The alteration of T lymphocyte functions as a consequence of human immunodeficiency virus (HIV) infection is a potential target for the genetic treatment of the acquired immunodeficiency syndrome (AIDS). One approach to the gene therapy for AIDS is to block the replication of HIV-1. Tat-dependent expression of forein gene and selective infection of CD4(+) cells by retroviral vector might be useful for abrogating the production of HIV-1 from cells. As part of studies to examine the feasibility of this concept, I constructed tat(+) and tat(-) HIV-1 proviral vectors that express all HIV-1 genes except for env and/or tat gene. When tat(+) or tat(-) HIV-1 particles were used for infection of HeLa T4 cells containing the endogenous beta-galactosidase (lacZ) gene under the control of the HIV-1 promoter and transactivation response element sequences, only the tat(+) HIV-1 particles transactivated the lacZ gene expression. This activation of lacZ expression following HIV infection of Tat(-) cells that stably contained but did not express the lacZ construct was determined to be an efficient process. I also constructed simple HIV-1 vectors that express the lacZ gene in a Tat-dependent manner or the hygromycin B phosphostransferase gene (Hyg(r)) under the control of the SV40 early promoter. The Tat-dependent vector conferring the lacZ(+) phenotype was assayed by beta-gal staining after infection of Tat(+) or Tat(-) cells. The activation of lacZ expression was observed only in tat(+) cells. Another simple HIV-1 vector containing the Hyg(r) gene was used for retroviral production from HeLa cells expressing the HIV-1 env gene and infection of CD4(+) or CD4(-) cells, but Hyg(r) colony was observed only from CD4(+) cells. These results provide a rationale for the use of HIV-1 retroviral vector system in the design of gene therapy of HIV infection.
Acquired Immunodeficiency Syndrome ; beta-Galactosidase ; CD4-Positive T-Lymphocytes ; Genes, env ; Genes, tat ; Genetic Therapy ; HeLa Cells ; HIV Infections ; HIV* ; HIV-1 ; Humans* ; Hygromycin B ; Lac Operon ; Lymphocytes ; Phenotype ; Response Elements ; Transcriptional Activation ; Zidovudine

OBJECTIVEChimeric human/bovine immunodeficiency virus (HBIV) cDNA was constructed by replacing HIV tat and LTR with bovine immunodeficiency virus (BIV) tat and LTR to study the activity of BIV tat and LTR in the chimerae.

METHODSThe target fragments of BIV tat, LTR and HIV gag, pol, env were respectively amplified by using PCR and sequentially inserted into pBluescript SK(+) vector. The chimeric clone was transfected into human MT4 cells. The transcript and gene expression of the HBIV chimeric virus were detected by using RT-PCR and a reverse transcriptase assay, respectively.

RESULTSBIV tat mRNA and HIV gag mRNA were detected. The reverse transcriptase activity of the chimeric virus was analyzed in the fluctuation curve.

CONCLUSIONSIn chimeric HBIV cDNA transfected MT?4 cells, BIV tat and HIV gag were transcripted. The reverse transcriptase of the chimeric virus had biological activity. These data suggest that in MT4 cells, BIV LTR had promoter activity and BIV tat had the function of transactivation in the chimeric virus. The study of the chimeric virus with infectivity is in progress.

AIDS Vaccines ; Animals ; Cattle ; Cloning, Molecular ; DNA, Complementary ; genetics ; DNA, Viral ; genetics ; Genes, gag ; genetics ; Genes, pol ; genetics ; Genes, tat ; genetics ; HIV-1 ; genetics ; Humans ; Immunodeficiency Virus, Bovine ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transcription, Genetic ; Transcriptional Activation ; Transfection ; Virus Replication

OBJECTIVE: The transfection efficiencies of gynecologic cancer cell lines were investigated by different mediated transfection methods using recombinant LacZ plasmid (pRcCMVLacZ and pAAVCMVLacZ). METHODS: In this study, the gynecologic cancer cell lines were used CaSki, SiHa (cervical, HPV16+, wild type p53 gene), HeLa, HeLa S3 (cervical, HPV18+, wild type p53 gene), C33A, HT3 (cervical, HPV-, p53 mutant), HckE6/E7 (cervical, HPV16 immortalized keratocyte), PA-1 (ovary, wild type p53), SKOV-3, A2774 (ovary, p53del) and OVCAR-3 (ovary, p53 mutant). The pRcCMVLacZ and pAAVCMVLacZ plasmid transfection were performed by using liposome system such as Ca2+-phosphate, Fugen6(TM), Lipofection(TM), Lipogen(TM) and N-stearyl lactobionamide (N-SLBA) with X-gal staining. The LacZ gene was used the reporter gene for the transfection efficiencies evaluation. RESULTS: Each of cell lines were showed different transfection efficiencies by Ca2+-phosphate, Fugen6(TM), Lipofectin(TM), Lipogen(TM) and N-SLBA. Each of cell were revealed that HeLa S3, HT3 and A2774 were high transfection efficiency using the pRcCMVLacZ by the Lipogen(TM), SiHa, HeLa, QGU, OVCAR-3 and PA-1 were high efficiency using the pAAVCMVLacZ by Lipofectin(TM), CaSki was high efficiency using the pRcCMVLacZ by the Lipogen(TM), A2774 and Cx16.2 were high efficiency using the pRcCMVLacZ by the Lipofectin(TM), SKOV-3 and HkcE6/E7 were high efficiency using pAAVCMVLacZ by the Lipogen(TM). CONCLUSION: As a result, We proved that each of cell lines differed trasnfection efficiencies according to mediated transfection and recombinant LacZ plasmid style. Above all, Lipofectin(TM) mediated transfection was showed high efficiency at the most of cell lines.
Cell Line* ; Genes, Reporter ; Humans* ; Lac Operon* ; Liposomes ; Plasmids* ; Transfection*

The genus Rhodococcus is of considerable interest in recent years, stemming from their diverse applications in biodegradation, bioremediation, biotransformation and biosurfactant. Using Nocardia/Rhodococcus-Escherichia coli shuttle plasmid pNV18.1 as the backbone vector, we tested the driven efficiency of promoters Ptac and PlacZ of E. coli and Pami-1/Pami-2 of R. ruber in host R. rhodochrous ATCC 33278 by overexpression of nitrile hydratase. Results showed that the specific activity of nitrile hydratase per dry cell weight in engineered Rhodococcus strains driven by Ptac, Pami-1, Pami-2 and PlacZ was 7.5, 6.3, 5.3 and 1.8 times of that in the wild, respectively. It indicated that these promoters could be well recognized by RNA polymerase of Rhodococcus. We further expressed the beta-galactosidase reporter gene (lacZ) in R. ruber driven by promoter PlacZ. Results indicated that lacZ was an appropriate reporter gene for genetic or metabolic engineering research of Rhodococcus.
Escherichia coli ; genetics ; Gene Expression Regulation, Bacterial ; Genes, Reporter ; genetics ; Lac Operon ; genetics ; Promoter Regions, Genetic ; genetics ; Rhodococcus ; enzymology ; genetics ; beta-Galactosidase ; genetics

To achieve efficient and stable expression of heterologous exogenetic protein or antigen in E. coli chromosome, the luciferase report gene was knocked in lacZ site of chromosome lac operon by using Red recombination system and selection-counterselection kan/sacB technology. The quantitative analysis of exogenous gene expression indicated that the target gene could be efficiently expressed at lacZ site of lac operon. The results confirmed the efficient screening and stable expression of heterologous protein or antigen on chromosome by using the recombinant engineering technique. This study demonstrated that the chromosome could be used as a vector for heterologous protein or antigen and the stable expression of exogenous gene on E. coli chromosome had no side effect on the bacterial growth and propagation.
Bacteriophage lambda ; genetics ; Chromosomes, Bacterial ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Knock-In Techniques ; methods ; Genes, Reporter ; genetics ; Lac Operon ; genetics ; Luciferases ; genetics ; Recombination, Genetic ; genetics

BACKGROUND AND OBJECTIVES: G207 virus is an ideal candidate of oncolytic viral therapy. It is a multi-gene mutant of HSV-1 with a deletion at the gamma34.5 loci and a LacZ gene insertion in the ICP6 gene, encoding the HSV ribonucleotide reductase. Ionizing radiation induces the growth arrest-inducible gene, Gadd34, a protein that correlates with apoptosis following radiation and has homology with the G207 gamma34.5 gene. It is hypothesized that the combination of radiotherapy with G207 virus may have a synergic effect on viral replication and efficacy. The purpose of this study is to evaluate the combination of the cytotoxic G207 virus with radiation therapy to treat head and neck tumors. MATERIALS AND METHOD: Five human SCCHN cell lines and one murine SCC cell line were utilized in this study. There were two groups of cells: control cells received no irradiation while the second group was irradiated with 400 cGy. Cells were infected with G207 vectors at a multiplicity of infection (MOI) of 0.1. Cytotoxicity assay was performed for 5 days. Cells and culture medium supernatant were collected and viral titers determined by plaque forming units on Vero cells. To evaluate infection efficiency, X-gal staining was performed at 24hr post infection. RESULTS: All head and neck squamous cancer cell lines tested demonstrated an increased susceptibility to the combination of G207 virus with radiation therapy when compared with each single modality (more than additive effect, p<0.05). Even though cell lines such as SCC25, MSKQLL2, or SCCVII were radioresistant, the combination of G207 with radiation therapy showed significantly increased cytotoxic effect. X-gal staining and viral growth curve studies demonstrated that G207 replications in radiated cell lines were not decreased as compared with non-radiated ones. CONCLUSION: The results provide preliminary support for the use of G207 oncolytic virus as a radiation adjuvant in treatment of head and neck cancer. The combination of radiation and oncolytic viral gene therapy may eventually be useful in treating patients with radio-resistant, non-resectable disease, and patients with a high probability of contracting postoperative microscopic residual diseases.
Apoptosis ; Carcinoma, Squamous Cell* ; Cell Line ; Genes, Viral ; Genetic Therapy ; Head and Neck Neoplasms ; Head* ; Herpes Simplex* ; Herpesvirus 1, Human ; Humans ; Lac Operon ; Neck* ; Oncolytic Viruses ; Radiation, Ionizing ; Radiotherapy ; Ribonucleotide Reductases ; Simplexvirus* ; Vero Cells

OBJECTIVETo study the epidemic status of human immunodeficiency virus type 1 (HIV-1) subtypes in Shenzhen and to study their transmission source and routes.

METHODSHIV-1 env and gag genes were amplified by nested PCR from uncultured peripheral blood mononuclear cells (PBMCs) obtained from 122 HIV-1 carriers confirmed in Shenzhen. The C2-V3 region (about 450 bp) of HIV-1 env and P17/ P24 region were sequenced.

RESULTSAmong 122 samples, 6 HIV-1 strains including 3 circulating recombinant forms (CRFs) of CRF01_AE, CRF08_BC, CRF07_BC and 3 subtypes of B', B, C were found in Shenzhen, and the percentages were 45.1% (55/122) for CRF01_AE, 31.1% (38/122) for CRF08_BC, 6.6% (8/122) for CRF07_BC, 14.8% (18/122) for B' subtype, 1.6% (2/122) for B subtype, and 0.8% (1/122) for C subtype. The intragroup genetic distances were (4.455 +/- 1.478)%, (2.997 +/- 1.345)%, (4.380 +/- 2.024)%, (5.186 +/- 2.487)%, and (4.869 +/- 2.638)%, respectively. In comparison with the sequence of respective international strains 01AE. TH. 90. CM240, 97CNGX-9F, CN. 97. C54A, B. US. 83. JRFL, and RLA2, the genetic distances were (5. 228 +/- 0.823)%, (3.634 +/- 1.073)%, (4.233 +/- 1.119)%, (4.950 +/- 2.564)%, and (5.795 +/- 2.198)%, respectively. The major subtypes found in injection drug users (IDUs) were CRF07_BC, CRF08_BC, and CRF01_AE strains. CRF01_AE and B' strains were epidemic mainly in sexual workers.

CONCLUSIONThere are 3 HIV-1 subtypes (B', B, C) and 3 CRFs (CRF01_AE, CRF08_BC, CRF07_BC) epidemics in Shenzhen. The predominant subtypes varies among different transmission routes. While CRF01_AE is predominant among sexual workers, CRF08_BC and CRF01_AE are major subtypes among IDU population.

Adolescent ; Adult ; China ; epidemiology ; Female ; Genes, env ; genetics ; Genes, gag ; genetics ; Genes, pol ; genetics ; HIV Infections ; epidemiology ; HIV-1 ; genetics ; Humans ; Male ; Middle Aged ; Molecular Epidemiology ; Polymerase Chain Reaction

Phylogenetic analysis was conducted to monitor transmission of HIV and to investigate the genetic structure of primary isolates from 12 HIV-1 subtype A infected Koreans. The individuals infected with subtype A viruses had been diagnosed as HIV-1 seropositives during the period 1987 to 1995 and blood samples have been collected from 1991 to 1997. DNA of each individual was isolated from uncultured or cultured peripheral blood mononuclear cells. V3-V5 (0.7 kb) fragment of HIV-1 rev gene was amplified by nested polymerase chain reaction and the PCR products were sequenced. The mean value of the divergence of nucleotide of HIV-1 euv V3-V5 fragment was 17.0+/-4.06% (8.6~25.8%) within HIV-1 subtype A isolates from Koreans. This diversity was higher than those of African isolates (13.7+/-2.66%). In the phylogenetic tree, Korean subtype A isolates were not grouped together, but intermingled into African isolates. The results of this study suggested that HIV-1 subtype A variants be introduced from multiple sites of Africa into Korea and the big genetic diversity of Korea HIV-1 subtype A isolates may be further influenced by the range of geographic locations in which the infection occurred rather than the elapsed time between infection and collection of samples and the disease progression.
Africa ; Disease Progression ; DNA ; Genes, env* ; Genes, rev ; Genetic Structures ; Genetic Variation ; Geographic Locations ; HIV ; HIV-1* ; Korea ; Polymerase Chain Reaction

Phylogenetic analysis was conducted to monitor transmission of HIV and to investigate the genetic structure of primary isolates from 12 HIV-1 subtype A infected Koreans. The individuals infected with subtype A viruses had been diagnosed as HIV-1 seropositives during the period 1987 to 1995 and blood samples have been collected from 1991 to 1997. DNA of each individual was isolated from uncultured or cultured peripheral blood mononuclear cells. V3-V5 (0.7 kb) fragment of HIV-1 rev gene was amplified by nested polymerase chain reaction and the PCR products were sequenced. The mean value of the divergence of nucleotide of HIV-1 euv V3-V5 fragment was 17.0+/-4.06% (8.6~25.8%) within HIV-1 subtype A isolates from Koreans. This diversity was higher than those of African isolates (13.7+/-2.66%). In the phylogenetic tree, Korean subtype A isolates were not grouped together, but intermingled into African isolates. The results of this study suggested that HIV-1 subtype A variants be introduced from multiple sites of Africa into Korea and the big genetic diversity of Korea HIV-1 subtype A isolates may be further influenced by the range of geographic locations in which the infection occurred rather than the elapsed time between infection and collection of samples and the disease progression.
Africa ; Disease Progression ; DNA ; Genes, env* ; Genes, rev ; Genetic Structures ; Genetic Variation ; Geographic Locations ; HIV ; HIV-1* ; Korea ; Polymerase Chain Reaction

Among a number of antigens characterized in M. leprae, an etiological agent of Leprosy, the 18 kDa antigen, is unique to M. leprae. We have previously determined a sequence specific element in the 18 kDa gene of M. leprae, which confers transcriptional repression. In this report, we have examined if the element could be applied to genes other than the 18 kDa gene of M. leprae. To identify the roles of the regulatory sequence in heterologous promoter, we have constructed pB3 vector series, which contains BCG hsp65 promoter and the M. leprae 18 kDa transcription repression responsive element in tandem using LacZ gene as a reporter gene. Cloning of hsp65 promoters of M. bovis BCG or M. smegmatis in front of LacZ gene resulted in normal beta- galactosidase activity as expected. However, when the sequence element was placed between the promoter and the LacZ gene, beta-galactosidase activity was reduced 10-fold less. Also we have examined with pB3(-) vector, that harbors the transcription repression responsive element in a reversed orientation, the beta-galactosidase activity was found to be similar to pB3(+) vector. Thus, these results further confirm that M. leprae 18 kDa transcription repression responsive element could regulate BCG hsp65 heterologous promoter and that the element could act as an operator for the transcription of mycobacteria.
beta-Galactosidase ; Clone Cells ; Cloning, Organism ; Galactosidases ; Genes, Reporter ; Lac Operon ; Leprosy ; Mycobacterium bovis* ; Mycobacterium leprae ; Repression, Psychology*