Showing papers on "Gene expression published in 1990"

Direct gene transfer into mouse muscle in vivo.

Abstract: RNA and DNA expression vectors containing genes for chloramphenicol acetyltransferase, luciferase, and beta-galactosidase were separately injected into mouse skeletal muscle in vivo. Protein expression was readily detected in all cases, and no special delivery system was required for these effects. The extent of expression from both the RNA and DNA constructs was comparable to that obtained from fibroblasts transfected in vitro under optimal conditions. In situ cytochemical staining for beta-galactosidase activity was localized to muscle cells following injection of the beta-galactosidase DNA vector. After injection of the DNA luciferase expression vector, luciferase activity was present in the muscle for at least 2 months.

Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans.

Abstract: We attempted to overexpress chalcone synthase (CHS) in pigmented petunia petals by introducing a chimeric petunia CHS gene. Unexpectedly, the introduced gene created a block in anthocyanin biosynthesis. Forty-two percent of plants with the introduced CHS gene produced totally white flowers and/or patterned flowers with white or pale nonclonal sectors on a wild-type pigmented background; none of hundreds of transgenic control plants exhibited such phenotypes. Progeny testing of one plant demonstrated that the novel color phenotype co-segregated with the introduced CHS gene; progeny without this gene were phenotypically wild type. The somatic and germinal stability of the novel color patterns was variable. RNase protection analysis of petal RNAs isolated from white flowers showed that, although the developmental timing of mRNA expression of the endogenous CHS gene was not altered, the level of the mRNA produced by this gene was reduced 50-fold from wild-type levels. Somatic reversion of plants with white flowers to phenotypically parental violet flowers was associated with a coordinate rise in the steady-state levels of the mRNAs produced by both the endogenous and the introduced CHS genes. Thus, in the altered white flowers, the expression of both genes was coordinately suppressed, indicating that expression of the introduced CHS gene was not alone sufficient for suppression of endogenous CHS transcript levels. The mechanism responsible for the reversible co-suppression of homologous genes in trans is unclear, but the erratic and reversible nature of this phenomenon suggests the possible involvement of methylation.

pEF-BOS, a powerful mammalian expression vector.

Abstract: Polypeptide chain elongation factor l a (EF-la) is an eukaryotic counterpart of E. coli EF-Tu which promotes the GTP-dependent binding of an aminoacyl-tRNA to ribosomes. EF-la is one of the most abundant proteins in eukaryotic cells, and expressed in almost all kinds of mammalian cells. Recently, we have isolated human chromosomal gene coding for EF-la, and shown that the promoter of EF-la chromosomal gene very efficiently stimulates the in vitro transcription (1). In this report, we have constructed a powerful mammalian expression vector, pEF-BOS, using the promoter of human EF-la chromosomal gene. As shown in Fig. 1, pEF-BOS carries the SV40 replication origin (311 bp of EcoRH G fragment), the promoter region of human EF-la chromosomal gene (1.2 kb), the stuffer fragment (450 bp) from CDM8 vector (2) and poly(A) adenylation signal from human G-CSF cDNA (700 bp EcoSll ~ EcoRl DNA fragment) (3) in HindUL-EcoRl site of pUC119. The promoter region of EF-la gene is from nucleotide position 373 to 1561 (1) which includes 203 bp 5' flanking region, 33 bp first exon, 943 bp first intron and 10 bp of the part of the second exon located at 20 bp upstream of the ATG initiation codon. The size of pEFBOS is 5.8 kb, and the cDNA to be expressed can be inserted at BstXl site using BstXl adapter, or Xbal site using Xbal linker. Human G-CSF cDNA (4) was inserted into BstXl site of pEFBOS or CDM8, or into BamhU site of pKCR vector containing SV40 early promoter (5). As shown in Table 1, when these plasmids were transfected into COS cells by DEAEdextran/chloroquine method, the construct in pEF-BOS has directed the synthesis of human G-CSF about 20 times more efficiently than the construct in CDM8, and 50 ~ 200 times more efficiently than the construct in pKCR. In addition, when E. coli chloramphenicol acetyltransferase (CAT) gene was inserted into pEF-BOS, the CAT activities observed with pEF-BOS-CAT were 1.5 ~ 50 times higher than that of pSV2-CAT or pRSV-CAT after transfection into various cell lines including murine L929, human HeLa, CHU-2 and simian COS cells (Table 2). The pEFBOS vector, therefore, will be used to produce a large amount of growth factors and proteins in mammalian cells, to express a high level of anti-sense RNA. Furthermore, the pEF-BOS-CAT will be an ideal positive control for CAT assay in various cell types.

Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction

Abstract: The expression of two cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3), has been investigated in MLA-144 cells before and after induction with phorbol 12-myristate 13-acetate. We describe an adaptation of the polymerase chain reaction (PCR) for highly accurate quantitation of mRNA or DNA from a small number of cells. Aliquots of the PCR mixture containing cDNA copies of the RNA to be assayed were added to serial dilutions of a competitor DNA fragment that differed from the cDNA of interest by having either a small intron or a mutated internal restriction enzyme site. Therefore, the same primers were used to coamplify the unknown and the competitor. The ratio of products remains constant through the amplification and can be readily quantitated. In unstimulated cells, no GM-CSF or IL-3 mRNA could be detected. However, with appropriate induction, mRNA for both cytokines was detected and quantitated in as few as 200 cells. Competitive PCR was also used to accurately quantitate the copy number of the human GM-CSF gene in normal human cells, in a clonal population of cells from a patient with 5q- syndrome, and in a human-hamster cell line known to have only one copy of the human GM-CSF gene.

Progressive development of the rat osteoblast phenotype in vitro: Reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix

Abstract: The relationship of cell proliferation to the temporal expression of genes characterizing a developmental sequence associated with bone cell differentiation was examined in primary diploid cultures of fetal calvarial derived osteoblasts by the combined use of autoradiography, histochemistry, biochemistry, and mRNA assays of osteoblast cell growth and phenotypic genes. Modifications in gene expression define a developmental sequence that has 1) three principle periods–;proliferation, extracellular matrix maturation, and mineralization–;and 2) two restriction points to which the cells can progress but cannot pass without further signal–;the first when proliferation is down-regulated and gene expression associated with extracellular matrix maturation is induced, and the second when mineralization occurs. Initially, actively proliferating cells, expressing cell cycle-and cell growth-regulated genes, produce a fibronectin/type I collagen extracel-lular matrix. A reciprocal and functionally coupled relationship between the decline in proliferative activity and the subsequent induction of genes associated with matrix maturation and mineralization is supported by 1) a temporal sequence of events in which there is an enhanced expression of alkaline phos-phatase immediately following the proliferative period, and later, an increased expression of osteocalcin and osteopontin at the onset of mineralization; 2) increased expression of a specific subset of osteoblast phenotype markers, alkaline phosphatase and osteopontin, when proliferation is inhibited by hydroxyurea; and 3) enhanced levels of expression of the osteoblast markers as a function of ascorbic acid-induced collagen deposition, suggesting that the extracellular matrix contributes to both the shutdown of proliferation and the development of the osteoblast phenotype.

Progressive Development of the Rat Osteoblast Phenotype In Vitro: Reciprocal Relationships in Expression of Genes Associated With Osteoblast Proliferation and Differentiation During Formation of the

Abstract: The relationship of cell proliferation to the temporal expression of genes characterizing a developmental sequence associated with bone cell differentiation was examined in primary diploid cultures of fetal calvarial derived osteoblasts by the combined use of autoradiography, histochemistry, biochemistry, and mRNA assays of osteoblast cell growth and phenotypic genes. Modifications in gene expression define a developmental sequence that has 1) three principle periodsproliferation, extracellular matrix maturation, and mineralization-and 2) two restriction points to which the cells can progress but cannot pass without further signals-the first when proliferation is down-regulated and gene expression associated with extracellular matrix maturation is induced, and the second when mineralization occurs. Initially, actively proliferating cells, expressing cell cycleand cell growth-regulated genes, produce a fibronectinhype I collagen extracellular matrix. A reciprocal and functionally coupled relationship between the decline in proliferative activity and the subsequent induction of genes associated with matrix maturation and mineralization is supported by 1) a temporal sequence of events in which there is an enhanced expression of alkaline phosphatase immediately following the proliferative period, and later, an increased expression of osteocalcin and osteopontin at the onset of mineralization; 2) increased expression of a specific subset of osteoblast phenotype markers, alkaline phosphatase and osteopontin, when proliferation is inhibited by hydroxyurea; and 3) enhanced levels of expression of the osteoblast markers as a function of ascorbic acid-induced collagen deposition, suggesting that the extracellular matrix contributes to both the shutdown of proliferation and the development of the osteoblast phenotype.

Amplified RNA synthesized from limited quantities of heterogeneous cDNA.

Abstract: The heterogeneity of neural gene expression and the spatially limited expression of many low-abundance messenger RNAs in the brain has made cloning and analysis of such messages difficult. To generate amounts of nucleic acids sufficient for use in standard cloning strategies, we have devised a method for producing amplified heterogeneous populations of RNA from limited quantities of cDNA. Whole cerebellar RNA was primed with a synthetic oligonucleotide containing the T7 RNA polymerase promoter sequence 5' to a polythymidylate region. After second-strand cDNA synthesis, T7 RNA polymerase was used to generate amplified antisense RNA (aRNA). Up to 80-fold molar amplification has been achieved from nanogram quantities of cDNA. The amplified material is similar in size distribution to the parent cDNA and shows sequence heterogeneity as assessed by Southern and Northern blot analysis. Specific messages for moderate-abundance mRNAs for actin and guanine nucleotide-binding protein (G-protein) alpha subunits have been detected in the amplified material. By using in situ transcription to generate cDNA, sequences for cyclophilin have been detected in aRNA derived from single cerebellar tissue sections. cDNA derived from a single cerebellar Purkinje cell also has been amplified and yields material that hybridizes to cognate whole RNA and mRNA but not to Escherichia coli RNA.

Activation of interleukin-6 gene expression through the NF-kappa B transcription factor.

Abstract: The promoter region of the interleukin-6 (IL-6) gene has a putative NF-kappa B-binding site. We found that a fragment of the IL-6 promoter containing the site specifically binds highly purified NF-kappa B protein and the NF-kappa B protein in nuclear extracts of phorbol ester-induced Jurkat cells. Mutations of the NF-kappa B site abolished complex formation with both purified NF-kappa B and the nuclear extract protein. Transient expression of chloramphenicol acetyltransferase (CAT) plasmids containing the IL-6 promoter revealed very little activity of the promoter in U-937 monocytic cells and in HeLa cells before stimulation. However, stimulation of U-937 and HeLa cells by inducers of NF-kappa B led to a dramatic increase in CAT activity. Mutations in the NF-kappa B-binding site abolished inducibility of IL-6 promoter-cat constructs in U-937 cells by lipopolysaccharide, tumor necrosis factor alpha, the double-stranded RNA poly(IC), or phytohemagglutinin and in HeLa cells by tumor necrosis factor alpha and drastically reduced but did not completely eliminate inducibility in HeLa cells stimulated by double-stranded RNA poly(IC) or phorbol 12-myristate 13-acetate. These results suggest that NF-kappa B is an important mediator for activation of the IL-6 gene by a variety of IL-6 inducers in both U-937 and HeLa cells and that alternative inducible enhancer elements contribute in a cell-specific manner to IL-6 gene induction. Because NF-kappa B is involved in the control of a variety of genes activated upon inflammation, NF-kappa B may play a central role in the inflammatory response to infection and tissue injury.

The t(15;17) translocation of acute promyelocytic leukaemia fuses the retinoic acid receptor α gene to a novel transcribed locus

Abstract: RETINOIC acid is a vitamin A derivative with striking effects on development and cell differentiation1–3. Several nuclear retinoic acid receptors (RARs), acting as ligand-inducible transcription factors, have been characterized4–8 and indirect evidence suggests that they have distinct roles9–11. One of the most intriguing properties of retinoic acid is its ability to induce in vivo differentiation of acute promyelocytic leukaemia (APL) cells into mature granulocytes, leading to morphological complete remissions12–13. Because the RARα gene maps to chromosome 17q21 (ref. 14), close to the t(15;17) (q21–qll–22) translocation specifically associated with APL15, we analysed RARα gene structure and expression in APL cells. We report here that, in one APL-derived cell line, the RARα gene has been translocated to a locus, myl, on chromosome 15, resulting in the synthesis of a myl/RARα fusion messenger RNA. Using two probes located on either side of the cloned breakpoint, we have found genomic rearrangements of one or other locus in six patients out of eight, demonstrating that the RARα and/or myl genes are frequently rearranged in APL and the breakpoints are clustered. These findings strongly implicate retinoic acid receptor α in leukaemogenesis.

Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression.

Abstract: To evaluate the effect of increased expression of genes involved in flower pigmentation, additional dihydroflavonol-4-reductase (DFR) or chalcone synthase (CHS) genes were transferred to petunia. In most transformants, the increased expression had no measurable effect on floral pigmentation. Surprisingly, however, in up to 25% of the transformants, a reduced floral pigmentation, accompanied by a dramatic reduction of DFR or CHS gene expression, respectively, was observed. This phenomenon was obtained with both chimeric gene constructs and intact CHS genomic clones. The reduction in gene expression was independent of the promoter driving transcription of the transgene and involved both the endogenous gene and the homologous transgene. The gene-specific collapse in expression was obtained even after introduction of only a single gene copy. The similarity between the sense transformants and regulatory CHS mutants suggests that this mechanism of gene silencing may operate in naturally occurring regulatory circuits.

The product of the H19 gene may function as an RNA.

Abstract: The mouse H19 gene was identified as an abundant hepatic fetal-specific mRNA under the transcriptional control of a trans-acting locus termed raf. The protein this gene encoded was not apparent from an analysis of its nucleotide sequence, since the mRNA contained multiple translation termination signals in all three reading frames. As a means of assessing which of the 35 small open reading frames might be important to the function of the gene, the human H19 gene was cloned and sequenced. Comparison of the two homologs revealed no conserved open reading frame. Cellular fractionation showed that H19 RNA is cytoplasmic but not associated with the translational machinery. Instead, it is located in a particle with a sedimentation coefficient of approximately 28S. Despite the fact that it is transcribed by RNA polymerase II and is spliced and polyadenylated, we suggest that the H19 RNA is not a classical mRNA. Instead, the product of this unusual gene may be an RNA molecule.

In vivo footprinting of a muscle specific enhancer by ligation mediated PCR

Abstract: In vivo protein-DNA interactions at the developmentally regulated enhancer of the mouse muscle creatine kinase (MCK) gene were examined by a newly developed polymerase chain reaction (PCR) footprinting procedure. This ligation mediated, single-sided PCR technique permits the exponential amplification of an entire sequence ladder. Several footprints were detected in terminally differentiated muscle cells where the MCK gene is actively transcribed. None were observed in myogenic cells prior to differentiation or in nonmuscle cells. Two footprints appear to correspond to sites that can bind the myogenic regulator MyoD1 in vitro, whereas two others represent muscle specific use of apparently general factors. Because MyoD1 is synthesized by undifferentiated myoblasts, these data imply that additional regulatory mechanisms must restrict the interaction between this protein and its target site prior to differentiation.

Isolation of an efficient actin promoter for use in rice transformation

Abstract: We have characterized the 5' region of the rice actin 1 gene (Act1) and show that it is an efficient promoter for regulating the constitutive expression of a foreign gene in transgenic rice. By constructing plasmids with 5' regions from the rice Act1 gene fused to the coding sequence of a gene encoding bacterial beta-glucuronidase, we demonstrate that a region 1.3 kilobases upstream of the Act1 translation initiation codon contains all of the 5'-regulatory elements necessary for high-level beta-glucuronidase (GUS) expression in transient assays of transformed rice protoplasts. The rice Act1 primary transcript has a noncoding exon separated by a 5' intron from the first coding exon. Fusions that lack this Act1 intron showed no detectable GUS activity in transient assays of transformed rice protoplasts. Deletion analysis of the Act1 5' intron suggests that the intron-mediated stimulation of GUS expression is associated, in part, with an in vivo requirement for efficient intron splicing.

p53 functions as a cell cycle control protein in osteosarcomas.

Abstract: Mutations in the p53 gene have been associated with a wide range of human tumors, including osteosarcomas. Although it has been shown that wild-type p53 can block the ability of E1a and ras to cotransform primary rodent cells, it is poorly understood why inactivation of the p53 gene is important for tumor formation. We show that overexpression of the gene encoding wild-type p53 blocks the growth of osteosarcoma cells. The growth arrest was determined to be due to an inability of the transfected cells to progress into S phase. This suggests that the role of the p53 gene as an antioncogene may be in controlling the cell cycle in a fashion analogous to the check-point control genes in Saccharomyces cerevisiae.

Overexpression of HER-2/neu is associated with poor survival in advanced epithelial ovarian cancer.

Abstract: Previous studies have suggested that overexpression of HER-2/neu oncogene occurs in 15-40% of breast cancers and that overexpression is associated with poor prognosis. In the present report, we have used an immunohistochemical technique involving a monoclonal antibody specifically reactive with the external domain of HER-2/neu to study expression of HER-2/neu in frozen sections of normal ovary and advanced epithelial ovarian cancer. The intensity of staining for HER-2neu was always moderate or less (0-2+) in normal ovarian epithelium. Among 73 ovarian cancers, 50 (68%) had staining similar to that for normal ovarian epithelium (0-2+) while 23 (32%) stained heavily (3+). Survival of the 23 patients with high HER-2/neu expression (median, 15.7 months) was significantly worse (P = 0.001) than that of the 50 patients (median, 32.8 months) with normal HER-2/neu expression. In addition, patients whose tumors had high HER-2/neu expression were significantly less likely to have a complete response to primary therapy (P less than 0.05) or have a negative second-look laparotomy when serum CA 125 levels were normal preoperatively (P less than 0.05). These findings suggest that HER-2/neu deserves further evaluation as a prognostic marker in epithelial ovarian cancer.

The cauliflower mosaic virus 35s promoter : combinatorial regulation of transcription in plants

Abstract: Appropriate regulation of transcription in higher plants requires specific cis elements in the regulatory regions of genes and their corresponding trans-acting proteins. Analysis of the cauliflower mosaic virus (CaMV) 35S promoter has contributed to the understanding of transcriptional regulatory mechanisms. The intact 35S promoter confers constitutive expression upon heterologous genes in most plants. Dissection into subdomains that are able to confer tissue-specific gene expression has demonstrated that the promoter has a modular organization. When selected subdomains are combined, they confer expression not detected from the isolated subdomains, suggesting that synergistic interactions occur among cis elements. The expression patterns conferred by specific combinations of 35S subdomains differ in tobacco and petunia. This indicates that a combinatorial code of cisregulatory elements may be interpreted differently in different species.

Antisense gene that inhibits synthesis of the hormone ethylene in transgenic plants

Abstract: ETHYLENE controls many physiological and developmental processes in higher plants, including ripening of fruit, abscission, senescence and responses to wounding1. Although the accumulation of messenger RNAs in ripening fruit and senescing leaves has been correlated with ethylene production and perception2–4, the regulatory mechanisms governing ethylene synthesis and the stimulation of gene expression by ethylene are not understood. We have previously shown that the complementary DNA, pTOM13, corresponds to an mRNA whose synthesis is correlated with that of ethylene in ripening fruit and wounded leaves5,6,8. The pTOM13 mRNA encodes a protein of relative molecular mass 35,0006. The cDNA and three related genomic clones have been sequenced, but the function of the protein is unknown7–9. We show here that antisense RNA, which has previously been used only to reduce the expression of genes of known function10–12, when applied to pTOM13, reduces ethylene synthesis in a gene dosage-dependent manner. Analysis of these novel mutants suggests that pTOM13 encodes a polypeptide involved in the conversion of 1-amino-cyclopropane-1-carboxylic acid to ethylene by the ethylene-forming enzyme (ACC-oxidase).

Different Temporal and Spatial Gene Expression Patterns Occur during Anther Development.

Abstract: We studied the temporal and spatial regulation of three mRNA sequence sets that are present exclusively, or at elevated levels, in the tobacco anther. One mRNA set accumulates in the tapetum and decays as the tapetum degenerates later in anther development. The second mRNA set accumulates after the tapetal-specific mRNAs, is localized within the stomium and connective, and also decays as these cell types degenerate during anther maturation. The third mRNA sequence set persists throughout anther development and is localized within most anther tissues. A tapetal-specific gene, designated as TA29, was isolated from a tobacco genome library. Runoff transcription studies and experiments with chimeric [beta]-glucuronidase and diphtheria toxin A-chain genes showed that the TA29 gene is regulated primarily at the transcriptional level and that a 122-base pair 5[prime] region can program the tapetal-specific expression pattern. Destruction of the tapetum by the cytotoxic gene had no effect on the differentiation and/or function of surrounding sporophytic tissues but led to the production of male-sterile plants. Together, our studies show that several independent gene expression programs occur during anther development and that these programs correlate with the differentiated state of specific anther cell types.

Metabolic repression of transcription in higher plants.

Abstract: Using freshly isolated maize mesophyll protoplasts and a transient expression method, I showed that the transcriptional activity of seven maize photosynthetic gene promoters is specifically and coordinately repressed by the photosynthetic end products sucrose and glucose and by the exogenous carbon source acetate. Analysis of deleted, mutated, and hybrid promoters showed that sugars and acetate inhibit the activity of distinct positive upstream regulatory elements without a common consensus. The metabolic repression of photosynthetic genes overrides other forms of regulation, e.g., light, tissue type, and developmental stage. Repression by sugars and repression by acetate are mediated by different mechanisms. The identification of conditions that avoid sugar repression overcomes a major obstacle to the study of photosynthetic gene regulation in higher plants.

Quantitative analysis of MDR1 (multidrug resistance) gene expression in human tumors by polymerase chain reaction.

Abstract: The resistance of tumor cells to chemotherapeutic drugs is a major obstacle to successful cancer chemotherapy. In human cells, expression of the MDR1 gene, encoding a transmembrane efflux pump (P-glycoprotein), leads to decreased intracellular accumulation and resistance to a variety of lipophilic drugs (multidrug resistance; MDR). The levels of MDR in cell lines selected in vitro have been shown to correlate with the steady-state levels of MDR1 mRNA and P-glycoprotein. In cells with a severalfold increase in cellular drug resistance, MDR1 expression levels are close to the limits of detection by conventional assays. MDR1 expression has been frequently observed in human tumors after chemotherapy and in some but not all types of clinically refractory tumors untreated with chemotherapeutic drugs. We have devised a highly sensitive, specific, and quantitative protocol for measuring the levels of MDR1 mRNA in clinical samples, based on the polymerase chain reaction. We have used this assay to measure MDR1 gene expression in MDR cell lines and greater than 300 normal tissues, tumor-derived cell lines, and clinical specimens of untreated tumors of the types in which MDR1 expression was rarely observed by standard assays. Low levels of MDR1 expression were found by polymerase chain reaction in most solid tumors and leukemias tested. The frequency of samples without detectable MDR1 expression varied among different types of tumors; MDR1-negative samples were most common among tumor types known to be relatively responsive to chemotherapy.

In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment.

Abstract: Chimeric chloramphenicol acetyltransferase and beta-galactosidase marker genes were coated onto fine gold particles and used to bombard a variety of mammalian tissues and cells. Transient expression of the genes was obtained in liver, skin, and muscle tissues of rat and mouse bombarded in vivo. Similar results were obtained with freshly isolated ductal segments of rat and human mammary glands and primary cultures derived from these explants. Gene transfer and transient expression were also observed in eight human cell culture lines, including cells of epithelial, endothelial, fibroblast, and lymphocyte origin. Using CHO and MCF-7 cell cultures as models, we obtained stable gene transfer at frequencies of 1.7 x 10(-3) and 6 x 10(-4), respectively. The particle bombardment technology thus provides a useful means to transfer foreign genes into a variety of mammalian somatic cell systems. The method is applicable to tissues in vivo as well as to isolated cells in culture and has proven effective with all cell or tissue types tested thus far. This technology may therefore prove to be applicable in various aspects of gene therapy.

Site-specific gene expression in vivo by direct gene transfer into the arterial wall.

Abstract: A recombinant beta-galactosidase gene has been expressed in a specific arterial segment in vivo by direct infection with a murine amphotropic retroviral vector or by DNA transfection with the use of liposomes. Several cell types in the vessel wall were transduced, including endothelial and vascular smooth muscle cells. After retroviral infection, a recombinant reporter gene was expressed for at least 5 months, and no helper virus was detected. Recombinant gene expression achieved by direct retroviral infection or liposome-mediated DNA transfection was limited to the site of infection and was absent from liver, lung, kidney, and spleen. These results demonstrate that site-specific gene expression can be achieved by direct gene transfer in vivo and could be applied to the treatment of such human diseases as atherosclerosis or cancer.

Expression of the HER-2/neu proto-oncogene in normal human adult and fetal tissues.

Abstract: The HER-2/neu proto-oncogene is homologous with, but distinct from, the epidermal growth factor receptor. Current evidence indicates that this gene is frequently amplified and/or overexpressed in some human breast and ovarian cancers and that these alterations may be clinically important; however, little is known about the expression pattern of the gene in normal tissues. Using immunohistochemistry and northern blot analyses to identify the HER-2/neu protein and transcript respectively, we have evaluated a variety of normal adult and fetal tissues for HER-2/neu expression. HER-2/neu protein was identified on cell membranes of epithelial cells in the gastro-intestinal, respiratory, reproductive, and urinary tract as well as in the skin, breast and placenta. Northern hybridization confirmed the presence of the 4.5 kb transcript encoding the protein in these tissues. The amount of HER-2/neu message and protein was generally higher in fetal tissues than in the corresponding normal adult tissues. HER-2/neu expression levels in these normal tissues were similar to the levels found in non-amplified, non-overexpressing breast cancers and breast cancer cell lines. Southern hybridization of extracted DNA showed that none of the normal tissues expressing HER-2/neu had amplification of the gene. These results confirm that HER-2/neu is normally a membrane constituent of a variety of epithelial cell types.