Structure and expression of a gene from Arabidopsis thaliana encoding a protein related to SNF1 protein kinase

The AKin10 gene from Arabidopsis thaliana encoding a putative Ser/Thr protein kinase (PK) has been isolated and characterized. The AKin10-encoding gene is located on a genomic 5.4-kb BamHI fragment and contains ten introns, one being located in the 5' untranslated region. The deduced amino acid sequence of AKin10 is 65% identical over the catalytic domain to the yeast PK (SNF1). SNF1 is essential for the derepression of many glucose-repressible genes, including Suc2 which encodes invertase. Southern blot hybridization experiments suggested the presence of one copy of the gene per haploid genome of A. thaliana. Northern hybridization experiments indicated that this gene is expressed in roots, shoots and leaves. AKin10 may play an important role in a signal transduction cascade regulating gene expression and carbohydrate metabolism in higher plants.     

Construction of yeast artificial chromosome (YAC) clone banks covering three genome equivalents and isolation of YACs containing the human gene encoding tumor necrosis factor receptor β

Yeast artificial chromosome (YAC) banks covering in total about three haploid genome equivalents were constructed using a human Epstein-Barr-virus-transformed B lymphocytic cell line. Two clone banks were made: 20 000 clones with average inserts of 350 kb in the pYAC4 vector and 9850 clones with average inserts of 180 kb using vectors pJS89 and pJS91. Direct comparison of pYAC4 with pJS89 and pJS91 showed pYAC4 to be the most suitable cloning vector. Two partial banks with average insert sizes of 220 kb for human endothelial cell DNA and epithelial HEp2 cell DNA were also constructed, each covering 10% of the haploid genome. A rapid, three-step PCR screening procedure for isolation of individual YAC clones was developed and used to identify two clones encoding TNF-Rβ. These clones cover about 200 kb and have 170 kb in common. TNF-Rβ is 9.3 kb long and contains two introns within the protein-coding sequence.     

cDNA cloning of the CEPUS, a secreted type of neural glycoprotein belonging to the immunoglobulin-like opioid binding cell adhesion molecule (OBCAM) subfamily.

GP55 is a family of glycoproteins distributed predominantly in the nervous system, and its previously characterized members, including the GP55A (EMBL Y08170) and E19S (EMBL Y08171) reveal a typical glycosyl phosphatidyl inositol (GPI)-anchored pattern for membrane proteins. CEPUS identified in this study appeared to represent the third member of GP55. This 3.2 kb long complete cDNA clone from the chicken brain exhibited 3 Ig-like domains. The open reading frame of CEPUS contains 313 amino acids, which can encode a 31.7 kDa core protein (pI 5.75) for the mature form. The signal peptide cleavage site was predicted at Gln25. The structural features of the CEPUS cDNA sequence represented a soluble counterpart to the recently identified cerebellar Purkinje cell specific antigen, CEPU-1. The sequence difference between CEPU-1 and CEPUS was only found in the C-terminus in which the CEPUS lacked the GPI-anchored binding site. It displays significant sequence homology to GP55-related molecules, including OBCAM, GP55A, E19S/LAMP, neurotrimin, and CEPU-1, which are all membrane attached types. The absence of the hydrophobic tail sequence in CEPUS may, therefore, suggest that CEPUS would represent the first identified secreted member in this group of genes. We defined that this molecule forms the opioid-binding cell adhesion molecule (OBCAM) subfamily in the molecular phylogeny. Structurally, these molecules represent acidic proteins (pI 5.47-6.09). Six cysteins, as well as 5 Asn-linked potential glycosylation sites were evolutionary-conserved, suggesting that this OBCAM subfamily resembles immunoglobulin-like and highly glycosylated molecules. The presence of CEPUS would probably suggest to us that the spatial/local expression of the CEPU gene may provide a favorable route for migrating CEPU-positive population of neurons to generate a neuron-specific guidance in developing neurons in vivo.     

Establishment of okadaic acid resistant cell clones using a cDNA expression library.

The mechanism whereby the universal apoptogen and serine/threonine phosphatase inhibitor okadaic acid (OA) kills cells, is still unclear. To create a novel tool for probing of OA action, fibroblasts were selected for OA-resistance after infection with a retroviral Jurkat T-cell cDNA expression library. Twenty-one clones were selected. Two of these (OAR1, OAR2) were studied in detail. OAR1 and 2 had each a retrovirally introduced short cDNA, corresponding to a human gene (oar1 and oar2, respectively) with unknown function. Reintroduction of oar1 or oar2 cDNA into wild-type cells reproduced the OA-resistant phenotype. OAR1 and 2 were cross-resistant to other phosphatase inhibitors (calyculin A, cantharidin), but not to staurosporine or microinjected Cytochrome c, thus, indicating a disturbance in a limited number of death pathways, upstream or independent of apaf-1/caspases-3/9. The action of OA involved caspase-dependent and caspase-independent components. Both components were less efficient in OAR1 and 2, than in wild-type cells. Subtle differences existed between OA-induced phosphoprotein patterns in wild-type cells, OAR1, and OAR2, indicating that a narrow selection of protein phosphorylation events had been targeted. We propose that the clones have defects in a hitherto non-elucidated signal pathway linking OA-induced protein phosphorylation to initiation of a death execution pathway provided with a caspase-dependent amplification loop. The novel OA-resistant cell clones will be used to elucidate the significance for apoptosis of oar1 and 2, their link to altered protein phosphorylation, and the potential link of the latter to initiation of apoptosis.     

生长抑制激素基因在大肠杆菌中的表达

从pSom5中提纯生长抑制激素基因片段,在体外与大肠杆菌pBD_2质粒DNA重组连接,转化受体细胞D_2A_1,获得7株工程菌,在IPTG诱导下进行表达。采用β-半乳糖苷酶底物亲和层析法纯化,得到一纯净的分子量为50000道尔顿左右的蛋白质。对此蛋白质及其经CNBr化学裂解的产物进行放射免疫分析,证实化学合成的生长抑制激素基因已在大肠杆菌D_2,A_1中成功地表达,每升培养基中获得生长抑制激素41.69mg,占菌体总蛋白质的0.71%。     

Characterization of N-glycans from mouse brain neural cell adhesion molecule.

The N-glycosylation pattern of the neural cell adhesion molecule (NCAM), isolated from brains of newborn mice, has been analyzed. Following digestion with trypsin, generated glycopeptides were fractionated by serial immunoaffinity chromatography using immobilized monoclonal antibodies specifically recognizing polysialic acid (PSA) units or the HNK1-carbohydrate epitope. Subsequent analyses of the resulting (glyco)peptides by Edman degradation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) revealed polysialylated glycans to be exclusively linked to glycosylation sites 5 (Asn(431)) and 6 (Asn(460)), whereas glycans carrying the HNK1-epitope could be assigned to sites 2 (Asn(297)), 5, 6, and, to a lesser extent, site 3 (Asn(329)). PSA-, HNK1-, and non-PSA/HNK1-glycan fractions were characterized by carbohydrate constituent and methylation analyses as well as MALDI-TOF-MS in conjunction with chromatographic fractionation techniques. The results revealed that the core structures of PSA-glycans represented predominantly fucosylated, partially sulfated 2,6-branched isomers of triantennary as well as tetraantennary complex-type glycans, whereas carbohydrate chains bearing the HNK1-epitope were dominated by diantennary species carrying in part bisecting GlcNAc residues. Non-PSA/HNK1-glycans exhibited a highly heterogeneous pattern of partially truncated, mostly diantennary structures being characterized by the presence of additional fucose, bisecting GlcNAc and/or sulfate residues. In conclusion, our results revealed that the glycosylation pattern of murine NCAM displays high structural and regional selectivity, which might play an important role in controlling the biological activities of this molecule.     

Molecular cloning and characterization of a novel human hydroxysteroid dehydrogenase-like 2 (HSDL2) cDNA from fetal brain

Hydroxysteroid dehydrogenases (HSDs) are responsible for the biosynthesis of steroid hormones and play a crucial role in mammalian physiology and development. By large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a novel human hydroxysteroid dehydrogenase-like cDNA (HSDL2). This cDNA is 3211 bp in length, encoding a 418–amino-acid polypeptide, which contains a typical motif for NAD(P)⁺-binding (TGxxxGxG), an SDR active site motif (S-Y-K) and a sterol carrier protein domain. HSDL2 shows high similarity with the homologues in the mouse and fruit fly. The HSDL2 gene is mapped to chromosome 9q32 and contains 11 exons. RT-PCR analysis shows that the HSDL2 gene is widely expressed in human tissues and the expression levels in liver, kidney, prostate, testis, and ovary are relatively high.     

Polymorphic (AAT)n trinucleotide repeats derived from a human brain cDNA library

Seven cDNA fragments containing polymorphic (AAT)n trinucleotide repeats were isolated from a human brain cDNA library and mapped by linkage to specific loci. These repeats may serve as gene markers or as candidates for diseases caused by expansion mutation.     

Cloning of the cDNA encoding neural adhesion molecule F3 from bovine brain

We cloned a bovine cDNA encoding the neural adhesion molecule F3 and analyzed its nucleotide sequence. The coding region consisted of 3054 bp encoding 1018 amino acid (aa) residues. The M_r calculated from the deduced as sequence was 113 383. Bovine F3 had 93, 94 and 77% as identity with the mouse, human and chicken homologs, respectively. Bovine F3, similar to those of chicken and human, was devoid of two as residues (Ile-Thr) in the sixth immunoglobulin type C2-like domain, as compared with the mouse homolog. Parts of bovine F3 protein were overproduced in Escherichia coli. The antibodies raised against the recombinant proteins in rabbits reacted specifically with F3. F3 protein was detected in cerebellum, cerebrum and spinal cord in Western blot analysis.     

Comparative characterization of a temperature responsive gene (lactate dehydrogenase-B,ldh-b) in two congeneric tropical fish,Lates calcarifer and Lates niloticus

The characterization of candidate loci is a critical step in obtaining insight into adaptation and acclimation of organisms. In this study of two non-model tropical (to sub-tropical) congeneric perciformes (Lates calcarifer and Lates niloticus) we characterized both coding and non-coding regions of lactate dehydrogenase-B (ldh-b), a locus which exhibits temperature-adaptive differences among temperate and sub-tropical populations of the North American killifish Fundulus heteroclitus. Ldh-b was 5,004 and 3,527 bp in length in L. calcarifer and L. niloticus, respectively, with coding regions comprising 1,005 bp in both species. A high level of sequence homology existed between species for both coding and non-coding regions of ldh-b (> 97% homology), corresponding to a 98.5% amino acid sequence homology. All six known functional sites within the encoded protein sequence (LDH-B) were conserved between the two Lates species. Ten simple sequence repeat (SSR) motifs (mono-, di-, tri- and tetranucleotide) and thirty putative microRNA elements (miRNAs) were identified within introns 1, 2, 5 and 6 of both Lates species. Five single nucleotide polymorphisms (SNPs) were also identified within miRNA containing intron regions. Such SNPs are implicated in several complex human conditions and/or diseases (as demonstrated by extensive genome-wide association studies). This novel characterization serves as a platform to further examine how non-model species may respond to changes in their native temperatures, which are expected to increase by up to 6°C over the next century.     

Expression of human dihydrofolate reductase cDNA and its induction by chloramphenicol in Bacillus subtilis

A bifunctional plasmid (pMP358) able to replicate and to express cloned human dihydrofolate reductase cDNA (cDHFR) in both Escherichia coli and Bacillus subtilis was constructed. The expression of cDHFR in B. subtilis was the result of a deletion that placed the cDNA fragment under the control of the chloramphenicol acetyltransferase (CAT) gene promoter of Staphylococcus aureus plasmid pC194. By sequence analysis of plasmid pMP358, we observed a gene fusion occurring between the cDHFR and the 32nd codon of the CAT gene. We report that such a “hybrid” gene is able to direct the synthesis of a 25-kDal “hybrid” protein, which was found to be inducible by supplementing B. subtilis cells with sublethal doses of chloramphenicol.     

The cDNA encoding Xenopus laevis heat-shock factor 1 (XHSF1): nucleotide and deduced amino-acid sequences, and properties of the encoded protein

We have isolated a cDNA encoding Xenopus laevis (Xl) heat-shock factor 1 (XHSF1). XHSF1, translated from the mRNA synthesized in vitro, will bind specifically to the X1 hsp70 promoter (hsp70). Microinjection of XHSF1 mRNA into Xl oocytes leads to synthesis of XHSF1 which accumulates in the nucleus and selectively activates Xl phsp70p activity at 18°C.     

The identification of nuclear and mitochondrial genes by sequencing randomly chosen clones from a marsupial mammary gland cDNA library

To increase the number of genes that can be mapped to the genome of the tammar wallaby (Macropus eugenii), we sequenced 100 randomly chosen clones from a mammary gland cDNA library. Provisional identifications were made of seven nuclear genes and one mitochondrial gene encoding two caseins, -galactosidase, acetyl-coenzyme A synthetase, lipoprotein lipase, inorganic pyrophosphatase, an ATP-dependent RNA helicase, and cytochromec oxidase I. Highly conserved genes, such as that encoding acetyl-coenzyme A synthetase, were easily identified even from cross-kingdom matches. Genes which are highly divergent, however, such as those encoding themature casein peptides, could not be aligned with homologues in the databases. Even in an organ where there is high mRNA species redundancy, the sequence characterization of expressed sequence tags provides a rapid means of gene identification for mapping purposes.