendrites of expressing ABCs by mechanisms that require the C-terminal domain of htau protein but do not require directed htau mRNA transport. htau becomes phosphorylated at the PHF-1 (Ser-396/404) and TAU-1/AT8 (Ser-199/202) epitopes throughout ABCs with heavy htau accumulations; many such cells also exhibit degenerative changes, which include the development of extracellular htau deposits. Finally, expression of htau protein fused to green fluorescent protein induced the somatodendritic accumulation of filaments containing htau when examined by immunoelectron microscopy. These results suggest that chronic expression of htau in lamprey ABCs may be useful for studying cellular mechanisms underlying tau hyperphosphorylation and filament formation in vertebrate central neurons in situ.

Alzheimer Disease↗

PubMed1997

Modeling, chemistry, and biology of the benzolactam analogues of indolactam V (ILV). 2. Identification of the binding site of the benzolactams in the CRD2 activator-binding domain of PKCdelta and discovery of an ILV analogue of improved isozyme selectivity.

Protein kinase C (PKC) is a complex enzyme system comprised of at least 11 isozymes that serves to mediate numerous extracellular signals which generate lipid second messengers. The discovery of isozyme-selective activators and inhibitors (modulators) of PKC is crucial to ascertaining the role of the individual isozymes in physiological and pathophysiological processes and to manipulating their function. The discovery of such small molecule modulators of PKC is at present a largely unmet pharmacological need. Herein we detail our modeling studies which reveal how the natural product indolactam V (ILV) and its 8-membered ring analogue, the benzolactam 15, bind to the CRD2 activator domain of PKC. These modeling studies reveal that not all PKC ligands possess a common pharmacophore, and further suggest an important role of specific hydrophobic contacts in the PKC-ligand interaction. The modeling studies find strong experimental support from mutagenesis studies on PKC alpha that reveal the crucial role played by the residues proline 11, leucine 20, leucine 24, and glycine 27. Next, we describe the synthesis of two 8-substituted benzolactams starting from L-phenylalanine and characterize their isozyme selectivity; one of the two benzolactams exhibits improved isozyme selectivity relative to the n-octyl-ILV. Lastly, we report inhibition of cellular proliferation of two different breast carcinoma cell lines by the benzolactam 5 and show that the compound preferentially down-regulates PKCbeta in both cell lines.

Antineoplastic Agents↗

PubMed1997

Kinetic characterization of the serralysins: a divergent catalytic mechanism pertaining to astacin-type metalloproteases.

Substrates HO2CCH2CH2CO- and HOCH2CHOHCHOHCO-Phe-Leu-Ala-5-nitro-2-pyridinamide are cleaved efficiently at the acylarenamide linkage, with a convenient spectrophotometric assay, by the Serratia and Pseudomonas approximately 50-kDa extracellular metalloproteases (serralysins). The pH range of catalytic activity extends uniformly from 4 to greater than 10 (k(cat)/Km approximately 10(3) s(-1) M(-1), similar profile for k(cat)). Substrate analogue hydroxamic acid Cbz-Leu-Ala-NHOH competitively inhibits serralysin (Ki 0.04 mM), with a pH dependence indicating that either a displaced metal-bound H2O or a similarly motile enzymic phenol residue (Tyr216) that is crystallographically found ligated to the active-site Zn2+ of the uncomplexed enzyme must have a pKa of approximately 5. A chemical catalytic mechanism of proteolysis consistent with the kinetic data is proposed, in which Tyr216-ArO-, in the course of being released from the active-site metal ion, deprotonates a water molecule attacking the Zn2+-activated substrate linkage, leading to a metal-coordinated tetrahedral oxyanion adduct that subsequently fragments.

Binding Sites↗

PubMed1997

Overexpression of the c-erbB-2 gene enhanced intrinsic metastasis potential in human breast cancer cells without increasing their transformation abilities.

Overexpression of the c-erbB-2 gene-encoded p185 has been reported in approximately 30% of human breast cancers and has been correlated with lymph node metastasis and poor prognosis in breast cancer patients. To investigate whether overexpression of p185 can enhance the metastatic potential of human breast cancer cells, we have introduced the human c-erbB-2 gene into the very low p185-expressing MDA-MB-435 human breast cancer cells and established 435.eB transfectants that express higher levels of p185. In this study, we compared the metastatic phenotypes of the parental MDA-MB-435 cells and the 435.eB transfectants. In vivo experimental metastasis assays in which we injected MDA-MB-435 parental cells or 435.eB transfectants into the tail veins of ICR-SCID mice demonstrated that mice injected with p185-overexpressing 435.eB transfectants formed significantly more metastatic tumors than the mice injected with parental and control cells. The changes in experimental metastatic potential in vivo were accompanied by increased invasiveness in vitro. In addition, the secretion of basement membrane-degradative enzymes, which is an important step in the invasion and metastasis process, was also increased in the p185-overexpressing 435.eB transfectants. These results indicated that p185 overexpression can enhance the metastatic potential of MDA-MB-435 human breast cancer cells. To investigate whether enhanced metastatic potential in the p185-overexpressing 435.eB transfectants was the result of increased cancer cell growth and transformation potential, we compared the growth rate, anchorage-independent growth ability, and tumorigenicity of the 435.eB transfectants with that of the parental cells. The transfectants and the parental cells all had similar growth rates and anchorage-independent growth abilities and demonstrated similar tumorigenic potential. These findings suggest that c-erbB-2 is a metastasis-promoting gene for breast cancers that is distinct from other tumor-promoting genes in that the c-erbB-2 gene can enhance the intrinsic metastatic potentials of MDA-MB-435 cells without increasing their transformation abilities.

Animals↗

PubMed1997

Lysosomal destabilization via increased potassium ion permeability following photodamage.

Isotonic K2SO4 solution protected lysosomes osmotically during a 20 min incubation, but lost its protective effect if the lysosomes were initially photooxidized after sensitization with Methylene blue. Increasing K2SO4 concentration promoted the latency loss of photodamaged lysosomes, but did not impair the integrity of unirradiated lysosomes. The results indicate that the photodamage enhanced lysosomal ionic permeability, with osmotic imbalance over the lysosomal membrane. Out of the decreased latency induced by the photodamage, 32% was prevented by the addition of 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid to the incubation solution, suggesting that electroneutral K+/SO4(2-) co-uptake plays a role in the lysosomal destabilization. The photooxidation increased lysosomal H+/K+ exchange, which was confirmed by monitoring the H+ leakage with the pH sensitive probe p-nitrophenol and examining the K+ entry by membrane potential measurements. Addition of K2SO4 to a lysosomal suspension lowered the delta pH of photodamaged lysosomes, presumably due to an increase in the exchange of internal H+ for external K+. Out of the photodamage-induced lysosomal latency loss, 50-60% was prevented by either lowering the external pH or preincubating the lysosomes with methylamine to elevate their internal pH. The results suggest that the photodamage-promoted K+/H+ exchange plays a major role in lysosomal osmotic destabilization.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1997

Induction of interleukin-1 beta-converting enzyme (ICE) in murine microglia by lipopolysaccharide.

Interleukin-1beta (IL-1beta) synthesized in the brain is thought to be involved in the behavioral response to LPS. In this study, we examined in microglia the ability of LPS to induce interleukin-1 beta-converting enzyme (ICE), the protease responsible for processing proIL-1beta to its mature biologically active form. The murine microglial cell line, N13, and primary microglia which had been previously isolated from brains of 2-d-old endotoxin-responsive C3H/HeOuJ mice and endotoxin-resistant C3H/HeJ mice, were cultured in the presence of various concentrations of LPS. Oligonucleotide primers for ICE and GAPDH were used in RT-PCR to determine the levels of ICE mRNA in microglia. ICE mRNA was constitutively expressed in N13 cells and primary microglia from both C3H/HeOuJ and C3H/HeJ mice. Upon exposure to LPS, ICE mRNA levels were markedly elevated in N13 cells and in microglia from C3H/HeOuJ mice, but not in microglia from endotoxin-resistant C3H/HeJ mice. Western immunoblotting was used to determine if the increase in ICE mRNA induced by LPS was accompanied by an increase in ICE protein. A modest increase in immunoreactivity for the p45 ICE precursor protein was evident in N13 cells and primary microglia from C3H/HeOuJ mice following exposure to LPS. Because corticosteroids inhibit the synthesis and secretion of IL-1beta, in a second experiment microglia were treated with LPS in the presence of dexamethasone (0, 10 and 100 microM). Dexamethasone inhibited the LPS-induced increase in ICE mRNA and protein in microglia from C3H/HeOuJ mice. These results indicate that LPS stimulates microglia to express ICE. That dexamethasone inhibited the expression of ICE, suggests that corticosteroids regulate the secretion of IL-1beta by microglial cells, in part, by regulating the expression of ICE.

Animals↗

PubMed1997

Cytoskeletal changes correlated with the loss of neuronal polarity in axotomized lamprey central neurons.

Axotomy within 500 microm of the soma (close axotomy) causes identified neurons (anterior bulbar cells or ABCs) in the lamprey hindbrain to lose their normal polarity and regenerate axons ectopically from dendritic tips, while axotomy at more distal sites (distant axotomy) results in orthotopic axonal regeneration from the axon stump. We performed immunocytochemical, electron microscopic and in situ hybridization analyses comparing ABCs subjected to close and distant axotomy to elucidate the mechanism by which neuronal polarity is lost. We show that polarity loss in ABCs is selectively and invariably preceded and accompanied by the following cellular changes: (1) a loss of many dendritic microtubules and their replacement with neurofilaments, (2) a loss of immunostaining for acetylated tubulin in the soma and proximal dendrites, and (3) an increase of immunostaining for phosphorylated neurofilaments in the distal dendrites. We also show that these changes do not depend on either the upregulation or spatial redistribution of neurofilament message, and thus must involve changes in the routing of neurofilament protein within axotomized ABCs. We conclude that close axotomy causes dendrites to undergo axonlike changes in the mechanisms that govern the somatofugal transport of neurofilament protein, and suggest that these changes require the reorganization of dendritic microtubules. We also suggest that the bulbous morphology and lack of f-actin in the tips of all regenerating sprouts supports the possibility that axonal regeneration in the lamprey CNS does not involve actin-mediated "pulling" of growth cones, but depends instead on the generation of internal extrusive forces.

Actins↗

PubMed1997

Comparative analysis of expression of the proprotein convertases furin, PACE4, PC1 and PC2 in human lung tumours.

Proprotein convertases mediate the production of a variety of peptidic mitogens by limited proteolysis of their precursors. These proteases may also participate in the autocrine production of such mitogens by cancer cells and thus contribute to the unchecked proliferation of these cells. As a step towards defining this contribution, we have examined the levels of four convertase mRNAs in human lung neoplasms using semiquantitative Northern blot analysis. Furin mRNA was expressed in all the tumours; its level in squamous cell carcinomas and adenocarcinomas was on average about threefold higher than in small-cell lung carcinomas (SCLCs). PACE4 transcripts were detected in eight of 14 adenocarcinomas and in seven of 17 squamous cell carcinomas; they were detectable in only two of seven SCLCs. PC1 mRNA was undetected in squamous cell carcinomas and in all but two adenocarcinomas; it was present in four of six SCLCs. PC2 mRNA was found in two adenocarcinomas, in one squamous cell carcinoma and in five of seven SCLCs. This preliminary survey indicates that SCLCs often carry more mRNA for the endocrine convertases PC1 and PC2 and less mRNA for the more ubiquitous furin and PACE4, suggesting inverse roles of these convertases in the development of this neoplasm.

Aspartic Acid Endopeptidases↗

PubMed1997

Adhesion molecules in the glomerular mesangium.

Experimental evidence indicates that extensive "cross-talk" exists between glomerular cells, extracellular matrix molecules and soluble mediator substances affecting the proliferative and secretory phenotype of glomerular mesangial cells. Both matrix and cytokines regulate mesangial cell behavior in vitro and in vivo after binding to specific cell surface receptors. It appears as if the concerted action of insoluble and soluble ligands on mesangial cells involves a reciprocal regulation of matrix molecules and cytokines as well as expression and affinity of their respective receptors. Elucidation of the potential biologic and clinical relevance of cell-matrix interactions in the glomerular mesangium represents a challenging goal in current kidney research. This brief review summarizes recent investigations concerning regulation of expression and function of adhesion molecules and matrix receptors in the mesangium. In addition to results from cell culture studies, descriptive findings on expression and regulation of adhesion molecules and their potential role for altered mesangial cell behavior in glomerular disease is considered.

Animals↗

PubMed1997

Do permanent pacemakers need an insulative coating? Results of a prospective randomized double-blind study.

During conventional manufacturing of implanted pulse generators (IPGs), an insulative coating is often applied to prevent local muscle stimulation and myopotential sensing in unipolar pacing. This can limit the orientation of the IPG into its pocket, be a potential source of muscle stimulation via coating scratches, and result in an increase in IPG production costs. We hypothesized that advances in the design and construction of current IPGs and leads obviates the need for an insulative coating of the IPG. Using a double-blind prospective randomized design, 39 patients were implanted with either coated or uncoated otherwise identical IPGs (19 dual, 20 single chamber). All testing was done in unipolar and bipolar mode in both channels. A strength-duration curve for muscle stimulation was constructed for all patients with muscle stimulation. Myopotential sensing was established during isometric exercise. At 6-month follow-up when tested in unipolar mode, 3 of 15 (20%) patients with coated IPGs and 3 of 20 (15%) with uncoated IPGs had muscle stimulation at 5.0 V/1.5 ms or lower (P = NS). No patients in either population had muscle stimulation at their normally programmed output. Myopotential sensing occurred in all patients in unipolar mode at a mean of 2.29 +/- 1.3 mV and 2.73 +/- 1.14 mV for coated versus uncoated, respectively (P = NS). The statistical power of these negative observations was 80%. An insulative coating for pacemakers does not appear to alter sensing performance or cause a significant difference in the occurrence or characteristics of muscle stimulation.

Adult↗

PubMed1997

Localization of BRCA1 and a splice variant identifies the nuclear localization signal.

Inherited mutations in BRCA1 confer susceptibility to breast and ovarian neoplasms. However, the function of BRCA1 and the role of BRCA1 in noninherited cancer remain unknown. Characterization of alternately spliced forms of BRCA1 may identify functional regions; thus, we constructed expression vectors of BRCA1 and a splice variant lacking exon 11, designated BRCA1 delta 672-4095. Immunofluorescence studies indicate nuclear localization of BRCA1 but cytoplasmic localization of BRCA1 delta 672-4095. Two putative nuclear localization signals (designated NLS1 and NLS2) were identified in exon 11; immunofluorescence studies indicate that only NLS1 is required for nuclear localization. RNA analysis indicates the expression of multiple, tissue-specific forms of BRCA1 RNAs; protein analysis with multiple antibodies suggests that at least three BRCA1 isoforms are expressed, including those lacking exon 11. The results suggest that BRCA1 is a nuclear protein and raise the possibility that splicing is one form of regulation of BRCA1 function by alteration of the subcellular localization of expressed proteins.

3T3 Cells↗

PubMed1997

Evolutionary conservation of regulatory strategies for the sex determination factor transformer-2.

Sex determination in Drosophila melanogaster is regulated by a cascade of splicing factors which direct the sex-specific expression of gene products needed for male and female differentiation. The splicing factor TRA-2 affects sex-specific splicing of multiple pre-mRNAs involved in sexual differentiation. The tra-2 gene itself expresses a complex set of mRNAs generated through alternative processing that collectively encode three distinct protein isoforms. The expression of these isoforms differs in the soma and germ line. In the male germ line the ratio of two isoforms present is governed by autoregulation of splicing. However, the functional significance of multiple TRA-2 isoforms has remained uncertain. Here we have examined whether the structure, function, and regulation of tra-2 are conserved in Drosophila virilis, a species diverged from D. melanogaster by over 60 million years. We find that the D. virilis homolog of tra-2 produces alternatively spliced RNAs encoding a set of protein isoforms analogous to those found in D. melanogaster. When introduced into the genome of D. melanogaster, this homolog can functionally replace the endogenous tra-2 gene for both normal female sexual differentiation and spermatogenesis. Examination of alternative mRNAs produced in D. virilis testes suggests that germ line-specific autoregulation of tra-2 function is accomplished by a strategy similar to that used in D. melanogaster. The similarity in structure and function of the tra-2 genes in these divergent Drosophila species supports the idea that sexual differentiation in D. melanogaster and D. virilis is accomplished under the control of similar regulatory pathways.

Alternative Splicing↗