Huarui Zheng

Cloning and Analysis of Human Apg16L

Autophagy is an intracellular bulk degradation system, which delivers cytoplasmic components to the lysosome/vacuole. In yeast and mammalian cells, the Apg12-Apg5 conjugate, together with Apg16, form a multimeric complex, which plays an essential role in autopihageosome formation. By large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a cDNA encoding a putative protein with 607 amino acid residues, which shows 90% identity and 93% similarity to mouse Apg16L. This protein, designated human Apg16L, contains a coiled-coil domain and a motif with seven WD repeats, which are also shared by mouse Apg16L. Database searching revealed that Apg16L is mapped to chromosome 2q37.1 and there exist at least four splice variants.

Cloning and Identification of a Novel Human Gene PDLIM5, a Homolog of AD-associated Neuronal Thread Protein (AD7c-NTP)

A novel human gene cDNA was successfully cloned from the human fetal brain cDNA library constructed by our lab, and this gene was termed PDLIM5 after acquiring the agreement of HUGO. BLASTX searching revealed that the hypothetical protein is a homolog of AD-associated neuronal thread protein (AD7c-NTP), which is over-expressed in Alzheimer disease (AD) beginning early in the course of disease, and over-expression of the AD7c-NTP gene would cause neuritic sprouting and cell death. SMART analysis showed that both our predicted protein and AD7c-NTP comprise BCL domain (only contains BH1 and BH2 regions). RT-PCR experiment revealed that the expression level of PDLIM5 in brain, skeletal muscle, prostate, colon and leukocyte is obviously higher than that in other tissues.

Molecular cloning and characterization of a novel human putative transmembrane protein homologous to mouse sideroflexin associated with sideroblastic anemia.

Sideroflexin1 (Sfxn1), the prototype of a novel family of evolutionarily conserved proteins present in eukaryotes, has been found mutated in mice with siderocytic anemia. It is speculated that this protein facilitates the transport of a component required for iron utilization into mitochondrial. During the large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a cDNA encoding a novel sideroflexin protein (SFXN4), which showed 59% identity and 71% similarity to mouse sideroflexin4. According to the search of the human genome database, SFXN4 gene is mapped to chromosome 10q25-26 and spans more than 24.7 kb of the genomic DNA. It is 1428 base pair in length and the putative protein contains 305 amino acids with a conserved predicted five-transmembrane-domains structure. RT-PCR result shows that the SFXN4 gene is expressed in many tissues.

A novel splice variant of human gene NPL, mainly expressed in human liver, kidney and peripheral blood leukocyte.

From the human fetal brain cDNA library constructed by our lab, a novel variant cDNA of a human gene was successfully cloned and identified. Because the gene has been named N-acetylneuraminate pyruvate lyase (NPL), accordingly we term our splice variant NPL_v2. The cDNA of NPL_v2 has a full-length open reading frame (ORF) from the nucleotide position 320 to 1225 that encodes a protein comprising 301 amino acids. SMART analysis showed that our hypothetical protein has one dihydrodipicolinate synthase (DHDPS) domain. Phosphorylation analysis of the deduced protein show that there are five phosphorylation sites including three “serine” and two “threonine” at the region that are not found in other splice variant. RT-PCR experiment revealed that our splice variant of the gene is mainly expressed in human placenta, liver, kidney, pancreas, spleen, thymus, ovary, small intestine and peripheral blood leukocyte.

A novel splice variant of human XRN2 gene is mainly expressed in blood leukocyte.

The XRN2 gene (XRN2a) is the human homologue of the Saccharomyces cerevisiae RAT1 gene, which encodes a nuclear 5′→3′ exoribonuclease, and is essential for RNA metabolism and cell viability. Xrn2p/Rat1p, product of XRN2/RAT1 gene, functions in the mRNA degradation and processing of rRNAs and small nucleolar RNAs (snoRNAs) in the nucleus. Here we describe the cloning and characterization of a novel splice variant of the human XRN2 gene (XRN2b). The 3271-bp cDNA encodes a putative protein with 907 amino acid residues, which shares high homology with mouse DHM1 protein. RT-PCR analysis showed that XRN2b was mainly expressed in blood leukocyte tissue, while XRN2a was detected in several human tissues and in human tumor tissues.

Molecular Cloning and Characterization of a Novel Human C4orf13 Gene, Tentatively a Member of the Sodium Bile Acid Cotransporter Family

By large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a novel human cDNA (C4orf13). This cDNA is 2706 bp in length, encoding a 340-amino-acid polypeptide that contains a typical SBF (sodium bile acid cotransporter family) domain and ten possible transmembrane segments. The putative protein C4orf13 shows high similarity with its orthologs in Mus musculus and Xenopus laevis. Human C4orf13 is mapped to chromosome 4q31.2 and contains 12 exons. RT-PCR analysis shows that human C4orf13 is widely expressed in human tissues, and the expression levels in liver and lung are relatively high, expression levels in placenta, kidney, spleen, and thymus are moderate, low levels of expression are detected in heart, prostate, and testis.

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.