Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “third generation sequencing”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,117 records · Page 62Linked to original sources
PubMed1994

Genetic evidence for IS1 transposition regulated by InsA and the delta InsA-B'-InsB species, which is generated by translation from two alternative internal initiation sites and frameshifting.

Insertion sequence IS1 contains two reading frames, insA and B'-insB, which are responsible for its transposition, and was previously shown to express two proteins. The first, InsA, is the product of insA. The second, InsA-B'-InsB is a fusion of InsA with the product of B'-insB. Synthesis of this protein occurs by a -1 frameshift from the 3' region of the insA frame to the open reading frame B', extending from the 5' end of the insB frame. Here, I have shown genetically that IS1 encodes the third species delta InsA-B'-InsB: delta InsA-B'-InsB uses two alternative initiation codons in the middle of the insA frame, and is produced by a frameshift mechanism similar to that used in InsA-B'-InsB expression. Deletion of the small region preceding these initiation codons resulted in decreased expression of delta InsA-B'-InsB, suggesting that the small region play some role in the translation initiation. Surprisingly, it was found that delta InsA-B'-InsB has a transposase-like function and InsA can stimulate the transposition promoted by delta InsA-B'-InsB, while delta InsA-B'-InsB seemed to bind to the left terminal inverted repeat (IRL) of IS1 and inhibit transposition when it was present in excess, as well as InsA represses transposition. It is likely that IS1 transposition activity depends on the ratio of InsA to delta InsA-B'-InsB. A double missense mutation of the internal initiation codons resulted in decreased cointegration activity, showing that delta InsA-B'-InsB is responsible for transposition but InsA-B'-InsB is probably not. Some IS elements, which also contain two tandem, out-of-phase, overlapping genes, appear to express deleted fusion proteins like delta InsA-B'-InsB, but the functions are unknown. The complex phenomena of transposition and its control found in IS1 may be more general in the other mobile DNAs.

Amino Acid Sequence↗
PubMed1997

Two two-gene macronuclear chromosomes of the hypotrichous ciliates Oxytricha fallax and O. trifallax generated by alternative processing of the 81 locus.

We describe the first know macronuclear chromosomes that carry more than one gene in hypotrichous ciliated protozoa. These 4.9- and 2.8-kbp chromosomes each consist almost exclusively of two protein-coding genes, which are conserved and transcribed. The two chromosomes share a common region that consists of a gene that is a member of the family of mitochondrial solute carrier genes (CR-MSC; [Williams and Herrick (1991): Nucleic Acids Res 19:4717-4724]. Each chromosome also carries another gene appended to its common region: The 4.9-kbp chromosome also carries a gene that encodes a protein that is rich in glutamine and charged amino acids and bears regions of heptad repeats characteristic of coiled-coils. Its function is unknown. The second gene of the 2.8 kbp chromosome is a mitochondrial solute carrier gene (LA-MSC); thus, the 2.8-kbp chromosomes consists of two mitochondrial solute carrier paralogs. Phylogenetic analysis indicates that the two genes were duplicated before ciliates diverged from the main eukaryotic lineage and were subsequently juxtaposed. The CR- and LA-MSC genes are each interrupted by three introns. The introns are not in homologous positions, suggesting that they may have originated from multiple group II intron transpositions. These chromosomes and their genes are encoded in the Oxytricha germline by the 81 locus. This locus is alternatively processed to generate a nested set of three macronuclear chromosomes, the 4.9- and 2.8-kbp chromosomes and a third (1.6 kbp) which consists almost exclusively of the shared common gene, CR-MSC. Such alternative processing is common in macronuclear development of O. fallax [Cartinhour and Herrick (1984): Mol Cell Biol 4:931-938]. Possible functions for alternative processing are considered; e.g., it may serve to physically link genes to allow co-regulation or co-replication by a common cis-acting sequence.

Animals↗
PubMed2005

Speculations on the pathogenesis of CHARGE syndrome.

To be seriously considered, a theory about the pathogenesis of a multiple congenital anomaly syndrome should meet three criteria: (1) it should explain all of the anomalies associated with the syndrome; (2) it should explain why certain anomalies are not associated with the syndrome; and (3) it should predict anomalies that could be associated with the syndrome, but have not yet been described. The theory must eventually pass the ultimate test, that is, molecular confirmation of the proposed mechanism. Several theories about the pathogenesis of CHARGE syndrome have been proposed, but none of these meet the three criteria stated above. In this study, the author proposes that CHARGE syndrome is due to a disruption of mesenchymal-epithelial interaction (epithelial includes ectoderm and endoderm). The theory is tested against the major, minor, and occasional anomalies that are used to make the clinical diagnosis of CHARGE syndrome. Review of the known embryology of the organs and tissues involved in CHARGE syndrome confirms that mesenchymal-epithelial interactions are necessary for proper formation of these organs and tissues. The presence of limb anomalies in approximately one-third of CHARGE syndrome patients fulfills criteria #3 above, in that limb anomalies were not felt to be a part of CHARGE syndrome until relatively recently. It is known that some patients with chromosomal abnormalities have a phenotype that overlaps with CHARGE syndrome. Given that critical developmental pathways must be robust and redundant in order to minimize errors, it may be that disruption of more than one gene is necessary to generate the CHARGE phenotype, as has been proposed for the holoprosencephaly sequence. Mutations and deletions of CHD7 have recently been identified as causing CHARGE syndrome in more than 50% of tested patients. Given this gene classes' putative role as a general controller of developmental gene expression as well as mesodermal patterning, it would fit the hypothesized mechanisms discussed in the study.

Abnormalities, Multiple↗
PubMed1997

Molecular cloning of anti-SS-A/Ro 60-kDa peptide Fab fragments from infiltrating salivary gland lymphocytes of a patient with Sjögren's syndrome.

Anti-SS-A/Ro antibodies are commonly found in systemic autoimmune diseases such as Sjögren's syndrome and systemic lupus erythematosus, and some of these antibodies appear to be responsible for certain pathological lesions including congenital heart block in neonatal lupus. In this study, we generated three human antibody Fab fragments that specifically bind to SS-A/Ro 60-kd peptide from salivary gland lymphocytes of a patient with Sjögren's syndrome by using a phage-display technique. Sequence analysis demonstrated that two of the three Fab clones (E-42 and E-60) used homologous heavy chains derived from the germline VH gene DP73 in combination with different light chains which were derived from germline V kappa gene L6 and V lambda gene DPL23. The third Fab clone (E-56) used another heavy chain derived from the germline VH gene DP31 in combination with the identical light chain as that of E-42. All three Fab clones revealed a high number of somatic mutations that likely occurred in the context of antigen selection. These findings suggest the restricted usage of VH and VL genes of anti-SS-A/Ro antibodies in salivary gland lymphocytes of the patient.

Amino Acid Sequence↗
PubMed2000

Extended-spectrum TEM- and SHV-type beta-lactamase-producing Klebsiella pneumoniae strains causing outbreaks in intensive care units in Italy.

The aim of the present study was to investigate the production of extended-spectrum beta-lactamases (ESbetaLs) and the epidemiological correlations in a total of 107 Klebsiella pneumoniae strains resistant to third- and fourth-generation cephalosporins. The strains were collected from patients in four intensive care units (3 neonatal and 1 general) in three hospitals in Italy between March 1996 and July 1997. All strains were found to produce ESbetaLs. Phenotypic (antibiotyping and ESbetaL patterns) and genotypic (plasmid profile and pulsed-field gel electrophoresis) analyses showed that a single strain had been responsible for each outbreak in each of the four intensive care units. Isoelectric focusing, activity on substrates and gene sequencing showed that the strains produced SHV-5, SHV-2a, SHV-12 and TEM-52 beta-lactamases. This is not only the first time that ESbetaL-producing Klebsiella pneumoniae strains have been reported as causing epidemics in Italian hospitals, it is also, to the best of our knowledge, the first time that an outbreak caused by a TEM-52 ESbetaL-producing Klebsiella pneumoniae strain has been reported. The data presented here illustrate the complexity of determining the epidemiological pattern of ESbetaL producers in large hospitals that do not have an ESbetaL-monitoring program.

Anti-Bacterial Agents↗
PubMed2004

The use of selective estrogen receptor modulators and selective estrogen receptor down-regulators in breast cancer.

Tamoxifen is one of the most effective treatments for breast cancer through its ability to antagonize estrogen-dependent growth by binding estrogen receptors (ERs) and inhibiting proliferation of breast epithelial cells. However, tamoxifen has estrogenic agonist effects in other tissues such as bone and endometrium due to liganded ER activating target genes in these different types of cell. Several novel anti-estrogen compounds have been developed which have a reduced agonist profile on breast and gynaecological tissues. These compounds offer the potential for enhanced efficacy and reduced toxicity compared with tamoxifen. In advanced breast cancer clinical data exist for two groups of agents: the selective estrogen receptor modulators (SERMs), further divided into "tamoxifen-like" (e.g. toremifene, droloxifene and idoxifene) and "fixed ring" compounds (e.g. raloxifene, arzoxifene and EM-800), and the selective estrogen receptor down-regulators (SERDs; e.g. fulvestrant (ICI 182780), SR 16234 and ZK 191703) also termed "pure anti-estrogens". In phase II trials in tamoxifen-resistant metastatic breast cancer the SERMs show low objective response rates (range 0-15%), suggesting cross resistance with tamoxifen. Randomized phase III trials for toremifene and idoxifene in over 1500 patients showed no significant difference compared with tamoxifen. Fewer clinical data exist for the "fixed ring" SERMs and it remains unclear whether any clinical advantage exists for the "fixed ring" SERMs over tamoxifen as first-line therapy. The main advantage for SERMs such as tamoxifen and raloxifene probably remains in early-stage disease (adjuvant therapy or prevention). Fulvestrant and the other SERDs have a high affinity for the estrogen receptor (ER) compared to tamoxifen, but none of its agonist activities. Of the SERDs, only fulvestrant has entered the clinic and this new agent is showing promising clinical activity in the treatment of advanced breast cancer. Recently published phase III studies have shown fulvestrant to be at least as effective as the third-generation aromatase inhibitor anastrozole in patients whose disease has relapsed or progressed on prior endocrine therapy. Surprisingly, however, in a phase III trial versus tamoxifen for the first-line therapy of advanced breast cancer fulvestrant did not attain the requirements for equivalence to tamoxifen, and in terms of time-to-treatment failure was inferior (5.9 versus 7.8 months for fulvestrant and tamoxifen, respectively; P=0.029). Future clinical studies will evaluate fulvestrant in the neoadjuvant setting together with its optimal sequencing in relation to tamoxifen and other endocrine therapies in advanced disease.

Antineoplastic Agents, Hormonal↗
PubMed2004

Analysis of serial CT scans to assess template and catheter movement in prostate HDR brachytherapy.

PURPOSE: As prostate high-dose-rate (HDR) brachytherapy becomes more prevalent, varying amounts of catheter displacement have been noted. To investigate the constancy of catheter position and its impact on dose distribution, we analyzed serial dosimetric CT scans. METHODS AND MATERIALS: The data from 50 patients were analyzed. During initial CT treatment planning, transverse images of the implant volume were collected, and all structures were digitized into the Nucletron Brachytherapy Planning System. Digitally reconstructed radiographs were generated with rendering of the catheter tips, ischial tuberosity, and perineal template. The distance from each catheter tip to the template and to the ischial tuberosity was measured. The distance between the ischial tuberosity and the template was similarly measured. A second CT set was obtained at different intervals and compared with the first measurement to assess catheter and/or template movement. In 10 patients, the second CT set was obtained before the third fraction in both 2-mm and 5-mm slice sequences, and the latter was used to re-create the HDR plan. RESULTS: Although no interfraction catheter movement relative to the template was found, the template-catheter unit moved in a caudal direction between HDR fractions. The amount of displacement was time dependent: 2 mm before the second fraction, 8 mm before the third, and 10 mm before the fourth. When comparing the first HDR treatment with the third, median decreases in the following dosimetric parameters were noted: dose to 90% of the prostate volume, 35% (r = 0-60); minimal dose to the base, 35% (r = 17-65); and maximal dose to 1 cm(3), 13% (r = 3-19%). CONCLUSION: The interstitial catheters did not slip within the template and were not caudally displaced independently but rather in conjunction with the template. The displacement occurred in a time-dependent fashion, and, without redress, significant dosimetric changes are encountered by the third fraction.

Aged↗
PubMed2000

Generation of longer cDNA fragments from serial analysis of gene expression tags for gene identification.

We have developed a technique called the generation of longer cDNA fragments from serial analysis of gene expression (SAGE) tags for gene identification (GLGI), to convert SAGE tags of 10 bases into their corresponding 3' cDNA fragments covering hundred bases. A primer containing the 10-base SAGE tag is used as the sense primer, and a single base anchored oligo(dT) primer is used as an antisense primer in PCR, together with Pfu DNA polymerase. By using this approach, a cDNA fragment extending from the SAGE tag toward the 3' end of the corresponding sequence can be generated. Application of the GLGI technique can solve two critical issues in applying the SAGE technique: one is that a longer fragment corresponding to a SAGE tag, which has no match in databases, can be generated for further studies; the other is that the specific fragment corresponding to a SAGE tag can be identified from multiple sequences that match the same SAGE tag. The development of the GLGI method provides several potential applications. First, it provides a strategy for even wider application of the SAGE technique for quantitative analysis of global gene expression. Second, a combined application of SAGE/GLGI can be used to complete the catalogue of the expressed genes in human and in other eukaryotic species. Third, it can be used to identify the 3' cDNA sequence from any exon within a gene. It can also be used to confirm the reality of exons predicted by bioinformatic tools in genomic sequences. Fourth, a combined application of SAGE/GLGI can be applied to define the 3' boundary of expressed genes in the genomic sequences in human and in other eukaryotic genomes.

Cloning, Molecular↗
PubMed1999

Alternative splicing generates multiple mRNA forms of the acetylcholine receptor gamma-subunit in rat muscle.

The fetal type acetylcholine receptor, composed of the alphabeta gammadelta subunits, has shown a highly variable channel kinetics during postnatal development. We examine the hypothesis whether such a variability could result from multiple channel forms, differing in the N-terminus of the gamma-subunit. RT-PCR revealed, in addition to the full-length mRNA, three new forms lacking exon 4. One of them in addition lacks 19 nucleotides from exon 5, predicting a complete subunit, with a 43 residues shorter N-terminus. A third one lacking the complete exon 5 predicts a subunit without transmembrane segments. These forms, generated by alternative splicing, may account for the kinetic variability of the acetylcholine receptor channel.

Alternative Splicing↗
PubMed2002

The role of aromatase inactivators in the treatment of breast cancer.

Third-generation aromatase inhibitors and inactivators have earned their place in the treatment of metastatic breast cancer. The third-generation aromatase inactivator exemestane was found to be superior to megestrol acetate as second-line endocrine therapy in postmenopausal women, with respect to time to progression as well as overall survival, and the results from an ongoing study comparing exemestane with tamoxifen in first-line treatment are promising. The finding that exemestane may also work in patients previously exposed to nonsteroidal aromatase inhibitors reveals lack of complete cross-resistance between the compounds. Currently, exemestane given as monotherapy, or in sequence with tamoxifen (2-3 + 3-2 years of tamoxifen-exemestane or 5 years of tamoxifen followed by 2 years of exemestane) is being compared with tamoxifen 5 years monotherapy in the adjuvant setting. In addition, we are currently addressing the toxicity of exemestane in a placebo-controlled trial in low-risk breast-cancer patients. The results from these studies will outline the potential role of exemestane in adjuvant treatment and, potentially, for breast-cancer prevention in postmenopausal women.

Androstadienes↗
PubMed1991

Selection of polyclonal antibodies to the N terminus of bean yellow mosaic potyvirus coat protein by induction of tolerance with monoclonal antibody.

Polyclonal antisera to potyviruses contain virus-specific as well as cross-reacting antibodies. The virus-specific antibodies are directed to the surface-located, N-terminal region of the coat protein, whereas cross-reacting antibodies are produced against multiple epitopes within the core region of the coat protein (minus N and C termini), which displays extensive sequence homology among distinct potyviruses. In the present study, immunological tolerance was induced in mice against the cross-reactive central core region of bean yellow mosaic virus (BYMV) using a rat monoclonal antibody (mAb) to the L3T4 molecule (the mouse equivalent of CD4). Generation of specific antisera reactive to the N terminus of BYMV was attained in tolerized mice by secondary immunization with whole viral coat protein from BYMV. This approach appears to be ideally suited to potyviruses where a two-third of the coat protein molecule contains immunogenic epitopes which can result in cross-reacting antibodies.

Animals↗
PubMed1998

Proprioceptive control of posture: a review of new concepts.

The assumption that proprioceptive inputs from the lower legs are used to trigger balance and gait movements is questioned in this review (an outgrowth of discussions initiated during the Neural Control of Movement Satellite meeting held in Cozumel, Mexico, April 1997). Recent findings presented here suggest that trunk or hip inputs may be more important in triggering human balance corrections and that proprioceptive input from the lower legs mainly helps with the final shaping and intermuscular coordination of postural and gait movements. Three major questions were considered. First, what role, if any, do lower-leg proprioceptive inputs play in the triggering of normal balance corrections? If this role is negligible, which alternative proprioceptive inputs then trigger balance corrections? Second, what is the effect of proprioceptive loss on the triggering of postural and gait movements? Third, how does proprioceptive loss affect the output of central pattern generators in providing the final shaping of postural movements? The authors conclude that postural and gait movements are centrally organized at two levels. The first level involves the generation of the basic directional-specific response pattern based primarily on hip or trunk proprioceptive input secondarily on vestibular inputs. This pattern specifies the spatial characteristics of muscle activation, that is which muscles are primarily activated, as well as intermuscular timing, that is, the sequence in which muscles are activated. The second level is involved in the shaping of centrally set activation patterns on the basis of multisensorial afferent input (including proprioceptive input from all body segments and vestibular sensors) in order that movements can adapt to different task conditions. Copyright 1998 Elsevier Science B.V.

Journal Article↗
PubMed1999

Novel biodegradable aromatic plastics from a bacterial source. Genetic and biochemical studies on a route of the phenylacetyl-coa catabolon.

Novel biodegradable bacterial plastics, made up of units of 3-hydroxy-n-phenylalkanoic acids, are accumulated intracellularly by Pseudomonas putida U due to the existence in this bacterium of (i) an acyl-CoA synthetase (encoded by the fadD gene) that activates the aryl-precursors; (ii) a beta-oxidation pathway that affords 3-OH-aryl-CoAs, and (iii) a polymerization-depolymerization system (encoded in the pha locus) integrated by two polymerases (PhaC1 and PhaC2) and a depolymerase (PhaZ). The complete assimilation of these compounds requires two additional routes that specifically catabolize the phenylacetyl-CoA or the benzoyl-CoA generated from these polyesters through beta-oxidation. Genetic studies have allowed the cloning, sequencing, and disruption of the genes included in the pha locus (phaC1, phaC2, and phaZ) as well as those related to the biosynthesis of precursors (fadD) or to the catabolism of their derivatives (acuA, fadA, and paa genes). Additional experiments showed that the blockade of either fadD or phaC1 hindered the synthesis and accumulation of plastic polymers. Disruption of phaC2 reduced the quantity of stored polymers by two-thirds. The blockade of phaZ hampered the mobilization of the polymer and decreased its production. Mutations in the paa genes, encoding the phenylacetic acid catabolic enzymes, did not affect the synthesis or catabolism of polymers containing either 3-hydroxyaliphatic acids or 3-hydroxy-n-phenylalkanoic acids with an odd number of carbon atoms as monomers, whereas the production of polyesters containing units of 3-hydroxy-n-phenylalkanoic acids with an even number of carbon atoms was greatly reduced in these bacteria. Yield-improving studies revealed that mutants defective in the glyoxylic acid cycle (isocitrate lyase(-)) or in the beta-oxidation pathway (fadA), stored a higher amount of plastic polymers (1.4- and 2-fold, respectively), suggesting that genetic manipulation of these pathways could be useful for isolating overproducer strains. The analysis of the organization and function of the pha locus and its relationship with the core of the phenylacetyl-CoA catabolon is reported and discussed.

Acetyl Coenzyme A↗
PubMed2001

Post-translational modification of the hepatitis C virus core protein by tissue transglutaminase.

The hepatitis C virus (HCV) core protein is a structural protein that packages the viral genomic RNA. In this study, we demonstrate that a stable core protein dimer could be produced in liver cells. The production of this protein could be enhanced by calphostin C and serum deprivation. This protein was determined to be the core protein dimer because of its reactivity with the anti-core antibody, its similar electrophoretic mobility compared with that of the core protein dimer generated by cross-linking with glutaraldehyde, and its increase in size by a hemagglutinin tag fused to the core protein sequence. This core protein dimer was highly stable and resistant to SDS and beta-mercaptoethanol. The enzyme that mediated the formation of this stable core protein dimer was determined to be the tissue transglutaminase (tTG) because, first, tTG could be activated by calphostin C and serum deprivation; second, the formation of this dimer was suppressed by monodansylcadaverine, a tTG inhibitor; and third, the core protein could be cross-linked by tTG in vitro. Thus, the HCV core protein represents the first known viral structural protein substrate of tTG. The post-translational modification by tTG reduced the RNA binding activity of the core protein, raising the possibility that tTG may regulate the biological functions of the HCV core protein.

Cell Line↗
PubMed1991

Identification of a cell retention signal in the B-chain of platelet-derived growth factor and in the long splice version of the A-chain.

The B-chain homodimer of platelet-derived growth factor (PDGF) is only very inefficiently secreted and remains largely associated with the producer cell; in contrast, the dimer of the short, and most common, splice variant of the A-chain is secreted. To identify the structural background to the differences in the secretory pattern between the different isoforms of PDGF, a set of chimeric PDGF A/B cDNAs was generated and expressed in COS cells. Analyses of the biosynthesis and processing of the corresponding products led to the identification of a determinant for cell association in the carboxy-terminal third of the PDGF B-chain precursor. Introduction of stop codons at various positions in the carboxy-terminal prosequence of the PDGF B-chain localized this determinant to an 11-amino-acid-long region (amino acids 219-229). This region contains an 8-amino-acid-long basic sequence that is homologous to a sequence present in an alternatively spliced longer version of the PDGF A-chain. In contrast to the short splice variant, the long splice A-chain version, like the B-chain, was found to remain predominantly cell associated. Thus, we have identified a conserved sequence that inhibits the secretion of some of the PDGF isoforms. Our data also suggest that switching of splicing patterns can be a mechanism to regulate the formation of secreted or cell-associated forms of PDGF-AA and possibly other growth factors.

Amino Acid Sequence↗
PubMed2001

Extension of resistance to cefepime and cefpirome associated to a six amino acid deletion in the H-10 helix of the cephalosporinase of an Enterobacter cloacae clinical isolate.

Enterobacter cloacae CHE, a clinical strain with overproduced cephalosporinase was found to be highly resistant to the new cephalosporins, cefepime and cefpirome (MICs> or =128 microg ml(-1)). The strain was isolated from a child previously treated with cefepime. The catalytic efficiency of the purified enzyme with the third-generation cephalosporins, cefepime and cefpirome, was 10 times higher than that with the E. cloacae P99 enzyme. This was mostly due to a decrease in K(m) for these beta-lactams. The clinical isolate produced large amounts of the cephalosporinase because introduction of the ampD gene decreased ampC expression and partially restored the wild-type phenotype. Indeed, MICs of cefepime and cefpirome remained 10 times higher than those for a stable derepressed clinical isolate (OUDhyp) transformed with an ampD gene. Sequencing of the ampC gene showed that 18 nucleotides had been deleted, corresponding to the six amino acids SKVALA (residues 289--294). According to the crystal structure of P99 beta-lactamase, this deletion was located in the H-10 helix. The ampR-ampC genes from the clinical isolates CHE and OUDhyp were cloned and expressed in Escherichia coli JM101. The MICs of cefpirome and cefepime of E. coli harboring ampC and ampR genes from CHE were 100--200 times higher than those of E. coli harboring ampC and ampR genes from OUDhyp. This suggests that the deletion, confirmed by sequencing of the ampC gene, is involved in resistance to cefepime and cefpirome. However, the high level of resistance to cefepime and cefpirome observed in the E. cloacae clinical isolate was due to a combination of hyperproduction of the AmpC beta-lactamase and structural modification of the enzyme. This is the first example of an AmpC variant conferring resistance to cefepime and cefpirome, isolated as a clinical strain.

Amino Acid Sequence↗
PubMed1998

HGF controls branched morphogenesis in tubular glands.

The c-MET gene encodes the tyrosine kinase p190MET, the receptor for a molecule known as Scatter Factor (SF) or Hepatocyte Growth Factor (HGF). This molecule has biological activities on epithelial sheets, including mitogenesis, cell-cell dissociation, stimulation of migration into the extracellular matrix, induction of cell polarization and branched tubulogenesis. The p190MET receptor is a heterodimer of two (alphabeta) disulfide-linked protein subunits. The RON and SEA genes encode tyrosine kinases that are structurally homologous to the HGF receptor. The three members of MET family elicit the same array of unusual biological responses that include "scattering", growth, and induction of polarized tubular structures. Recently, we identified another family of genes (SEX genes) encoding putative receptors with structural similarities to the extracellular domain of the HGF receptor and large cytoplasmic domains with unknown functions. Receptor autophosphorylation triggers the signal transduction pathways inside the target cells. The phosphorylation of an intracellular site made of a specific two-tyrosine containing sequence, mediates interactions with multiple SH2-containing intracellular signaling molecules. The multifunctional docking site is conserved and functional in the receptors encoded by RON and SEA. Substitution of N1358, critical for recruiting the Grb2/SoS complex and activating the Ras pathway, results in the abrogation of the growth response, while mutation of H1351 into N, generating a docking site inefficient to activate the PI3-kinase pathway, stimulates growth but fails to support the induction of scattering and branched tubulogenesis. Activation of a third pathway, mediated by direct phosphorylation on tyrosine of STAT-3 plays a critical role in induction of cell polarization and formation of tubular structures.

Animals↗
PubMed1995

The Saccharomyces cerevisiae Leu3 protein activates expression of GDH1, a key gene in nitrogen assimilation.

The Leu3 protein of Saccharomyces cerevisiae has been shown to be a transcriptional regulator of genes encoding enzymes of the branched-chain amino acid biosynthetic pathways. Leu3 binds to upstream activating sequences (UASLEU) found in the promoters of LEU1, LEU2, LEU4, ILV2, and ILV5. In vivo and in vitro studies have shown that activation by Leu3 requires the presence of alpha-isopropylmalate. In at least one case (LEU2), Leu3 actually represses basal-level transcription when alpha-isopropylmalate is absent. Following identification of a UASLEU-homologous sequence in the promoter of GDH1, the gene encoding NADP(+)-dependent glutamate dehydrogenase, we demonstrate that Leu3 specifically interacts with this UASLEU element. We then show that Leu3 is required for full activation of the GDH1 gene. First, the expression of a GDH1-lacZ fusion gene is three- to sixfold lower in a strain lacking the LEU3 gene than in an isogenic LEU3+ strain. Expression is restored to near-normal levels when the leu3 deletion cells are transformed with a LEU3-bearing plasmid. Second, a significant decrease in GDH1-lacZ expression is also seen when the UASLEU of the GDH1-lacZ construct is made nonfunctional by mutation. Third, the steady-state level of GDH1 mRNA decreases about threefold in leu3 null cells. The decrease in GDH1 expression in leu3 null cells is reflected in a diminished specific activity of NADP(+)-dependent glutamate dehydrogenase. We also demonstrate that the level of GDH1-lacZ expression correlates with the cells' ability to generate alpha-isopropylmalate and is lowest in cells unable to produce alpha-isopropylmalate. We conclude that GDH1, which plays an important role in the assimilation of ammonia in yeast cells, is, in part, activated by a Leu3-alpha-isopropylmalate complex. This conclusion suggests that Leu3 participates in transcriptional regulation beyond the branched-chain amino acid biosynthetic pathways.

Base Sequence↗