RESUMO
The luciferase gene from the firefly, Photinus pyralis, was used as a reporter of gene expression by light production in transfected plant cells and transgenic plants. A complementary DNA clone of the firefly luciferase gene under the control of a plant virus promoter (cauliflower mosaic virus 35S RNA promoter) was introduced into plant protoplast cells (Daucus carota) by electroporation and into plants (Nicotiana tabacum) by use of the Agrobacterium tumefaciens tumor-inducing plasmid. Extracts from electroporated cells (24 hours after the introduction of DNA) and from transgenic plants produce light when mixed with the substrates luciferin and adenosine triphosphate. Light produced by the action of luciferase was also detected in undisrupted leaves or cells in culture from transgenic plants incubated in luciferin and in whole transgenic plants "watered" with luciferin. Although light was detected in most organs in intact, transgenic plants (leaves, stems, and roots), the pattern of luminescence appeared to reflect both the organ-specific distribution of luciferase and the pathway for uptake of luciferin through the vasculature of the plant.
RESUMO
The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six introns, all less than 60 bases in length. The 5' end of the luciferase mRNA was determined by both S1 nuclease analysis and primer extension. Although the luciferase cDNA clone lacked the six N-terminal codons of the open reading frame, we were able to reconstruct the equivalent of a full-length cDNA using the genomic clone as a source of the missing 5' sequence. The full-length, intronless luciferase gene was inserted into mammalian expression vectors and introduced into monkey (CV-1) cells in which enzymatically active firefly luciferase was transiently expressed. In addition, cell lines stably expressing firefly luciferase were isolated. Deleting a portion of the 5'-untranslated region of the luciferase gene removed an upstream initiation (AUG) codon and resulted in a twofold increase in the level of luciferase expression. The ability of the full-length luciferase gene to activate cryptic or enhancerless promoters was also greatly reduced or eliminated by this 5' deletion. Assaying the expression of luciferase provides a rapid and inexpensive method for monitoring promoter activity. Depending on the instrumentation employed to detect luciferase activity, we estimate this assay to be from 30- to 1,000-fold more sensitive than assaying chloramphenicol acetyltransferase expression.
Assuntos
Luciferases/genética , Animais , Sequência de Bases , Linhagem Celular , Chlorocebus aethiops , Clonagem Molecular , Besouros/genética , DNA/genética , Luciferina de Vaga-Lumes/metabolismo , Regulação da Expressão Gênica , Genes , Luciferases/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/genéticaAssuntos
Besouros/enzimologia , Luciferases/genética , Vírus do Mosaico/genética , Plantas/genética , Regiões Promotoras Genéticas , Transfecção , Animais , Sequência de Bases , Linhagem Celular , Códon , Besouros/genética , DNA Recombinante/metabolismo , Expressão Gênica , Técnicas Genéticas , Vetores Genéticos , Glucuronidase/genética , Glucuronidase/metabolismo , Indicadores e Reagentes , Luciferases/metabolismo , Medições Luminescentes , Mamíferos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Plasmídeos , Proteínas Recombinantes/metabolismo , Mapeamento por RestriçãoRESUMO
We discuss the utility of visual assays for the expression of genes introduced into plant cells. Such assays are valuable for both transient and stable gene expression studies. We review the properties of three visual assays that are already in use or under development phase for maize and other cereal crops. These assays depend on the expression of beta-galactosidase, luciferase, or structural genes required for anthocyanin pigment biosynthesis.
Assuntos
Plantas/genética , Transformação Genética , Antocianinas/genética , Regulação da Expressão Gênica , Luciferases/genética , Rhizobium/genética , beta-Galactosidase/genéticaRESUMO
A map of bacteriophage lambda was constructed, including accurate positions for all 41 cut sites made by 12 different restriction enzymes. Over 100 fragments from single, multiple, and partial enzyme digestions were measured versus standards that were calibrated with respect to DNA molecules of known sequence. The data were subjected to least-squares analysis to assign map coordinates. In no case did a fragment size predicted from the map differ from the measurement of the fragment by more than +/- 5%. This low error rate was consistent in all size ranges of fragments. The total length of lambda was calculated as 49,133 nucleotide pairs. This probably is accurate to within 500 base pairs.
Assuntos
Bacteriófago lambda/genética , DNA Viral/genética , Genes Virais , Bacteriófago lambda/análise , Mapeamento Cromossômico , Enzimas de Restrição do DNA/metabolismo , DNA Viral/análiseRESUMO
A cDNA library was constructed from firefly (Photinus pyralis) lantern poly(A)+ RNA, using the Escherichia coli expression vector lambda gt11. The library was screened with anti-P. pyralis luciferase (Photinus luciferin:oxygen 4-oxidoreductase, EC 1.13.12.7) antibody, and several cDNA clones expressing luciferase antigens were isolated. One clone, lambda Luc1, contained 1.5 kilobase pairs of cDNA that hybridized to a 1.9- to 2.0-kilobase band on a nitrocellulose blot of electrophoretically fractionated lantern RNA. Hybridization of the cloned cDNA to lantern poly(A)+ RNA selected an RNA that directed the in vitro synthesis of a single polypeptide. This polypeptide comigrated with luciferase on NaDodSO4/PAGE and produced bioluminescence upon the addition of luciferin and ATP. A 1.8-kilobase-pair cDNA was isolated by probing the firefly cDNA library with the cDNA from lambda Luc1. This cDNA contained sufficient coding information to direct the synthesis of active firefly luciferase in E. coli.
Assuntos
Luciferases/genética , Animais , Clonagem Molecular , Besouros/genética , DNA/genética , Escherichia coli/genética , Regulação da Expressão Gênica , Plasmídeos , Regiões Promotoras Genéticas , Biossíntese de Proteínas , RNA Mensageiro/genéticaRESUMO
A lambda gt11 human hepatoma cDNA expression library was screened with antibodies to human alpha-L-fucosidase, a lysosomal enzyme whose activity is deficient in the human autosomal recessive disease fucosidosis. Three positive clones were identified after screening 9 X 10(6) plaques. One of these was sequenced and found to be spurious, probably representing an out-of-frame cDNA that gave rise to amino acid sequences of unknown length that crossreacted with alpha-L-fucosidase. A second clone, lambda AF3, was isolated which, after establishment in Escherichia coli BNN103, gave rise to a fusion protein of Mr 154,000 containing a human fragment of Mr 40,000 that represented 80% of the mature processed enzyme (Mr 50,000). Southern blot analysis of mouse and human chromosomal DNA confirmed the human origin of insert AF3. The nucleotide sequence of AF3 was determined and colinearity was established between 270 nucleotides and 90 amino acids in alpha-L-fucosidase. AF3 was found to contain 1058 base pairs and to code for 347 amino acids of alpha-L-fucosidase. Four potential glycosylation sites were identified. The frequency of lambda AF3 in the hepatoma library was 0.0018%.
Assuntos
DNA/genética , alfa-L-Fucosidase/genética , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Sequência de Bases , Carcinoma Hepatocelular/genética , Clonagem Molecular , Fucosidose/genética , Humanos , Neoplasias Hepáticas , RNA Mensageiro/genética , alfa-L-Fucosidase/imunologiaRESUMO
Poly (A)+ RNA was isolated from the lanterns of adult fireflies, Photinus pyralis. The Poly (A)+ RNA was translated in a cell-free translation mixture from rabbit reticulocytes and the synthesis of enzymatically active firefly luciferase was demonstrated. The translation products were immunoprecipitated with anti-luciferase and then subjected to SDS gel electrophoresis. It was shown that a newly synthesized polypeptide exhibited an identical electrophoretic mobility as the purified enzyme.
Assuntos
Besouros/enzimologia , Luciferases/genética , Poli A/isolamento & purificação , Biossíntese de Proteínas , RNA/isolamento & purificação , Animais , Sistema Livre de Células , Cinética , Luciferases/isolamento & purificação , Luciferases/metabolismo , Poli A/genética , RNA/genética , RNA Mensageiro , Coelhos , Reticulócitos/metabolismoRESUMO
A human hepatoma cDNA library was constructed in lambda gt11, a bacteriophage vector that was designed to express cDNA-encoded antigenic determinants in Escherichia coli. The cDNA expression library contained approximately 8 X 10(6) recombinant phages with an average insert size of 780 bp. The feasibility of using a chromogenic immunodetection procedure to isolate cDNA clones was proved by isolating a human serum albumin (HSA) cDNA clone. An approximately 1.0-kb cDNA clone was then isolated by screening the library with rabbit anti-human alpha-L-fucosidase antibodies. The identity of the alpha-L-fucosidase cDNA clone was confirmed by DNA sequence analysis and a comparison of the predicted amino acid sequence to the amino acid sequences of human alpha-L-fucosidase tryptic peptides.
Assuntos
Carcinoma Hepatocelular/genética , DNA/genética , Albumina Sérica/genética , alfa-L-Fucosidase/genética , Bacteriófago lambda/genética , Sequência de Bases , Carcinoma Hepatocelular/metabolismo , Clonagem Molecular , Escherichia coli/genética , Humanos , Imunoquímica , Neoplasias HepáticasRESUMO
The mapping of the sites of cleavage of nine restriction endonucleases (EcoRI, HindIII, BamHI, SalI, KpnI, SstI, BglII, XhoI, and XbaI) on 21 Charon phage vectors is described. Maps of individual subsections were obtained and then combined to assemble the complete vector maps. Calculations of maximum and minimum sizes of inserts which may be carried by the vectors using different restriction endonucleases or pairs of restriction endonucleases are presented. The regions mapped include several parts of phi 80 that had not been mapped previously.
Assuntos
Bacteriófago lambda/genética , DNA Viral/genética , Genes Virais , Vetores Genéticos , Bacteriófago lambda/análise , Mapeamento Cromossômico , Enzimas de Restrição do DNA/metabolismo , Elementos de DNA Transponíveis , DNA Viral/análise , Recombinação GenéticaRESUMO
Human alpha-L-fucosidase, a lysosomal enzyme, hydrolyzes alpha-L-fucose from glycolipids and glycoproteins. Its activity is deficient in human fucosidosis an autosomal recessive disease. In order to understand the molecular basis of this lysosomal storage disorder we have cloned several cDNAs coding for human alpha-L-fucosidase from a human hepatoma and a human liver cDNA library constructed in lambda gt11. Compiling the cDNA sequences of these clones we have identified 1,829 base pairs (bp) encoding human alpha-L-fucosidase. This includes an open reading frame of 1,172 bp, a consensus polyadenylation signal AAT AAA and a poly(A)+ tail. The sequence is incomplete at the 5'-end, and clones encoding the amino terminus of the native protein, the propeptide and leader signal have not yet been isolated. The open reading frame encodes for 390 amino acids with a calculated Mr of 45,557. This represents 78-95% of the mature processed alpha-L-fucosidase. The availability of these cDNA clones has enabled us to map the structural gene for alpha-L-fucosidase to chromosome 1p34.1-1p36.1 by Southern blot analysis of DNA from human-rodent somatic cell hybrids and by in situ hybridization. Furthermore, a Pvu II restriction fragment length polymorphism (RFLP) has been identified at the human alpha-L-fucosidase gene locus. Analysis of mRNA by Northern blotting gives a major species of 2.25 kb. In 4 patients with fucosidosis no mRNA signal was detected and Western blots gave no immunoreactive enzyme. Southern blotting after Eco RI digestion in two fucosidosis families revealed a banding abnormality (extra 6-kb band).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Fucosidose/genética , alfa-L-Fucosidase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Colódio , DNA Circular/genética , Eletroforese em Gel de Poliacrilamida , Humanos , Dados de Sequência Molecular , Polimorfismo de Fragmento de Restrição , RNA Mensageiro/genéticaRESUMO
To study the role interleukin (IL)-5 may play in altering airway function in asthma, we have produced recombinant protein for exogenous administration to guinea pigs. The guinea pig IL-5 (gpIL-5) cDNA was cloned by polymerase chain reaction (PCR) amplification of guinea pig spleen RNA and expressed as a secretion product from recombinant baculovirus-infected Sf9 insect cell cultures. The protein was purified to homogeneity by a four-step procedure that included immunoaffinity chromatography using polyclonal antipeptide antibodies against a region of the mature secreted cytokine. The cytokine was properly processed after the signal sequence by the Sf9 cells, was glycosylated with terminal mannose-containing oligosaccharide, and had proper disulfide-linked dimer structure as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified preparation was active in vitro and in vivo as determined by its ability to prime human basophils to release leukotriene C4 in the presence of C5a and to induce airway eosinophilia in naive guinea pigs.