Specific modification of a single cysteine residue in both bovine liver glutamate dehydrogenase and yeast glyceraldehyde-3-phosphate dehydrogenase. Difference in the mode of modification by pyrene maleimide.

Pyrene maleimide is shown to be a 'half of the sites' reagent for glutamate dehydrogenase and for glyceraldehyde-3-phosphate dehydrogenase. The modified residues are identified as cysteine-115 for glutamate dehydrogenase and cysteine-149 for glyceraldehyde-3-phosphate dehydrogenase. The two enzymes react differently with pyrene maleimide. Whereas the hydrophobic environment of cysteine-115 directs the modification of glutamate dehydrogenase, the high reactivity of cysteine-149 determines the specific modification of glyceraldehyde-3-phosphate dehydrogenase. Glutamate dehydrogenase activity is unaltered by the modification: glyceraldehyde-3-phosphate dehydrogenase activity in inhibited.

A histone H4-specific methyltransferase. Properties, specificity and effects on nucleosomal histones.

A histone H4-specific methyltransferase was purified 80-100-fold from nuclei of calf lymphocytes and from calf thymus. Some biochemical properties of the enzyme are described. The enzyme transfers in vitro methyl groups from S-adenosylmethionine preferentially to the lysine residue 20 of histone H4. This is the major in vivo methylation site of H4. DNA-bound or nucleosomal H4 is not methylated in vitro. We have used methylated and unmodified H4 (in the presence of sufficient quantities of the other core histones) for nucleosome reconstitution in vitro and have not found significant differences in the efficiencies of assembly.

Pore-forming properties of the adsorption protein of filamentous phage fd.

The gene 3-encoded adsorption protein (g3p) of filamentous phage fd has been purified to homogeneity by using high-performance liquid chromatography. Removal of SDS from the SDS-solubilized g3p results in spontaneous oligomerization of the g3p. Reconstitution into artificial lipid bilayer membranes shows that the oligomer forms large aqueous pores that remain open for seconds and are insensitive to changes in membrane potential. The estimated diameter of the pores suggest that they are large enough to allow passage of phage single-stranded DNA. The implications of these findings for phage infection are discussed.

Purification and characterization of a novel acid-soluble nuclear protein from developing embryos of the camel tick Hyalomma dromedarii (Acarina: Ixodidae).

A novel acid-soluble protein has been extracted from nuclei of developing embryos of H. dromedarii ticks and purified to homogeneity. This tick embryo basic protein (TEBP) was predominant during the cleavage stage of tick embryogenesis, whereas the complete set of histones was detectable at the late cleavage stage. The amount of TEBP reaches a maximum value at day 9 after oviposition. Thereafter, the original N-terminal dipeptide (leucine-serine) is eliminated. This coincides with the start of organogenesis. In spite of its low molecular mass, TEBP seems to be related to histone H1 in some properties such as solubility in perchloric acid and binding affinity to DNA. A task for the future will be to define the role of this protein as a counterpart of the histones for the genome organization during embryogenesis.

The inhibitory effect of dithiothreitol on the assembly of the filamentous phage fd.

Assembly of the filamentous phage fd is preceded by the formation of a complex between the viral single-stranded (ss) DNA and the virally coded gene 5 protein (gene 5 protein-ssDNA complex). The presence of 5 mM dithiothreitol in the growth medium prevents phage production; however, phage infection, phage DNA replication and phage genome expression are still observed. In contrast, the gene 5 protein-ssDNA complex is not formed in the presence of dithiothreitol in vivo, although the complex is not affected by the disulfide reducing agent in vitro. Furthermore, host lipid composition is altered by growth in the presence of dithiothreitol. The zwitterionic lipid, phosphatidylethanolamine, increases while the cationic phospholipid content, cardiolipin and phosphatidylglycerol, decreases. This suggests a role for lipids or membranous structures in the process of gene 5 protein-ssDNA complex formation.

Structural characterization and biological activity of recombinant human epidermal growth factor proteins with different N-terminal sequences.

The primary structures and molecular homogeneity of recombinant human epidermal growth factors from different suppliers were characterized and their biological activities evaluated by a standard DNA synthesis assay. Molecular weight determinations using 252Cf-plasma-desorption and electrospray mass spectrometry in combination with N- and C-terminal sequence analysis and determination of intramolecular disulfide bridges revealed that one recombinant protein had the correct human-identical structure (54 aa residues; 6347 Da). In contrast, a second recombinant protein (7020 Da) was found to contain a pentapeptide (KKYPR) insert following its N-terminal methionine. This structural variant showed a significant reduction in its capacity to stimulate DNA synthesis.

Dissection of functional domains in phage fd adsorption protein. Discrimination between attachment and penetration sites.

We constructed a set of deletion mutants in the attachment protein of phage fd. These mutants lack sequences coding for sections in the amino-terminal half. All the mutants that comprise a leader sequence are incorporated into phage particles. Our data strongly suggest a bipartite organization of the amino-terminal domain with (1) a region for receptor recognition and (2) a region that is necessary for penetration of the DNA into the host cell. These regions were mapped. Some evidence suggesting different roles for gene 3 protein in penetration of the outer and inner membrane are discussed. We demonstrate that the phenotypes caused by gene 3 protein in host cells can be subdivided into two groups with different sequence requirements: (1) phenotypes related to outer membrane disturbance; and (2) phenotypes related to the tolQRA transport system.

Versatile kanamycin-resistance cartridges for vector construction in Escherichia coli.

To generate polylinker sequences which can be transferred together with an adjacent selectable marker, two plasmids (pWW-84 and pWW-97) were constructed which contain a kanamycin-resistance gene (KmR) flanked by various restriction sites. From these plasmids KmR-cartridges can be obtained as EcoRI, BamHI, SalI, AccI or HincII fragments for insertion into the appropriate restriction site of any plasmid. The following restriction sites can be introduced with these cartridges: BamHI, SalI (AccI, HincII), EcoRI, SacI, SphI and KpnI (Asp718) all adjacent to KmR, XhoI and HindIII, both within KmR. If desired, KmR can be removed by PstI digestion and religation, creating a single PstI site and leaving all adjacent sites intact.

Release of periplasmic proteins induced in E. coli by expression of an N-terminal proximal segment of the phage fd gene 3 protein.

We used the enzymes beta-lactamase and alkaline phosphatase to quantitatively evaluate the release of periplasmic proteins from E. coli cells transformed by plasmids harboring gene 3 of phage fd. Different deletion mutants of gene 3 released varying fractions of the enzymes. From these results we conclude that essentially the amino-terminal proximal part, upstream of the first glycine-rich region but not this region itself, is responsible for the excretion of periplasmic proteins in E. coli cells expressing the gene 3 protein of phage fd.

Quantitative data on peroxidatic markers for electron microscopy. With a note on actin identification in Paramecium cells.

Several important points of heme-peptide cytochemistry were quantitatively analyzed, with particular regard to their use in electron microscopic immunocytochemistry. A simple procedure is presented for the preparation of heme-octapeptide (H-8-P) microperoxidase. H-8-P, hemenonapeptide (H-9-P), and various horseradish peroxidase (HRP) isoenzymes were used for coupling with immunoglobulin (Ig)G or the papain-cleavage fragments from IgG (Fab) molecules. Ultracentrifugation and spectrophotometric analyses revealed the following characteristics of the conjugates: a) They are of a uniform size class; b) their diameters were calculated, and ranged from 5.6 (Fab-H-8-P; H-9-P) to 10.5 (IgG-HRP); c) the persistence of antigen binding capacity was ascertained; d) the deactivation of the marker peroxidase activity due to coupling was as low as 20-30%; e) optimal conditions for use of the electron microscopic (EM) with 3,3'-diaminobenzidine media were elaborated (with a pH optima somewhat different from some standard methods in current use); and f) on the basis of the quantitative data presented, an optimal compromise (either in favor of higher peroxidase activity with HRP conjugates or of smaller size with microperoxidase-Fab conjugates) can be achieved. Finally, the identification of isolated purified actin and of actin in cortical microfilament bundles and ciliary basal bodies of Paramecium cells served as a test object for the usefulness of conjugation products and optimized assay conditions for EM immunocytochemistry.

Genetic analysis of the transfer region of the IncN plasmid N3.

Using lambda::Tn5 insertion mutagenesis and screening for conjugation, the boundaries of the IncN plasmid N3 transfer region were determined. Sensitivity to phage IKe infection was used to monitor that part of the N3 transfer region which harbours genes for pilus synthesis and assembly. We cloned this region, creating plasmid pBG21. Escherichia coli cells transformed with pBG21 became sensitive to phage IKe and produced pili, as shown by electron microscopy. Various plasmid constructions containing parts of the pilus-encoding region were used for expression in a minicell system and for expression in an in vitro translation system, thus characterizing for the first time some of the gene products of domain I (Winans and Walker, 1985a) of the transfer region.

Convergent evolution of amino acid usage in archaebacterial and eubacterial lineages adapted to high salt.

Chemical composition and physical properties of the total protein of Haloferax mediterranei, a halophilic archaebacterium requiring high salt concentration for growth, of Halomonas elongata, a halotolerant eubacterium able to grow at any concentration of salt, and of Escherichia coli B, a eubacterium related to H. elongata, unable to grow at high salt concentration, were compared using robust standard biochemical methods. The distribution of amino acid abundancies in the bulk protein from H. elongata was found to be intermediate between that from H. mediterranei and that from E. coli. The two high-salt-adapted organisms displayed an enrichment in aspartic acid and glutamic acid together with an impoverishment in lysine as compared to E. coli. This signature in amino acid usage is reflected in the charge distribution of proteins, as revealed by anion exchange chromatography of crude cell extracts. Since H. elongata diverged from H. mediterranei more than three billion years ago, the resemblance of their amino acid usages can be interpreted as a convergent imprint of their common habitats onto the chemical constitution of their proteins.

The role of the adsorption complex in the termination of filamentous phage assembly.

The adsorption complex of filamentous phage fd consists of two minor coat proteins, g3p and g6p, and is considered to be not only a structural entity, but also a functional unit to terminate phage assembly. Cells were infected with phage M13am8H1, which cannot assemble because it lacks the major coat protein g8p, although producing all of the other minor coat proteins. The membranes of infected cells were solubilized and analysed by non-denaturing PAGE and gel filtration. The data suggest the presence of the adsorption complex in these membranes. Furthermore, the non-polar gene 3 amber-mutant phage R171 was shown to lack g6p in the phage coat as well. The termination of assembly of this phage is disturbed, resulting in synthesis of polyphages. Electron micrographs and transient electrical birefringence show that these polyphages are eight times longer as compared to unit length phage. From these results, we conclude that the formation of the g3p-g6p complex is essential for correct termination of filamentous phage assembly.

The lysine residues implicated in the gene 5 protein association sites.

Gene 5 protein, a DNA unwinding protein encoded by the bacteriophage fd, is self-associative in presence of DNA or oligonucleotides. The lysine residues implicated in the protein-protein binding domains have been identified after modification with acetimidate by means of peptide and amino acid analyses. These residues are Lys-7 and Lys-69.

Purification and Characterization of Extracellular Pectinesterases from Phytophthora infestans.

Constitutively produced extracellular pectinesterases from culture filtrates of the potato late blight fungus Phytophthora infestans were purified and characterized. One enzyme (PE II) was purified to homogeneity. Sodium dodecyl sulfate electrophoresis of the second enzyme (PE I) revealed two protein bands; there are indications that both proteins are pectinesterases, which were not separable by a number of different techniques. Thus, P. infestans might produce three pectinesterases in vitro. Enzyme activities were optimal in the neutral pH range and were largely dependent on the presence of NaCl or CaCl(2) in the reaction medium. The molecular weight of the PE I-complex was between 45 and 48 kilodaltons, and the one of PE II was between 35 and 40 kilodaltons. Further investigations will help us to clarify the role of these enzymes during pathogenesis.