摘要:

ABSTRACTBasic helix–loop–helix (bHLH) transcription factors play diverse roles in controlling many developmental events. Although a great deal is understood about how bHLH factors activate gene transcriptionviaE-box DNA consensus sequences, studies of bHLH factor function in higher eukaryotes often have been hindered by the presence of multiple family members. As a first step in developing a simplifiedin vivosystem to examine bHLH factor activities, we examined whether the bHLH muscle regulatory factors MRF4 and MyoD function appropriately in yeast. We show that Gal4–MRF4 fusion proteins, or native MRF4 proteins, activate expression of an E-boxHIS3reporter gene whereas MyoD proteins remain inactive. Deletion of the MRF4 transcription activation domain (TAD) or point mutations that abolish MRF4 DNA interactions inhibitHIS3expression. Substitution of the MRF4 TAD with the Gal4 TAD also produces a functional protein, demonstrating that these transcription activation domains are functionally equivalent in yeast. Replacement of the MRF4 TAD with the related MyoD TAD, however, generates an inactive protein, suggesting that some specificity exists between bHLH family members. Using this experimental system, we also demonstrate that mammalian cDNA libraries can be screened successfully for cDNAs encoding novel bHLH proteins that interact with E-box targets. Thus, thisin vivoyeast system provides a novel approach to facilitate functional studies of bHLH factor regul

ISSN:1044-5498    

DOI:10.1089/dna.1996.15.1

年代:1996

数据来源: MAL

摘要:

ABSTRACTProtein disulfide isomerase (PDI) catalyzes protein folding and thiol-disulfide interchange reactions. The enzyme is localized in the lumen of endoplasmic reticulum (ER) and is abundant in secretory cells of various tissues. In this study we describe the isolation and characterization from human pancreas of a new protein, PDIp, that is structurally and functionally related to PDIs. PDIp cDNA is 1,659 bp in length and predicts a protein with an open reading frame of 511 amino acids. PDIp amino acid sequence shows 46% identity and 66% similarity to that of human PDI. PDIp possesses two thioredoxin-like active sites (WCGHCQ and WCTHCK) and an endoplasmic reticulum retention signal sequence, KEEL, at the carboxyl terminus. Northern analysis of normal human tissues and various human tumor cell lines revealed PDIp mRNA (2.0 kb) expression only in the normal pancreas. Recombinant PDIp protein catalyzed reductive cleavage of insulin and renaturation of reduced RNaseA. Somatic cell genetics and fluorescencein situhybridization localized the PDIp gene to the short arm of human chromosome 16. It is concluded that PDIp is a new member of the PDI family and is highly expressed in human pancreas.

ISSN:1044-5498    

DOI:10.1089/dna.1996.15.9

年代:1996

数据来源: MAL

摘要:

ABSTRACTThiopurine methyltransferase (TPMT) catalyzes theS-methylation of thiopurine drugs. Individual variation in the toxicity and therapeutic efficacy of these drugs is associated with a common genetic polymorphism that controls levels of TPMT activity and immunoreactive protein in human tissues. Because of the clinical significance of the "pharmacogenetic" regulation of this enzyme, it would be important to clone the gene for TPMT in humans and to study the molecular basis for the genetic polymorphism. As a first step toward cloning the gene for TPMT, we used the rapid amplification of genomic DNA ends to obtain aTPMT-specific intron sequence. That DNA sequence was used to design primers for the polymerase chain reaction (PCR), which made it possible to determine that the active gene for TPMT is located on human chromosome 6. ATPMT-positive cosmid clone was then isolated from a human chromosome 6-specific genomic DNA library, and the gene was sublocalized to chromosome band 6p22.3 by fluorescencein situhybridization. The gene for TPMT was found to be approximately 34 kb in length and consisted of 10 exons and 9 introns. On the basis of the results of 5′-rapid amplification of cDNA ends, transcription initiation occurred at or near a point 89 nucleotides upstream from the translation initiation codon of previously reported TPMT cDNAs. Once the structure of the TPMT gene had been determined, it was possible to perform the PCR with primers complementary to the sequences of introns flanking each exon that encodes enzyme protein with template DNA obtained from subjects with known phenotypes for the TPMT genetic polymorphism. This DNA was isolated from blood samples from 4 unrelated subjects with genetically low TPMT activity and 4 unrelated subjects with high TPMT activity. All subjects with low TPMT activity were homozygous for two point mutations— a G → A transition at nucleotide 460 in exon 7 and an A → G transition at nucleotide 719 in exon 10. Both mutations resulted in alterations in amino acid sequence, with Ala-154 → Thr and Tyr-240 → Cys, respectively. All DNA samples isolated from the blood of subjects with high TPMT activity contained "wild-type" sequence. Results obtained with these blood samples were confirmed when DNA from four human liver samples with high TPMT activity were found to have wild-type sequence at nucleotides 460 and 719, while three liver samples with intermediate enzyme activity (i.e., samples presumed to be heterozygous for the polymorphism) were heterozygous for the exon 7 and exon 10 mutations present in the blood samples of homozygous low subjects. Transient expression in COS-1 cells of TPMT expression constructs that contained both of the mutations in exons 7 and 10, as well as each independently, demonstrated that each mutation, as well as both together, resulted in decreased expression of TPMT enzymatic activity and immunoreactive protein. Molecular cloning and structural characterization of the TPMT gene as well as elucidation of the molecular basis for a common TPMT genetic polymorphism will help make it possible to develop DNA-based diagnostic tests for the polymorphism and to determine the mechanism by which it results in decreased expression of this important drug-metaboli

ISSN:1044-5498    

DOI:10.1089/dna.1996.15.17

年代:1996

数据来源: MAL

摘要:

ABSTRACTThe functional consequences of heterodimer formation between the epidermal growth factor receptor (EGFr) and the p185c-ncu e"receptor tyrosine kinase include increased mitogenic and transformation potencies. To determine the possible alteration of signal transduction pathways resulting from this heteromeric complex, the capacity of several signaling proteins to associate with the heterodimeric receptors has been assayed. Thein vivointeraction with the EGF1/pl-neu"c"heterodimer of several signal transduction proteins, including phospholipasγ1C-yl (γ1C-yl), the p85 subunit of phosphotidylinositol 3-kinase, therasGTPase activating protein, SHC, NCK, p72RAF, and the tyrosine phosphatase SHPTP2, was measured by coimmunoprecipitation. The binding of these signaling proteins to a complex composed of EGFr and a kinase-inactive form 1f pl81 (pl85K757M) was not impaired, even though the mitogenic and transformation activity of this complex had been abrogated. In addition, the EGF-induced phosphorylation of GAP, p85, and γ1C-yl did not correlate with the dominant-negative action 1f pl85K757M on EGFr function. Thus, substrate association and phosphorylation do not correlate stringently with the mitogenic and transforming activity of this receptor complex, suggesting additional pathways or mechanisms vital to EG1r/p-neuc""e"heterodimeric signali

ISSN:1044-5498    

DOI:10.1089/dna.1996.15.31

年代:1996

数据来源: MAL

摘要:

ABSTRACTHomozygous deletions of the tumor suppressor gene p16/MTS1 were reported in a wide variety of tumors and tumor cell lines. Its product inhibits the phosphorylation of the retinoblastoma protein (pRb) by CDK4 and CDK6. Because phosphorylation of pRb is a major regulatory event in the activation of the transcription factor E2F, a role for p16 in the regulation of E2F-dependent transcription was presumed. We investigated the effect of the loss of p16 on E2F-mediated transcription in a tumor progression model consisting of three cell lines originating from a common precursor cell—one p16-positive cell line established from the primary biopsy and two lines derived from more advanced stages of the tumor representing the same cell clone after loss of p16. We observed up- and deregulation of E2F-dependent transcription during the cell cycle of the p16-negative cell clones, which returned to normal after transient expression of p16. This p16-dependent regulation affects a set of enzymes necessary for the activation of all four DNA precursors; it is paralleled by the interconversion of transcriptionally active free E2F and transcriptionally inactive higher molecular complexes of E2F and is dependent on the existence of endogenous pRb. Furthermore, we show that p16-negative cell clones exhibit a growth advantage compared to their p16-positive counterparts. One might speculate that one feature of tumor progression could be deregulation of E2F-dependent transcription caused by loss of p1

ISSN:1044-5498    

DOI:10.1089/dna.1996.15.41

年代:1996

数据来源: MAL

摘要:

ABSTRACTWe have examinedcis-elements andtrans-acting factors that regulate transcription of the human cholecystokinin (CCK) gene. Transient expression of CCK promoter deletion constructs in human SK-N-MC neuroblastoma cells depicted positivecis-elements between the positions −100 to −92, −84 to −74, and −58 to −37, 5′ to the transcription initiation site. Correspondingly, DNase I protection analysis showed thattrans-acting factors bound to elements within these regions. The sequences encompass a putative basic helix–loop–helix leucine zipper (bHLH-ZIP) element, an Sp1 element, and a combined cAMP- and TPA-responsive element (CRE/TRE) at positions −97 to −92, −39 to −34, and −80 to −73, respectively. Mobility and supershift assays demonstrated that upstream stimulatory factor (USF) and Sp1 bind to the former elements and competition experiments confirmed that CREB/ATF and AP-1 bind to the CRE/TRE element. Mutation of the bHLH-ZIP and CRE/TRE elements decreased the activity of the promoter by 65% and 42%, respectively. The activity of the promoter was increased six- and two-fold after stimulation with forskolin and TPA, respectively. Stimulation was eliminated after mutation of the CRE/TRE element. Co-transfection experiments with pRSV-c-jun, pSV-fos, and pRC-RSV-CREB constructs showed that jun, CREB, and AP-1 stimulate transcription. We conclude that USF, Sp1, and members of the CREB/ATF and AP-1 family of transcription factors are the major determina

ISSN:1044-5498    

DOI:10.1089/dna.1996.15.53

年代:1996

数据来源: MAL

摘要:

ABSTRACTKeratin K17, while not present in healthy skin, is expressed under various pathological conditions, including psoriasis and cutaneous allergic reactions. The regulatory circuits involved in transcription of the human keratin K17 gene are poorly understood. To begin an analysis of the molecular mechanisms that regulate K17 gene transcription, we have studied the interactions between the nuclear proteins and the promoter region of the human K17 gene. That promoter region comprised 450 bp upstream from the translation initiation site. For these studies, we used electrophoretic mobility-shift assays, computer analysis, site-directed mutagenesis, and DNA-mediated cell transfection. In addition to the previously characterized interferon-γ-responsive elements, we identified eight protein binding sites in the promoter. Five of them bind the known transcription factors NF1, AP2, and Sp1 and three others bind still unidentified proteins. Using site-directed mutagenesis, we have demonstrated the importance of the protein binding sites for the promoter function involved in both constitutive and interferon-induced expression of the K17 keratin gene

ISSN:1044-5498    

DOI:10.1089/dna.1996.15.65

年代:1996

数据来源: MAL

摘要:

ABSTRACTCathepsin B cysteine protease enzymes have been shown to be involved in a variety of different biological processes in eukaryotes. We have isolated and characterized four distinct cathepsin B-like genes from the genetically tractable nematode,Caenorhabditis elegans. This is the first reported finding of a cathepsin B-like multigene family within a nonparasitic metazoan. The four genes possess distinct genomic architectures, with variations in the position, number, and size of introns. The predicted amino acid sequences of the four genes are highly diverged. Phylogenetic analysis indicates the divergence of this multigene family withinC. elegansis as great as the interspecies divergence between the vertebrates and nematode cathepsin B-like genes. In addition, each of the four genes described here shows a distinct temporal pattern of expression duringC. elegansdevelopment.

ISSN:1044-5498    

DOI:10.1089/dna.1996.15.75

年代:1996

数据来源: MAL

摘要:

ABSTRACTThe human OZF gene encodes a protein consisting essentially of zinc finger motifs of theKrüppeltype. Evolutionary studies revealed amplification of the bovine OZF gene in cattle. Domestic cattle includes two major subspecies: taurine (Bos primigenius taurus) and zebu (B. p. indicus). Amplified sequences were found in both subspecies and mapped to the same locus of chromosome X in band q11. No amplification was found in other bovids and in particular inBison, the closest related genus to the genusBos. One copy of the amplified locus was cloned and sequenced. It encodes a protein sharing 95% identity with the human OZF protein. The bovine OZF gene was detected in an additional locus on chromosome 18 in bothBosandBison, in band q23–24, which is likely to represent the ancestral position of the OZF gene. This is the first evidence of gene amplification in a mammalian species during evoluti

ISSN:1044-5498    

DOI:10.1089/dna.1996.15.83

年代:1996

数据来源: MAL