Molecular cloning and characterization of a novel human gene (ANP32E alias LANPL) from human fetal brain

Leucine-rich acidic nuclear protein (LANP) is a member of the leucine-rich repeats (LRRs) superfamily. Here we report on a human homologue of LANP, encoded by the gene ANP32E (alias LANPL). The gene was cloned and identified during large-scale sequencing analysis of a human fetal brain cDNA library. The human protein shared 70% amino acid identity with rat LANP. According to bioinformatics analysis, ANP32E is located on chromosome 1q22. RT-PCR analysis indicates that ANP32E was expressed in human peripheral blood leukocytes, colon, small intestine, prostate, thymus, spleen, skeletal muscle, liver and kidney.     

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.     

Molecular cloning and characterization of a novel human protein phosphatase,LMW-DSP3

Reversible phosphorylation is recognized to be a major mechanism for the control of intracellular events in eukaryotic cells. From a human fetal brain cDNA library, we isolated a cDNA clone encoding a novel dual specificity protein phosphatase, which showed 88% identity with previously reported mouse LMW-DSP3 at the amino acid level. The deduced protein had a single dual-specificity phosphatase catalytic domain, and lacked a cdc25 homology domain. LMW-DSP3 was expressed in the heart, lung, liver, and pancreas, and the expression level in the pancreas was highest. The LMW-DSP3 gene was located in human chromosome 2q32, and consisted of five exons spanning 21kb of human genomic DNA. LMW-DSP3 fused to GST showed phosphatase activity towards p-nitrophenyl phosphate which was optimal at pH 7.0 and 40 degrees C, and the activity was enhanced by Ca(2+) and Mn(2+). The phosphatase activity of LMW-DSP3 was inhibited by orthovanate. LMW-DSP3 showed phosphatase activity toward oligopeptides containing pSer/Thr and pTyr, indicating that LMW-DSP3 is a protein phosphatase with dual substrate specificity.     

Molecular cloning and characterization of a novel peptidylprolyl isomerase (cyclophilin)-like gene (PPIL3) from human fetal brain.

During the large-scale sequencing analysis of a human fetal brain cDNA library, we isolated two cDNA clones encoding two novel proteins, which show 52% and 72% identity to the cyclophilin isoform 10 of C. elegans, respectively. Sequence analysis revealed these two cDNA clones are two different splicing variants of a novel cyclophilin-like gene (PPIL3). The PPIL3 gene was identified on a completely sequenced BAC (GenBank accession AC005037) from chromosome 2q33 between STS markers stSG2762 (proximal) and SHGC-3074 (distal), oriented toward the telomere. The PPIL3 gene consisted of eight exons spanning more than 18 kb of genomic DNA. RT-PCR analysis indicated that PPIL3 was ubiquitously expressed in adult human tissues.     

Molecular cloning and characterization of a novel Dehydrogenase/reductase (SDR family) member 1 genea from human fetal brain

Short-chain dehydrogenases/reductases (SDR) constitute a large protein family of NAD(P)(H)-dependent oxidoreductase. They are defined by distinct, common sequence motifs and show a wide range of substrate specialisms. By large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a novel human SDR-type dehydrogenase/reductase gene named Dehydrogenase/reductase (SDR family) member 1 (DHRS1). The DHRS1 cDNA is 1411 base pair in length, encoding a 314-amino-acid polypeptide which has a SDR motif. Northern blot reveals two bands, of about 0.9 and 1.4 kb in size. These two forms are expressed in many tissues. The DHRS1 gene is localized on chromosome 14q21.3. It has 9 exons and spans 9.2 kb of the genomic DNA.     

Molecular cloning and characterization of a novel WRKY gene from Brassica chinensis

A new WRKY gene was cloned from Brassica chinensis by rapid amplification of cDNA ends (RACE). The full-length cDNA of BcWRKY was 1175 bp long and contained a 924-bp open reading frame (ORF) encoding a putative W-box-binding protein of 308 amino acids. The predicted BcWRKY protein was found to have a potential bipartite nuclear localization sequence (NLS-PB) in its N-terminal region followed by a WRKY DNA-binding domain. Bioinformatic analysis revealed that BcWRKY resembled other WRKY domain-containing proteins from Arabidopsis thaliana (AtWRKY18), tobacco (WIZZ), parsley (PcWRKY4), and wild oat (ABF2). Expression of the BcWRKY gene could be induced by salicylic acid (SA) and influenced by Pseudomonas syringae pv. tomato strain DC3000 infection and wounding treatment. Our study implies that BcWRKY might have similar functions possessed by other WRKY genes, such as inducing the expression of some defense-related genes and increasing plants’ disease resistance ability. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 5, pp. 816–824. The text was submitted by the authors in English.     

Molecular cloning and characterization of a novel WRKY gene from Brassica chinensis

A new WRKY gene was cloned from Brassica chinensis by rapid amplification of cDNA ends (RACE). The full-length cDNA of BcWRKY was 1175 bp long and contained a 924 bp open reading frame (ORF) encoding a putative W-box-binding protein of 308 amino acids. The predicted BcWRKY protein was found to have a potential bipartite nuclear localization sequence (NLS-BP) in its N-terminal region followed by a WRKY DNA-binding domain. Bioinformatic analysis revealed that BcWRKY resembled other WRKY domain-containing proteins from Arabidopsis (AtWRKY18), tobacco (WIZZ), parsley (PcWRKY4) and wild oat (ABF2). Expression of the BcWRKY gene could be induced by salicylic acid (SA), and influenced by Pseudomonas syringae pv. tomato strain DC3000 infection and wounding treatment. Our study implies that BcWRKY might have similar functions possessed by other WRKY genes such as inducing the expression of some defense-related genes and increasing plant's disease resistance ability.     

Molecular cloning and characterization of a novel stem-specific gene from Camptotheca acuminata

In higher plants, P450s participate in the biosynthesis of many important secondary metabolites. Here we reported for the first time the isolation of a new cytochrome P450 cDNA that expressed in a stem-specific manner from Camptotheca acuminata (designated as CaSS), a native medicinal plant species in China, using RACE-PCR. The full-length cDNA of CaSS was 1735 bp long containing a 1530 bp open reading frame (ORF) encoding a polypeptide of 509 amino acids. Bioinformatic analysis revealed that CASS contained a heme-binding domain PFGXGRRXCX and showed homology to other plant cytochrome P450 monooxygenases and hydroxylases. Southern blotting analysis revealed that there was only one copy of the CaSS present in the genome of Camptotheca acuminata. Northern blotting analysis revealed that CaSS expressed, in a tissuespecific manner, highly in stem and lowly in root, leaf and flower. Our study suggests that CaSS is likely to be involved in the phenylpropanoid pathway.     

Molecular cloning and characterization of a novel calcium channel from rabbit brain.

The complete amino acid sequence of a novel calcium channel (designated BII) from rabbit brain has been deduced by cloning and sequencing the cDNA. The BII calcium channel is structurally more closely related to the BI calcium channel than to the cardiac and skeletal muscle L-type calcium channels. Blot hybridization analysis of RNA from different tissues and from different regions of the brain shows that the BII calcium channel is distributed predominantly in the brain, being abundant in the cerebral cortex, hippocampus and corpus striatum.     

Molecular cloning and characterization of a novel mannose-binding lectin gene from Amorphophallus konjac

A new lectin gene was cloned from Amorphophallus konjac. The full-length cDNA of Amorphophallus konjac agglutinin (aka) was 736 bp and contained a 474 bp open reading frame encoding a 158 amino acid protein. Homology analysis revealed that the lectin from this Araceae species belonged to the superfamily of monocot mannose-binding proteins. Molecular modeling of AKA indicated that the three-dimensional structure of AKA strongly resembles that of the snowdrop lectin. Southern blot analysis of the genomic DNA revealed that aka belonged to a low-copy gene family. Northern blot analysis demonstrated that aka expression was tissue-specific with the strongest expression being found in root.     

Molecular cloning and characterization of a novel human gene containing 4 ankyrin repeat domains

Ankyrin repeat, one of the most important protein motifs, plays a wide variety of roles in protein-protein interactions and in the signal pathways. Via large-scale sequencing, a novel 941-bp gene was isolated from an 18-week old human fetal brain cDNA library. It encodes a putative protein of 158 amino acid residues with four conserved ankyrin repeat domains. It displays a high degree of homology with rat low-density lipoprotein receptor-related protein 2-binding protein (Lrp2bp), and was therefore was named hLrp2bp (human Lrp2bp). The hLrp2bp gene was located in chromosome 4q35 and the conserved ankyrin repeat domains were located between amino acid residues 10 and 116. RT-PCR revealed that hLrp2bp was mainly expressed in the human testis, small intestine, colon and blood leukocytes, and in human pancreatic adenocarcinoma cells. A HEK293 cell was transfected with the ORF of hLrp2bp, and analyses showed that the protein was distributed both in the cytoplasm and nucleus.     

Molecular cloning and characterization of a novel cold-regulated gene from Capsella bursa-pastoris.

A novel cor gene was cloned from Capsella bursa-pastoris (designated Cbcor15b) by RACE-PCR. The full-length cDNA of Cbcor15b was 652bp and contained a 417bp open reading frame (ORF) encoding a 139-amino acid hydrophilic protein. Multiple alignments showed that Cbcor15b had high similarity with other cold-regulated genes from Arabidopsis thaliana (cor15b, cor15a), Brassica napus (bn115, bn19 and bn26) and genes encoding late embryogenesis abundant (LEA) proteins. The predicted CbCOR15B protein was found to have a potential chloroplast signal sequence cleavage site, two cAMP- and cGMP-dependent protein kinase (PKA and PKG) phosphorylation sites. Cold acclimation assay showed that Cbcor15b was relevant to cold acclimation. Our study implies that Cbcor15b might have similar functions possessed by other cor genes in increasing plants' freezing tolerance.     

Molecular cloning and characterization of a novel human kinase gene,PDIK1L

We isolated a 4301-bp cDNA from a human foetal brain cDNA library by high-throughput cDNA sequencing. It encodes a protein of 341 amino acids, which shows 69% identity with the human kinase CLIK1 (AAL99353), which was suggested to be the CLP-36 interacting kinase. Bioinformatics analysis suggests that the putative kinase may interact with PDZ and LIM domain proteins. Therefore the protein and its cDNA were named ’PDLIM1 interacting kinase 1 like’ (PDIK1L; nomenclature approved by the HUGO Gene Nomenclature Committee). Ensembl Genome Browser locatedPDIK1L to human chromosome 1p35.3. It spans about 13.7 kb and consists of four exons and three introns. Multiple-tissue cDNA panel PCR revealed that the gene is expressed widely in human tissues: liver, kidney, pancreas, spleen, thymus and prostate. The protein appears to be localized to the nucleus.