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The stable expression of antibodies in plants is one recent strategy for the unconventional control of plant viruses that is undergoing development. The advantages of this approach are its wide applicability and intrinsic safety; however, to be successful, the 'genetic immunization' of plants requires careful antibody design, efficient expression and targeting to appropriate cell compartments.
Retroviral transduction of antifolate-resistant variants of human dihydrofolate reductase (hDHFR) into cells can increase their resistance to the cytotoxic effects of these drugs. We evaluated the ability of wild-type hDHFR and 20 mutant enzymes (13 with single-amino acid substitutions, 7 with two substitutions) to prevent growth inhibition in antifolate-treated CCRF-CEM cells. The wild-type enzyme and all of the variants significantly protected transduced cells from trimetrexate (TMTX)-induced growth inhibition. However, only half of the variants conferred more protection than does the wild-type enzyme. For the variants tested, the observed protective effect was higher for TMTX than for methotrexate (< or =7.5-fold increased resistance), piritrexim (< or =16-fold), and edatrexate (negligible). Transduction of the variants L22Y-F31S and L22Y-F31R led to the greatest protection against TMTX (approximately 200-fold). Protection from loss of cell viability was similar to protection from growth inhibition. The protection associated with a particular mutant hDHFR did not result from the level of expression: Efficient protection resulted from low affinity of the variant for antifolates, reasonable catalytic activity, and good thermal stability. Clones isolated from a polyclonal population of transduced cells varied by as much as 30-fold in their resistance to TMTX, the resistance differences depending on hDHFR expression levels.
The cis-acting sequences that mediate insulin gene expression exclusively in pancreatic islet beta-cells are localized within the 5'-flanking region between nucleotides -340 and -91. We have identified an evolutionarily conserved, A+T-rich element at -201/-196 basepairs in the rat insulin II gene that is essential for efficient expression in beta-cells. Affinity-purified antibody to the XIHbox 8 protein super-shifted the major beta-cell-activator factor complex binding to the -201/-196 element. XIHbox 8 is a Xenopus endoderm-specific homeodomain protein whose expression is restricted to the nucleus of endodermal cells of the duodenum and developing pancreas. Antibody to XIHbox 8 specifically interacts with a 47-kilodalton protein present in this DNA complex. Immunohistochemical studies revealed XIHbox 8-like proteins within the nucleus of almost all mouse islet beta-cells and a subset of islet alpha- and beta-cells. These results are consistent with the proposal that an XIHbox 8-related homeoprotein of 47 kilodalton is required for expression of the mammalian insulin gene in beta-cells. Experiments conducted with antiserum raised to somatostatin transcription factor-1 (STF-1), a recently isolated mammalian XIHbox 8-related homeoprotein, indicate that the STF-1 protein is the mammalian homolog of Xenopus XIHbox 8.
The rat branched-chain-2-oxo-acid dehydrogenase (BCOD) kinase mRNA is transcribed from a TATA-less promoter that has GC-rich sequences and two putative Sp1 binding sites near the transcription start site. We demonstrated previously that the 5' region of the kinase gene, base pairs -128 to +264, contained promoter activity when assayed using luciferase as a reporter (Huang and Chuang (1996) Biochem. J. 313, 603-609). To define DNA elements required for efficient expression of the kinase gene, nested deletion constructs of the above promoter region fused with a luciferase reporter gene were transfected into cultured H4IIE (hepatoma) and NRK-52E (kidney) cells. The results showed that the region between nucleotides -58 and +21 was indispensable for the kinase basal promoter activity. Methylation-interference and mutagenesis-promoter assays identified nucleotides -50 to -40 (ACAACTCCCA) as cis-acting DNA sequences that are required for nuclear protein binding and efficient promoter activity. Gel-supershift analysis with anti-Sp1 antibody suggested that the nuclear protein capable of binding to the -58 oligonucleotide (bp -58 to -34) was immunologically related to the Sp1 protein. The -58 oligonucleotide formed a DNA-protein complex with recombinant Sp1 protein with an affinity approximately ten-fold lower than that of the consensus Sp1 oligonucleotide. Co-transfection of the Sp1 expression plasmid and the -58 promoter construct into Drosophila Schneider cells revealed that Sp1 contributed to the kinase basal promoter activity by binding to the non-consensus site in the -58 region. Deletion of two consensus Sp1 binding sites (bases -150 to -140 and bases +29 to +38) in the kinase gene did not affect the basal promoter activity. Therefore binding of Sp1 or Sp1-like proteins to the above single non-consensus Sp1 sequence in the -58 region plays a major role of transactivating basal expression of the BCOD kinase.
BACKGROUND: Sindbis virus, a blood-borne virus transmitted by mosquitoes, has been used as a vector to efficiently express exogenous genes in vitro and in vivo and to induce apoptosis. Because Sindbis virus infects mammalian cells by interacting with the high-affinity laminin receptors, which are expressed at higher levels in several human cancers than in normal cells, we determined whether a Sindbis viral vector could be used to target cancers in vivo. METHODS: C.B-17-SCID mice with established xenografts were given daily intraperitoneal injections of the Sindbis viral vector SinRep/LacZ containing the bacterial beta-galactosidase gene. Control mice were untreated or received injections with phosphate-buffered saline. Tumor size was measured daily. Expression of beta-galactosidase and Factor VIII (a marker for endothelial cells) was determined by immunohistochemical staining of tumor sections. Apoptosis was analyzed by TUNEL (terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end labeling) staining. C.B-17-SCID beige mice, which lack natural killer (NK) cells, were used to assess the importance of NK cells in antitumor efficacy of Sindbis viral vectors. RESULTS: Tumors from mice treated with SinRep/LacZ were statistically significantly smaller than tumors from control mice. This effect was observed for tumor xenografts derived from BHK (kidney, hamster), LS174T (colon, human), HT29 (colon, human), and CFPAC (pancreas, human) cells. Expression of beta-galactosidase co-localized with that of Factor VIII in tumor sections. Tumors from SinRep/LacZ-treated mice contained more apoptotic cells than tumors from control mice. Complete tumor regression was observed in three of five C.B-17-SCID mice but in none of five C.B-17-SCID beige mice treated with SinRep/LacZ. CONCLUSION: Sindbis viral vectors efficiently targeted tumors in vivo, were apparently delivered through the circulation, and were more effective in the presence of NK cells.
The nucleotide sequence of a 968 bp DNA fragment spanning the promoter and the 5' upstream sequence of the LEU2 coding sequence of the yeast Yarrowia lipolytica has been determined. A LEU2::lacZ fusion has been constructed and expressed in transformed yeast cells, showing that as few as 232 bp of the LEU2 promotor were sufficient to direct gene expression. In order to develop new markers for transformation of this yeast, the LEU2 initiation codon was destroyed by in vitro mutagenesis and replaced by a cloning site. A gene conferring phleomycin resistance in E. coli was attached to the LEU2 promoter and shown to be efficiently expressed in yeast: direct selection of phleomycin resistant transformants was possible.
The F11-receptor (F11R) (a.k.a. JAM-1, JAM-A, CD321) is a cell adhesion molecule of the immunoglobulin superfamily involved in platelet adhesion, secretion and aggregation. In addition, the F11R plays a critical role in the function of endothelial cells and in platelet adhesion to inflamed endothelium. In the present study, we used partial sequences of the human F11R gene, F11R cDNAs, and information in unannotated human genome databases, to delineate the F11R gene. We found that the F11R gene is composed of 13 exons (E1a, 1b, 1c, E1-E10) encoding two groups of mRNAs differing in length and sequence at their 5' UTRs, referred to as type 1 and type 2 messages. Type 1 cDNAs are shorter at the 5' end and contain a region not found within type 2 messages. Type 1 mRNAs are present in endothelial cells (EC), platelets, white blood cells and in the cell lines CMK, HeLa, K562, HOG and A549, while type 2 messages are limited to EC. Type 1 messages contain exons E1-E10 whereas type 2 messages usually contain exons E1a, 1c, part of E1 and E2-E10. The translation start site is localized in the 3' end of E1, common for both type 1 and type 2 messages. Expression of these messages is regulated by two alternative promoters, P1 and P2. P1 is a TATA-less promoter containing an initiator element, multiple transcription start sites, several GC and CCAAT boxes, and GATA, NF-kappaB and ets consensus sequences. The cloned P1 drives efficient expression of the luciferase reporter gene. A high level of similarity between human P1 and its rat and mouse counterparts was observed. Promoter P2, located upstream of P1, contains a TATA box, GC boxes, a CCAAT box and GATA and ets consensus sequences. 3' RACE provided evidence for variability in the 3' UTR due to the presence of two polyadenylation signals. The finding of multiple regulatory sites in the promoters supplements the biochemical evidence that the F11R has several different roles in the functional repertoire of endothelial cells, platelets and other cells. In particular, the presence of NF-kappaB provides additional evidence to the significance of the F11R function in the initiation of inflammatory thrombosis.
Expression of the more than 80 individual genes of herpes simplex virus 1 (HSV-1) takes place in a tightly regulated sequential manner that was first described over 20 years ago. Investigations since that time have focused on understanding the mechanisms that regulate this orderly and efficient expression of viral genes. This review examines recent findings that have shed light on how this process is regulated during productive infection of the cell. Although the story is still not complete, several aspects of HSV gene expression are now clearer as a result of these findings. In particular, several new functions have recently been ascribed to some of the known viral regulatory proteins. The results indicate that the viral gene expression is regulated through transcriptional as well as post-transcriptional mechanisms. In addition, it has become increasingly clear that the virus has evolved specific functions to interact with the host cell in order to divert and redirect critical host functions for its own needs. Understanding the interactions of HSV and the host cell during infection will be essential for a complete understanding of how viral gene expression is regulated. Future challenges in the field will be to develop a complete understanding of the mechanisms that temporally regulate virus gene expression, and to identify and characterize the relevant interactions between the virus and the distinctive cell types normally infected by the virus.
In this paper we study the effect of the chromophores' beta tensor active components on the diffraction efficiency of a high T(g) photorefractive polymer. In particular, we study the two simplest structures with nonvanishing dipole moment, the one-dimension push-pull systems, and the Lambda-shaped chromophores. We have developed a model that relate the diffraction efficiency expression with experimental conditions and microscopic properties of the molecules used. Using this model we determine the optimum experimental conditions for both kinds of chromophores and the criteria for the design of chromophores with improved microscopic properties. The model was also used to evaluate the diffraction efficiency of the chromophore Disperse Red 1 (DR1) with a good agreement with experimental data present in bibliography, and of other chromophores selected with the criteria derived from the model, using quantum mechanical calculations to obtain the microscopic properties. Using the designed chromophores diffraction efficiencies more than one order of magnitude higher than that calculated for DR1 with the experimental conditions has been obtained in simulations. These chromophores also exhibit a low dependency of eta on the electric field polarization in contrast to the DR1 or the low T(g) photoreactive materials.
We present a comparative study of the cis- and trans-acting elements governing the expression of the human transferrin (Tf) gene in two tissues, liver and testis, where Tf is expressed at various levels. We have previously identified the elements of the promoter, negative, and enhancer regions involved in the liver-specific expression of the gene. By transfection experiments of primary cultured rat Sertoli cells compared with hepatoma cells, DNase I footprinting, and gel retardation studies, we have analyzed 3.6 kilobase pairs of the Tf regulatory region. The far upstream enhancer functional in Hep3B cells is inactive in Sertoli cells; in the two cell types, different nuclear factors appear to bind to a DNA domain crucial for enhancer activity. Similar negative- and positive-acting elements are present in the distal promoter in both tissues. However different combinations of proximal promoter elements control tissue-specific expression. Liver-specific transcription is governed by the interaction of the Tf-LF1 protein and a C/EBP-related factor with the -125 to -45 region. In Sertoli cells, a -34 to -18 TATA box-binding factor is sufficient to initiate basal-level transcription. Efficient expression is achieved by the association of two factors binding either to the (-82, -1) or to the (-153, -52) region. The addition of a third adjacent element decreases the promoter activity, suggesting that the balance of three factors binding to the proximal sites regulates testis-specific expression.
OBJECTIVE: To construct an cell penetrating peptide-based expression vector capable of targeted delivery of proteins into the cell nuclei and study its function of protein transduction. METHODS: The fusion protein expression vector pET14b-HC(L)NE (pET14-b-His-CPP-Linker-NLS-EGFP) incorporating cell penetrating peptide (CPP), nuclear localization signal(NLS), linker and enhanced green fluorescent protein (EGFP) was constructed based on His-tagged pET14b-HE (pET14b-His-EGFP) by site-directed mutagenesis PCR method. After identification by enzyme digestion and DNA sequencing, the recombinant plasmid was transformed into BL21(DE(3)) strain. The HC(L)NE fusion protein was expressed following IPTG induction and purified with Ni(2+)-NTA affinity chromatography. After dialysis and filtration, the HC(L)NE fusion protein was added into cultured eukaryotic cells. The protein transduction in the living cells was observed under fluorescence microscope and analyzed by Western blotting. RESULTS: Enzyme digestion and DNA sequencing confirmed successful construction of the pET14b-HC(L)NE vector, and the fusion protein efficiently expressed in E. coli. Protein transduction experiments in eukaryotic cells revealed that the fusion protein could rapidly penetrate the cell membrane and reach the cell nucleus, and this internalization was time- and concentration-dependent. CONCLUSION: The cell penetrating peptide-based expression vector for targeted protein delivery to the cell nucleus has been successfully constructed, and a transport system that can delivery exogenous proteins or polypeptides into the cytoplasm and cell nucleus is established, which provides an economical and efficient means for functional study of the proteins and polypeptide in cells and targeted drug delivery.
17beta-hydroxysteroid dehydrogenase from the filamentous fungus Cochliobolus lunatus (17beta-HSDcl) has recently been characterized. Since its function is still unclear, we performed substrate specificity studies to obtain some indications about its physiological function. Different steroids were studied as putative substrates of recombinant 17beta-HSDcl, androgens and estrogens, brassinosteroids, and the fungal steroid herbarulid. Among these androgens and estrogens were most efficiently converted. The following substrates in decreasing order were best reduced: 4-estrene-3,17-dione, 5alpha-androstane-3,17-dione, 4-androstene-3,17-dione and estrone. Two typical inhibitors were tested: carbenoxolone--a representative inhibitor of the SDR family and quercetin--a diagnostic inhibitor of carbonyl reductases. Among these two quercetin was more efficient. Expression studies revealed that 17beta-HSDcl is mainly expressed in the stationary phase of growth indicating its possible involvement in secondary metabolism.
Early B cell factor (EBF) and E47 participate in the transcriptional control of early B lymphocyte differentiation. With the aim of identifying genetic targets for these transcription factors, we stably transfected cDNAs encoding EBF or a covalent homodimer of E47, individually or together, into immature hematopoietic Ba/F3 cells, which lack both factors. In combination, EBF and E47 induce efficient expression of the endogenous immunoglobulin surrogate light chain genes, lambda5 and VpreB, whereas other pre-B cell-specific genes remain silent. Multiple functionally important EBF and E47 binding sites were identified in the lambda5 promoter/enhancer region, indicating that lambda5 is a direct genetic target for these transcription factors. Taken together, these data suggest that EBF and E47 synergize to activate expression of a subset of genes that define an early stage of the B cell lineage.
Monoclonal polyreactive antibodies can bind to many structurally dissimilar self and non-self antigens. Neither the precise antigen-binding site on the polyreactive antibody molecule nor the molecular basis of polyreactivity has been elucidated. The present study was initiated to see whether antibody genes encoding the Fab fragment of a human monoclonal polyreactive IgM antibody (MoAb 67) could be efficiently expressed in Escherichia coli, and whether the bacterially expressed Fab fragments possessed biological activity. cDNA encoding the variable domains of the heavy and light chains of MoAb 67 were cloned, amplified by polymerase chain reaction (PCR) and expressed in E. coli. Neither the recombinant heavy nor light chain showed antigen-binding activity. In contrast, the recombinant Fab 67 fragment showed the same antigen-binding reactivity profile as the native IgM antibody. It is concluded that the antigen-binding activity of polyreactive antibodies resides in the Fab fragment, and that both the heavy and light chains are required for activity.
A cloned putative promoter region upstream of the 16S rRNA gene of the western X-disease phytoplasma was inserted behind the promoterless chloramphenicol acetyltransferase gene of plasmid pPL603. The DNA construct was used to transform Bacillus subtilis cells. The transformants were assayed for chloramphenicol acetyltransferase activity, showing that the phytoplasma promoter is efficiently expressed in a B. subtilis background.
EBNA-1 is a nuclear antigen of lymphocytes infected by Epstein-Barr virus and whose size polymorphism correlates only with the strain of infecting virus and the length of the glycine-alanine copolymer encoded by the third internal repeat of the viral genome. The major antigenic determinant(s) also appear to reside in this region. We have been able to obtain efficient expression of this nuclear antigen in cultured Drosophila cells transfected with a cosmid carrying the EBNA-1 coding region, indicating that insect mechanisms recognise control sequences and transcripts of the herpes virus. The association of a vimertin-like protein of mol. wt. 46,000 with Drosophila cell nuclei has been found to vary with culture conditions and heat shock. We now find that the level and nuclear association of this protein also increase after transfection with either EBNA-1 or yolk protein DNA.
The petE gene encoding plastocyanin precursor protein from the cyanobacterium Anabaena PCC 7937 was introduced in the cyanobacterial host strain Synechococcus PCC 7942. The host normally only uses cytochrome c553 as Photosystem I (PS I) donor. The heterologous gene was efficiently expressed using the inducible Escherichia coli trc promoter. Accumulation of plastocyanin protein depended on the presence of Cu2+. The protein was accurately targeted to the thylakoid lumen, from which it could be isolated in the mature form. Redox difference spectroscopy proved the presence of a Cu2+ ion in the holoenzyme. Isolated heterologous plastocyanin was functional in reconstitution of in vitro electron transfer to PS I. The presence of Anabaena plastocyanin in Synechococcus thylakoid membranes increased PS I electron transfer rate 2.5 times. Analysis of P700 redox and PS II fluorescence transients in vivo showed a faster electron transfer through PS I because of enhanced electron supply in the presence of plastocyanin. In addition, the distribution of electrons between photosynthetic and respiratory electron transfer changed. Plastocyanin preferentially donates electrons to PS I rather than to the respiratory cytochrome-c oxidase complex and is not functionally equivalent to cytochrome c553.
The amphicrine AR42J acinar cell line is an excellent model to study both exocrine and neuroendocrine exocytotic mechanisms. As a first step toward this goal, we determined the specific isoforms of the v- and t-SNARE and Munc18 families expressed in these cells. In addition, we show that dexamethasone-induced differentiation toward the exocrine phenotype causes an upregulation of several of these proteins. AR42J is notoriously difficult to transfect, limiting its usefulness as a model. However, we have now overcome this obstacle by acheiving high efficiency expression of a beta-galactosidase reporter gene and truncated SNAP-25 gene using adenoviral infection techniques. The AR42J cells can now be used to pursue and elucidate the distinct functions of individual SNARE isoforms used in endocrine and exocrine secretion within a single cell line.