The folding of ovalbumin. Renaturation in vitro versus biosynthesis in vitro.

Hen ovalbumin, the major secretory product of oviduct cells, is a 43 000-dalton glycoprotein. Many studies have led to controversy over the question of whether ovalbumin (OA) can be fully renatured after chemical denaturation. We have studied the renaturation of OA after denaturation with guanidinium chloride, urea or alkaline pH. Denatured OA displays an intrinsic viscosity consistent with nearly complete unfolding of the protein. Removal of the denaturant results in a complete reversal of the changes in intrinsic viscosity. However, closer examination of the renatured protein reveals major differences from the native form. Renatured OA (OAR) can be completely separated from the native form (OAN) by affinity chromatography on phenyl-Sepharose. OAR displays altered tryptophan fluorescence, u.v.-absorption and c.d. spectra. Only OAR binds anilinonaphthalenesulphonate (as measured by fluorescence enhancement). OAR, but not OAN, binds about 2 mol of the covalent hydrophobic affinity probe phenyl isothiocyanate/mol. Renaturation, and the production of OAR, occurs regardless of the oxidation state of the disulphide bonds, of phosphorylation of the protein, and of the presence or the absence of the single carbohydrate chain. OAR may be either monomeric or an irreversible aggregate. Which of these two states is formed depends on the protein concentration during renaturation. Monomeric and aggregated OAR can be distinguished on the basis of some spectroscopic characteristics, but they share the essential hydrophobic characteristics that distinguish them from OAN. OAN and OAR do not spontaneously interconvert. Antibodies raised to each can be made monospecific by immunoabsorption. Thus two stable forms of OA can be obtained, one of which, OAR, displays hydrophobic characteristics. OAN, but not OAR, is formed when OA is synthesized in vitro in a translation system.     

Protein folding in vitro.

It is becoming increasingly evident that intermediates observed in protein folding in vitro may be closely related to conformational states that are important in various intracellular processes. This review focuses on recent advances in in vitro protein-folding studies with particular reference to the molten globule state, which is purported to be a common and distinct intermediate of protein folding.     

An in vitro DNA virus for in vitro protein evolution.

In vitro virus is a molecular construct for in vitro protein evolution, which requires some mechanism to link phenotype to genotype. The first in vitro virus was realized by bonding a nascent protein with its coding mRNA via puromycin in in vitro translation. We report a new construct of in vitro DNA virus. The virion was a covalent cDNA-protein fusion, and virion formation did not require any modification of mRNA. Due to intactness of mRNA, this type of in vitro DNA virus will take the next step toward in vitro autonomous evolution, just like in vivo viral evolution in a cellstat.     

Labelling of cardiolipin in vitro.

A method for labelling the polar head groups of cardiolipin is described. Labelling was carried out on sonicated cardiolipin/water suspensions. The free hydroxyl group of cardiolipin was oxidised with an excess of p-(diazonium) benzenesulfonic acid (DABS) and then reduced with NaB3H4. Isopropanol was oxidised in the presence of DABS to test the reactivity of the diazonium salts, and the reaction product was analysed by means of gas-chromatography. Labelled cardiolipin, identified by thin-layer chromatography (TLC), was chromatographically pure and identical to untreated cardiolipin. The hydrolysis of cardiolipin confirmed that the labelling was at the level of polar head groups.     

Xer site-specific recombination in vitro.

Two related recombinases, XerC and XerD, belonging to the lambda integrase family of enzymes, are required for Xer site-specific recombination in vivo. In order to understand the roles of these proteins in the overall reaction mechanism, an in vitro recombination system using a synthetic Holliday junction-containing substrate has been developed. Recombination of this substrate is efficient and requires both XerC and XerD. However, only exchange of one pair of strands, the one corresponding to the conversion of the Holliday junction intermediate back to the substrate, has been observed. Recombination reactions using XerC and XerD derivatives that are mutant in their presumptive catalytic residues, or are maltose-binding fusion recombinase derivatives, have demonstrated that this pair of strand exchanges is catalysed by XerC. The site of XerC-mediated cleavage has been located to between the last nucleotide of the XerC binding site and the first nucleotide of the central region. Cleavage at this site generates a free 5'-OH and a covalent complex between XerC and the 3' end of the DNA.     

Plant ribosome shunting in vitro.

It has been proposed that cauliflower mosaic virus 35S RNA with its 600 nt long leader uses an unusual translation process (the translational shunt). A wheat germ in vitro translation assay was used to improve the study of this mechanism. Deletions, the introduction of stable stem-loop structures, and the inhibitory effect of antisense oligonucleotides on gene expression were used to determine the roles of various parts of the leader. It was found that the 5'- and 3'-ends of the leader are absolutely required for translation whereas the middle part is apparently dispensable. These results confirm the data already reported from transient expression experiments with protoplasts. However, the in vitro data suggest in contrast to protoplast experiments that only two relatively short regions at both ends, approximately 100 nt each, are required. The in vitro system provides tools for further studying the shunt model at the molecular level and for examining the involvement of proteins in this mechanism. Shunting was also found to occur with the rice tungro bacilliform virus leader. As wheat is neither a host plant of cauliflower mosaic virus nor rice tungro bacilliform virus, the shunt seems to be host independent, a finding that deviates from earlier studies in protoplasts.     

Xer-mediated site-specific recombination in vitro.

The Xer site-specific recombination system acts at ColE1 cer and pSC101 psi sites to ensure that these plasmids are in a monomeric state prior to cell division. We show that four proteins, ArgR, PepA, XerC and XerD are necessary and sufficient for recombination between directly repeated cer sites on a supercoiled plasmid in vitro. Only PepA, XerC and XerD are required for recombination at psi in vitro. Recombination at cer and psi in vitro requires negative supercoiling and is exclusively intramolecular. Strand exchange at cer produces Holliday junction-containing products in which only the top strands have been exchanged. This reaction requires the catalytic tyrosine residue of Xer C but not that of XerD. Recombination at psi gives catenated circular resolution products. Strand exchange at psi is sequential. XerC catalyses the first (top) strand exchange to make a Holiday junction intermediate and XerD catalyses the second (bottom) strand exchange.     

In vitro packaging of bacteriophate T7 DNA synthesized in vitro.

An in vitro DNA packaging system was used to encapsulate T7 DNA that had been synthesized by extracts prepared from gently lysed Escherchia coli infected with bacteriophage T7 carrying amber mutations in gene 3 or in both genes 3 and 6. Isopycnic centrifugation of density-labeled wild-type DNA was employed in an effort to separate product from template; suppressor-free indicator bacteria were used to eliminate contributions from endogenous DNA or contaminating phage. Additional controls indicated that fragmented DNA is packaged in vitro only with very low efficiency and that the frequency of recombination during packaging is too low to affect interpretation of these experiments. T7 DNA replicated by extracts prepared using T7 mutants deficient in both genes 3 and 6 could be packaged in vitro with an efficiency comparable to that found when highly purified virion T7 DNA was used. When T7 deficient in the gene 3 endonuclease but with normal levels of the gene 6 exonuclease was used, fast-sedimentingconcatemer-like DNA structures were formed during in vitro DNA synthesis. Electron microscopy revealed many branched and highly complex DNA structures formed during this reaction. This concatemer-like DNA was encapsulated in vitro with an efficiency significantly greater than that found for DNA the length of a single T7 genome.     

Actinidia deliciosa C.F. Liang in vitro

In vitro growth of Actinidia deliciosa C.F. Liang, cv Hayward and changes in mineral composition of the medium and in the different parts of the explants (callus, stem and leaves), were analyzed after 0, 15, 30, 45 and 60 days of culturing in each of three successive 60 days subcultures.Fresh (FW) and dry weight (DW) of the explants increased mainly during the first 30 days of culturing, with a predominant increase of FW in leaves and an equal distribution in DW in callus and leaves. Stem FW and DW changes were lower than those observed with callus and leaves. As FW and DW of the explants increased the FW and DW of the medium decreased.The presence of the explants induced a large decrease of medium pH during the first 15 days of culturing followed by a return to the initial level at the end of the culturing.The initial P content of the MS medium was insufficient for the long term culturing, as after 30 days of culturing almost all (94.5%) the P present in the medium was absorbed by the explants and evenly distributed in their different parts. During the first month, 85% of the initial N was absorbed. At the end of the culture only 2% of the initial P and 5% of N remained in the medium. These two elements were equally distributed in callus and leaves during the first month of culturing, while in the last 30 days they increased only in the callus.MS medium initial concentrations of K, Mg, Ca, Fe, Zn, Mn and Cu were sufficient for 60 days explants growth. Almost all these elements were absorbed during the first 30 days of culturing. Their distribution in the different parts of the explant was uneven throughout the culture period. Callus tissue was the main site for accumulation of all these mineral elements.     

In vitro secondary MLR. I. Kinetics of proliferation and specificity of in vitro primed responder cells.

We have examined the kinetics and specificity of secondary in vitro mixed lymphocyte reactions (MLR). With limited numbers of primed responder cells (PRC) in the presence of "excess antigen" it was possible to obtain proliferative responses that were proportional to the number of PRC initially placed in culture. The responding cells, after an initial lag period, seem to grow exponentially until day 3 of culture. The responses of PRC (with the strain combinations and culture conditions described in this report) seemed to be directed toward stimulator cell determinants whose expression was determined by genes in the I region of the MHC. In one case, the relevant incompatibilities could be further restricted to the I-A region. Although PRC responded best to stimulator cells sharing the I region with the priming stimulator cell, apparent cross-reactivity could be observed by restimulating PRC with stimulator cells that did not carry the MHC haplotype of the priming stimulator cell. The rate of proliferation (measured as 3H-thymidine incorporation) in these apparent cross-reactions was reproducible and comparable to the rate observed in response to the priming stimulator cell. It was possible, therefore, to estimate the proportion of PRC that reacted in the presence of third party stimulator cells compared to the response of these PRC to the priming stimulator cells. We have estimated that the response of A (B6) PRC against H-2d and H-2s haplotype stimulator cells is about half of the response of these PRC to H-2b, the priming stimulator cell.     

In vitro growth of cytotoxic human lymphocytes. I. Growth of cells sensitized in vitro to alloantigens.

Human lymphocytes sensitized to allogeneic determinants in vitro were continuously cultured on medium prepared from phytohemagglutinin-(PHA-P) stimulated human mixed lymphocyte cultures. With such human-conditioned medium (HCM), human peripheral lymphoid cells could be grown in culture for over 90 days with a doulbing time of about 54 hr. Cytotoxic lymphocytes could be grown in culture for this time with no loss of cytotoxicity or change in cytotoxic specificity.