Expression and detection of green fluorescent protein (GFP).
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
P Kitts
Publications and source records attributed to P Kitts.
Dual color microscopic imagery of cells expressing the green fluorescent protein and a red-shifted variant.
The green fluorescent protein (GFP) from the jellyfish, Aequorea victoria, has become a versatile reporter for monitoring gene expression and protein localization in a variety of cells and organisms. GFP emits bright green light (lambda max = 510 nm) when excited with ultraviolet (UV) or blue light (lambda max = 395 nm, minor peak at 470 nm). The chromophore in GFP is intrinsic to the primary structure of the protein, and fluorescence from GFP does not require additional gene products, substrates or other factors. GFP fluorescence is stable, species-independent and can be monitored noninvasively using the techniques of fluorescence microscopy and flow cytometry [Chalfie et al., Science 263 (1994) 802-805; Stearns, Curr. Biol. 5 (1995) 262-264]. The protein appears to undergo an autocatalytic reaction to create the fluorophore [Heim et al., Proc. Natl. Acad. Sci. USA 91 (1994) 12501-12504] in a process involving cyclization of a Tyr66 aa residue. Recently [Delagrave et al., Bio/Technology 13 (1995) 151-154], a combinatorial mutagenic strategy was targeted at aa 64 through 69, which spans the chromophore of A. victoria GFP, yielding a number of different mutants with red-shifted fluorescence excitation spectra. One of these, RSGFP4, retains the characteristic green emission spectra (lambda max = 505 nm), but has a single excitation peak (lambda max = 490 nm). The fluorescence properties of RSGFP4 are similar to those of another naturally occurring GFP from the sea pansy, Renilla reniformis [Ward and Cormier, Photobiochem. Photobiol. 27 (1978) 389-396]. In the present study, we demonstrate by fluorescence microscopy that selective excitation of A. victoria GFP and RSGFP4 allows for spectral separation of each fluorescent signal, and provides the means to image these signals independently in a mixed population of bacteria or mammalian cells.
Amino Acid Sequence↗
Human adenovirus serotype 3 (Ad3) and the Ad3 fiber protein bind to a 130-kDa membrane protein on HeLa cells.
The fiber protein of adenovirus mediates the interaction of adenovirus with cell membrane receptors. We have produced the Ad3 fiber protein in the baculovirus expression system. Biochemical, morphological and functional analyses showed that the recombinant fiber was properly folded and functionally competent. The specific binding of Ad3 virus to two HeLa membrane proteins of 130 and 100 kDa was demonstrated with an overlay protein binding assay. In the same assay, Ad3 fiber only recognized the 130-kDa protein. Divalent cations seemed to be important for the interaction of both virus and fiber with these proteins.
Adenoviruses, Human↗
Green fluorescent protein as a reporter of gene expression and protein localization.
The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is rapidly becoming an important reporter molecule for monitoring gene expression and protein localization in vivo, in situ and in real time. GFP emits bright green light (lambda max = 509 nm) when excited with UV or blue light (lambda max = 395 nm, minor peak at 470 nm). The fluorescence excitation and emission spectra of GFP are similar to those of fluorescein, and the conditions used to visualize this fluorophore are also suitable for GFP. Unlike other bioluminescent reporters, the chromophore in GFP is intrinsic to the primary structure of the protein, and GFP fluorescence does not require a substrate or cofactor. GFP fluorescence is stable, species-independent and can be monitored non-invasively in living cells and, in the case of transparent organisms, whole animals. Here we demonstrate GFP fluorescence in bacterial and mammalian cells and introduce our Living Colors line of GFP reporter vectors, GFP protein and anti-GFP antiserum. The reporter vectors for GFP include a promoterless GFP vector for monitoring the expression of cloned promoters/enhancers in mammalian cells and a series of six vectors for creating fusion protein to either the N or C terminus of GFP.
3T3 Cells↗
Cell-binding domain of adenovirus serotype 2 fiber.
The adenovirus fiber appears as a long, thin projection terminated by a knob (head). The fiber consists of a trimeric protein whose head domain is thought to interact with cell receptors. The head part (amino acids 388 to 582) of adenovirus type 2 fiber was produced in a baculovirus expression system. The purified protein was shown to cross-link into trimers. It was very resistant to proteolytic attack and seemed to attain a high degree of compactness. The head domain efficiently inhibited attachment of adenovirus to receptors on the surface of HeLa cells, thereby confirming the hypothesis that the head domain interacts with viral receptors.
Adenoviruses, Human↗
Lambda integrative recombination: supercoiling, synapsis, and strand exchange.
We have presented the first results of three ongoing experiments on the biochemical mechanism of lambda site-specific recombination. First, we have shown that under the tension induced by negative supercoiling, a segment of attP and attR undergoes a transition to a novel conformation. The basis for detecting this new structure implies that it contains unpaired bases, perhaps arising as a result of the transition of an inverted repeat to a cruciform structure. Since the segment undergoing transition lies outside the homologous core region, the unpairing of DNA that we have detected cannot be directly involved in synapsis. It will be of interest to learn whether recombination proteins alter this conformational change, possibly to extend its effect into the core region. Second, we have demonstrated that replacing phosphate groups in attachment site DNA with phosphorothioates interferes with recombination. Our first experiments indicate that the effect depends on locating phosphorothioates precisely at the site of recombination crossover. We take this to mean that interference is primarily at the step of breakage and reunion; we plan to exploit this inhibition in an attempt to trap synaptic intermediates in recombination. Finally, we have found that the rare cleavage of attP DNA by Int can involve either one or both strands of the duplex. This indicates that the degree of coordination between Int action at the two halves of the core region can be variable. Although we do not know whether this variability affects binding of Int, breakage of DNA, or resealing of broken strands, our data suggest that this variability can be regulated by sulfhydryl reagents.
Bacteriophage lambda↗
Transposon-specified site-specific recombination.
Cointegrate DNA molecules containing two copies of a transposable element appear to be intermediates in the transposition process. These structures are resolved by site-specific recombination to yield the normal end products of transposition. The transposable element gamma delta (Tn1000) synthesizes a product interchangeable with the Tn1/3tnpR protein in promoting Tn1/3 site-specific recombination. These data support the hypothesis that cointegrates containing directly repeated copies of Tn1/3 are obligatory intermediates in interreplicon transposition of Tn1/3. In addition, we show here that the reaction is independent of the element-encoded tnpA gene product. Tn501, which specifies mercury resistance, also produces cointegrates as intermediates in interreplicon transposition. The appearance of Tn501-specified recombination activity that can act on these cointegrates requires growth of cells in the presence of Hg2+.
DNA Transposable Elements↗