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.

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.

ISOBUTYRAMIDE ACTIVATES TRANSCRIPTION OF HUMAN FETAL γ-AND MURINE EMBRYONIC εy-GLOBIN GENES

Objective. To examine the effect of isobutyramide synthesized in our laboratory on human and murine globin gene expression and to test cell toxicity ofthe drug.Methods. MEL cells were transfected with the recombinant construct μLCRAγψβδβand the stable transformants were cultured in the medium with different concentrations of isobutyramide. The experimental mice and rabbit were injected with different doses of isobutyramide. The globin mRNAs were analyzed by RNase protection assay. The hematological toxicity and electrolyte toxicity ofthe drug were tested.Results. An inducible and dose dependent expression of the human γ , β and mouse α globin gene was observed in the transfected MEL cells. The induction of the human γ globin gene is significant stronger than that of the β globin gene. With 2.5～5 mmol/L isobutyramide, the induction of the human γ globin gene is even more effective than that of mouse α globin gene. After a 15 day injection under the doses of 500～900mg·kg-1·d-1, the level of the mouse embryonic εy globin mRNA could be significantly induced up to 3～4 fold of that of uninjected controls. The changes of hemoglobin(Hb), RBC, hematocrit(HCT), WBC, derived from mice injected with different doses of isobutyramide at the interval of 24 hours for 2～4 weeks, were generally within the normal range. In rabbits injected with isobutyramide in the same regiment for 2 weeks, the concentration of blood K+, Na+, Cl-and CO2 were all within normal range and serum ionic osmotic pressure remained stable as well. Conclusion. Our results suggested that isobutyramide is a weak inducer ofcell differentiation, but it can selectively activate transcription of human γ globin gene at a certain degree, and it can act on early stages of erythroid progenitor differentiation in adult mice and activate transcription of embryonic εy-globin gene and have no hematological toxicity. Our results have further proved the potential value of isobutyramide in treatment of β-thalassemia and sickle cell disease.

Variation and evolution of NP genes of human avian H_5N_1 virus strains

In order to reveal variation and revolution of NP genes of human avian H5 N1 influenza virus strains, the NP gene of a human avian H5 N1 influenza virus strain in Guangdong was sequenced and the global NP genes of strains were retrieved. The sequences were analyzed by DNAStar 5.0, and the evolutionary speed was studied with reference to the epidemiological data. It was found that NP genes of 45 strains during 1997-2006 were homologically classified into three groups： strains in 1997-1998, strains in 2004-2005 and strains from 2003 to 2006. There were 35 substitutions in NPs in all strains accounting for a ratio of 7.03% （35/498）. An additional glycoprotein domain （NGT430-432） was found in NP genes in the strains of 2003-2006, the mutation of N370S in GD-01-06 resulted in occurrence of one more glycoprotein domain （NES368-370）. In the synonymous variation, Ks values in NP were 2.03 × 10^-5-2.55 × 10^-5 Nt/d and K. values in NP were 1.58 × 10^-6-3.10 × 10^-6 Nt/d. There didn＇t exist obviously selective pressure. An additional glycoprotein domain in every strain of 2003-2006 and one more in strain GD-01-06 might change the antigenicity of human avian H5 N1 influenza virus. The variation on human avian H5 N1 influenza strains occurred frequently in the natural world, which would result in high probability of human-human transmission along with the natural evolution of the virus.

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.

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.

Disease Prevention and Alleviation by Human Myoblast Transplantation

Myoblast implantation is a unique, patented technology of muscle regeneration being tested in Phase III clinical trials of muscular dystrophy, ischemic cardiomyopathy, Phase II trial of cancer, and Phase I trial of Type II diabetes. Differentiated and committed, myoblasts are not stem cells. Implanted myoblasts fuse spontaneously among themselves, replenishing genetically normal myofibers. They also fuse with genetically abnormal myofibers of muscular dystrophy, cardiomyopathy, or Type II diabetes, transferring their nuclei containing the normal human genome to provide stable, long-term expression of the missing gene products. They develop to become cardiomyocytes in the infracted myocardium. Myoblasts transduced with VEGF165 allow concomitant regeneration of blood capillaries and myofibers. They are potent biologics for treating heart failure, ischemic cardiomyopathy, diabetic ischemia, erectile dysfunction, and baldness. Myoblasts, because of their small size, spindle shape, and resilience, can grow within wrinkles and on skin surfaces, thus enhancing the color, luster and texture of the skin “plated” with them. They can be injected subcutaneously as a cellular filler to reduce wrinkles. Intramuscular injection of myoblasts can augment the size, shape, consistency, tone and strength of muscle groups, improving the lines, contours and vitality to sculpt a youthful appearance. This highly promising technology has great social economic values in treating hereditary, fatal and debilitating disease conditions.

EFFECT OF MATRINE ON EXPRESSION OF HCCR1 AND HCCR2 PROTEINS IN CULTURAL HUMAN HEPATOCELLULAR CARCINOMAS CELLS

Objective： To explore the effects of matrine on HCCR1 and HCCR2 expression in cultural human hepatocellular carcinomas （HCC） cells at the level of gene and protein. Methods： Three methods, representational difference analysis （RDA） of cDNA, microarrays and fluorescence in situ hybridization （FISH） were used to detect levels of mRNA and protein expression of HCCR1 and HCCR-2 before and after treatment of matrine. Results： Matrine had inhibitory effect on the mRNA and protein expression of HCCR1 and HCCR2 in cultural HCC cells. Conclusion： Matrine has inhibitory effect on gene transcription, protein expression of HCCR 1 and HCCR2 in cultural HCC cells.

Ceruloplasmin or Fibronectin Synergism with Quartz Dust on Stimulating Collagen Gene Transcription in Human 2BS Fibroblast

Human α1(Ⅰ), α2(Ⅰ) and α1(Ⅲ) cDNA probes and RNA dot hybridization were employed to quantitate collagen mRNA changes after adding silica dust into the media of human 2BS fibroblasts. At all dosages used (100, 200, 500 and 1000μg), the α1(Ⅰ), α2(Ⅰ)and α1(Ⅲ) mRNA levels increased one day after dusting. At the same dosage of silica (100μg), α1(Ⅲ) mRNA increased earlier than type Ⅰ collagen mRNA did. The type Ⅰ and type Ⅲ collagen mRNA contents in the experimental groups were higher than those in control on days 3, 5, 7 and 9. The effect of ceruloplasmin (Cp) and fibronectin (Fn) on collagen mRNA synthesis was also studied, after adding silica dust, Cp or Fn into the media of human 2BS fibroblast. The results showed that Cp and Fn have stimulating effect on collagen mRNA production. When both Cp and silica dust were added into cell culture media, the collagen mRNA level was increased more than those of adding either Cp or silica dust alone. Similar situations were found for Fn. Cp (or Fn) synergism with silica dust on stimulating transcription of human collagen gene was suggested

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.

Identification of an ｋB-like motif at the 5' upstream region of human lymphotoxin gene

Lymphotoxin(LT) is a glycoprotein secreted by activated T cell. The expression of LT gene is mainly regulated at the level of transcription. By using human LT DNA as a probe, we carried out a RNA dot blotting test and found that the longer the time of Jurkat human Tlymphoma cells exposed to the PMA and PHA, the more endogenous LT mRNA could be produced. Results of gel retardation assay showed that the nuclear extract from Jurkat cells treated with PMA and PHA formed different DNA-protein complexes. Changes in complex patterns were observed at various time intervals of PMA and PHA induction. A specific protein-binding site was mapped out to be a 22-bp sequence at the 5’upstream regioll of human LT gene by DNase I footprinting analysis. This region was similar to the sequence recognized by the proteins of NFｋB family The results of fragment competition and homology analysis indicated that the 22-bp sequence contains a ｋB-like motif only which is located at the base pairs -100 to -90 (5’-GGGGGCTTCCC-3’). Thus, the NF-ｋBlike factors were involved in the protein-DNA interaction.Furthermore, there were more than one retarded bands appearing in the gel retardation assay. It suggested that there may be several NF-ｋB-like factors involved in theregulation of LT gene transcription at the same site.

Studies on DNA-protein interactions in the upstream regulatory region of the human ε-globin gene promoter

The erythroid- and developmental stage-specific expression of the human ε-globin gene is controlled, in part,by the 5’-flanking DNA sequence of this gene. In the present study, we have used DNA-protein binding assays to identify trans-acting factors which regulate the temporal expression of the human ε-globin gene during development. Using gel mobility shift assays and DNasel footprinting assays, a nuclear protein factor (termed ε-SSF1) in the nuclear extracts from mouse haematopoietic tissues at d 11 and d 13 of gestation was identified. It could specifically bind to the positive control region (between -535 and -453bp) of the human ε-globin gene. We speculated that the E-SSF1 might be an erythroid- and developmental stage-specific activator. In addition, we found another nuclear protein factor (termed ε-R1) in the nuclear extract from mouse fetal liver at d 18 of gestation, which could strongly bind to the silencer region (between -392 and -177bp) of this gene. Therefore, we speculated that the ε-R1 might be an erythroid- and developmental stagespecific repressor. Our data suggest that both ε-SSF1 and ε-R1 might play important roles in developmental regulation of the human ε-globin gene expression during the early embryonic life. On the other hand, we observed that the binding patterns of nuclear proteins from three cell lines (K562, HEL and Raji) to these regulatory regions were partially different. These results suggest that different trans-acting factors in K562, HEL and Raji cells might be responsible for activating or silencing the human ε-globin gene in three different cell lines.

Molecular cloning and structural analysis of human norepinephrine transporter gene(NETHG)

A cDNA molecule encoding a major part of the hu-man Norepinephrine transporter(hNET) was synthesized by means of Polymerase Chain Reaction(PCR) technique and used as a probe for selecting the human genomic NET gene. A positive clone harbouring the whole gene was ob-tained from a human lymphocyte genomic library through utilizing the "genomic walking" technique. The clone, des-ignated as phNET, harbours a DNA fragment of about 59 kb in length inserted into BamH Ⅰ site in cosmid pWE15.The genomic clone contains 14 exons encoding all amino acid residues in the protein. A single exon encodes a dis-tinct transmembrane domaill, except for transmembrane domain 10 and 11, which are encoded by part of two ex-ons respectively, and exon 12, which encodes part of do-main 11 and all of domain 12. These results imply that there is a close relationship between exon splicing of a gene and structural domains of the protein, as is the case for the human γ-aminobutyric acid transporter(hGAT) and a number of other membrane proteins.

The 5'-flanking cis-acting elements of the human ε-globin gene associates with the nuclear matrix and binds to the nuclear matrix proteins

The nucleax mains attachment regions(MARs) and the binding nuclear matrix proteins in the 5’-flalildng cisacting elements of the humanε-globin gene have been examined. Using in vitro DNA-matrix binding assay,it has been shown that the positive stage-specific regulatory element (ε-PREII, -446bp-419bp) upstream of this gene could specifically associate with the nuclear matrix from K562 cells, indicating thatε-PREII mad be an erythroidspecilic facultstive MAR. In gel mobility shift assay and Southwestern blotting assal an eothroid-specific nuclear matrix protein (ε-NMPk) in K562 cells has been revealed to bind to this positive regulatory element (E-PREII). Furthermore, we demonstrated that the silencer (-392hp -177bp) uP8tream of the humanε-globin gene could associate with the nuclear matrices from K562, HEL and Raji cells. In addition, the nucleax matrix proteins prepared from these three cell lines could also bind to this silencer, suggesting that this silencer element linght be a constitutive nuclear mains attachment region (constitutive MAR). Our results demonstrated that the nucleax madrid and nuclear mains proteins lxilght play an important role in the regulation of the human 5-globin gene expression.

A DNA-binding protein factor in K562 nuclear extract interacts with positive control region (PCR) in the 5'flanking sequence of human β-globin gene

It has been known that there are at least three regulatory regions (NCR1, NCR2 and PCR) in the 5'-flanking sequence (from -610 bp to +1bp) of human β-globin gene and that the function of PCR is unique to the human erythroleukemia (K562) cells. Here we have detected a DNA-binding protein factor (termed NFEa) in K562 cells, which can bind specifically to the PCR of human β-globin gene. The sequence of the binding site is 5'ACTGATG3' (between -222 bp and -216 bp). The NFEa is erythroid-specific and perhaps specific for K562 cells. It seemed that this factor differed from the erythroid-specific tran-scriptional factor (NFE-1) using competition assay. The presence of the NFEa further supported that the function of the cis-acting element PCR was specific for K562 cells, and helps us to understand the mechanism of the regulation of the expression of human β-globin gene in the human K562 cells.

Proteins binding to the 5'-flanking regulatory elements of the human β-globin gene

The binding of nuclear proteins prepared from mouse erythroid tissue in different developmental stages to the 5'-flanking regulatory elements of human β-globin gene, two negative control regions(NCR1,-610 to -490 bp;NCR2, -338 to-233bp), was identified.Two stage specific protein factors corresponding to embryonic and fetal stages were found to be capable of binding to NCR2.These data provided evidence that the cis acting elements of the 5'-flanking region might be involved in the developmental control of β-globin gene and NCR2 might be responsible in part for the silence of β-globin gene in the embryonic and fetal stages.