A large-scale functional approach to uncover human genes and pathways in Drosophila

We demonstrate the feasibility of performing a systematic screen for human gene functions in Drosophila by assaying for their ability to induce overexpression phenotypes. Over 1 500 transgenic fly lines corresponding to 236 human genes have been established. In all, 51 lines are capable of eliciting a phenotype suggesting that the human genes are functional. These heterologous genes are functionally relevant as we have found a similar mutant phenotype caused either by a dominant negative mutant form of the human ribosomal protein L8 gene or by RNAi downregulation of the Drosophila RPL8. Significantly, the Drosophila RPL8 mutant can be rescued by wild-type human RPL8. We also provide genetic evidence that Drosophila RPL8 is a new member of the insulin signaling pathway. In summary, the functions of many human genes appear to be highly conserved, and the ability to identify them in Drosophila represents a powerful genetic tool for large-scale analysis of human transcripts in vivo.

A systematic survey of LU domain-containing proteins reveals a novel human gene,LY6A,which encodes the candidate ortholog of mouse Ly-6A/Sca-1 and is aberrantly expressed in pituitary tumors

The Ly-6 and uPAR(LU)domain-containing proteins represent a large family of cell-surface markers.In particular,mouse Ly-6A/Sca-1 is a widely used marker for various stem cells;however,its human ortholog is missing.In this study,based on a systematic survey and comparative genomic study of mouse and human LU domain-containing proteins,we identified a previously unannotated human gene encoding the candidate ortholog of mouse Ly-6A/Sca-1.This gene,hereby named LY6A,reversely overlaps with a lncRNA gene in the majority of exonic sequences.We found that LY6A is aberrantly expressed in pituitary tumors,but not in normal pituitary tissues,and may contribute to tumorigenesis.Similar to mouse Ly-6A/Sca-1,human LY6A is also upregulated by interferon,suggesting a conserved transcriptional regulatory mechanism between humans and mice.We cloned the full-length LY6A cDNA,whose encoded protein sequence,domain architecture,and exon‒intron structures are all well conserved with mouse Ly-6A/Sca-1.Ectopic expression of the LY6A protein in cells demonstrates that it acts the same as mouse Ly-6A/Sca-1 in their processing and glycosylphosphatidylinositol anchoring to the cell membrane.Collectively,these studies unveil a novel human gene encoding a candidate biomarker and provide an interesting model gene for studying gene regulatory and evolutionary mechanisms.

人类“绿色基因”假说(Human"Green-Gene"Hypothesis):核心内容、科学佐证与实践意义

首先,对人类“绿色基因”的形成进行探讨,包括人类(动物)与植物共同维持大气中碳氧平衡、人类(动物)与植物互为食物链关系、人体功能中存在植物性功能、绿色是人类眼睛最易看到的颜色、人类血红蛋白的结构相似于叶绿素结构、作为人体呼吸器官的肺部相似于树木地上部形状(树形)、人类肠道绒毛相似于植物根系毛细根等方面。进而对支持早期人类生活的植物类型与栽培植物诞生进行研究。在此基础上,提出人类“绿色基因”假说及其核心内容,归纳该假说的科学佐证。人类“绿色基因”假说从整体的、发展的思维解释人类与植物关系,奠定了园艺疗法、园林康养、森林康养的坚实基础,并指出接触植物、进行园艺操作与田园劳作是人们实现接地、激发触觉潜在功能的途径。

Gene therapy for type 1 diabetes mellitus in rats by gastrointestinal administration of chitosan nanoparticles containing human insulin gene

AIM:To study the expression of human insulin gene in gastrointestinal tracts of diabetic rats. METHODS: pCMV.Ins, an expression plasmid of the human insulin gene, wrapped with chitosan nanoparticles, was transfected to the diabetic rats through lavage and coloclysis, respectively. Fasting blood glucose and plasma insulin levels were measured for 7 d. Reverse transcription polymerase chain reaction (RT-PCR) analysis and Western blot analysis were performed to confirm the expression of human insulin gene. RESULTS: Compared with the control group, the fasting blood glucose levels in the lavage and coloclysis groups were decreased significantly in 4 d (5.63 ± 0.48 mmol/L and 5.07 ± 0.37 mmol/L vs 22.12 ± 1.31 mmol/L, respectively, P < 0.01), while the plasma insulin levels were much higher (32.26 ± 1.81 μIU/mL and 32.79 ± 1.84 μIU/mL vs 14.23 ± 1.38 μIU/mL, respectively, P < 0.01). The human insulin gene mRNA and human insulin were only detected in the lavage and coloclysis groups. CONCLUSION: Human insulin gene wrapped with chitosan nanoparticles can be successfully transfected to rats through gastrointestinal tract, indicating that chitosan is a promising non-viral vector.

Is there a close relationship between synonymous codon bias and codon-anticodon binding strength in human genes?

Synonymous codon bias has been examined in 78 human genes (19967 codons) and measured by relative synonymous codon usage (RSCU). Relative frequencies of all kinds of dinucleotides in 2,3 or 3,4 codon positions have been calculated, and codon-anticodon binding strength has been estimated by the stacking energies of codon-anticodon bases in Watson-Crick pairs. The data show common features in synonymous codon bias for all codon families in human genes: all C-ending codons, which possess the strongest codon-anticodon binding energies, are the most favored codons in almost all codon families, and those codons with medium codon-anticodon binding energies are avoided. Data analysis suggests that besides isochore and genome signature, codon-anticodon binding strength may be closely related to synonymous codon choice in human genes. The join-effect of these factors on human genes results in the common features in codon bias.

Trans-acting factors from the human fetal liver bindingto the human ε-globin gene silencer

The developmental stage-specific silencing of the human ε-globin gene during embryonic life is controlled, inpart, by the silencer (-392bp～-177bp) upstream of thisgene. In order to elucidate its role, the nuclear extractfrom the human fetal liver has been prepared and the interactions between trans-acting factors and this silencerelement have been examined. By using DNasel footprinting assay, a major protected region from -278bp to -235bpwithin this silencer element was identified. Furthermore,we found in gel mobility shift assay and Southwestern blotting assay that there were at least four trans-acting factors (MW ≈ 32, 28, 26 and 22kD) in the nuclear extractisolated from the human fetal liver, which could specifically bind to this region. Our results suggested that thesetrans-acting factors might play an important role in silencing the human embryonic ε-globin gene expression at thefetal stage through the interactions with this silencer.

PREDOMINANT EXPRESSION OF HUMAN Aγ-IN CONTRAST WITH β-GLOBIN GENE IN MEL CELLS TRANSFECTED WITH THE CONSTRUCT μLCRAγψβδ

A cosmid construct μLCRAγψβδβ were induced into mouse erythroleukemia cell lines 585 that expresses murine adult globin only and MEL GM 979 that expresses both murine embryonic and adult globins.Similar patterns of human globin gene expression were displayed in the two MEL cell lines transfected with the construct.Inducible expression of the Aγ and β gene was observed during induced cell differentiation.However,the expression level of the Aγ globin gene is much higher than that of the β globin gene in either uninduced or induced MEL transformants.No γ to β switching happened in the stable MEL transformants following a continuous culture.The much more effective enhance of the μLCR on the Aγ globin gene than that on the β globin gene is resulted probably from the fact that the distance between the LCR and the β globin gene is much longer than that between the LCR and the Aγ globin gene in the construct,in comparison with other constructs containing HS2 or μLCR linked to both of γ and β globin genes in different order.Two suggestions can be derived from these results:1) A competition between the γ and β globin gene for interaction with the LCR may indeed present,but only an enough long distance difference between the LCR to the γ and to the β gene can effectively influence the competition;2) Unlike transgenic mice,MEL cells are incapable of reconstructing the regulatory information involved in developmental control when it is provided by a fragment of the β globin gene cluster with limited length.

Expression of human yrdC gene promotes proliferation of gastric carcinoma cells

Objective： The aim of the research was to study the function of human yrdC gene in the gastric carcinoma cells. Methods： Human yrdC gene was isolated from human spleen tissue by RT-PCR. Anti-human yrdC monoclonal antibody was prepared by hybridoma cell technique. Recombinant adenovirus Ad.yrdC carrying yrdC gene was constructed by using the AdEasy adenoviral vector system. Recombinant adenovirus Ad.yrdCshRNA mediated yrdCshRNA was prepared by RNA interference technology. Gastric adenocarcinoma BGC-823 cells of moderate differentiation were transfected and absorbance of the transfected cells was calculated at 490 nm by methyl thiazolyl tetrazolium （MTT） method. Results： A value of the transfected Ad.yrdC group was significantly greater than that of the non-transfected and transfected Ad.Null groups, and A value of Ad.yrdCshRNA group was significantly lower than that of the non-transfected and transfected Ad.Null groups. Conclusion： Expression of yrdC gene has a function of promoting the proliferation of gastric carcinoma cells.

Analysis of Single Nucleotide Polymorphism in Human Paraoxonase 1 Gene(Q192R) with Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry

Introduction Single nucleotide polymorphisms （SNPs） are the most abundant DNA markers in the human genome occurring at a frequency of one in every 500--1000 nucleotides. A variety of methods have been used for the analysis of single nucleotide polymorphisms, including restriction fragment length polymorphism （RFLP）, direct sequencing by using laser-induced fluorescence detectionTM, fluorescence energy transfer, MALDI-TOF MS combined with primer extension or invasive cleavage, and fluorescence polarization. During the past two decades, mass spectrometry has become a very popular tool in the analysis of biomolecules and is perfectly suited to the analysis of single nucleotide polymorphisms （SNPs） due to its speed, low cost, and accuracy. In this work, we used MALDI TOF mass spectrometry to detect the fragments of restriction endonuclease hydrolysis of PCR products flanking a SNP located at paraoxonase 1（Q192R）. Compared with electrophoresis, this method requires less time of analysis and possess a higher accuracy.

Identification and Validation of Candidate Radiation-responsive Genes for Human Biodosimetry

The aim of the present study is to analyze the global research trend of radiation-responsive genes and identify the highly reproducible radiation-responsive genes. Bibliometric methods were applied to analyze the global research trend of radiation-responsive genes. We found 79 publications on radiation-responsive genes from 2000 to 2017. A total of 35 highly reproducible radiation-responsive genes were identified. Most genes are involved in response to DNA damage, cell proliferation, cell cycle regulation, and DNA repair.The p53 signal pathway was the top enriched pathway. The expression levels of 18 genes in human B lymphoblastoid cell line（AHH-1） cells were significantly up-regulated in a dose-dependent manner at 24 h after exposure to 0-5 Gy ^60 Coγ-ray irradiation. Our results indicate that developing a gene expression panel with the 35 high reproducibility radiation-responsive genes may be necessary for qualitative and quantitative assessment after exposure.

Cloning and expression of MXR7 gene in human HCC tissue

AIM To clone and identify the whole cDNA ofMXR7 gene and to find out its expression inhuman HCC,and normal tissues.METHODS The DNA primers were designed andsynthesized according to the whole cDNAsequence of MXR? gene.The cDNA of humanHCC was taken as the template while the cDNA ofMXR7 gene was synthesized by polymerasechain reaction（PCR）.Recombinant DNAconforming to reading frame was constructed byconnecting purified PCR product of the cDNA ofMXR? gene with expression vector pGEX-5X-1 offusion protein.The plasmid MXRT/pGEX-5X-1was identified by sequencing.Using 32p labeledMXR? cDNA as probe,MXR7 mRNA expressionwas detected by Northern blot analysis in 12different human normal tissues,7 preoperativelyuntreated non-liver tumor tissues,30preoperatively untreated HCC,theparacancerous liver tissues and 12 normal livertissues samples.RESULTS Restriction enzyme and sequenceanalysis confirmed that the insertion sequence invector pGEX-5X-1 was the same as the cDNAsequence of MXR7 gene.Northern blot analysisshowed no expression of MXR? mRNA in 12 kindsof normal human tissues including liver,7 tumortissues in other sites and 12 normal livertissues,the frequencies of MXR7 mRNA expression in HCC and paracancerous livertissues were 76.6% and 13.3%,respectively.The frequency of MXR7 mRNA expression in HCCwithout elevation of serum AFP and in HCC【5cm was 90%（9/10）and 83.3%（5/6）,respectively.CONCLUSION MXR7 mRNA is highly expressedin human HCC,which is specific and occurs at anearly stage of HCC,suggesting MXR7 mRNA canbe a tumor biomarker for HCC.The detection ofMXR7 mRNA expression in the biopsied livertissue is helpful in discovering early subclinicalliver cancer in those with negative serum AFP.

SIMULTANEOUS OVER-EXPRESSION OF INSULIN-LIKE GROWTH FACTOR- Ⅱ (IGF- Ⅱ ) AND IGF- Ⅱ RECEPTOR(IGF- Ⅱ R) GENES IN HUMAN PRIMARY CANCER-IMPLICATION OF AUTOCRINE AND PARACRINE MECHANISM IN AUTONOMOUS GROWTH OF HEPATIC CANCER

This is first report about the simultaneous over-expression of both Insulin-like growth factor (IGF- I ) and its receptor (IGF- I R) at mRNA level in human primary hepatic Cancer (PHC). In 10 PHC samples from China, IGF-I and IGF- I R were both over-expressed, whereas only a background signal was detected in normal liver. In 5 pairs of PHC and its non- tumorous adjacent liver tissues from South Africa, IGF- I and IGF- I R were also over-expressed in PHC. mRNA expression of IGF- I in all 5 cases and IGF- I R in 4 of 5 cases were higher in cancer than non- tumorous adjacent liver tissues. These results strongly implicate that an autocrine and/ or paracrine mechanism might be Involved in formation and progression of PHC.

Human Papillomavirus 16E6 Oncogene Mutation in Cervical Cancer

Objective： Cervical cancer （CC） is the second most common type of cancer in women worldwide, after breast cancer. High-risk human papillomaviruses （HR-HPVs） are considered to be the major causes of cervical cancer. HPV16 is the most common type of HR-HPVs and HPV16 E6 gene is one of the major oncogenes. Specific mutations are considered as dangerous factors causing CC. This study was designed to find mutations of HPV16 E6 and the relationship between the mutations and the happening of CC. Methods： The tissue DNA was extracted from 15 biopsies of CC. Part of HPV16 E6 gene （nucleotide 201-523） was amplified by polymerase chain reaction （PCR） from the CC tissue DNA. The PCR fragments were sequenced and analyzed. Results： The result of PCR showed that the positive rate of HPV16 E6 was 93.33% （14/13）. After sequencing ana analyzing, in the 13 out of 14 PCR fragments, 4 maintained prototype （30.77%）, 8 had a same 350G mutation （61.54%）, and 1 had a 249G mutation （7.69%）. Conclusion： This study suggest that there is a high infection rate of HPV in cervical cancer and most of the HPV16 E6 gene has mutations. Those mutations may have an association with the development of cervical cancer.

Radioiodide uptake in melanoma cells after transfer of human NaI symporter gene

To obtain human sodium/iodide symporter gene cDNA for studying its potential ability as a radioiodide treatment for melanoma, the hNIS gene cDNA was amplified with total RNA from human thyroid tissue by RT-PCR. The hNIS cDNA was inserted into cloning vector pUCm-T and subcloned into eukaryotic expression vector pc-DNA3. The pc-DNA3-hNIS and pc-DNA3 were transduced into melanoma cells (B16) by electroporation, and two cell lines termed B16-A and B16-B respectively were established. The uptake and efflux of iodide was examined in vitro. The three cell lines (B16-A, B16-B, B16) were injected subcutaneously into the right flank of C57 mice. Biodistribution study and tumor imaging were performed when the tumor reached approximately 10mm in diameter. The cloned hNIS cDNA sequence was identical with the published sequence. Two novel cell lines named 16-A containing pc-DNA3-hNIS and B16-B containing pc-DNA3 only were established. The resultant cell line B16-A accumulated 17 and 19 times more radioiodide in vitro than B16 and B16-B respectively. The iodide uptake reached the half-maximal level within 10 min, and reached a plateau at 30 min. The efflux of iodide was also rapid (T1/2eff=10min). The imaging shows in vivo uptake in expected sites including the salivary glands, thyroid, stomach, and hNIS-transduced tumor, whereas the nontransduced tumor was not visualized. The %ID/g of B16-A tumors at 1, 2, 4, 12, and 24h after injec- tion of 125I were 12.22±0.71, 10.91±0.72, 8.73±0.99, 1.24±0.29, and 0.19±0.03, respectively, which were signifi- cantly higher percentages than those for controlling tumors, p<0.01. However, biologic T1/2 was about 6 h. Our pre- liminary data indicate that the transduction of the hNIS gene per se is sufficient to induce iodide transport in mela- noma cells both in vitro and in vivo, but T1/2eff is short.

Enhancement of mouse germ cell-associated genes expression by injection of human umbilical cord mesenchymal stem cells into the testis of chemical-induced azoospermic mice

Various methods are currently under investigation to preserve fertility in males treated with high-dose chemotherapy and radiation for malignant and nonmalignant disorders. Human umbilical cord mesenchymal stem cells （HUC-MSCs）, which possess potent immunosuppressive function and secrete various cytokines and growth factors, have the potential clinical applications. As a potential alternative, we investigate whether injection of HUC-MSCs into the interstitial compartment of the testes to promote spermatogenic regeneration efficiently. HUC-MSCs were isolated from different sources of umbilical cords and injected into the interstitial space of one testis from 10 busulfan-treated mice （saline and HEK293 cells injections were performed in a separate set of mice） and the other testis remained uninjected. Three weeks after MSCs injection, Relative quantitative reverse transcription polymerase chain reaction was used to identify the expression of 10 of germ cell associated, which are all related to meiosis, demonstrated higher levels of spermatogenic gene expression （2-8 fold） in HUC-MSCs injected testes compared to the contralateral uninjected testes （five mice）. Protein levels for germ cell-specific genes, miwi, vasa and synaptonemal complex protein （Scp3） were also higher in MSC-treated testes compared to injected controls 3 weeks after treatment. However, no different expression was detected in saline water and HEK293 cells injection control group. We have demonstrated HUC-MSCs could affect mouse germ cell-specific genes expression. The results also provide a possibility that the transplanted HUC-MSCs may promote the recovery of spermatogenesis. This study provides further evidence for preclinical therapeutic effects of HUC-MSCs, and explores a new approach to the treatment of azoospermia.

Metallothionein 1 Isoform Gene Expression Induced by Cadmium in Human Peripheral Blood Lymphocytes

Objective To study the gene expression of metallothionein 1 （MT-1） isoforms in human peripheral blood lymphocytes （HPBLs）. Methods The expression of mRNA representing the seven active MT-I genes was determined in HPBLs by quantitative RT-PCR before and after exposure to cadmium. Results Basal expressions of MT-IX, and MT-1A in HPBLs were similar to expression of housekeeping gene. In contrast, the basal gene expressions of MT- 1 H, IF, 1E, and 1G were a little transcripts in human HPBLs. No signal was detected for MT-1B. There was a sex difference （P〈0.05）. in basal gene expression of MT-1E. The levels of gene expression of MT-1A, 1E, IF, 1G, 1H, and 1X increased, but the level of MT-1B did not increase after exposure to cadmium. Conclusions Gene expressions of MT-1 G, MT-1 H, MT-1 F, and MT-1X in HPBLs can be used as a potential biomarker of cadmium exposure.

Bioinformatic prediction and functional characterization of human KIAA0100 gene

Our previous study demonstrated that human KIAA0100 gene was a novel acute monocytic leukemia-associated antigen (MLAA) gene. But the functional characterization of human KIAA0100 gene has remained unknown to date. Here, firstly, bioinformatic prediction of human KIAA0100 gene was carried out using online softwares; Secondly, Human KIAA0100 gene expression was downregulated by the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system in U937 cells. Cell proliferation and apoptosis were next evaluated in KIAA0100-knockdown U937 cells. The bioinformatic prediction showed that human KIAA0100 gene was located on 17q11.2, and human KIAA0100 protein was located in the secretory pathway. Besides, human KIAA0100 protein contained a signalpeptide, a transmembrane region, three types of secondary structures (alpha helix, extended strand, and random coil) , and four domains from mitochondrial protein 27 (FMP27). The observation on functional characterization of human KIAA0100 gene revealed that its downregulation inhibited cell proliferation, and promoted cell apoptosis in U937 cells. To summarize, these results suggest human KIAA0100 gene possibly comes within mitochondrial genome; moreover, it is a novel anti-apoptotic factor related to carcinogenesis or progression in acute monocytic leukemia, and may be a potential target for immunotherapy against acute monocytic leukemia.

Synthesis of Codon-optimized Human Interleukin-18 Gene by Combination of Chemical and Enzymatic Method

According to the amino acid sequence and codon preference of E. coli, the human interleukin-18（IL-18） gene was optimized to avoid the rare codons. The total length of the synthesized gene is 571 bp; 18 oligonucleotides, DNA fragments were designed and synthesized by the phosphoramidite four-step chemical method. The whole DNA sequence was synthesized by a one-step total gene synthesis method, and then inserted in pUC18 vector. Five positive clones identified by blue-white colony screening were sent to Shanghai Sangon Biological Engineering Technology and Service Co., Ltd. for sequencing. The sequencing result shows that one clone contained the complete correct gene in all the five positive clones.

Correlation between human seizure-related gene 6 variants and idiopathic generalized epilepsy in a Southern Chinese Han population

This study sought to analyze the genotype and gene mutations of human seizure-related gene 6 in 98 patients with idiopathic generalized epilepsy （non-febrile seizures）, who were selected from three generations of the Chinese Han population living in Shanghai, Zhejiang Province, Wuxi of Jiangsu Province, and Jiangxi Province of Southern China. Twenty-six patients＇ parents were available as a first-degree relatives group and 100 biologically unrelated healthy controls were collected as the control group. Based on the age of onset and seizure type, the patients were divided into six subgroups. Polymerase chain reaction and DNA direct sequencing analysis showed that the most frequent mutations c. 1249dupC （p.Gly418Argfx31 ） and c.1636A 〉 G （p.Thr546Ala） were detected in some idiopathic generalized epilepsy patients and tl^eir asymptomatic first-degree relatives （30.6% vs. 19.2% and 11.2% vs. 26.9%）. A novel mutation c.1807G 〉A （p.Val603Met） was found in a patient with late-onset idiopathic generalized epilepsy. There was no significant difference in the incidence of these three mutations among the different subgroups of idiopathic generalized epilepsy and controls. Thus, further analysis of a larger population is needed to confirm the assumption that human seizure-related gene 6 is a susceptibility gene for idiopathic generalized epilepsy with various sub-syndromes.

Seizure-related 6,a brain-specific expression gene,is highly expressed in the human cerebellum

Epilepsy is a complex, Mendelian disease, and most cases are sporadic. Genomic comparisons of tissue from identified monogenic epilepsies with multigenic and acquired syndromes could ultimately reveal crucial molecular neuropathology for an epileptic phenotype. In the present study, a novel gene, human seizure-related （hSEZ）-6, was isolated from a human brain cDNA library. hSEZ-6 comprises 17 exons and spans a region of at least 55.6 kb, which was localized to 17q 12 by radiation hybridization, hSEZ-6 exhibits two isoform types, hSEZ-6A and hSEZ-6B, which encode 996 and 995 amino acids, respectively. The two putative hSEZ-6 proteins contain similar motifs and share 82% and 84% identity with mouse SEZ-6A protein, whose expression level increased in mouse cerebral cortex-derived cells treated with a convulsant drug, pentylentetrazole. Northern blot analysis demonstrated that hSEZ-6 is expressed highly in the cerebellum and in nucleus of the extrapyramidal system, such as the caudate nucleus and putamen. Reverse transcription polymerase chain reaction revealed that hSEZ-6 is expressed in neurons rather than gliocytes, which suggests that hSEZ-6 is a seizure-related gone.