Expression and Chromosomal Localization of the Human α4/IGBP1 Gene, the Structure of Which Is Closely Related to the Yeast TAP42 Protein of the Rapamycin-Sensitive Signal Transduction Pathway

To study the function of the B cell signal transduction molecule α4 (IGBP1), we isolated a human α4 (IGBP1) gene that has sequence similarity to the yeast protein (TAP42) involved in the rapamycin-sensitive signal transduction pathway. The human α4 has sequence identities with murine α4 of 83.4% nucleotide and 82.9% amino acid sequence, and a stretch of consensus motifs in the carboxyl terminal is conserved among the related genes of human, mouse, yeast, and rice. The gene is expressed as a 1.4-kb mRNA in the spleen, lymph node, thymus, appendix, peripheral blood leukocytes, bone marrow, fetal liver, heart, brain, placenta, skeletal muscle, kidney, and pancreas. The anti-human α4 antibody detected a 45-kDa protein in human lymphoid cell lines. Moreover, human α4 (IGBP1) gene is located at q13.1–q13.3 on chromosome X.     

Organization, Structure, and Evolution of the Nonadult Rat β-Globin Gene Cluster

The β-globin gene cluster of Wistar rat was extensively cloned and the embryonic genes were mapped and sequenced. Four overlapping λ Dash recombinant clones cover about 31 kb and contain four nonadult β-globin genes, 5′–ε1–γ1–γ2–ψγ3–3′. The ε1 and γ2 are active genes, since their protein products were detected in the fetal stage of the rat (Iwahara et al., J Biochem 119:360–366, 1996). The γ1 locus might be a pseudogene, since the ATA box in the promoter region is mutated to GTA; however, no other defect is observed. The ψγ3 locus is a truncated pseudogene because a 19-base deletion, which causes a shift of the reading frame, is observed between the second nucleotide of the putative codon 68 and codon 76. A sequence comparison suggests that the ψγ3 might be produced by a gene conversion event of the proto-γ-globin gene set. Possible histories of the evolution of rat nonadult β-globin genes are discussed. Received: 6 August 1998 / Accepted: 12 February 1999     

Expression and characterization of a thermostable β-xylosidase from the hyperthermophile, Thermotoga maritima

A thermostable -xylosidase from a hyperthermophilic bacterium, Thermotoga maritima, was over-expressed in Escherichia coli using the T7 polymerase expression system. The expressed -xylosidase was purified in two steps, heat treatment and immobilized metal affinity chromatography, and gave a single band on SDS-PAGE. The maximum activity on p-nitrophenyl -d-xylopyranoside was at 90 °C and pH 6.1. The purified enzyme had a half-life of over 22-min at 95 °C, and retained over 57% of its activity after holding a pH ranging from 5.4 to 8.5 for 1 h at 80 °C. Among all tested substrates, the purified enzyme had specific activities of 275, 50 and 29 U mg^–1 on pNPX, pNPAF, and pNPG, respectively. The apparent Michaelis constant of the -xylosidase was 0.13 mm for pNPX with a V _max of 280 U mg^–1. When the purified -xylosidase was added to xylanase, corncob xylan was hydrolized completely to xylose.     

Variants of the α6 β1 Laminin Receptor in Early Murine Development: Distribution,Molecular Cloning and Chromosomal Localization of the Mouse Integrin α6 Subunit

Laminin (A:B1:B2) is a major component of the first basement membrane to appear in the developing mouse embryo. Its effects on morphogenesis and differentiation are mediated by interaction with cell surface receptors that are members of the integrin family. We have studied the expression of the α₆ subunit of murine α₆β₁ and its ligand, laminin, in preimplantation mouse embryos, embryo outgrowths and in embryonic stem (ES) cells and embryonal carcinoma (EC) cells. The α₆ subunit is present in the oocyte and throughout preimplantation development. Laminin A chain appears later than α₆ and has a more restricted distribution until the late blastocyst stage. α₆β₁ is strongly expressed in ES and EC cells; the levels of mRNA expression are not altered by differentiation. Molecular cloning of cDNA for the murine integrin α₆ subunit from a mammary gland γt11 library showed, as in man, an open reading frame encoding two variants of α₆, α₆ and α_6B. The identity of the α₆ amino acid sequence to that in man and chicken is 93% and 73%, respectively. The gene for murine α₆ was mapped to chromosome 2. While undifferentiated ES and EC cells express only α_6B, α_6A is co-expressed in ES cells after differentiation is induced by retinoic acid. α_6B is also the only variant expressed in blastocyst stage embryos, but when blastocysts have grown out in culture both α_6A and α_6B are expressed reflecting the results in the cell lines. We suggest that the deposition of laminin in the embryo is a receptor-mediated process and that the shift in the expression of the variants, as the inner cell mass forms its first differentiated progeny, reflects a change in functional properties.     

Expression and Localization of Type II Diacylglycerol Kinase Isozymes δ and η in the Developing Mouse Brain

The functions of type II diacylglycerol kinase (DGK) δ and -η in the brain are still unclear. As a first step, we investigated the spatial and temporal expression of DGKδ and -η in the brains of mice. DGKδ2, but not DGKδ1, was highly expressed in layers II–VI of the cerebral cortex; CA–CA3 regions and dentate gyrus of hippocampus; mitral cell, glomerular and granule cell layers of the olfactory bulb; and the granule cell layer in the cerebellum in 1- to 32-week-old mice. DGKδ2 was expressed just after birth, and its expression levels dramatically increased from weeks 1 to 4. A substantial amount of DGKη (η1/η2) was detected in layers II–VI of the cerebral cortex, CA1 and CA2 regions and dentate gyrus of the hippocampus, mitral cell and glomerular layers of the olfactory bulb, and Purkinje cells in the cerebellum of 1- to 32-week-old mice. DGKη2 expression reached maximum levels at P5 and decreased by 4 weeks, whereas DGKη1 increased over the same time frame. These results indicate that the expression patterns of DGK isozymes differ from each other and also from other isozymes, and this suggests that DGKδ and -η play distinct and specific roles in the brain.     

Comparative Analysis of Gene Regulation by the Transcription Factor PPARα between Mouse and Human

Background

Studies in mice have shown that PPARα is an important regulator of hepatic lipid metabolism and the acute phase response. However, little information is available on the role of PPARα in human liver. Here we set out to compare the function of PPARα in mouse and human hepatocytes via analysis of target gene regulation.

Methodology/Principal Findings

Primary hepatocytes from 6 human and 6 mouse donors were treated with PPARα agonist Wy14643 and gene expression profiling was performed using Affymetrix GeneChips followed by a systems biology analysis. Baseline PPARα expression was similar in human and mouse hepatocytes. Depending on species and time of exposure, Wy14643 significantly induced the expression of 362–672 genes. Surprisingly minor overlap was observed between the Wy14643-regulated genes from mouse and human, although more substantial overlap was observed at the pathway level. Xenobiotics metabolism and apolipoprotein synthesis were specifically regulated by PPARα in human hepatocytes, whereas glycolysis-gluconeogenesis was regulated specifically in mouse hepatocytes. Most of the genes commonly regulated in mouse and human were involved in lipid metabolism and many represented known PPARα targets, including CPT1A, HMGCS2, FABP1, ACSL1, and ADFP. Several genes were identified that were specifically induced by PPARα in human (MBL2, ALAS1, CYP1A1, TSKU) or mouse (Fbp2, lgals4, Cd36, Ucp2, Pxmp4). Furthermore, several putative novel PPARα targets were identified that were commonly regulated in both species, including CREB3L3, KLF10, KLF11 and MAP3K8.

Conclusions/Significance

Our results suggest that PPARα activation has a major impact on gene regulation in human hepatocytes. Importantly, the role of PPARα as master regulator of hepatic lipid metabolism is generally well-conserved between mouse and human. Overall, however, PPARα regulates a mostly divergent set of genes in mouse and human hepatocytes.     

Human γ-Aminobutyraldehyde Dehydrogenase (ALDH9): cDNA Sequence, Genomic Organization, Polymorphism, Chromosomal Localization, and Tissue Expression

The cDNA and the gene (ALDH9) for a human aldehyde dehydrogenase isozyme, which has a high activity for oxidation of γ-aminobutyraldehyde and other amino aldehydes, were cloned and characterized. The cDNA has an open reading frame of 1479 bp encoding 493 amino acid residues. The gene is about 45 kb and consists of 10 coding exons interrupted by nine introns. The gene was assigned to chromosome 1q22–q23, using fluorescencein situhybridization. Northern blot hybridization indicated that the size of the mRNA is about 2.4 kb and that the gene is expressed at high levels in adult liver, skeletal muscle, and kidney and low levels in heart, pancreas, lung, and brain. The gene is polymorphic, i.e., C or T at nt 327 and C or G at nt 344.     

Cloning,Sequencing, and Expression of the Gene Encoding an Intracellular β-D-Xylosidase from Streptomyces thermoviolaceus OPC-520

The intracellular β-xylosidase was induced when Streptomyces thermoviolaceus OPC-520 was grown at 50°C in a minimal medium containing xylan or xylooligosaccharides. The 82-kDa protein with β-xylosidase activity was partially purified and its N-terminal amino acid sequence was analyzed. The gene encoding the enzyme was cloned, sequenced, and expressed in Escherichia coli. The bxlA gene consists of a 2,100-bp open reading frame encoding 770 amino acids. The deduced amino acid sequence of the bxlA gene product had significant similarity with β-xylosidases classified into family 3 of glycosyl hydrolases. The bxlA gene was expressed in E. coli, and the recombinant protein was purified to homogeneity. The enzyme was a monomer with a molecular mass of 82 kDa. The purified enzyme showed hydrolytic activity towards only p-nitrophenyl-β-D-xylopyranoside among the synthetic glycosides tested. Thin-layer chromatography analysis showed that the enzyme is an exo-type enzyme that hydrolyze xylooligosaccharides, but had no activity toward xylan. High activity against pNPX occurred in the pH range 6.0-7.0 and temperature range 40-50°C.     

Expression of the Cloned Hemagglutinin–Neuraminidase Gene of the Newcastle Disease Virus in Mouse Myeloma Cells

Vaccination with autologous cancer cells expressing a potent foreign antigen is promising for immunotherapy of tumors. A construct was obtained to transfect cancer cells with the hemagglutinin–neuraminidase (HN) gene of the Newcastle disease virus (NDV). Specific primers were designed, and the HN cDNA was amplified from RNA isolated from the allantoic fluid of NDV-infected embryonated chicken eggs. The amplified fragment was cloned in pCR2.1, sequenced, and recloned in expression vector pCDNA3.1/Zeo(+). The resulting construct was used to transfect mouse myeloma cells SP2/0. Production of HN was checked by ELISA and by a neuraminidase activity assay. Cell agglutination on ice was proposed as a test for surface HN.     

Conformational Analysis of the β-amyloid Peptide Fragment, β(12–28)

Abstract

NMR and CD spectroscopy have been used to examine the conformation of the peptide, β(12–28), (VHHQKLVFFAEDVGSNK) in aqueous and 60% TFE/40% H₂0 solution at pH 2.4. In 60% TFE solution, the peptide is helical as confirmed by the CD spectrum and by the pattern of the NOE cross peaks detected in the NOESY spectrum of the peptide. In aqueous solution, the peptide adopts a more extended and flexible conformation. Broadening of resonances at low temperature, temperature-dependent changes in the chemical shifts of several of the CH_α resonances and the observation of a number of NOE contacts between the hydrophobic side-chain protons of the peptide are indicative of aggregation in aqueous solution. The behavior of β(12–28) in 60% TFE and in aqueous solution are consistent with the overall conformation and aggregation behavior reported for the larger peptide fragment, β(1–28) and the parent β-amyloid peptide.     

Conformational Analysis of the β-amyloid Peptide Fragment, β(12-28)

Abstract NMR and CD spectroscopy have been used to examine the conformation of the peptide, β(12-28), (VHHQKLVFFAEDVGSNK) in aqueous and 60% TFE/40% H(2)0 solution at pH 2.4. In 60% TFE solution, the peptide is helical as confirmed by the CD spectrum and by the pattern of the NOE cross peaks detected in the NOESY spectrum of the peptide. In aqueous solution, the peptide adopts a more extended and flexible conformation. Broadening of resonances at low temperature, temperature-dependent changes in the chemical shifts of several of the CH(α) resonances and the observation of a number of NOE contacts between the hydrophobic side-chain protons of the peptide are indicative of aggregation in aqueous solution. The behavior of β(12-28) in 60% TFE and in aqueous solution are consistent with the overall conformation and aggregation behavior reported for the larger peptide fragment, β(1-28) and the parent β-amyloid peptide.