[Coding region of far-red fluorescent protein katushka contains a strong donor splice site].

Computer analysis predicted a strong donor splice site within the 3'-part of the far-red fluorescent protein Katushka coding region. To test the functional activity of this site a model vector has been constructed. This vector encoded Katushka and green fluorescent protein TagGFP2 with a gene fragment of tafazzin in between. Normal splicing of this pre-mRNA should result in a frameshift between Katushka and TagGFP2. Alternatively, after splicing at internal katushka donor splice site appearance of Katushka-TagGFP2 fusion protein was expected. Expression of this construct in a mammalian cell culture led to bright red and green fluorescence. Therefore, katushka-specific donor splice site is functional. Disruption of this splice site by several silent substitutions resulted in red-only fluorescent cells that corresponded to normal splicing. The mutant katushka can be used for visualization of pre-mRNA splicing at single cell level by fluorescence microscopy and flow cytometry.

[Spectral diversity among the members of the family of Green Fluorescent Protein in hydroid jellyfish (Cnidaria, Hydrozoa)].

The cDNAs encoding the genes of new proteins homologous to the well-known Green Fluorescent Protein (GFP) from the hydroid jellyfish Aequorea victoria were cloned. Two green fluorescent proteins from one un-identified anthojellyfish, a yellow fluorescent protein from Phialidium sp., and a nonfluorescent chromoprotein from another unidentified anthojellyfish were characterized. Thus, a broad diversity of GFP-like proteins among the organisms of the class Hydrozoa in both spectral properties and primary structure was shown.

Diversity and evolution of the green fluorescent protein family.

The family of proteins homologous to the green fluorescent protein (GFP) from Aequorea victoria exhibits striking diversity of features, including several different types of autocatalytically synthesized chromophores. Here we report 11 new members of the family, among which there are 3 red-emitters possessing unusual features, and discuss the similarity relationships within the family in structural, spectroscopic, and evolutionary terms. Phylogenetic analysis has shown that GFP-like proteins from representatives of subclass Zoantharia fall into at least four distinct clades, each clade containing proteins of more than one emission color. This topology suggests multiple recent events of color conversion. Combining this result with previous mutagenesis and structural data, we propose that (i) different chromophore structures are alternative products synthesized within a similar autocatalytic environment, and (ii) the phylogenetic pattern and color diversity in reef Anthozoa is a result of a balance between selection for GFP-like proteins of particular colors and mutation pressure driving the color conversions.

GFP-like chromoproteins as a source of far-red fluorescent proteins.

We have employed a new approach to generate novel fluorescent proteins (FPs) from red absorbing chromoproteins. An identical single amino acid substitution converted novel chromoproteins from the species Anthozoa (Heteractis crispa, Condylactis gigantea, and Goniopora tenuidens) into far-red FPs (emission lambda(max)=615-640 nm). Moreover, coupled site-directed and random mutagenesis of the chromoprotein from H. crispa resulted in a unique far-red FP (HcRed) that exhibited bright emission at 645 nm. A clear red shift in fluorescence of HcRed, compared to drFP583 (by more than 60 nm), makes it an ideal additional color for multi-color labeling. Importantly, HcRed is excitable by 600 nm dye laser, thus promoting new detection channels for multi-color flow cytometry applications. In addition, we generated a dimeric mutant with similar maturation and spectral properties to tetrameric HcRed.

Novel fluorescent protein from Discosoma coral and its mutants possesses a unique far-red fluorescence.

A novel gene for advanced red-shifted protein with an emission maximum at 593 nm was cloned from Discosoma coral. The protein, named dsFP593, is highly homologous to the recently described GFP-like protein drFP583 with an emission maximum at 583 nm. Using the remarkable similarity of the drFP583 and dsFP593 genes, we performed a 'shuffling' procedure to generate a pool of mutants consisting of various combinations of parts of both genes. One 'hybrid gene' was chosen for subsequent random mutagenesis, which resulted in a mutant variant with a uniquely red-shifted emission maximum at 616 nm.

Natural animal coloration can Be determined by a nonfluorescent green fluorescent protein homolog.

It is generally accepted that the colors displayed by living organisms are determined by low molecular weight pigments or chromoproteins that require a prosthetic group. The exception to this rule is green fluorescent protein (GFP) from Aequorea victoria that forms a fluorophore by self-catalyzed protein backbone modification. Here we found a naturally nonfluorescent homolog of GFP to determine strong purple coloration of tentacles in the sea anemone Anemonia sulcata. Under certain conditions, this novel chromoprotein produces a trace amount of red fluorescence (emission lambda(max) = 595 nm). The fluorescence demonstrates unique behavior: its intensity increases in the presence of green light but is inhibited by blue light. The quantum yield of fluorescence can be enhanced dramatically by single amino acid replacement, which probably restores the ancestral fluorescent state of the protein. Other fluorescent variants of the novel protein have emission peaks that are red-shifted up to 610 nm. They demonstrate that long wavelength fluorescence is attainable in GFP-like fluorescent proteins.

Fluorescent proteins from nonbioluminescent Anthozoa species.

We have cloned six fluorescent proteins homologous to the green fluorescent protein (GFP) from Aequorea victoria. Two of these have spectral characteristics dramatically different from GFP, emitting at yellow and red wavelengths. All the proteins were isolated from nonbioluminescent reef corals, demonstrating that GFP-like proteins are not always functionally linked to bioluminescence. The new proteins share the same beta-can fold first observed in GFP, and this provided a basis for the comparative analysis of structural features important for fluorescence. The usefulness of the new proteins for in vivo labeling was demonstrated by expressing them in mammalian cell culture and in mRNA microinjection assays in Xenopus embryos.

Identification and localization of differences between Escherichia coli and Salmonella typhimurium genomes by suppressive subtractive hybridization.

The availability of bacterial genome sequences raises an important new problem - how can one move from completely sequenced microorganisms as a reference to the hundreds and thousands of other strains or isolates of the same or related species that will not be sequenced in the near future? An efficient way to approach this task is the comparison of genomes by subtractive hybridization. Recently we developed a sensitive and reproducible subtraction procedure for comparison of bacterial genomes, based on the method of suppression subtractive hybridization (SSH). In this work we demonstrate the applicability of subtractive hybridization to the comparison of the related but markedly divergent bacterial species Escherichia coli and Salmonella typhimurium. Clone libraries representing sequence differences were obtained and, in the case of completely sequenced E. coli genome, the differences were directly placed in the genome map. About 60% of the differential clones identified by SSH were present in one of the genomes under comparison and absent from the other. Additional differences in most cases represent sequences that have diverged considerably in the course of evolution. Such an approach to comparative bacterial genomics can be applied both to studies of interspecies evolution - to elucidate the "strategies" that enable different genomes to fit their ecological niches - and to development of diagnostic probes for the rapid identification of pathogenic bacterial species.

[Cloning of region-specific genetic markers of planaria using a new method--ordered differential display]. / Klonirovanie region-spetsifichnykh geneticheskikh markerov planarii s pomoshch'iu novogo metoda--uporiadochennogo differentsional'nogo displeiia.

A new method for finding differentially expressed genes, termed ordered differential display of mRNAs (ODD), was used in the search for region-specific molecular markers of freshwater planarian Dugesia tigrina. In this method, the effect of selective suppression of a polymerase chain reaction (PCR) is used for the differential amplification of a pool of 3'-terminal cDNA fragments generated by digestion of cDNAs with a restriction endonuclease. In the resulting amplified cDNAs, every mRNA is represented by a cDNA fragment whose length is determined by the position of the restriction site nearest to the 3'-terminus. Subsequent PCR with primers 3'-extended by two random nucleotides allowed the amplification of 1/192 part of all cDNA molecules present in the sample. The comparison of the generated pools of cDNA molecules separated by PAGE leads to the identification of differentially expressed sequences. The systematic study of the total mRNA pool is achieved by the successive use of all possible combinations of extended primers. Some sequences preferentially expressed along the anterior-posterior axis of planarian were identified using ODD.

[Biosynthesis of human calcitonin and mini-proinsulin in bacterial cells in the form of hybrid proteins with corresponding antisense peptides and a metal-binding peptide]. / Biosintez v bakterial'nykh kletkakh kal'tsitonina i mini-proinsulina cheloveka v vide gibridnykh belkov s sootvetstvuiushchimi antismyslovymi peptidami i metallsviazyvaiushchim peptidom.

To verify experimentally the molecular recognition theory, plasmids were constructed that provided the efficient synthesis of hybrid proteins composed of human calcitonin or miniproinsulin, the corresponding antisense peptides, and a histidine-rich metal-binding peptide. A method for isolation of the hybrid proteins by metal-chelating chromatography, cleavage, and renaturation was developed.

[An artificial gene, biosynthesis and properties of the recombinant Fc fragment of human immunoglobulin G1]. / Iskusstvennyi gen, biosintez i svoistva rekombinantnogo Fc-fragmenta immunoglobulina G1 cheloveka.

Chemicoenzymatic synthesis and cloning of a gene encoding the Fc domain of human immunoglobulin G1 were carried out. The artificial gene was expressed in Escherichia coli cells in plasmid vectors under control of a late T7 promoter. The recombinant protein isolated from the bacterial cells is capable of forming dimers and binding protein A from Staphylococcus aureus.

Expression of the barley psbA gene in Escherichia coli yields a functional in vitro photosystem II protein D1.

The barley chloroplast psbA gene encoding D1 protein, one of the main photosystem II components, has been over-expressed in E. coli cells. The existance of two in vivo expression products, a protein with M(r) about 33.5 kDa, corresponding to the full-length precursor of the 32 kDa D1 mature form, and a truncated 29 kDa polypeptide was revealed. A modified D1 protein containing six histidine residues at the carboxy-terminus was also obtained. After isolation and renaturation, the ability of the recombinant D1 protein to bind atrazine and pigments from barley thylakoids was demonstrated.

[Synthesis and cloning of genes for the antisense peptides for human calcitonin and miniproinsulin]. / Sintez i klonirovanie genov antismyslovykh peptidov kal'tsitonina i miniproinsulina cheloveka.

Because of the potential significance of the 'molecular recognition' theory for studies in molecular biology and biotechnology, the theory is worth being examined using methods of chemical-enzymatic gene synthesis, recombinant DNA construction and microbiological peptide synthesis. We therefore undertook the synthesis of human Va18-calcitonin, miniproinsulin, and the corresponding antisense peptides as model compounds. In designing an experimental system the idea was to combine sense and antisense polypeptides into a single chain and to examine their intramolecular interaction. In this paper the chemical-enzymatic synthesis, cloning and expression of the genes for calcitonin, miniproinsulin, the corresponding antisense peptides and their combinations are described. The recombinant DNAs obtained were able to direct in vivo expression of the target polypeptides as hybrid proteins with the IgG-binding domain of the staphylococcal A protein in bacterial cells.

[Expression of the gene coding for the D1-protein of barley photosystem II in Escherichia coli]. / Ekspressiia gena, kodiruiushchego D1-belok fotosistemy II iachemnia, v Escherichia coli.

Previously characterized by us barley chloroplast psbA gene, which encodes one of the main Photosystem II components--D1 protein, has been inserted in a set of special plasmid vectors and its expression in vitro and in vivo has been investigated. Experiments on the in vitro expression in the rabbit reticulocyte lysate system revealed a major product with a molecular weight ca. 33.5 kD, which corresponds to the unprocessed D1 barley protein. A lower molecular weight protein (about 29 kD) was also observed. These results are in agreement with the existence of two potential translation start sites in the psbA gene in the same reading frame, the second one starting from Met37 residue. The results fully correlate with the earlier data on the in vitro expression of psbA genes of maize, pea, and tobacco. Experiments on the in vivo expression of psbA gene in E. coli cells with the above constructions also revealed proteins with m. w. about 33.5 and 29 kD. The yield of the target recombinant protein in some cases was about 25-30% of the total E. coli cellular protein. The correspondence of the bands to the desired products was proved by the immunoenzyme analysis with the use of polyclonal antibodies. The data obtained show for the first time the construction of E. coli strains producing recombinant D1 protein of cereals in a high level.