摘要:

ABSTRACTA condition called thymine uracilurea has been described that is due to a lack of dihydropyrimidine dehydrogenase (DPD) activity. Cancer patients experiencing acute 5-fluorouracil toxicity also have lower-than-normal DPD activities. However, to date, the molecular basis of this disorder has not been addressed. In this study, the phenotype and genotype of a family that presents a patient showing no DPD activity was determined. Fibroblast mRNAs from the patient and four family members were subjected to reverse transcriptase polymerase chain reaction (RT-PCR) using primers generated from the human DPD cDNA sequence. DPD mRNA from the patient was found to lack a segment of 165 nucleotides that results from exon skipping. DPD mRNA from the parents and a sibling were found to be heterozygous for the deleted and the normal mRNA, while a brother had two normal transcripts. DPD activities and levels of DPD protein correlated with genotype; the deficient patient had no detectable DPD protein. PCR analysis of the genomic DNA from this family revealed that the defective mRNA is not due to a deletion of a portion of the gene that contains the exon, thus implying that the mutation is the result of an as yet nonidentified point mutation that causes faulty splicing.

ISSN:1044-5498    

DOI:10.1089/dna.1995.14.1

年代:1995

数据来源: MAL

摘要:

ABSTRACTCell line-dependent expression of foreign genes in the baculovirus system was investigated using a recombinantvAcβhCG-lucvirus carrying two reporter genes—β subunit of human chorionic gonadotropin (βhCG) and luciferase (luc)—placed under the transcriptional control of theAutographa californicanuclear polyhedrosis virus (AcNPV) polyhedrin gene promoters. Five different lepidopteran cell lines derived fromSpodoptera frugiperda(Sf21 and Sf9),Bombyx mori(BmN and Bm5), andTrichoplusia ni(TN368) were used as host cells. TN368 expressed both βhCG and LUC to maximum levels, followed by BmN, Sf21, and Sf9 in descending order. Bm5 did not show any evidence of synthesis of the two proteins. Dot blot analysis of DNA from thevAcβhCG-luc-infected cells revealed that the level of entry of viral DNA was the same for all the five cell lines. After the completion of viral DNA replication (18 hr post infection), the level of viral DNA was the same for all the cell lines except for Bm5 where viral DNA replication did not take place and the residual virus was cleared from the cells. Analysis of RNA from the four expressing cell lines revealed a direct correlation between protein levels and levels of mRNA, suggesting transcriptional control. Differences in mRNA stability between cell lines was also evident. Gel retardation analysis of a host factor binding to transcriptionally important sequence motifs within the AcNPV polyhedrin gene promoter revealed an inverse correlation between the levels of this polyhedrin promoter-binding protein (PPBP) and reporter gene expression. Cold competition and mutation analyses of the DNA–protein complexes indicated that PPBP present in different cell lines recognized the same DNA sequence motifs present within the polyhedri

ISSN:1044-5498    

DOI:10.1089/dna.1995.14.7

年代:1995

数据来源: MAL

摘要:

ABSTRACTWe have generated various mammalian expression constructs that produce fusion proteins of human immunodeficiency virus type 1 (HIV-1) protease (PR) with the HIV-1 Nef protein. The expression of these proteins is inducible by the HIV-1 Tat protein. High-level expression of proteolytically active PR was produced from PR imbedded into Nef coding sequences, flanked by PR cleavage sites. The fusion protein was cleaved nearly to completion and did not exhibit the regulated processing that is seen with the virally encoded PR. No cytotoxic effect of PR expression was detected. The self-cleavage of PR could be inhibited by a specific inhibitor of HIV-1 PR (U75875). Elimination of the aminoterminal PR cleavage site did not have a measurable effect on cleavage of the precursor fusion protein. The cleaved fusion proteins appeared to be extremely unstable in the transfected cells. These findings demonstrate the intrinsic activity of HIV-1 PR in mammalian cells, in the context of a heterologous fusion protein.

ISSN:1044-5498    

DOI:10.1089/dna.1995.14.15

年代:1995

数据来源: MAL

摘要:

ABSTRACTWe have cloned and mapped the chromosomal location of three novel human genes encoding G protein-coupled receptors that we have named GPR6, GPR5, and GPR4. The entire coding region for each of these genes was contained on single exons. Gene GPR6 encoded a receptor that shared closest identity (71% in the transmembrane regions) with the human orphan receptor GPR3 and was localized to chromosome 6 (q21–q22.1). Northern blot analysis revealed that GPR6 transcripts were abundant in the human putamen and to a lesser extent in the frontal cortex, hippocampus, and hypothalamus. Gene GPR5 encoded a receptor that most closely resembled the orphan receptor RBS11 (48% in the transmembrane regions) and the MIP 1α/RANTES receptor (45% in the transmembrane regions) and was localized to chromosome 3 (p21.3–p21.1). Gene GPR4 shared identity (40% in the transmembrane regions) with the human platelet-activating factor receptor and was localized to chromosome 19 (q13.2–

ISSN:1044-5498    

DOI:10.1089/dna.1995.14.25

年代:1995

数据来源: MAL

摘要:

ABSTRACTN-Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, removesN-alkylpurines and other purine lesions induced in DNA by simple alkylating carcinogens. A mouse MPG cDNA clone was isolated from a λ recombinant phage library of BALB/c mouse lung cell and characterized. Using the mouse MPG cDNA as a probe, the complete mouse MPG gene was isolated in two overlapping λ recombinant genomic clones. The 6-kb gene has four exons containing 1,002 bp of coding sequence. The transcription start site was identified in the genomic sequence by primer extension of MPG mRNA from a mouse lung fibroblast cell line. The location of this transcription start site was confirmed byin vitrotranscription with the promoter-containing plasmid template. Promoter function of the sequence 5′ upstream of the transcription initiation site was shown by transient expression of the firefly luciferase reporter gene under the control of this sequence in transfected human and mouse cells. The mouse MPG promoter contains no TATA box, but has a CAAT element and is G·C-rich with putative AP2 elements and SP1-complementary seque

ISSN:1044-5498    

DOI:10.1089/dna.1995.14.37

年代:1995

数据来源: MAL

摘要:

ABSTRACTA common feature of cells selectedin vitrofor the multidrug resistance (MDR) phenotype is the amplification and concomitant overexpression of themdrgenes. In murine macrophage-like J774.2-derived MDR cell lines, there is a good correlation between levels of amplification and expression for themdr1b gene, but not for the other two gene family members,mdr1a andmdr2. To understand this phenomenon better, a study of the amplification and expression of themdr2 gene was undertaken. Southern blotting of genomic DNAs from a series of six MDR cell lines revealed that five of these lines had 5′-end amplification ofmdr2, whereas only three contained 3′-end amplification. The analysis also suggested the involvement of a recombination hot-spot in this phenomenon. Despite the observation that the ratio between the number of copies of the 5′ and 3′ ends of the gene differs among cell lines, the ratio of 5′ to 3′ end transcription ofmdr2 was approximately 1 in all cell lines. An analysis of promoter methylation in MDR cell lines demonstrated that this mechanism may play a role in regulating the transcription ofmdr2, but not ofmdr1b. Long-range mapping of themdrlocus in parental and amplified cell lines suggested that the threemdrgenes are oriented in the same direction, and also revealed the presence of a number of rearrangement events. Models for the murinemdrgene locus in wild-type cells and in a cell line containing a rearrangement ar

ISSN:1044-5498    

DOI:10.1089/dna.1995.14.47

年代:1995

数据来源: MAL

摘要:

ABSTRACTWe have analyzed amino acid sequence relationships among soluble and microsomal epoxide hydrolases, haloacid dehalogenases, and a haloalkane dehalogenase. The amino-terminal residues (1–229) of mammalian soluble epoxide hydrolase are homologous to a haloacid dehalogenase. The carboxy-terminal residues (230–554) of mammalian soluble epoxide hydrolase are homologous to haloalkane dehalogenase, to plant soluble epoxide hydrolase, and to microsomal epoxide hydrolase. The shared identity between the haloacid and haloalkane dehalogenases does not indicate relatedness between these two types of dehalogenases. The amino-terminal and carboxy-terminal homologies of mammalian soluble epoxide hydrolase to the respective dehalogenases suggests that this epoxide hydrolase, but not the soluble epoxide hydrolase of plant or the microsomal epoxide hydrolase, derives from a gene fusion. The homology of microsomal to soluble epoxide hydrolase suggests they derive from a gene duplication, probably of an ancestral bacterial (epoxide) hydrolase gene. Based on homology to haloalkane dehalogenase, the catalytic residues for the soluble and microsomal epoxide hydrolases are predicted. A nomenclature system based on divergent molecular evolution is proposed for these epoxide hydrola

ISSN:1044-5498    

DOI:10.1089/dna.1995.14.61

年代:1995

数据来源: MAL

摘要:

ABSTRACTA cluster of genes of theCYP6family was found in a series of overlapping λDASH clones from a genomic library of the house fly,Musca domestica. Four complete genes,CYP6A3,CYP6A4,CYP6A5, andCYP6C1, and fragments of two other genes,CYP6A6andCYP6C2, were closely linked on a 24-kb segment of DNA. Restriction fragment length polymorphism (RFLP) analysis of PCR-amplified segments of two of the genes showed that the cluster is localized on chromosome V of the house fly. Each gene contained a short intron of 57 to 125 bp interrupting a conserved Glu codon, as in the previously describedCYP6A1gene. The gene fragmentCYP6A6consisted only of the coding region downstream from this intron, i.e., about one-third of the complete P450. The gene fragmentCYP6C2was missing a short amino-terminal part of the coding region, and may represent the two last exons of a larger gene. Gene duplication and chromosomal inversion events may explain the origin of this cluster. The P450 proteins deduced from the nucleotide sequences shared 39–71% amino acid identity with each other. This low identity and the lack of evidence of recent gene conversion events suggested that this cluster may be evolutionarily ancient and that homologous clusters may be found in other holometabolous insects. Evidence for transcription of the genes and for correct splicing of the introns was obtained by northern blotting and reverse transcription polymerase chain reaction (RT-PCR) experiments. No overexpression was observed in any of three insecticide-resistant house fly strains. RT-PCR and sequencing also revealed the existence of other genes or alleles closely related to the members of this clust

ISSN:1044-5498    

DOI:10.1089/dna.1995.14.73

年代:1995

数据来源: MAL

摘要:

ABSTRACTA great amount of genomic DNA in multcellular eukaryotic organisms is regarded as junk because it has no real function in protein coding. However, there is growing evidence that noncoding DNA can play a vital role in the regulation of gene expression during development (Leeet al., 1993). This indicates that the so-called junk DNA may have essential functions that are yet to be found (Nowak, 1994). A novel binary model of noncoding repetitive DNA sequence is proposed to illustrate its possible structure and implications in genome organization and development.

ISSN:1044-5498    

DOI:10.1089/dna.1995.14.83

年代:1995

数据来源: MAL

摘要:

ABSTRACTSeveral techniques are currently available for detecting point mutations in DNA. The most widely used methods either use hazardous chemicals (chemical mismatch cleavage) or can detect mutations only in short (200- to 500-bp) fragments (single-stranded conformational polymorphism and denaturing gradient gel electrophoresis). In an effort to develop a sensitive and reliable method for the detection of mutations in large segments of DNA, a novel RNase protection assay using RNase I was developed. In this method, the desired portion of the gene is amplified by the polymerase chain reaction (PCR) using specific oligonucleotides and hybridized to a32P-labeled RNA probe containing the wild-type sequence. The RNA/DNA hybrid is subsequently digested with RNase I, which cleaves the RNA at the mismatch sites. The protected RNA fragments are separated on a denaturing polyacrylamide-urea gel and detected by autoradiography. Four different RNA probes from two protein tyrosine phosphatases (PTP1C and PTP2C) were assayed using this procedure. Several mutants of the two enzymes were tested using wild-type RNA probes. Single-base changes involving all four bases at the mismatch site could be detected efficiently. The ability of this method to detect insertions and single-base deletions was also demonstrated. Using a PCR-based RNase protection assay, a single-base deletion in PTP1C in themotheatenmutation in mice could be detected. Using fragments amplified from genomic DNA, mice that were heterozygous for themotheatenmutation could be distinguished from wild type and homozygotes for this mutation. The assay outlined in this paper should prove to be very useful in screening for mutations due to the possibility of using large DNA segments (>1 kb) without compromising either the sensitivity or reliability.

ISSN:1044-5498    

DOI:10.1089/dna.1995.14.87

年代:1995

数据来源: MAL