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At least 487 records · Page 27Linked to original sources
PubMed1994

Mammalian mitogen-activated protein kinase kinase kinase (MEKK) can function in a yeast mitogen-activated protein kinase pathway downstream of protein kinase C.

Mitogen-activated protein kinase cascades are conserved in fungal, plant, and metazoan species. We expressed murine MAP kinase kinase kinase (MEKK) in the yeast Saccharomyces cerevisiae to determine whether this kinase functions as a general or specific activator of genetically and physiologically distinct MAP-kinase-dependent signaling pathways and to investigate how MEKK is regulated. Expression of MEKK failed to correct the mating deficiency of a ste11 delta mutant that lacks an MEKK homolog required for mating. MEKK expression also failed to induce expression of a reporter gene controlled by the HOG1 gene product (Hog1p), a yeast MAP kinase homolog involved in response to osmotic stress. Expression of MEKK did correct the cell lysis defect of a bck1 delta mutant that lacks an MEKK homolog required for cell-wall assembly. MEKK required the downstream MAP kinase homolog in the BCK1-dependent pathway, demonstrating that it functionally replaces the BCK1 gene product (Bck1p) rather than bypassing the pathway. MEKK therefore selectively activates one of three distinct MAP-kinase-dependent pathways. Possible explanations for this selectivity are discussed. Expression of the MEKK catalytic domain, but not the full-length molecule, corrected the cell-lysis defect of a pkc1 delta mutant that lacks a protein kinase C homolog that functions upstream of Bck1p. MEKK therefore functions downstream of the PKC1 gene product (Pkc1p). The N-terminal noncatalytic domain of MEKK, which contains several consensus protein kinase C phosphorylation sites, may, therefore, function as a negative regulatory domain. Protein kinase C phosphorylation may provide one mechanism for activating MEKK.

Animals↗
PubMed1994

Engineered biosynthesis of novel polyketides: influence of a downstream enzyme on the catalytic specificity of a minimal aromatic polyketide synthase.

To identify the minimum set of polyketide synthase (PKS) components required for in vivo biosynthesis of aromatic polyketides, combinations of genes encoding subunits of three different aromatic PKSs--act from Streptomyces coelicolor A3(2) (an actinorhodin producer), fren from Streptomyces roseofulvus (a frenolicin and nanaomycin producer), and tcm from Streptomyces glaucescens (a tetracenomycin producer)--were expressed in a recently developed Streptomyces host-vector system. The "minimal" components (ketosynthase/putative acyltransferase, chain length-determining factor, and acyl carrier protein) were produced with and without a functional polyketide ketoreductase and/or cyclase, and the polyketide products of these recombinant strains were structurally characterized. Several previously identified polyketides were isolated in addition to two previously unidentified polyketides, dehydromutactin and SEK 15b, described here. The results proved that the act cyclase is not required for the biosynthesis of several aberrantly cyclized products that have been previously reported. They are also consistent with earlier conclusions that the minimal PKS controls chain length as well as the regiospecificity of the first cyclization and that it can do so in the absence of both a ketoreductase and a cyclase. However, the ability of the minimal tcm PKS to synthesize two different singly cyclized intermediates suggests that it is unable to accurately control the course of this reaction by itself. In the presence of a downstream enzyme, the flux through one branch of the cyclization pathway increases relative to the other. We propose that these alternative specificities may be due to the ability of downstream enzymes to associate with the minimal PKS and to selectively inhibit a particular branch of the cyclization pathway.

DNA Mutational Analysis↗
PubMed1997

An N-linked glycosylation motif from the noncleaving luteinizing hormone receptor substituted for the homologous region (Gly367 to Glu369) of the thyrotropin receptor prevents cleavage at its second, downstream site.

The thyrotropin receptor (TSHR) exists in two forms (single polypeptide and two subunits), whereas the lutropin/chorionic gonadotropin receptor (LH/CGR) is a single chain. Recent data suggest that the TSHR cleaves at two sites. We mutagenized selected chimeric TSH-LH/CGR to localize the cleavage sites in the TSHR. All 23 receptors mutated in the estimated vicinity of the upstream site cleaved into two subunits as determined by 125I-TSH cross-linking to intact cells. In contrast, in a series of mutations homologous to the noncleaving LH/CGR, the downstream TSHR cleavage site localized to three amino acids (GQE367-369). Remarkably, group substitution of these residues, but not substitution of individual residues, abolished cleavage. Moreover, the mutation that prevented cleavage (GQE367-369NET) transposed a motif (NET291-293) that is glycosylated in the LH/CGR. TSHR cleavage or noncleavage after substitution of GQE367-369 with other triplets (AAA, NQE, and NQT) was consistent with a role for N-linked glycosylation at this site. In summary, our data (i) support the concept that the TSHR cleaves at two sites, (ii) relate TSHR residues GQE367-369 to cleavage at the second, downstream site, and (iii) suggest that cleavage or noncleavage at site two is related to N-linked glycosylation. These findings provide new insight into the evolutionary divergence of two closely related receptors.

Amino Acid Sequence↗
PubMed1998

Guanine nucleotide exchange factors regulate specificity of downstream signaling from Rac and Cdc42.

The Rac and Cdc42 GTPases regulate diverse cellular behaviors involving the actin cytoskeleton, gene transcription, and the activity of multiple protein and lipid kinases. All of these pathways can potentially become activated when GTP-Rac or GTP-Cdc42 is formed in response to external cell signals, yet it is evident that each activity must also be able to be controlled individually. The mechanisms by which such specificity of GTPase signaling in response to upstream stimuli is achieved remains unclear. We investigated the action of several well characterized guanine nucleotide exchange factors (GEFRho) to activate Rac- and/or Cdc42-dependent kinase pathways. Coexpression studies in COS-7 cells revealed that the ability of individual guanine nucleotide exchange factors (GEFs) to activate the p21-activated kinase PAK1 could be dissociated from activation of c-Jun amino-terminal kinase, even though activation of both pathways requires the action of the GEFs on Rac and/or Cdc42. In contrast, expression of constitutively active forms of Rac or Cdc42 effectively stimulated both downstream kinases. We conclude that GEFs can be important determinants of downstream signaling specificity for members of the Rho GTPase family.

Animals↗
PubMed1998

Constitutive apoptosis in human neutrophils requires synergy between calpains and the proteasome downstream of caspases.

Programmed cell death invariably requires the activation of proteolytic cascades that are not yet well defined but are initiated after apical caspase activation. We provide evidence that calpains and the proteasome function synergistically downstream of caspases to assist the constitutive apoptotic program of aging neutrophils, which plays an important role in resolution of inflammatory responses. Inhibitor studies indicated that "tethering" of preapoptotic senescent neutrophils to human macrophages required caspase activity. However, the development of morphological features characteristic of apoptosis, including nuclear morphology, PS exposure, surface protein shedding, and the capacity to be ingested by macrophages, required the downstream action of either calpains or the proteasome. Calpain activities were constitutively active in freshly isolated neutrophils and responsible for rearrangements in the protein composition and structure of the plasmalemmal cytoskeleton as they aged in culture and underwent apoptosis. This included a dissociation of protein(s) from F-actin, a candidate mechanism for increased susceptibility to cleavage, and a loss in immunodetectable alpha-actinin and ezrin, two actin-binding, membrane-anchoring proteins. These results clarify roles for different classes of proteases in a physiologically important form of constitutive apoptosis.

Actinin↗
PubMed1999

Characterization of the human elk-1 promoter. Potential role of a downstream intronic sequence for elk-1 gene expression in monocytes.

To characterize the human elk-1 promoter, we mapped the transcriptional start site and isolated elk-1-specific genomic phage clones that contained extensive upstream and downstream sequences. A TATA-like motif was identified immediately upstream of the transcriptional start site. Functional analyses of DNA fragments containing the TATA element and the identification of a DNase I-hypersensitive chromatin site (HS 1) in close proximity to the TATA box suggest that the identified TATA motif is important for elk-1 transcription in vivo. Sequences upstream and downstream from the TATA box were found to contribute to elk-1 promoter activity. A second hypersensitive site (HS 2) was identified within the first intron in pre-monocytic cells, which express Elk-1 only when differentiating to monocytes. In a variety of other cell types, which display a constitutive Elk-1 expression, HS 2 did not exist, suggesting that inducibility of elk-1 expression is associated with the presence of HS 2. Egr-1 and the serum response factor were found to interact specifically with the intronic sequence at +265 and +448, respectively. Because Egr-1 mRNA and protein levels were observed to increase significantly before induction of elk-1 expression, we propose that Egr-1 is important for the regulation of elk-1 transcription in differentiating monocytes.

Animals↗
PubMed1999

Transmembrane segment (TMS) VIII of the Na(+)/Citrate transporter CitS requires downstream TMS IX for insertion in the Escherichia coli membrane.

The amino acid sequence of the sodium ion-dependent citrate transporter CitS of K. pneumoniae contains 12 hydrophobic stretches that could form membrane-spanning segments. A previous analysis of the membrane topology in Escherichia coli using the PhoA gene fusion technique indicated that only nine of these hydrophobic segments span the membrane, while three segments, Vb, VIII and IX, were predicted to have a periplasmic location (Van Geest, M., and Lolkema, J. S. (1996) J. Biol. Chem. 271, 25582-25589). A topology study of C-terminally truncated CitS molecules in dog pancreas microsomes revealed that the protein traverses the endoplasmic reticulum membrane 11 times. In agreement with the PhoA fusion data, segment Vb was predicted to have a periplasmic location, but, in contrast, segments VIII and IX were found to be membrane-spanning (Van Geest, M., Nilsson, I., von Heijne, G., and Lolkema, J. S. (1999) J. Biol. Chem. 274, 2816-2823). In the present study, using site-directed Cys labeling, the topology of segments VIII and IX in the full-length CitS protein was determined in the E. coli membrane. Engineered cysteine residues in the loop between the two segments were accessible to a membrane-impermeable thiol reagent exclusively from the cytoplasmic side of the membrane, demonstrating that transmembrane segments (TMSs) VIII and IX are both membrane-spanning. It follows that the folding of CitS in the E. coli and endoplasmic reticulum membrane is the same. Cysteine accessibility studies of CitS-PhoA fusion molecules demonstrated that in the E. coli membrane segment VIII is exported to the periplasm in the absence of the C-terminal CitS sequences, thus explaining why the PhoA fusions do not correctly predict the topology. An engineered cysteine residue downstream of TMS VIII moved from a periplasmic to a cytoplasmic location when the fusion protein containing TMSs I-VIII was extended with segment IX. Thus, downstream segment IX is both essential and sufficient for the insertion of segment VIII of CitS in the E. coli membrane.

Alkaline Phosphatase↗
PubMed2000

Integrins regulate the linkage between upstream and downstream events in G protein-coupled receptor signaling to mitogen-activated protein kinase.

Receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) can both activate mitogen-activated protein kinase (MAPK), a critical intermediate in the transduction of proliferative signals. Numerous observations have demonstrated that integrin-mediated cell anchorage can regulate the efficiency of signaling from RTKs to MAPK. Recently, a relationship between integrins and GPCR signaling has also emerged; however, little is understood concerning the mechanisms involved. Here, we investigate integrin regulation of GPCR signaling to MAPK, focusing on the P2Y class of GPCRs that function through activation of phospholipase Cbeta. P2Y receptor signaling to the downstream components mitogen-activated protein kinase kinase and MAPK is highly dependent on integrin-mediated cell anchorage. However, activation of upstream events, including inositol phosphate production and generation of calcium transients, is completely independent of cell anchorage. This indicates that integrins regulate the linkage between upstream and downstream events in this GPCR pathway, just as they do in some aspects of RTK signaling. However, the P2Y pathway does not involve cross-activation of a RTK, nor a role for Shc or c-Raf; thus, it is quite distinct from the classical RTK-Ras-Raf-MAPK cascade. Rather, integrin-modulated P2Y receptor stimulation of MAPK depends on calcium and on the activation of protein kinase C.

Adenosine Triphosphate↗
PubMed2000

Activation of downstream signals by the long form of the leptin receptor.

The adipocyte-derived hormone leptin signals the status of body energy stores by activating the long form of the leptin receptor (LRb). Activation of LRb results in the activation of the associated Jak2 tyrosine kinase and the transmission of downstream phosphotyrosine-dependent signals. We have investigated the signaling function of mutant LRb intracellular domains under the control of the extracellular erythropoietin (Epo) receptor. By using this system, we confirm that two tyrosine residues in the intracellular domain of murine LRb become phosphorylated to mediate LRb signaling; Tyr(985) controls the tyrosine phosphorylation of SHP-2, and Tyr(1138) controls STAT3 activation. We furthermore investigated the mechanisms by which LRb controls downstream ERK activation and c-fos and SOCS3 message accumulation. Tyr(985)-mediated recruitment of SHP-2 does not alter tyrosine phosphorylation of Jak2 or STAT3 but results in GRB-2 binding to tyrosine-phosphorylated SHP-2 and is required for the majority of ERK activation during LRb signaling. Tyr(985) and ERK activation similarly mediate c-fos mRNA accumulation. In contrast, SOCS3 mRNA accumulation requires Tyr(1138)-mediated STAT3 activation. Thus, the two LRb tyrosine residues that are phosphorylated during receptor activation mediate distinct signaling pathways as follows: SHP-2 binding to Tyr(985) positively regulates the ERK --> c-fos pathway, and STAT3 binding to Tyr(1138) mediates the inhibitory SOCS3 pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Distant downstream sequence determinants can control N-tail translocation during protein insertion into the endoplasmic reticulum membrane.

We have studied the membrane insertion of ProW, an Escherichia coli inner membrane protein with seven transmembrane segments and a large periplasmic N-terminal tail, into endoplasmic reticulum (ER)-derived dog pancreas microsomes. Strikingly, significant levels of N-tail translocation is seen only when a minimum of four of the transmembrane segments are present; for constructs with fewer transmembrane segments, the N-tail remains mostly nontranslocated and the majority of the molecules adopt an "inverted" topology where normally nontranslocated parts are translocated and vice versa. N-tail translocation can also be promoted by shortening of the N-tail and by the addition of positively charged residues immediately downstream of the first trasnmembrane segment. We conclude that as many as four consecutive transmembrane segments may be collectively involved in determining membrane protein topology in the ER and that the effects of downstream sequence determinants may vary depending on the size and charge of the N-tail. We also provide evidence to suggest that the ProW N-tail is translocated across the ER membrane in a C-to-N-terminal direction.

ATP-Binding Cassette Transporters↗
PubMed2000

An intronic downstream enhancer promotes 3' splice site usage of a neural cell-specific exon.

The human nonmuscle myosin heavy chain B gene contains a 30-nucleotide alternative exon, N30, that is included in the mRNA from neural cells but is skipped in all other cells. We have previously identified an intronic distal downstream enhancer (IDDE) region that is required for neural cell-specific inclusion of N30. In this study, we investigated the mechanism by which the IDDE promotes N30 exon usage. In vitro splicing analysis using neural cell nuclear extracts and two-exon pre-mRNA substrates, which consist of the N30 exon and either the upstream (E5) or downstream (E6) exon, demonstrates that the IDDE activates upstream E5-N30 splicing by facilitating early prespliceosome complex formation. The IDDE has no effect on N30-E6 splicing where the IDDE resides. Inspection of splice site consensus sequences shows that a polypyrimidine (Py) tract preceding N30 is suboptimal for U2AF binding. Optimizing the Py tract completely relieves the requirement for the IDDE in E5-N30 splicing in vitro. In transfected cells, the wild-type minigene transcripts, which consist of three exons, E5, N30, and E6, undergo neural cell-specific and IDDE-dependent alternative splicing of N30. Optimizing the Py tract in minigenes also completely relieves the requirement for the IDDE in N30 inclusion. Furthermore, overexpression of the truncated U2AF65, which contains the arginine and serine dipeptide-rich domain and linker domain, but lacks the RNA binding domain, selectively inhibits the IDDE-mediated N30 inclusion in mRNA from the wild-type minigene in a dominant negative fashion. These results support the hypothesis that the IDDE facilitates the recognition of the 3' splice site preceding N30 by a network of protein-protein interactions implicated in the recruitment of U2AF to a suboptimal Py tract.

Enhancer Elements, Genetic↗
PubMed2001

Forkhead family transcription factor FKHRL1 is expressed in human megakaryocytes. Regulation of cell cycling as a downstream molecule of thrombopoietin signaling.

FKHRL1, a member of the Forkhead transcription factor family, is one of the downstream molecules of phosphatidylinositol 3-kinase-Akt. This molecule is a mammalian homolog of DAF-16, which plays an important role in the longevity of Caenorhabditis elegans. In this study we found that Akt and FKHRL1 proteins were detectable in highly purified normal human megakaryocytes and that these molecules were actually phosphorylated by thrombopoietin (TPO). To clarify the functional role of FKHRL1 in TPO signaling, we established a tetracycline-inducible system in the human TPO-dependent leukemia cell line UT-7/TPO. Induced expression of active FKHRL1 led to cell cycle arrest at G0/G1 phase in this cell line. These results suggest that FKHRL1 plays an important role in the cell cycle of megakaryocytic cells as one of the downstream target molecules of phosphatidylinositol 3-kinase-Akt, presumably mediated through the activation or inactivation of cell cycle-associated gene(s).

Cell Cycle↗
PubMed2001

The insulin receptor substrate (IRS)-1 pleckstrin homology domain functions in downstream signaling.

The pleckstrin homology (PH) domain of the insulin receptor substrate-1 (IRS-1) plays a role in directing this molecule to the insulin receptor, thereby regulating its tyrosine phosphorylation. In this work, the role of the PH domain in subsequent signaling was studied by constructing constitutively active forms of IRS-1 in which the inter-SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase was fused to portions of the IRS-1 molecule. Chimeric molecules containing the PH domain were found to activate the downstream response of stimulating the Ser/Thr kinase Akt. A chimera containing point mutations in the PH domain that abolished the ability of this domain to bind phosphatidylinositol 4,5-bisphosphate prevented these molecules from activating Akt. These mutations also decreased by about 70% the amount of the constructs present in a particulate fraction of the cells. These results indicate that the PH domain of IRS-1, in addition to directing this protein to the receptor for tyrosine phosphorylation, functions in the ability of this molecule to stimulate subsequent responses. Thus, compromising the function of the PH domain, e.g. in insulin-resistant states, could decrease both the ability of IRS-1 to be tyrosine phosphorylated by the insulin receptor and to link to subsequent downstream targets.

3T3 Cells↗
PubMed2001

Membrane proximal ERK signaling is required for M-calpain activation downstream of epidermal growth factor receptor signaling.

Localization of signaling is critical in directing cellular outcomes, especially in pleiotropic signaling pathways. The extracellular signal-regulated kinase (ERK)/microtubule-associated protein kinase, which promotes cell migration, proliferation, and differentiation is found in the nucleus and throughout the cytoplasm. Recently, it has been shown that nuclear translocation of ERK is required for transcriptional changes and cell proliferation. However, the cellular consequences, of cytoplasmic signaling have not been defined. We explored whether cytoplasmic, specifically membrane-proximal, ERK signaling is involved in growth factor-induced cell motility. We previously have demonstrated that increased M-calpain activity downstream of epidermal growth factor receptor (EGFR)-mediated ERK activation is necessary for epidermal growth factor (EGF)-induced motility. Calpain isoforms also have been found in nuclear, cytosolic, and plasma membrane-associated compartments in a variety of cell types. We now employ cell engineering approaches to control localization of the upstream EGFR and ERK activities to examine the spatial effect of upstream signal locale on downstream calpain activity. With differential ligand-induced internalization and trafficking-restricted receptor variants, we find that calpain activity is triggered only by plasma membrane-restricted activated EGFR, not by internalized (although still active) EGFR. Cells transfected with membrane-targeted ERK1 and ERK2, which sequester endogenous ERKs, exhibited normal EGF-induced calpain activity. Transfection of an inactive ERK phosphatase (MKP-3/Pyst1) that sequesters ERK in the cytoplasm prevented calpain activation as well as de-adhesion. These data strongly suggest that EGF-induced calpain activity can be enhanced near sites of membrane-proximal EGFR-mediated ERK signaling, providing insights about how calpain activity might be regulated and targeted to enhance its effects on adhesion-related substrates.

Animals↗
PubMed2001

Maf and Jun nuclear oncoproteins share downstream target genes for inducing cell transformation.

The Maf oncoprotein is a basic leucine zipper (bZip)-bearing transcriptional activator that recognizes the Maf recognition element (MARE) DNA sequence. In this study, we investigated the role of Maf's transactivation function in cell transformation. Replacement of the conserved amino terminus transactivator domain of Maf by a heterologous and stronger transactivator domain (the acidic transactivator domain of VP16) resulted in enhanced transformation of chicken embryo fibroblast cells. In contrast, the fusing of a transcriptional repressor domain (Sin3 interaction domain of Mxi1) with the whole Maf protein masked the transactivator function of Maf, which in turn inhibited its transforming activity. Furthermore, the leucine zipper domain of Maf, which defines its dimer-forming specificity, was exchangeable with that of GCN4 yeast protein in terms of its transactivating and cell transforming activities. Thus, heterodimer formation with other bZip factors is not required for Maf's ability to transform. These results together suggest that transactivation through MARE is necessary for Maf-induced transformation and that there exist downstream target gene(s) for transformation. Since the MARE sequence overlaps with the recognition element of another bZip oncoprotein Jun, we assessed whether Jun and Maf induce cell transformation through activating the same genes. We thus constructed a mutated version of Jun that has a GCN4 leucine zipper and lacks the transactivator domain. This mutant repressed the cell transformation not only by Jun but also by Maf. Thus, Maf and Jun share downstream target gene(s) that are involved in cell transformation.

Animals↗
PubMed2001

Gene expression profiling leads to identification of GLI1-binding elements in target genes and a role for multiple downstream pathways in GLI1-induced cell transformation.

The zinc finger transcription factor GLI1, which mediates Sonic hedgehog signaling during development, is expressed in several human cancers, including basal cell carcinoma, medulloblastoma, and sarcomas. We identified 147 genes whose levels of expression were significantly altered in RNA obtained from cells demonstrating a transformed phenotype with stable GLI1 expression or stable Ha-ras expression. Comparison of expression profiles from GLI1- and Ha-ras-expressing cells established a set of genes unique to GLI1-induced cell transformation. Thirty genes were altered by stable GLI1 expression, and 124 genes were changed by stable Ha-ras expression. Seven genes had altered expression levels in both GLI1- and Ha-ras-expressing cells. Genes whose expression was altered by GLI1 included cell cycle genes, cell adhesion genes, signal transduction genes, and genes regulating apoptosis. GLI1 consensus DNA-binding sequences were identified in the 5' regions of cyclin D2, IGFBP-6, osteopontin, and plakoglobin, suggesting that these genes represent immediate downstream targets. Gel shift analysis confirmed the ability of the GLI1 protein to bind these sequences. Up-regulation of cyclin D2 and down-regulation of plakoglobin were demonstrated in GLI1-amplified compared with non-amplified human rhabdomyosarcoma cells. Many of the GLI1 targets with known function identified in this study increase cell proliferation, indicating that GLI1-induced cell transformation occurs through multiple downstream pathways.

Amino Acid Motifs↗
PubMed2001

mDia-interacting protein acts downstream of Rho-mDia and modifies Src activation and stress fiber formation.

The formin homology protein mDia is a Rho GTPase effector protein that participates in stress fiber formation, cytokinesis, and transcriptional activation of the serum response factor. Although the function of another effector of Rho, Rho-associated kinase, is well established, relatively little is known about the functional mechanism and the downstream targets of mDia. Our recent report of a Rho-mDia-Src-tyrosine kinase pathway suggested an important role for mDia in cell adhesion turnover. We identified a new mDia-interacting protein which is expressed ubiquitously. The new protein mainly binds to the proline-rich region of mDia through its Src homology 3 domain and also binds to Grb2 through its proline-rich domain. The protein is localized at the cell periphery and membrane ruffles and co-localizes with mDia. Co-expression of vSrc and the mDia-interacting protein induces significant morphological changes at focal contacts and activation of vSrc. Furthermore, we found that the mDia-interacting protein plays an important role in stress fiber formation induced by active mDia1. Our results suggest that this new protein regulates actin polymerization and cell adhesion turnover in the downstream portion of the Rho-mDia pathway by interacting with Grb2 and Src.

3T3 Cells↗
PubMed2002

Characterization of the murine hyaluronidase gene region reveals complex organization and cotranscription of Hyal1 with downstream genes, Fus2 and Hyal3.

Hyaluronidases are required for the breakdown of hyaluronan (HA), an abundant component of the extracellular matrix of vertebrate tissues. Multiple hyaluronidase genes have been identified, but the only clue to the function of their products has come from the identification of hyaluronidase 1 deficiency in a single patient with a mild clinical phenotype. As a first step in the generation of mice with hyaluronidase deficiency, we have used experimental and bioinformatic approaches to examine the organization of the mouse chromosome 9 region containing, in order, Hyal2, Hyal1, and Hyal3. This region was found to be complex, with Fus2 partially embedded in Hyal3, and Ifrd2 immediately downstream from Hyal3. The Hyal genes were all found to have four exons, and exons 2-4 exhibited the highest sequence conservation. Northern blot analysis demonstrated that the tissue expression profile for Hyal1 was similar in mice and humans, but a greater number of transcripts was detected in mouse tissues. Hyal3 was expressed more broadly in mice compared with humans and again exhibited additional transcripts. Reverse transcription-PCR demonstrated that some of the larger Hyal1 transcripts, seen on the Northern blot, were the result of cotranscription of Hyal1 with downstream genes, Fus2 or Hyal3. In vitro transcription/translation of one of the high abundance bicistronic transcripts produced Hyal 1, suggesting that Hyal 1 could be produced from all of the bicistronic transcripts. Characterization of the region including mouse Hyal1 and Hyal3 revealed complex organization and transcription that must be considered in the development and interpretation of mouse models involving genes in this region.

Amino Acid Sequence↗