Liu Wang

Identification of a novel human doublecortin-domain-containing gene ( DCDC1 ) expressed mainly in testis

AbstractMutations in the X-linked gene doublecortex (DCX) result in lissencephaly in males or subcortical laminar heterotopia (double cortex) in females. Recently, an evolutionarily conserved doublecortin (DC) domain important for microtubule binding and microtubule polymerization was defined according to detailed sequence analysis of DCX and DCX-like proteins. Subsequently we cloned a novel human cDNA that contained a DC domain during large-scale DNA sequencing of the human fetal brain cDNA library, and termed it doublecortin-domain-containing 1 (DCDC1). According to a search against the human genome database, DCDC1 was mapped to 11p13. Expression analysis showed that DCDC1 was mainly expressed in adult testis. Furthermore, the expression level of DCDC1 in fetal brain was much higher than in adult brain.

Molecular cloning and characterization of a novel dual-specificity phosphatase18 gene from human fetal brain.

Dual-specificity protein phosphatases (DSPs), a new family of protein tyrosine phosphatases (PTPs), are characterized by the ability to dephosphorylate both phospho-tyrosyl and phospho-seryl/threonyl residues. It has been known that most of the enzymes play important roles in the regulation of mitogenic signal transduction and control the cell cycle in response to extracellular stimuli. In this study, a novel human DSP gene named Dual-specificity Phosphatase18 (DUSP18) was isolated by large-scale sequencing analysis of a human fetal brain cDNA library. DUSP18 is localized at Chromosome 22 q12.1. Its cDNA is 2450 base pairs in length, encoding a 188-amino acid polypeptide in which a DSP motif but not a CH2 domain is included. RT-PCR revealed that the DUSP18 was widely expressed in different tissues. GST-DUSP18 fusion protein showed distinctive phosphatase activity toward p-nitrophenyl phosphate (pNPP), as well as oligopeptides containing pThr and pTyr, indicating that DUSP18 is a protein phosphatase with dual substrate specificity. The optimal condition for the reaction was pH 6.0 and 55 degrees C. Addition of Mn(2+) ions was able to enhance the enzyme activity while the activity was strongly inhibited by iodoaretic acid. Mutations in selected sites showed the importance of Asp-73, Cys-104, Arg-110 and Ser-111 in phosphatase activity of DUSP18.

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.

Identification of a novel human DDX40 gene, a new member of the DEAH-box protein family

AbstractThe DExH/D-box superfamily of RNA helicases seems to play key roles during RNA metabolism, such as pre-mRNA splicing, ribosome biogenesis, and others. We have cloned a new gene of the DEAH-box protein subgroup, designated DDX40 (DEAD/H-box polypeptide 40 gene). DDX40 contains 3656 nucleotides and codes for a putative 779-amino-acid protein. Sequence analysis of the cDNA product revealed that it contained a DEAH (Asp-Glu-Ala-His) sequence motif and other conserved motifs. The DDX40 protein shared 53% and 43% amino acid identity with human DDX8 and yeast Drh1, respectively, in the conserved region. Northern blot analysis showed that DDX40 was expressed ubiquitously in the eight tissues examined, implying a general physiological function of the protein. We speculate that, like other members of the DExH/D-box superfamily, DDX40 may play roles in pre-mRNA splicing, ribosome biogenesis and other RNA processing functions.

Cloning and characterization of a novel human homolog* of mouse U26, a putative PQQ-dependent AAS dehydrogenase

Mouse U26 has been defined as a 2-aminoadipic 6-semialdehyde dehydrogenase. It was speculated to be a PQQ-dependent AAS dehydrogenase due to the research of demonstrating PQQ as a new B vitamin. We isolated a novel human cDNA from the human fetal brain cDNA library we constructed. Its deduced protein was most related to mouse U26. Thus, we termed it human U26. This putative protein contains an AMP-binding domain, a Phosphopantetheine-binding domain and six PQQ-binding motifs. Human U26 mRNA is ubiquitously expressed in adult tissues and is highly expressed in colon adenocarcinoma (CX-1) and colon adenocarcinoma (GI-112) cell lines. Further study should be made to clarify the precise function of human U26.

Cloning and characterization of a novel human STAR domain containing cDNA KHDRBS2.

KHDRBS2, KH domain containing, RNA binding, signal transduction associated 2, is an RNA-binding protein that is tyrosine phosphorylated by Src during mitosis. It contains a KH domain,which is embedded in a larger conserved domain called the STAR domain. This protein has a 99% sequence identity with rat SLM-1 (the Sam68-like mammalian protein 1) and 98% sequence identity with mouse SLM-1 in its STAR domain. KHDRBS2 has the characteristic Sam68 SH2 and SH3 domain binding sites. RT-PCR analysis showed its transcript is ubiquitously expressed. The characterization of KHDRBS2 indicates it may link tyrosine kinase signaling cascades with some aspect of RNA metabolism.

Characterization of a novel splicing variant of KLHL5*, a member of the kelch protein family

The kelch-repeat protein family is a recently found new kind of actin-binding protein. It is characterized by tandemly arranged motifs of about 50 amino acids [7]. Previous study showed that most members of the kelch-repeat family were cytoskeletal proteins implicated in various cellular processes, such as actin cytoskeleton interaction, cytoplasmic sequestration of transcription factors and cell morphology [1]. And some of the family members play important roles in tissue development, such as human ENC-1, NRP/B, etc. Another characteristic of the kelch family is that most members have another conserved BTB domain at the extreme amino terminus. The BTB domain is also found at the N-terminus of 5–10% of zinc-finger transcription factor types and is a conserved protein-protein interaction motif [15]. During the large-scale sequencing analysis of a human fetal brain cDNA library we found a novel kelch-like protein gene 5, KLHL5[11], KLHL5 has high identity with Drosophila kelch protein and many other family members. Here we report a novel splicing variants of KLHL5, named KLHL5b and the expression pattern of KLHL5b in many tissues.

Molecular Cloning and Characterization of SGT1.2, a Novel Splice Variant of Homo sapiens SGT1

SGT1.2, a novel splice variant of Homo sapiens SGT1 was isolated from a human fetal brain cDNA library. This cDNA is 1404 bp and contains an open reading frame from 68 to 1165 encoding a putative protein of 365 amino acids (SGT1.2) that share 90% identities to Homo sapiens SGT1, suppressor of G2 allele of SKP1 at protein level. RPS-BLAST searching revealed that SGT1.2 have a TPR domain, a p23 domain, a SGS domain and a CS domain. According to the search of the human genome database, SGT1.2 was mapped to human chromosome 13q14.13. Reversed transcription-polymerase chain reaction analysis indicated that it widely expressed in human adult tissues.

Cloning and expression of a novel human C5orf12 gene, a member of the TMS_TDE family

We report here cloning and characterization of a novel human gene, termed C5orf12, which is a putative membrane protein belonging to the TMS_TDE family. The cDNA encodes 42 animo acid with a putative molecular weight of about 47 KDa. Secondary structure prediction showed that C5orf12 contained 10 putative transmembrane helices, which has high identity with other family members. We performed RT-PCR to examine its expression pattern. The result showed that C5orf12 was highly expressed in placenta, skeletal muscle, spleen, thymus, testis and peripheral leukocyte while expressed weakly in heart and liver. C5orf12 has high identity with the rat TPO1, so we speculate that C5orf12 may also have a role in the brain development.

Cloning and sequence analysis of the human cDNA encoding the synaptoporin (Δ), a highly conservative synaptic vesicle protein

Synaptoporin is a membrane protein of synaptic vesicles, which belongs to the synaptophysin family. It was first isolated from rat brain. Here we report the cloning and characterization of the human cDNA that encodes the human homologue of synaptoporin. It has two splicing variants, which is in accordance with its ortholog in chicken. Its deduced amino acid sequence displays 97% and 81% identity with the synaptoporin of rat and chicken, respectively. This gene was mapped to the chromosome 3p14.3 by matching against the Human Genome Sequence Database. 16-tissue RT-PCR analysis shows that human synaptoporin is specifically expressed in the brain.