Molecular characterization of two lipoxygenases from barley.

Two full-length lipoxygenase cDNA sequences (LoxB and LoxC) from barley (Hordeum distichum cv. L. Triumph) are described. The cDNAs share high homology with the barley LoxA cDNA. Southern blotting experiments indicate single copy numbers of the three lipoxygenase genes. RFLP mapping revealed the presence of single lipoxygenase loci. LoxA and LoxB map on chromosome 4 and LoxC on chromosome 7. Two isoenzymes, LOX1 and LOX2, have been purified previously from germinating barley and characterized. LOX1 is encoded by LoxA, while LOX2 is encoded by LoxC. The product related to the third cDNA (loxB) has not been identified so far, suggesting a low protein abundance for the corresponding isoform in barley. Transcripts corresponding with these LOX genes are predominantly observed in grain and in seedling, whereas transcripts corresponding to LoxB and LoxC are also observed in mature vegetative tissue. No lipoxygenase mRNA could be detected in aleurone layer of germinating grain. No significant differences in lipoxygenase mRNA levels were observed in developing grains grown under dormant or non-dormant conditions, suggesting that LOX is not directly involved in induction of grain dormancy.

Primary structure of a lipoxygenase from barley grain as deduced from its cDNA sequence.

A full length cDNA sequence for a barley grain lipoxygenase was obtained. It includes a 5' untranslated region of 69 nucleotides, an open reading frame of 2586 nucleotides encoding a protein of 862 amino acid residues and a 3' untranslated region of 142 nucleotides. The molecular mass of the encoded polypeptide was calculated to be 96.392. Its amino acid sequence shows a high homology with that of other plant lipoxygenases identified to date.

Purification and characterization of two lipoxygenase isoenzymes from germinating barley.

Two lipoxygenase isoenzymes (linoleate: oxygen oxidoreductase, EC 1.13.11.12) present in the embryo of germinating barley seed have been purified to homogeneity and characterized. Both isoenzymes are monomeric proteins with a molecular mass of approx. 90 kDa and crossreact on Western blots with antibodies raised against pea lipoxygenase. They have an apparent Km of approx. 16 microM for linoleic acid. The isoenzymes differ in the product formed upon incubation with linoleic acid. One of the isoenzymes (lipoxygenase 1) solely forms the 9-HPOD as a product whereas the 13-HPOD is the major product formed by the other isoenzyme (lipoxygenase 2). Lipoxygenase 1 shows a pH-optimum of 6.5, is active in a broad pH range and has an isoelectric point of 5.2-5.3. Lipoxygenase 2 has the same pH optimum, but is active in a narrow pH range and has a significantly higher pI, namely 6.8-6.9. The occurrence of two isoenzymes was confirmed by peptide analysis of the proteins. Amino acid sequence data obtained from proteolytic fragments of lipoxygenase 1 show up to 50% identity with other plant lipoxygenases.

Binding of tissue-type plasminogen activator to lysine, lysine analogues, and fibrin fragments.

Human tissue-type plasminogen activator (t-PA) consists of five domains designated (starting from the N-terminus) finger, growth factor, kringle 1, kringle 2, and protease. The binding of t-PA to lysine-Sepharose and aminohexyl-Sepharose was found to require kringle 2. The affinity for binding the lysine derivatives 6-aminohexanoic acid and N-acetyllysine methyl ester was about equal, suggesting that t-PA does not prefer C-terminal lysine residues for binding. Intact t-PA and a variant consisting only of kringle 2 and protease domains were found to bind to fibrin fragment FCB-2, the very fragment that also binds plasminogen and acts as a stimulator of t-PA-catalyzed plasminogen activation. In both cases, binding could completely be inhibited by 6-aminohexanoic acid, pointing to the involvement of a lysine binding site in this interaction. Furthermore, the second site in t-PA involved in interaction with fibrin, presumably the finger, appears to interact with a part of fibrin, different from FCB-2.

Transient expression in mammalian cells of the bacterial reporter gene encoding mercuric reductase: effects of various regulatory elements.

The effect of several transcriptional regulatory elements on gene expression in mammalian cells was investigated. As a reporter gene we have used the bacterial gene merA coding for the enzyme mercuric reductase. Several plasmids were constructed with different promoter/enhancer sequences (pSV/E, pSV/L, pMT, pRSV or pAd) at the 5' end and different splicing (small intron of the T antigen of SV40 or the second intron of the rabbit beta-globin gene) and/or polyadenylation signals (AEn, ALn or AR beta Gn) at the 3' end of the merA gene. Expression was measured in five different mammalian cell lines. In COS cells the highest level of expression is obtained with pSV/L and the lowest level with pSV/E. In HeLa, CV-1, Ltk-, and CHO cells merA expression is relatively high, under control of pRSV and pMT and relatively low under control of pSV/L and pAd. The introns studied have a negative effect on the expression of merA. The presence of a polyadenylation signal downstream from the gene is essential for its expression. The three different polyadenylation signals studied give a similar stimulatory effect on the level of expression of the merA gene.

Poliovirus protein 3CD is the active protease for processing of the precursor protein P1 in vitro.

A transcription/translation system for generating poliovirus proteins in vitro has been used to assess the proteolytic activity of various polypeptides containing the virus-coded 3C region towards the poliovirus precursor protein P1. Plasmids containing a phage T7 promoter followed by either the complete poliovirus P1 sequence or various sequences containing the 3C region were used for this purpose. We showed that all except one of the 3C-containing polypeptides had a very restricted activity towards P1, generating only a small amount of VP1 and no VP0 or VP3. The only polypeptide capable of fully processing P1 into VP0, VP3 and VP1 in vitro was protein 3CD, consisting of the complete 3C and 3D sequences.

Fusion proteins with multiple copies of the major antigenic determinant of foot-and-mouth disease virus protect both the natural host and laboratory animals.

Proteins consisting of one, two or four copies of the amino acid sequence 137 to 162, which contains the major immunogenic site of VP1 of foot-and-mouth disease virus, attached to the N-terminus of beta-galactosidase have been expressed in Escherichia coli cells. In guinea-pigs the protein containing one copy (P71) of the viral determinant elicited only low levels of neutralizing antibody whereas protective levels were elicited by the proteins containing two (P72) or four (P74) copies of the determinant. Single inoculations of the P72 and P74 proteins containing as little as 2 micrograms or 0.8 micrograms of peptide respectively were sufficient to protect all the animals against challenge infection. Moreover, the equivalent of 40 micrograms of peptide in P74 protected pigs against challenge infection after one inoculation.

Construction of expression plasmids for Saccharomyces cerevisiae: application for synthesis of poliovirus protein VP2.

A series of expression plasmids was constructed to compare the usefulness of various promoters for the synthesis of a given protein in the Saccharomyces cerevisiae. The plasmids pMBL212, -213, -214, -215 and -216 can be used to synthesize the protein of interest directly as a non-fused protein or, if the protein is difficult to detect, indirectly as an enzymatically active beta-galactosidase fusion protein. The plasmids were employed to identify which yeast promoter and strain are suitable for the synthesis of poliovirus protein VP2. It was concluded that the GAL7 and PGK promoters in combination with strain X904 can be used for efficient synthesis of a VP2 in the form of a N-terminally fused VP2-beta-galactosidase protein.

Involvement of finger domain and kringle 2 domain of tissue-type plasminogen activator in fibrin binding and stimulation of activity by fibrin.

Human tissue-type plasminogen activator (t-PA) catalyses the conversion of inactive plasminogen into active plasmin, the main fibrinolytic enzyme. This process is confined to the fibrin surface by specific binding of t-PA to fibrin and stimulation of its activity by fibrin. Tissue-type plasminogen activator contains five domains designated finger, growth factor, kringle 1, kringle 2 and protease. The involvement of the domains in fibrin specificity was investigated with a set of variant proteins lacking one or more domains. Variant proteins were produced by expression in Chinese hamster ovary cells of plasmids containing part of the coding sequence for the activator. It was found that kringle 2 domain only is involved in stimulation of activity by fibrin. In the absence of plasminogen and at low concentration of fibrin, binding of t-PA is mainly due to the finger domain, while at high fibrin concentrations also kringle 2 is involved in fibrin binding. In the presence of plasminogen, fibrin binding of the kringle 2 region of t-PA also becomes important at low fibrin concentrations.

Synthesis of fusion proteins containing antigenic determinants of foot-and-mouth disease virus.

Part of the genome of foot-and-mouth disease virus (FMDV) type 01,BFS, including the sequence encoding the capsid polypeptide VP1, was cloned in Escherichia coli following a new cloning strategy. The clone containing the VP1 sequence was used for the construction of two expression plasmids encoding VP1 fusion proteins. Subsequently, substantial amounts of the two VP1-beta-galactosidase fusion proteins, containing either one (amino acid region 140-160) or two (amino acid regions 140-160 and 200-213) antigenic determinants of the virus, were synthesized by E. coli bacteria. The protein containing the amino acid region 140-160 of VP1 fused to beta-galactosidase efficiently induced antibodies in rabbits specifically reacting with FMDV type 01,BFS. The same protein was also capable of eliciting neutralizing antibodies. The fusion protein containing both antigenic determinants did not efficiently induce antibodies reacting with FMDV.

Synthesis of fusion proteins with multiple copies of an antigenic determinant of foot-and-mouth disease virus.

A series of four expression plasmids coding for fusion proteins containing foot-and-mouth disease virus (FMDV) sequences was constructed. The fusion proteins contain a large part of beta-galactosidase from Escherichia coli preceded (N-terminal) by 1, 2, 4 or 8 repeats of the antigenic determinant of FMDV consisting of amino acids 137-162 of the capsid polypeptide VP1. All four fusion proteins were efficiently produced in E. coli host bacteria. Immunization of rabbits resulted in FMDV-specific, neutralizing antibodies, the response being dependent on the number of repeats. With enzyme-linked immunosorbent-assay techniques it was shown that the FMDV antigenic determinants are exposed on the surface of the fusion proteins under non-denaturing conditions.

A system to determine basepair substitutions at the molecular level, based on restriction enzyme analysis; influence of the muc genes of pKM101 on the specificity of mutation induction in E. coli.

A system has been developed for the analysis of basepair substitutions that are involved in the reversion of a specific missense mutation. The method is based on the ability of restriction enzymes to recognize and cut specific DNA sequences. Wild-type revertants arising from AT----GC transitions, pseudo wild-type revertants arising from AT-transversions and second site revertants can be distinguished. 4 mutagenic agents have been used, 2,6-diaminopurine, MMS, EMS and ENU, which differ in the types of damage they cause in DNA and in the susceptibility of the damage to repair. All 4 mutagens effectively enhanced the reversion of the mutation studied, trpA223, particularly by increasing the fraction of AT----GC transitions. In this system the influence of the muc genes of plasmid pKM101 was investigated. The presence of these genes reduced the fraction of AT----GC transitions and enhanced the fraction of AT-transversions as well as the fraction of second-site mutations. This change in mutation specificity is found irrespective whether mutation induction occurs mainly via SOS repair (MMS, ENU) or via mainly misreplication (2,6-diAP, EMS). These data suggest that the muc genes are involved in the induction of mutations not only during SOS repair, but also during misreplication. The change in mutation specificity may be caused by a change in the selection and insertion of nucleotides by the DNA-polymerising complex, or by interference with the repair of mismatched bases.

Construction of new vectors for cloning of promoters.

Vectors for cloning promoter-DNA fragments were derived from plasmid pBR313 (Bolivar et al., 1977). These have several unique restriction sites and carry the trpA gene from Escherichia coli as a selective marker. The selection is based on an enhancement of the growth rate of those bacteria in which the expression of trpA is directed by the cloned promoter. The expression of trpA can be determined quantitatively, independently of the copy number of the vector, and should reflect the apparent strength of the promoter, since the DNA segment located before trpA contains translational stop signals in all three reading frames.

The construction of new vehicles for the cloning of transcription termination signals.

We have constructed new plasmids that can be used to clone transcription terminator containing DNA fragments between the first gene of the tryptophan (trp) operon and the tetracycline resistance (tet) gene. Both genes are under control of the trp promotor. Therefore the presence of a transcription termination signal on cloned fragments can be monitored by a decrease in expression of the tet gene. The plasmids contain cloning sites at different distances from the translation start signal. Consequently a cloned DNA fragment can be translated in the three possible reading frames, offering the opportunity to distinguish terminators from translation polarity (pseudo-terminators). The usefulness of the plasmids was shown by the cloning of the trp terminator and of a pseudo-terminator located in the trpB gene.

Construction of new cloning vehicles with genes of the tryptophan operon of Escherichia coli as genetic markers.

In vitro recombination techniques were used to construct a series of new cloning vehicles with genes of the tryptophan (trp) operon of Escherichia coli as selective marker. To construct these plasmids we have made a restriction cleavage map of the trp operon for the enzymes AosI, AvaI, BglI, BglII, HindIII, HpaI, PvuII, SalI, SstI and XhoI. The constructed plasmids pHP39, pEP392, pEP3921 and pEP3923 are derived from the amplifiable plasmid pBR345 and carry two or more genes of the trp operon, which are controlled by the trp regulatory elements. Plasmid pEP3921 (7.0 kb) carries intact trpE and trpA genes and contains single BglII and SstI sites in trpE, a single HindIII site located between trpE and trpA, and single EcoRI, SalI and XhoI sites located outside the trp genes. Plasmid pEP121 (12 kb) is similar to pEP3921, but has an extra selective marker conferring bacterial resistance to ampicillin. Plasmid pEP3923 (7.4 kb) comprises intact trpB and trpA genes and single BglII, HindIII, EcoRI, SalI and XhoI sites. Plasmids pHP39 (9.8 kb) and pEP392 (9.8 kb) carry an intact trp operon and have two and one EcoRI site, respectively. Plasmid pHP3 (18 kb) carries an intact trp operon and markers for tetracycline and ampicillin resistance.

Studies on the binding of FMN by apoflavodoxin from Peptostreptococcus elsdenii, pH and NaCl concentration dependence.

1. The pH and ionic strength dependence of the interaction of FMN with apoflavodoxin has been studied by fluorometry in the pH region 2-5, at 22 degrees C. 2. The rate constant of dissociation and the dissociation constant were experimentally determined; the rate constants of association were claculated at a given pH value. These constants depend on the ionic strength. The plots of these constants against the square root of the ionic strength are straight. 3. Our data have been interpreted in terms of the Brönsted theory, which relates chemical reaction rates to ionic strength. The data indicate that the apoenzyme reaches its maximum net positive charge at pH 2.0-2.6. The calculated net charge in this pH region is between 11 and 12 and is in agreement with the theoretical value of 12 as deduced from the primary structure of the protein. The isoelectric point of the holoenzyme is about 4. 4. The rate constant of association extrapolated to zero ionic strength is 3.2-10(5)M-1-s-1 and is pH-independent. 5. The rate constant of dissociation and the dissociation constant extrapolated to zero ionic strength depend on the pH. The results are explained by assuming that there are two protein ionizations with a pK value of 3.4; these ionizing groups are possibly close to the FMN binding site.