Femtosecond spectroscopy probes the folding quality of antibody fragments expressed as GFP fusions in the cytoplasm.

Time-resolved femtosecond spectroscopy can improve the application of green fluorescent proteins (GFPs) as protein-folding reporters. The study of ultrafast excited-state dynamics (ESD) of GFP fused to single chain variable fragment (scFv) antibody fragments, allowed us to define and measure an empirical parameter that only depends on the folding quality (FQ) of the fusion. This method has been applied to the analysis of genetic fusions expressed in the bacterial cytoplasm and allowed us to distinguish folded and thus functional antibody fragments (high FQ) with respect to misfolded antibody fragments. Moreover, these findings were strongly correlated to the behavior of the same scFvs expressed in animal cells. This method is based on the sensitivity of the ESD to the modifications in the tertiary structure of the GFP induced by the aggregation state of the fusion partner. This approach may be applicable to the study of the FQ of polypeptides over-expressed under reducing conditions.

Production of fluorescent single-chain antibody fragments in Escherichia coli.

We describe a novel vector-host system suitable for the efficient preparation of fluorescent single-chain antibody Fv fragments (scFv) in Escherichia coli. The previously described pscFv1F4 vector used for the bacterial expression of functional scFv to the E6 protein of human papillomavirus type 16 was modified by appending to its C-terminus the green fluorescent protein (GFP). The expression of the scFv1F4-GFP fusion proteins was monitored by analyzing of the typical GFP fluorescence of the transformed cells under UV illumination. The brightest signal was obtained when scFv1F4 was linked to the cycle 3 GFP variant (GFPuv) and expressed in the cytoplasm of AD494(DE3) bacteria under control of the arabinose promoter. Although the scFv1F4 expressed under these conditions did not contain disulfide bridges, about 1% of the molecules were able to bind antigen. Fluorescence analysis of antigen-coated agarose beads incubated with the cytoplasmic scFv-GFP complexes showed that a similar proportion of fusions retained both E6-binding and green-light-emitting activities. The scFv1F4-GFPuv molecules were purified by affinity chromatography and successfully used to detect viral E6 protein in transfected COS cells by fluorescence microscopy. When an anti-beta-galactosidase scFv, which had previously been adapted to cytoplasmic expression at high levels, was used in this system, it was possible to produce large amounts of functional fluorescent antibody fragments. This indicates that these labeled scFvs may have many applications in fluorescence-based single-step immunoassays.

In vivo biotinylated recombinant antibodies: high efficiency of labelling and application to the cloning of active anti-human IgG1 Fab fragments.

In vivo biotinylation of antibody fragments with a gene fusion approach is a realistic alternative to conventional in vitro chemical labelling. We have previously reported the construction of a vector system suitable for the bacterial expression of the binding fragment of antibody (Fab) genetically linked to the C-terminal domain of Escherichia Coli biotin carboxy carrier protein (BCCP*). A minor fraction of the expressed hybrids was biotinylated in vivo and therefore able to interact with streptavidin. We now show that the large majority of bacterially-expressed Fab-BCCP* fusions are labelled with biotin when plasmid-encoded biotin holoenzyme synthetase (BirA) is co-expressed. The yield of biotinylated Fab is maximal when overexpression of BirA is driven by a second compatible plasmid. We took advantage of this property to develop a novel filter assay for the rapid identification of recombinant Fab reacting with immunoglobulin. Starting with total RNA of two newly established murine hybridoma cell lines producing anti-human IgG1 antibodies, we selected in a single experiment the bacterial clones that expressed in vivo biotinylated anti-IgG1 Fab. Sequence analysis of the isolated Fabs showed that they did not derive from a single B clone. In addition, we found that these recombinant Fabs labelled with biotin in vivo are useful for the specific detection of human IgG1 by a solid-phase immunoassay.

5-[[(carboxymethyl)amino]methyl]uridine is found in the anticodon of yeast mitochondrial tRNAs recognizing two-codon families ending in a purine.

The modified nucleoside (U*) present in the wobble position of Saccharomyces cerevisiae mitochondrial tRNA(Leu) and tRNA(Trp) was isolated by thin-layer chromatography and HPLC. Its chromatographic, UV spectral, and mass spectrometric properties were shown to be identical with those of 5- [[(carboxymethyl)amino]methyl]uridine (cmnm5U). This nucleoside found in yeast mitochondrial tRNAs reading two-codon families ending in a purine permits the selective recognition of A and G in the third codon position.

Codon reading patterns in Saccharomyces cerevisiae mitochondria based on sequences of mitochondrial tRNAs.

The sequences of Saccharomyces cerevisiae mitochondrial tRNA Arg1, tRNA Arg2, tRNA Gly, tRNA Lys2, tRNA Leu amd tRNA Pro are reported. Special structural features were found in tRNA Pro, which has A8, C21, A48 instead of the constant residues U8, A21 and pyrimidine 48, and in tRNA Lys2, which has a U excluded from base-paring and bulging out from the TpsiC stem. The tRNA Arg1, tRBA Lys2 and tRNA Leu, which belong to two-codon families ending in a purine, have a modified uridine in the wobble position, which prevents misreading of C and U. It is likely to be 5-carboxymethylaminomethyluridine. tRNA Gly and tRNA Pro have an unmodified uridine in the wobble position allowing the reading of all four codons of a four-codon family. However, tRNA Arg2, which is a minor species and belongs to the CGN four-codon family, has an unmodified A in the wobble position. This unusual feature raises the problem of the mechanism by which the codons CGA, CGG and CGC are recognized.

Yeast mitochondrial tRNAIle and tRNAMetm: nucleotide sequence and codon recognition patterns.

The nucleotide sequence of yeast mitochondrial isoleucine- and methionine-elongator tRNA have been determined. Interestingly, long stretches of almost identical nucleotide sequences are found within these two tRNAs and also within the yeast mt tRNAMetf, suggesting that the 3 tRNAs may have arisen from a common ancestor. Both mt tRNAMetm and tRNAIle contain all the structural characteristics which are present in the standard cloverleaf, except that the mt tRNAMetm contains an extra unpaired nucleotide within the base-paired T psi C stem. This rather unusual feature may have an influence on the decoding properties of the C-A-U anticodon of mt tRNAMetm by conferring the ability to translate not only the codon A-U-G but also A-U-A.

[Yeast mitochondrial transfer RNA. Structure, coding properties and genome organization]. / Transportnye RNK mitokhondrii drozhzhei. Struktura, kodovye svoistva i organizatsiia genoma.

The up-to-date data on mitochondrial tRNAs of yeast, their structures and peculiarities of these structures, anomalies of the mitochondrial genetic code and anticodons of tRNAs, the structure and number of tRNA genes are reviewed in the present paper. New information concerning 17 types of yeast mitochondrial tRNAs, deciphered by the authors of the paper are given; among them 8 types are first published. The likeness and differences of yeast mitochondrial tRNAs from their cytoplasmic counterparts are discussed by comparison with other organisms.

The primary structure of yeast mitochondrial tyrosine tRNA.

The mitochondrial tyrosine tRNA from Saccharomyces cerevisiae has been sequenced. It has two interesting structural features: (i) it lacks two semi-invariant purine residues in the D-loop which are involved in tertiary interactions in the yeast cytoplasmic tRNAPhe; (ii) it has a large variable loop and therefore resembles procaryotic tRNAsTyr rather than eucaryotic cytoplasmic ones.

The primary structures of three yeast mitochondrial serine tRNA isoacceptors.

Yeast mitochondria contain several isoaccepting species of serine-tRNA. The relative amount of these isoacceptors varies according to the conditions used to grow the yeast cells. In order to gain insight into the structural differences among these isoacceptors, the three mitochondrial tRNAsSer, which are present in derepressed yeast cells, have been sequenced. The primary structure of tRNASer1 differs considerably from that of tRNASer2; these two isoacceptors have only 39 nucleotides in common. In contrast, tRNASer3 differs from tRNASer2 by only one post-transcriptional modification: the psi residue in position 28 of tRNASer2 is replaced by a normal U in tRNASer3. Unlike tRNASer2 and tRNASer3, the primary sequence of tRNASer1 shows two unusual structural features: it has a D in position 14 instead of the "universal" A14 of the standard tRNA cloverleaf and it contains two G residues between the D-stem and the anticodon-stem. Considering their respective anticodons, tRNASer1 should recognize the two serine codons A-G-C and A-G-U, whereas both tRNASer2 and tRNASer3 should recognize all four serine codons of the U-C-N series.

[Primary structure of yeast mitochondrial tryptophan-tRNA capable of translating the termination U-G-A codon]. / Structure primaire d'un tryptophane-tRNA de mitochondrie de levure capable de traduire le codon de terminaison U-G-A.

The primary structure of Yeast Saccharomyces cerevisiae mitochondrial tryptophane-tRNA, isolated by reversed phase chromatography and two-dimensional gel electrophoresis has been determined. It is as follows: pA-A-G-G-A-U-A-U-A-G-U-U-U-A-A-D-G-G-D-A-A-A-A-C-A-G-U-U-G-A-psi-U-U-C-A-i6 A (ms2i6 A)-A-psi-C-A-A-U-C-A-U-U-A-G-G-A-G-T-psi-C-G-A-A-U-C-U-C-U-U-U-A-U-C-C-U-U-G-C-C-A. Owing to its anticodon U-C-A-, where U represents a modified uridine, this tRNA can translate the opal codon U-G-A, which is usually a protein synthesis termination codon in cytoplasmic systems.

Primary structure of yeast mitochondrial DNA-coded phenylalanine-tRNA.

Mitochondrial tRNAPhe from Saccharomyces cerevisiae isolated by two-dimensional gel electrophoresis was sequenced by fingerprinting uniformly labeled 32 P-tRNA as well as by 5'-end postlabeling techniques. Its sequence was found to be: pG-C-U-U-U-U-A-U-A-G-C-U-U-A-G-D-G-G-D-A-A-A-G-C-m22G-A-U-A-A-A-phi-U-G-A-A-m1G-A-phi-U-U-A-U-U-U-A-C-A-U-G-U-A-G-U-phi-C-G-A-U-U-C-U-C-A-U-U-A-A-G-G-G-C-A-C-C-A. The secondary structure we propose, in order to maximize base pairing in the phiC stem and to allow tertiary interaction between G15 and C46, excludes U50 from base pairing giving a bulge in the phiC stem. No conclusion can be drawn concerning the endosymbiotic theory of mitochondria evolution by comparing the primary structure of mt. tRNAPhe with other sequenced tRNAsPhe. This mt.tRNAPhe lacks some of the structural elements reported to be involved in the yeast cytoplasmic phenylalanyl-tRNA ligase recognition site and cannot be aminoacylated by purified yeast cytoplasmic phenylalanyl-tRNA ligase.

[Nucleotide sequence determination of yeast mitochondrial phenylalanine-tRNA]. / Etablissement de la séquence des nucléotides du phénylalanine-tRNA de mitochondries de Levure.

The primary structure of mitochondrial tRNAPhe from Saccharomyces cerevisiae, purified by two-dimensional polyacrylamide gel electrophoresis, was determined using, standard procedures on in vivo 32P-labeled tRNA, as well as the new 5'-end postlabeling techniques. We propose a cloverleaf model which allows for tertiary interaction between cytosine in position 46 and guanine in position 15 and maximizes base pairing in the psi C stem, thus excluding the uracile in position 50 from base pairing in the psi C stem. Comparison of the primary structure of this tRNA with all other known procaryotic, chloroplastic or cytoplasmic tRNAsPhe sequences does not lead to any conclusion about the endosymbiotic theory of mitochondria evolution.