Expression of two related nonstructural proteins of bluetongue virus (BTV) type 10 in insect cells by a recombinant baculovirus: production of polyclonal ascitic fluid and characterization of the gene product in BTV-infected BHK cells.

In vitro translation of bluetongue virus (BTV) double-stranded RNA in the rabbit reticulocyte lysate system has shown segment 10 (S10) to code for two related proteins, NS3 and NS3A. The presence of both products in vivo, however, has remained unconfirmed owing to the very low level of synthesis of the S10 gene product(s) in BTV-infected BHK cells. In the present work, a cDNA copy of BTV type 10 (BTV-10) S10 RNA was inserted into Autographa californica nuclear polyhedrosis baculovirus (AcNPV) in lieu of the 5' coding region of the AcNPV polyhedrin gene. Spodoptera frugiperda cells infected with the recombinant baculovirus synthesized two polypeptides, which were shown to represent NS3 and NS3A by Western blot (immunoblot) and peptide map analysis. Antibodies raised to the expressed NS3 by immunization of mice detected both NS3 and NS3A in BTV-10-infected BHK cells but not in purified BTV-10 virus particles. In contrast to in vitro translation of BTV S10 RNA in which NS3 and NS3A are synthesized in equimolar amounts, NS3 was the principle product both in the baculovirus expression system and in vivo in BTV-infected cells. The results indicate the caution which should be exercised when using the rabbit reticulocyte lysate system to predict the pattern of protein synthesis from a gene with alternative start codons. The expressed NS3 and NS3A proteins reacted strongly with sera from sheep infected with homologous and heterologous BTV serotypes, suggesting that the S10 gene products are highly conserved group-specific antigens.     

Molecular cloning, sequencing, and expression of mouse ferrochelatase

The cDNA encoding mouse ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) was isolated from a mouse erythroleukemia (MEL) cell cDNA library in lambda gt11 expression vector, by immunoscreening with a polyclonal antibody. Two full-length clones containing cDNA inserts of 2.2 and 2.90 kilobases were obtained. These clones have the same entire enzyme coding region, but alternative putative polyadenylation sites in the 3'-noncoding regions. From the deduced primary structure, a putative leader sequence of 53 amino acid residues resulted in a precursor protein of 420 amino acid residues (Mr 47,130) and a mature protein of 367 residues (Mr 41,692). The cDNA allows for the expression of active ferrochelatase by transfected culture cells. RNA blot analysis showed two species of ferrochelatase mRNA consistent with findings of two polyadenylation sites. Both the mRNAs increased by treatment of the MEL cells with dimethyl sulfoxide. The band pattern of the RNA of the mouse liver was the same as that of the MEL cells. Based on these results, we deduce that ferrochelatase in erythroid and hepatic cells can be only of one type.     

Isolation and characterization of cDNA clones encoding ADP-glucose pyrophosphorylase (AGPase) large and small subunits from chickpea (Cicer arietinum L.)

Four cDNA clones encoding two large subunits and two small subunits of the starch regulatory enzyme ADP-glucose pyrophosphorylase (AGPase) were isolated from a chickpea (Cicer arietinum L.) stem cDNA library. DNA sequence and Southern blot analyses of these clones, designated CagpL1, CagpL2 (large subunits) and CagpS1 and CagpS2 (small subunits), revealed that these isoforms represented different AGPase large and small subunits. RNA expression analysis indicated that CagpL1 was expressed strongly in leaves with reduced expression in the stem. No detectable expression was observed in seeds and roots. CagpL2 was expressed moderately in seeds followed by weak expression in leaves, stems and roots. Similar analysis showed that CagpS1 and CagpS2 displayed a spatial expression pattern similar to that observed for CagpL2 with the exception that CagpS1 showed a much higher expression in seeds than CagpS2. The spatial expression patterns of these different AGPase subunit sequences indicate that different AGPase isoforms are used to control starch biosynthesis in different organs during chickpea development.     

马尾松毛虫质型多角体病毒多角体蛋白基因的cDNA克隆及序列分析

通过对马尾松毛虫质型多角体病毒的增殖、纯化,获得一株单一类型的质型多角体病毒。提纯的病毒粒子经SDS－酚抽提,琼脂糖凝胶电泳分离基因组dsRNDA,回收纯化第十片段S10。S10经DMSO变性,逆转录合成cDNA第一链,PCR扩增后,克隆在pGEM-T载体上,对重组子进行限制性内切酶分析及序列测定。结果表明,克隆片段全长763bp,起始密码AUG位于3－5残基,终止密码UGA位于747－749残基。推测DpCPV多角体蛋白基因编码248个氨基酸的多肽,分子量28kD。和家蚕质型多角体病毒（BmCPV）多角体蛋白基因相比较,核苷酸和编码氨基酸序列同源性分别为89.3%和97.6%。     

Cloning and expression of a cDNA encoding mouse indoleamine 2,3-dioxygenase

The depletion of an essential amino acid (aa), tryptophan, caused by interferon-gamma (IFN-gamma)-mediated induction of indoleamine 2,3-dioxygenase (IDO) in mouse allografted tumor cells, has been suggested as a reason for the allograft rejection. To elucidate the mechanism of this IDO induction, attempts were made to isolate cDNA clones encoding mouse IDO. In seven of 25 mouse cell lines, IDO was induced by IFN-gamma, and the highest IDO induction was observed in the case of rectal cancer (CMT-93) cells, which were further stimulated two- to threefold by the simultaneous addition of dibutyryl cyclic AMP (Bt2cAMP). A cDNA library was prepared from poly(A)+ RNA isolated from CMT-93 cells treated with IFN-gamma/Bt2cAMP. The cDNA clones were isolated using the cDNA encoding human IDO as a probe. The mouse IDO cDNA encodes a 407-aa protein with an Mr of 45,639. The deduced aa sequence agreed with partial aa sequences derived from endopeptidase digestion of purified mouse IDO and revealed 61% homology with that of human IDO. Transient expression of the mouse IDO cDNA in COS-7 cells yielded a high level of IDO activity in the cells. Northern hybridization analysis of RNA in CMT-93 cells indicated that IFN-gamma induced the IDO mRNA, and that the level of RNA was increased by simultaneous addition of Bt2cAMP, while Bt2cAMP itself had no effect on mRNA induction.     

Molecular cloning and expression of the murine interleukin-5 receptor

Murine interleukin-5 (IL-5) is known to play an essential role in Ig production of B cells and proliferation and differentiation of eosinophils. Here, we have isolated cDNA clones encoding a murine IL-5 receptor by expression screening of a library prepared from a murine IL-5 dependent early B cell line. A cDNA library was expressed in COS7 cells and screened by panning with the use of anti-IL-5 receptor monoclonal antibodies. The deduced amino acid sequence analysis demonstrates that the receptor is a glycoprotein of 415 amino acids (Mr 45,284), including an N-terminal hydrophobic region (17 amino acids), a glycosylated extracellular domain (322 amino acids), a single transmembrane segment (22 amino acids) and a cytoplasmic tail (54 amino acids). COS7 cells transfected with the cDNA expressed a 60 kd protein that bound IL-5 with a single class of affinity (KD = 2-10 nM). FDC-P1 cells transfected with the cDNA for murine IL-5 receptor showed the expression of IL-5 binding sites with both low (KD = 6 nM) and high affinity (KD = 30 pM) and acquired responsiveness to IL-5 for proliferation, although parental FDC-P1 cells did not show any detectable IL-5 binding. In addition, several cDNA clones encoding soluble forms of the IL-5 receptor were isolated. Northern blot analysis showed that two species of mRNAs (5.0 kb and 5.8 kb) were detected in cell lines that display binding sites for murine IL-5. Homology search for the amino acid sequence of the IL-5 receptor reveals that the IL-5 receptor contains a common motif of a cytokine receptor family that is recently identified.     

A cloned cDNA encoding MAP1 detects a single copy gene in mouse and a brain-abundant RNA whose level decreases during development

Screening of a bacteriophage lambda gt11 cDNA expression library with a polyclonal anti-microtubule associated protein (MAP) antiserum resulted in the isolation of two non-cross-hybridizing sets of cDNA clones. One set was shown to encode MAP2 (Lewis, S. A., A. Villasante, P. Sherline, and N. J. Cowan, 1986, J. Cell Biol., 102:2098-2105). To determine the specificity of the second set, three non-overlapping fragments cloned from the same mRNA molecule via a series of "walking" experiments were separately subcloned into inducible plasmid expression vectors in the appropriate orientation and reading frame. Upon induction and analysis by immunoblotting, two of the fusion proteins synthesized were shown to be immunoreactive with an anti-MAP1-specific antibody, but not with an anti-MAP2-specific antibody. Since these MAP1-specific epitopes are encoded in non-overlapping cDNAs cloned from a single contiguous mRNA, these clones cannot encode polypeptides that contain adventitiously cross-reactive epitopes. Furthermore, these cDNA clones detected an abundant mRNA species of greater than 10 kb in mouse brain, consistent with the coding requirement of a 350,000-D polypeptide and the known abundance of MAP1 in that tissue. The MAP1-specific cDNA probes were used in blot transfer experiments with RNA prepared from brain, liver, kidney, stomach, spleen, and thymus. While detectable quantities of MAP1-specific mRNA were observed in these tissues, the level of MAP1 expression was approximately 500-fold lower than in brain. The levels of both MAP1-specific and MAP2-specific mRNAs decline in the postnatal developing brain; the level of MAP1-specific mRNA also increases slightly in rat PC12 cells upon exposure to nerve growth factor. These surprising results contrast sharply with reported dramatic developmental increases in the amount of MAP1 in brain and in nerve growth factor-induced PC12 cells. The cDNA clones encoding MAP1 detect a single copy sequence in mouse DNA, even under conditions of low stringency that would allow the detection of related but mismatched sequences. The cDNAs cross-hybridize with genomic sequences in rat, human, and chicken DNA, but not with DNA from frog, Drosophila, or sea urchin. These data are discussed in terms of the evolution and possible biological role of MAP1.     

Isolation and characterization of variant cDNAs encoding mouse tyrosinase

Two different cDNA clones encoding mouse tyrosinase (monophenol oxygenase, E.C. 1.14.18.1) were isolated from B16 melanoma cells, and their primary structure was determined. One of the cDNAs consists of 3309 nucleotides with an open reading frame coding for a peptide of 533 amino acids. The other cDNA is approximately 1600 nucleotides long, with a shorter 3'-untranslated region and a deduced in-frame deletion of 77 amino acid residues with respect to the former clone. Neither of these clones is structurally identical to other described mouse tyrosinase cDNAs (1-3). RNA blotting analysis demonstrates that multiple tyrosinase mRNA species are not only present in B16 melanoma, but also in normal skin melanocytes.     

Thionin genes specifically expressed in barley leaves

Complementary-DNA (cDNA) clones encoding thionin were identified as one of the most frequent types of clones in a cDNA library constructed from total polyadenylated RNA from young barley leaf cells. One full-length clone codes for a precursor protein that starts with a signal peptide (28 amino acids) followed by the mature thionin (46 amino acids) and terminated by a long acidic extension (63 amino acids). The amino-acid sequence of the leaf thionin is 52% homologous to thionins from barley endosperm and in the C-terminal extension the homology decreases to 41%. In contrast, the leaf thionin is 72% homologous to viscotoxin from mistletoe leaves. Leaf thionin is coded by a multigene family with an estimated nine to eleven genes and analysis of the cDNA clones showed that at least two extremely homologous genes are expressed. Northern hybridization experiments indicate that the leaf thionin genes are not expressed in endosperm and roots. In leaves, the expression of the thionin genes is strongly repressed by light.Abbreviations cDNA complementary DNA - poly(A)RNA polyadenylated RNA     

Cyclin mRNA and protein expression in recombinant interleukin 2-stimulated cloned murine T lymphocytes

Expression of cyclin, a non-histone nuclear protein, during recombinant interleukin 2 (rIL2)-driven cell-cycle progression of cloned T lymphocytes has been assessed. We found that expression of cyclin protein, as detected by immunofluorescence, is tightly associated with proliferation, and not merely S-phase, of L2 cells stimulated with rIL2. Cyclin immunofluorescence was detected in all cell-cycle phases (G1/S/G2/M, as detected by flow cytometry) of proliferating L2 cells. Accumulation of cyclin mRNA levels was induced as early as 1 h after stimulation, was maximal at 25-49 h, and remained elevated throughout stimulation, as detected by Northern blot analysis. A cDNA-encoding murine cyclin was cloned from a cDNA library prepared from IL2-stimulated cloned T cells. The sequence of the 5' end of the murine cyclin cDNA was determined and found to be 88% and 82% similar to the sequences of cDNA clones encoding rat and human cyclin, respectively. The present studies demonstrate that cyclin protein and mRNA accumulation are highly regulated during IL2-induced proliferation of a cloned T cell. These data provide a framework for addressing the molecular mechanisms regulating cyclin gene expression during cellular proliferation.     

Determination of the Nucleotide Sequence of Bombyx mori Cytoplasmic Polyhedrosis Virus Segment 9 and Its Expression in BmN4 Cells

Cloning and sequencing of segment 9 of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) strains H and I were performed. The segment consisted of 1,186 bp harboring 5′ and 3′ noncoding regions and an open reading frame from positions 75 to 1037, encoding a protein with 320 amino acids, termed NS5. Comparison of the nucleotide sequences of NS5 for the two strains indicated 37 point differences resulting in only six amino acid replacements. Homology search showed that NS5 has localized similarities to human poliovirus RNA-dependent RNA polymerase and human rotavirus NS26. By Western blot analysis, NS5 was found in BmCPV-infected midgut cells, but not in polyhedra or virus virions, and was mainly detectable in the nucleus in BmCPV-infected BmN4 cells. Immunoblot analysis with anti-NS5 and antipolyhedrin antibodies displayed marked differences in the period of expression of NS5 and polyhedrin: the polyhedrin molecule was first detected 2 or 3 days after infection with BmCPV, whereas the expression of NS5 was initiated within a few hours. In addition, the level of polyhedrin increased as the infection developed, whereas the amount of NS5 remained essentially constant. When segment 9 was expressed with a baculovirus expression system, the resulting NS5 protein possessed the ability to bind to the double-stranded RNA genome. These results suggest that NS5 is expressed in early stages of infection and contributes to regulation of genomic RNA function.     

Identification of differentially expressed genes in Con A-activated carp (Cyprinus carpio L.) leucocytes

A cDNA library was constructed from the message RNA (mRNA) obtained from Con A-induced head kidney (HK) leucocytes of carp (Cyprinus carpio L.). Differential screening of the cDNA was carried out by hybridization against the total cDNA probes from normal, Con A-uninduced HK leucocytes or Con A-induced HK leucocytes of carp. The differential expression patterns of certain cDNA clones were confirmed by Southern-blot and Northern-blot analysis. Single-pass of the sequencing analysis and homology search in Genbank (EMBL) revealed those differentially expressed cDNA clones encode for cytochrome c oxidase sub-unit II and III (COII and COIII), elongation factor-1 beta (EF-1 beta), bleomycin hydrolase (BH), heat shock cognate protein 70 (HSC70) and 16S ribosomal RNA (16S rRNA).     

Cytokine-enhanced expression of glycoprotein Ib alpha in human endothelium

Platelet glycoprotein Ib is a major platelet membrane protein composed of two disulfide-linked chains, termed the alpha and beta chains. The larger alpha chain (GpIb alpha), a platelet receptor for von Willebrand factor, plays a major role in mediating platelet adhesion to the subendothelium. Our laboratories have previously reported synthesis of a protein in human endothelial cells that is immunoprecipitated with polyclonal and monoclonal antibodies to platelet GpIb alpha. Lopez et al. (Lopez, J. A., Chung, D. W., Fujikawa, K., Hagan, F. S., Papayannopoulou, T., and Roth, G. J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5615-5619) have reported the cloning of GpIb alpha from a human erythroleukemia (HEL) cell cDNA library. Using this clone as probe, we have isolated two partial GpIb alpha clones from a human umbilical vein endothelial cell lambda gt11 cDNA library. These clones were localized within HEL-derived GpIb alpha cDNA by sequence and restriction enzyme analysis. Additionally, they detected the same message species in HEL and tonsilar RNA that was detected with the HEL GpIb alpha cDNA. Low level GpIb alpha mRNA expression was detected in cultured human umbilical vein endothelial cells, which was increased by treatment of the cells with tumor necrosis factor-alpha. This effect was enhanced by pretreatment with interferon-gamma. Additionally, localization of GpIb alpha in endothelium of fresh tonsilar tissue was demonstrated by immunohistochemistry and in situ hybridization. GpIb alpha may play a role in mediating platelet or other effector cell adhesion to activated endothelium.     

Structure, inheritance, and expression of hybrid poplar (Populus trichocarpa x Populus deltoides) phenylalanine ammonia-lyase genes.

A heterologous probe encoding phenylalanine ammonia-lyase (PAL) was used to identify PAL clones in cDNA libraries made with RNA from young leaf tissue of two Populus deltoides x P. trichocarpa F1 hybrid clones. Sequence analysis of a 2.4-kb cDNA confirmed its identity as a full-length PAl clone. The predicted amino acid sequence is conserved in comparison with that of PAL genes from several other plants. Southern blot analysis of popular genomic DNA from parental and hybrid individuals, restriction site polymorphism in PAL cDNA clones, and sequence heterogeneity in the 3' ends of several cDNA clones suggested that PAL is encoded by at least two genes that can be distinguished by HindIII restriction site polymorphisms. Clones containing each type of PAL gene were isolated from a poplar genomic library. Analysis of the segregation of PAL-specific HindIII restriction fragment-length polymorphisms demonstrated the existence of two independently segregating PAL loci, one of which was mapped to a linkage group of the poplar genetic map. Developmentally regulated PAL expression in poplar was analyzed using RNA blots. Highest expression was observed in young stems, apical buds, and young leaves. Expression was lower in older stems and undetectable in mature leaves. Cellular localization of PAL expression by in situ hybridization showed very high levels of expression in subepidermal cells of leaves early during leaf development. In stems and petioles, expression was associated with subepidermal cells and vascular tissues.     

Isolation, characterization, and recombinant expression of multiple serpins from the cat flea, Ctenocephalides felis

Several clones encoding serine protease inhibitors were isolated from larval and adult flea cDNA expression libraries by immunoscreening and PCR amplification. Each cDNA contained an open reading frame encoding a protein of approximately 45 kDa, which had significant sequence similarity with the serpin family of serine protease inhibitors. The thirteen cDNA clones isolated to date encode serpin proteins, which share a primary structure that includes a nearly identical constant region of about 360 amino acids, followed by a C-terminal variable region of about 40-60 amino acids. The variable C-terminal sequences encode most of the reactive site loop (RSL) and are generated by mutually exclusive alternative exon splicing, which may confer unique protease selectivity to each serpin. Utilization of an alternative exon splicing mechanism has been verified by sequence analysis of a flea serpin genomic clone and adjacent genomic sequences. RNA expression patterns of the cloned genes have been examined by Northern blot analysis using variable region-specific probes. Several putative serpins have been overexpressed using the cDNA clones in Escherichia coli and baculovirus expression systems. Two purified baculovirus-expressed recombinant proteins have N-terminal amino acid sequences identical to the respective purified native mature flea serpins indicating that appropriate N-terminal processing occurred in the virus-infected insect cells.     

Osteonectin mRNA: distribution in normal and transformed cells.

Overlapping cDNA clones encoding bovine osteonectin were isolated from a lambda gt11 expression library constructed from bovine bone cell mRNA. The longest clone, lambda On 17 (insert size 2.0 kb) was studied in detail. The clone was shown to encode osteonectin by hybrid select translation experiments and by DNA sequence analysis. Northern analysis of bone cell RNA showed the length of the osteonectin mRNA to be 2.0 kb. Osteonectin message was found in bone but not in soft tissue (liver and brain) preparations consistent with the distribution of the protein in these tissues. On the other hand, osteonectin message was observed in tendon, a tissue in which little or no osteonectin protein is found in vivo. Hybridization of osteonectin cDNA was detected in cells from a number of species including human, rat, mouse and chick. The level of osteonectin mRNA was drastically decreased in chick embryo fibroblasts transformed by Rous sarcoma virus.