lacⅠ 靶基因突变子的一种正向选择系统的建立

目的：建立一种能正向选择ｌａｃⅠ靶基因突变子的选择系统。方法：比较了分别携带有ｌａｃⅠ－ ／ｌａｃＺα型质粒和ｌａｃⅠ＋ ／ｌａｃＺα型质粒的ＤＨ１０Ｂ菌在ＬＢ完全培养基、以葡萄糖为碳源的Ｍ９ 基本培养基和以乳糖为碳源的Ｍ９／Ｌ基本培养基内的生长情况。结果：组成型ＤＨ１０Ｂ菌在Ｍ９／Ｌ固体培养基中只需培养２３ ｈ 便可观察到生长，而诱导型ＤＨ１０Ｂ菌则需培养８８ ｈ 才可观察到生长，但在ＬＢ和Ｍ９ 培养基内两者的生长速度却相同；当将少量的组成型ＤＨ１０Ｂ菌与大量的诱导型ＤＨ１０Ｂ菌混合接种于Ｍ９／Ｌ固体培养基上培养时， Ｍ９／Ｌ固体培养基能从约２．７×１０６ 的ｌａｃⅠ＋ 型ＤＨ１０Ｂ菌中将少量的ｌａｃⅠ－ 型ＤＨ１０Ｂ菌筛选出来。结论：本研究建立了一种在特定时间内只选择生长携带有ｌａｃⅠ－ ／ｌａｃＺα型质粒的ＤＨ１０Ｂ菌落。

SOD_1基因突变子对AD转基因小鼠β-淀粉样蛋白表达影响的实验研究

目的 :探讨Cu/ZnSOD1基因表达对Aβ产生的影响。 方法 :建立APP C10 0和G93ASOD1基因共表达的转双基因小鼠 ,应用免疫细胞化学方法测定不同鼠龄转基因鼠海马神经元Aβ表达 ,Westernblot定量测定转基因鼠脑SOD1、APP、APP C10 0和Aβ表达水平。结果 :低龄转双基因鼠与转单基因鼠之间Aβ水平无差异 ,与年龄匹配的转单基因鼠比较 ,高龄转双基因鼠脑Aβ水平升高 ,经统计处理差异无显著性意义 (P >0 0 5 )。但高龄转双基因鼠Aβ水平比低龄转双基因鼠明显升高 (P <0 0 5 )。 结论 :转双基因鼠脑APP C10 0和G93ASOD1基因共表达虽然未导致老年斑形成 ,但引起Aβ含量增加 。

β地中海贫血杂合子基因突变及Gγ珠蛋白基因-158位点SNP与HbF的关系

目的 探讨 β地中海贫血 (简称 β地贫 )杂合子基因突变类型和 Gγ珠蛋白基因启动子 - 15 8位点 (Gγ- 15 8)单核苷酸多态性与胎儿血红蛋白 (fetal hemoglobin,Hb F)水平的关系。方法 抗碱 -比色法测定 Hb F水平 ;PCR-寡核苷酸斑点杂交法检测β地贫基因型 ;限制性内切酶 Xmn 消化经 PCR扩增的Gγ基因启动子 DNA片段 ,分析Gγ- 15 8位点的单核苷酸多态性。结果 6 3例受检的轻型β地贫中 15例 Hb F≥ 2 % (2 .0 6 %～ 10 .4 4 % )。共检出 6种β地贫基因突变 ,分别是 :CD4 1/42 (- TTCT)、CD17(A→T)、nt- 2 8(A→ G)、CD71/72 (+A)、IVS- II- 6 5 4 (C→ T)、IVS- I- 1(G→ T)。 CD4 1/42、CD17、CD71/72、IVS- II-6 5 4的杂合子在 15例 Hb F升高组和 4 8例 Hb F正常组各自所占比例相同。 6 3例个体中有 10例为Gγ-15 8(C→T)突变的杂合子 ,总检出率为 15 .9% ;其中 15例高 Hb F个体中检出 8例 (检出率 5 3.33% ) ,HbF正常的 4 8例检出 2例 (检出率 4 .17% ) ,两组检出率差异有显著性 (P<0 .0 0 1)。结论 β地贫基因突变CD4 1/42、CD17、CD71/72、IVS- II- 6 5 4与 β地贫杂合子的 Hb F水平无关 ;而 Gγ- 15 8(C→ T)突变与广西地区 β地贫杂合子 Hb F升高密切相关。

2a型磷酸钠协同转运子基因突变导致低磷血症的研究

我们对20个病例NPT2a基因的13个外显子和启动子区域进行检测,探讨研究该基因突变与低磷血症的发生和临床表型的关系. 一、材料与方法 1.实验组共选择20例病例,其中男性11例,女性9例,平均年龄(47.00±2.33)岁.肾结石患者14例,其中男性8例,女性6例;骨质疏松患者6例,男女各3例.均伴有持续低磷血症,伴有降低的最大肾磷酸盐重吸收率,全部病例肾小球滤过率正常[(90±14)ml/min/1.73 m2体表面积].标准的离子血清钙浓度,正常血浆甲状旁腺浓度.均无佝偻病史.对照组选择20例健康志愿者,平均年龄(42.00±3.32)岁,其中,男性志愿者12例,女性8例.

Fine Mapping of AST Gene in Arabidopsis

The ast ( anthocyanin spotted testa) mutant, which was induced by carbon ion radiation, was a single recessive gene mutant of Arabidopsis thaliana (L.) Heynh. with spotted pigment in seed coats, and involved in the anthocyanin biosynthesis. To clone the AST gene by map-based cloning strategy, a series of molecular markers were designed according to the SNPs (single nucleotide polymophisms) and insertion/deletion polymophisms in the Arabidopsis database. With these molecular markers, the fine-structure mapping of the AST gene was finished, the AST locus was located in BAC clone T13M11. It was suggested that the AST candidate gene was T13M11. 8 in the T13M11 This gene was 1432 bp long with 6 exons and 5 introns. The putative protein of T13M11. 8 gene was similar to dihydroflavonol 4-reductase (DFR), which was an important enzyme in the anthocyanin biosynthesis pathway.

Accumulation of Mutant Genes and Its Pathway with Reproductive Isolation

According to the fitness of heterozygote was lower than homozygote among panmictic population,the process of generational accumulate of mutant gene r was considered.Branch point of r＇s frequency by generational evolution which revealed the hereditary incompatibility between R and r,was worked out,and it was found that genetic drift can make r have higher frequency to surpass the branch point to form reproductive isolation.It was not enough to have the three conditions of mutation,genetic drift and natural selection to be the drive of biological evolution;hybrid weakness,the repelling interaction between the genetic background of original population and the new mutation,were also needed.

Molecular mechanisms of ＂off-on switch＂ of activities of human IDH1 by tumor-associated mutation R132H

Human cytosolic NADP-1DH （IDH1） has recently been found to be involved in tumorigenesis. Notably, the tumorderived IDH1 mutations identified so far mainly occur at Arg132, and mutation R132H is the most prevalent one. This mutation impairs the oxidative IDH activity of the enzyme, but renders a new reduction function of converting a-ketoglutarate （aKG） to 2-hydroxyglutarate. Here, we report the structures of the R132H mutant IDH1 with and without isocitrate OCT） bound. The structural data together with mutagenesis and biochemical data reveal a previ- ously undefined initial ICT-binding state and demonstrate that IDH activity requires a conformational change to a closed pre-transition state. Arg132 plays multiple functional roles in the catalytic reaction; in particular, the R132H mutation hinders the conformational changes from the initial ICT-binding state to the pre-transition state, leading to the impairment of the IDH activity. Our results describe for the first time that there is an intermediate conformation that corresponds to an initial ICT-binding state and that the R132H mutation can trap the enzyme in this conforma- tion, therefore shedding fight on the molecular mechanism of the ＂off switch＂ of the potentially tumor-suppressive IDH activity. Furthermore, we proved the necessity of Tyr139 for the gained aKG reduction activity and propose that Tyr139 may play a vital role by compensating the increased negative charge on the C2 atom of aKG during the trans- fer of a hydride anion from NADPH to aKG, which provides new insights into the mechanism of the ＂on switch＂ of the hypothetically oncogenic reduction activity of IDH1 by this mutation.

Mutations in carboxy-terminal part of E2 including PKR/eIF2αphosphorylation homology domain and interferon sensitivity determining region of nonstructural 5A of hepatitis C virus 1b:Their correlation with response to interferon monotherapy and viral load

AIM： To study the amino acid substitutions in the carboxy （C）-terminal part of E2 protein and in the interferon （IFN） sensitivity determining region （ISDR） and their correlation with response to IFN and viral load in 85 hepatitis C virus （HCV）-lb-infected patients treated with IFN. METHODS： The C-terminal part of E2 （codons 617-711） including PKR/eIF2α phosphorylation homology domain （PePHD） and ISDR was sequenced in 85 HCV-1b-infected patients treated by IFN monotherapy. RESULTS： The amino acid substitutions in PePHD detected only in 4 of 85 patients were not correlated either with response to iFN or with viral load. The presence of substitutions in a N-terminal variable region （codons 617-641） in the C-terminal part of E2 was significantly correlated with both small viral load （33.9% vs 13.8%, P = 0.0394） and sustained response to iFN （25.0% vs 6.9 %, P = 0.0429）. Four or more substitutions in ISDR were significantly correlated with both small viral load （78.6% vs 16.2%, P 〈 0.0001） and sustained response to iFN （85.7% vs 2.9%, P 〈 0.0001）. In multivariate analysis, ISDR in nonstructural （NS） 5A （OR = 0.39, P 〈 0.0001） and N-terminal variable region （OR = 0.51, P = 0.039） was selected as the independentpredictors for small viral load, and ISDR （OR = 39.0, P 〈 0.0001） was selected as the only independent predictor for sustained response. CONCLUSION： The N-terminal variable region in the C-terminal part of E2 correlates with both response to IFN monotherapy and viral load and is one of the factors independently associated with a small viral load.

Frequency of primary iron overload and HFE gene mutations (C282Y,H63D and S65C) in chronic liver disease patients in north India

AIM:To identify the frequency of iron overload and study the three mutations in the HFE gene (C282Y,H63D,and S65C) in patients with chronic liver disorders (CLD) and controls. METHODS:To identify patients with iron overload (transferrin saturation > 45% in females and > 50% in males and serum ferritin > 1000 ng/mL) we evaluated 236 patients with CLD,including 59 with non-alcoholic steatohepatitis (NASH),22 with alcoholic liver disease (ALD),19 of cirrhosis due to viruses (HBV,HCV),and 136 with cryptogenic cirrhosis. Mutations of the HFE gene were analyzed by PCR-RE. hundred controls were screened for iron status and the mutations. RESULTS:Seventeen patients with CLD showed evidence of iron overload. Fifteen cases of iron overload had cryptogenic cirrhosis and two had ALD. None of the controls showed iron overload. We did not find any individual with 282Y or 65C either in the cases or in the controls. The prevalence of H63D heterozygosity was 12% in normal individuals,14.8% in 236 patients (16.9% in NASH,13.6% in ALD,26.3% in viral and 12.5% in cryptogenic cirrhosis) and the overall prevalence was 13.98%. Only two of the 17 patients with primary iron overload were heterozygous for H63D. One patient with NASH and one normal individual who were homozygous for H63D showed no iron overload.CONCLUSION:Primary iron overload in Indians is nonHFE type,which is different from that in Europeans and further molecular studies are required to determine the defect in various iron regulatory genes.

Germline mutation analysis of MLH1 and MSH2 in Malaysian Lynch syndrome patients

AIM: To investigate the protein expression profi le of mismatch repair (MMR) genes in suspected cases of Lynch syndrome and to characterize the associated germline mutations. METHODS: Immunohistochemical analysis of tumor samples was performed to determine the protein expression profile of MMR protein. Germline mutation screening was carried out on peripheral blood samples. The entire exon regions of MLH1 and MSH2 geneswere amplifi ed by polymerase chain reaction, screened by denaturing high performance liquid chromatography (dHPLC) and analyzed by DNA sequencing to characterize the germline mutations. RESULTS: Three out of 34 tissue samples (8.8%) and four out of 34 tissue samples (11.8%) showed loss of nuclear staining by immunohistochemistry, indicating the absence of MLH1 and MSH2 protein expression in carcinoma cells, respectively. dHPLC analysis followed by DNA sequencing showed these samples to have germline mutations of MSH2 gene. However, no deleterious mutations were identifi ed in any of the 19 exons or coding regions of MLH1 gene, but we were able to identify MLH1 promoter polymorphism, -93G > A (rs1800734), in 21 out of 34 patients (61.8%). We identified one novel mutation, transversion mutation c.2005G > C, which resulted in a missense mutation (Gly669Arg), a transversion mutation in exon 1, c.142G > T, which resulted in a nonsense mutation (Glu48Stop) and splice-site mutation, c.2006-6T > C, which was adjacent to exon 13 of MSH2 gene. CONCLUSION: Germline mutations were identified in four Malaysian Lynch syndrome patients. Immunohistochemical analysis of tumor tissue proved to be a good pre-screening test before proceeding to germline mutation analysis of DNA MMR genes.

Prevalence of hepatitis B virus precore stop codon mutations in chronically infected children

AIM： To find out whether there is a significant difference in the prevalence of the precore stop codon mutation between HBeAg positive and anti-HBe positive children. METHODS： We investigated a large pediatric population of 155 European children （mean age 10.9 years） with chronic hepatitis B by PCR and direct sequencing. Ninety were HBeAg positive and 65 had seroconversion to anti-HBe. Additionally genotyping was performed. RESULTS： Seventy-four （48%） of the sequenced HBV strains were attributed to genotype D and 81 （52%） to genotype A. In the group of 90 HBeAg positive patients, 2 （2.2%） 1896-G-to-A transitions leading to precore stop codon mutation were found, and in the group of 65 anti-HBe positive children, 5 （7.7%） were identified harbouring HBeAg-minus mutants. The difference was not statistically significant （P= 0.13）. CONCLUSIONS： HBeAg minus variants as predominant viral HB strains play a minor role in the course of chronic hepatitis B in European children.

Epidemiological aspects of Budd-Chiari in Egyptian patients:A single-center study

AIM： TO describe the socio-demographic features, etiology, and risk factors for Budd-Chiari syndrome （BCS） in Egyptian patients. METHODS： Ninety-four Egyptian patients with confirmed primary Budd-Chiari syndrome were presented to the Budd-Chiari Study Group （BCSG） and admitted to the Tropical Medicine Department of Ain Shams University Hospital （Cairo, Egypt）. Complete clinical evaluation and laboratory investigations, including a thrombophilia workup and full radiological assessment, were performed to determine underlying disease etiologies.RESULTS： BCS was chronic in 79.8% of patients, acute or subacute in 19.1%, and fulminant in 1.1%. Factor V Leiden mutation （FVLM） was the most common etiological cause of disease （53.1%）, followed by mutation of the gene encoding methylene tetrahydrofolate reductase （MTHFR） （51.6%）. Current or recent hormonal treatment was documented in 15.5% of females, and BCS associated with pregnancy was present in 17.2% of females. Etiology could not be determined in 8.5% of patients. Males had significantly higher rates of MTHFR gene mutation and Behcet＇ s disease, and females had significantly higher rates of secondary antiphospholipid antibody syndrome. A highly significant positive relationship was evident between the presence of Behcet＇s disease and inferior vena caval occlusion, either alone or combined with occlusion of the hepatic veins （,0 〈 0.0001）. CONCLUSION： FVLM is the most common disease etiology and MTHFR the second most common in Egyptian BCS patients. BCS etiology tends to vary with geographic region.

Serrated polyposis syndrome:Molecular,pathological and clinical aspects

Hyperplastic polyps have traditionally been considered not to have malignant potential.New pathological classification of serrated polyps and recent discoveries about the serrated pathway of carcinogenesis have revolutionized the concepts and revitalized the research in this area.Until recently,it has been thought that most colorectal cancers arise from conventional adenomas via the traditional tumor suppressor pathway initiated by a mutation of the APC gene,but it has been found thatthis pathway accounts for only approximately 70%-80% of colorectal cancer(CRC)cases.The majority of the remaining colorectal cancer cases follow an alternative pathway leading to CpG island methylator phenotype carcinoma with BRAF mutation and with or without microsatellite instability.The mechanism of carcinomas arising from this alternative pathway seems to begin with an activating mutation of the BRAF oncogene.Serrated polyposis syndrome is a relatively rare condition characterized by multiple and/or large serrated polyps of the colon.Clinical characteristics,etiology and relationship of serrated polyposis syndrome to CRC have not been clarified yet.Patients with this syndrome show a high risk of CRC and both sporadic and hereditary cases have been described.Clinical criteria have been used for diagnosis and frequent colonoscopy surveillance should be performed in order to prevent colorectal cancer.In this review,we try to gather new insights into the molecular pathogenesis of serrated polyps in order to understand their possible clinical implications and to make an approach to the management of this syndrome.

Detection of Frameshift Mutations of the Transforming Growth Factor p ReceptorⅡin Gastric Cancers with Microsatellite Instability

OBJECTIVE To study the relationship among microsatellite instability （MSI）, frameshift mutations （FM） of the transforming growth factor β receptor Ⅱ （TGF β R Ⅱ）, methylation state of the hMLH1 promoter and hMLH1 protein expression level in gastric cancers, and to explore their relationship to gastric carcinogenesis. METHODS DNA was isolated from 101 gastric specimens and 5 microsatellite loci were detected. PCR, electrophoresis on denatured polyacrylamide gels and silver staining were performed to detect the MSI. The FMs of TGFβR Ⅱ were also screened with the same method. HMLH1 methylation was analyzed by methylation specific PCR （MSP） and sequencing. HMLH1 protein expression was detected using immunohistochemistry. RESULTS The incidence of MSIs was 53.7% and 0% in the cancers and normal tissues, respectively, with the frequency of MSIs being significantly higher in the gastric cancers compared to the normal gastric tissues （P〈0.05）. The frequency of hMLH1 methylation was 41.5%（17/41） in the gastric cancers and 0%（0/60） in the normal group. Decreased hMLH1 expression was observed in 94.1%（16/17） of cases exhibiting methylation. FMs of TGFβR Ⅱ were identified in 5 （62.5%） of the 8 samples with MSIH. In contrast, FMs were not found in MSI-L or microsatellite stable （MSS） cases. CONCLUSION MSIs and FMs of TGFβR Ⅱ may play an important role in gastric carcinogenesis. HMLH1 methylation is an important modification of the DNA which results in inactivation of hMLH1 and mismatch repair defects which lead to MSls and FMs of TGFβR Ⅱ.

Adult hereditary fructose intolerance

Hereditary fructose intolerance(HFI) is an underrecognized,preventable life-threatening condition.It is an autosomal recessive disorder with subnormal activity of aldolase B in the liver,kidney and small bowel.Symptoms are present only after the ingestion of fructose,which leads to brisk hypoglycemia,and an individual with continued ingestion will exhibit vomiting,abdominal pain,failure to thrive,and renal and liver failure.A diagnosis of HFI was made in a 50-year-old woman on the basis of medical history,response to fructose intolerance test,demonstration of aldolase B activity reduction in duodenal biopsy,and molecular analysis of leukocyte DNA by PCR showed homozygosity for two doses of mutant gene.HFI may remain undiagnosed until adult life and may lead to disastrous complications following inadvertent fructose or sorbitol infusion.Several lethal episodes of HFI following sorbitol and fructose infusion have been reported.The diagnosis can only be suspected by taking a careful dietary history,and this can present serious complications.

Homozygous lethality and heterozygous spotting due to a novel missense mutation in the mouse Kit gene

N-ethyl-N-nitrosourea （ENU） mutagenesis in mice can be used to study gene function in vivo and to establish genetic mouse models of human disease. In this study, a white spotted mouse （named Kit^W-1 Bao） was obtained by ENU-induced mutagenesis. Inheritance testing showed a single-gene dominant mutation and lethality in the Kit^W-1 Bao homozygous mice. The mutation was mapped to Chromosome 5 between markers DSMit356 and DSMit308. The region contains the Kit gene, whose mutations are known to lead to pigmentation defects in mice. Sequence analysis of the Kit cDNA from Kit^W-1 Bao heterozygotes revealed an A to T missense mutation resulting in an amino acid substitution of Asp （D） by Val （V） at amino acid position 849 within a highly conserved tyrosine kinase domain. The combined phenotype displayed by the Kit^W-1 Bao heterozygous and homozygous mutant mice demonstrates the critical function of the highly conserved aspartie acid residue at position 849 in the Kit gene product

Differential roles of EPS8 in carcinogenesis:Loss of protein expression in a subset of colorectal carcinoma and adenoma

AIM:To analyze the epidermal growth factor receptor pathway substrate 8(EPS8) expression status and role in colorectal carcinogenesis given that EPS8 has a conserved actin barbed-end capping function that is required for proper maturation in intestinal cells.METHODS:We studied 8 colon cancer cell lines and 58 colorectal tumors(19 adenomas and 39 carcinomas).We performed expression microarray analysis of colon cancer cell lines followed by loss of heterozygosity(LOH)analysis and immunohistochemistry for EPS8 expression in colon tumors.Subsequently,we performed mutation analysis by direct sequencing and methylation analysis by bisulfite sequencing and methylation-specific polymerase chain reaction assays.RESULTS:Expression microarray analysis of colon cancer cell lines showed overexpression of EPS8 transcript in all lines but RKO.Genome wide loss of heterozygosity(LOH) analysis of colon tumors,showed considerable LOH at the EPS8 gene locus.Immunohistochemically,EPS8 was constitutively expressed in normal colonic mucosa with a dot-like supranuclear localization with accentuation at the luminal surface supporting its proposed role in epithelial maturation.Nineteen colon tumors(4 adenoma,15 carcinoma) out of 51(37%) showed strikingly tumor specific EPS8 protein loss.Of the remaining tumors,5/51(2 adenoma,and 3 carcinoma,10%) showed marked overexpression,while 27/51 tumors(53%) showed retained expression.Mutation analysis revealed a missense mutation(c.794C>T,p.R265C) in exon 8 in RKO.The EPS8 promoter was also methylated in RKO,but there was no significant methylation in other cell lines or carcinoma specimens.CONCLUSION:The loss of EPS8 expression in colorectal adenomas and carcinomas suggests that down regulation of this gene contributes to the development of a subset of colorectal cancers,a finding which could have applications in diagnosis and treatment.

Mutations in Exons of the CYP17-Ⅱ Gene Affect Sex Steroid Concentration in Male Japanese Flounder(Paralichthys olivaceus)

As a specific gene of fish,cytochrome P450c17-Ⅱ(CYP17-Ⅱ) gene plays a key role in the growth,development and reproduction level of fish.In this study,the single-stranded conformational polymorphism(SSCP) technique was used to characterize polymorphisms within the coding region of CYP17-Ⅱ gene in a population of 75 male Japanese flounder(Paralichthys olivaceus).Three single nucleotide polymorphisms(SNPs) were identified in CYP17-Ⅱ gene of Japanese flounder.They were c.G594A(p.G188R),c.G939A and c.G1502A(p.G490D).SNP1(c.G594A),located in exon 4 of CYP17-Ⅱ gene,was significantly associated with gonadosomatic index(GSI).Individuals with genotype GG of SNP1 had significantly lower GSI(P < 0.05) than those with geno-type AA or AG.SNP2(c.G939A) located at the CpG island of CYP17-Ⅱ gene.The mutation changed the methylation of exon 6.Indi-viduals with genotype AA of SNP2 had significantly lower serum testosterone(T) level and hepatosomatic index(HSI) compared to those with genotype GG.The results suggested that SNP2 could influence the reproductive endocrine of male Japanese flounder.How-ever,the SNP3(c.G1502A) located in exon 9 did not affect the four measured reproductive traits.This study showed that CYP17-Ⅱ gene could be a potentially useful candidate gene for the research of genetic breeding and physiological aspects of Japanese flounder.

Unexpected discovery of 2 cases of hepatocyte nuclear factor 1α-mutated infracentimetic adenomatosis

We present 2 cases of hepatocyte nuclear factor 1α (HNF1α)-mutated adenomatosis, discovered for reasons unrelated to this disease, and identified using immunohistochemical methods. These new tools may further our understanding of the link between adenomas/adenomatosis subtypes and their complications, and their association with other abnormalities.

Specific VpU Codon Changes were Significantly associated with gp120 V3 Tropic Signatures in HIV-1 B-subtype

After infection and integration steps, HIV-1 transcriptions increase sharply and singly-spliced mRNAs are produced. These encode Env （gpl20 and gp41） and auxiliary proteins Vif, Vpr and VpU. The same localization within the unique structure of the mRNAs suggests that the VpU sequence prior to the Env could affect the Env polyprotein expression.The HIV-I infection process begins when the gpl20 subunit of the envelope glycoprotein complex interacts with its receptor（s） on the target cell. The V3 domain of gpl20 is the major determinant of cellular co-receptor binding. According to phenotypic information of HIVol isolates, sequences from the VpU to V3 regions （119 in R5- and 120 X4-tropic viruses; one per patient） were analysed. The binomial correlation phi coefficient was used to assess covariation among VpU and gpl20v3 signatures. Subsequently, average linkage hierarchical agglomerative clustering was performed. Beyond the classical V3 signatures （R5-viruses： SI1, E25D; X4-viruses： SllKR, E25KRQ）, other specific V3 and novel VpU signatures were found to be statistically associated with co-receptor usage. Several statistically significant associations between V3 and VpU mutations were also observed. The dendrogram showed two distinct large clusters： one associated with R5-tropic sequences （bootstrap=0.94）, involving： （a） H13NPv3, E25Dv3, Sllv3, T22Av3 and Q61Hvpu, （b） E25Av3 and L12Fvpu, （c） D44Evpu, R18Qv3 and D80Nvpu; and another associated with X4-tropic sequences （bootstrap=0.97）, involving： （i） E25Iv3 and V10Avpu, （ii） 0-1insVvpc, H13Rv3, I46Lvpc, I30Mv3 and 60-62delvpu, （iii） SllKRv3 and E25KRQv3. Some of these pairs of mutations were encoded always by one specific codon. These data indicate the possible VpU mutational patterns contributing to regulation of HIV-I tropism.