In vitro fenoprofenyl–coenzyme a thioester formation: Interspecies variations

In vitro coenzyme A thioester formation from (?)-(R)-fenoprofen (FPF) and palmitic acid has been studied using liver microsomes from rat, guinea pig, sheep, and dog. In every species with both palmitic acid or (?)-(R)-fenoprofen, the Lineweaver–Burk plot was linear in the substrate concentration range used and as a consequence agrees with the involvement of only one isoenzyme (or different isoenzymes of similar K_m values). The V_max values for the thioesterification of (?)-(R)-fenoprofen present large species variations from 2.1 ± 1.0 with sheep liver microsomes to 60.6 ± 11 nmol/min/mg with dog liver microsomes. These values statistically significantly correlate (r = 0.94) to the V_max values observed when palmitic acid was used as a substrate. Furthermore palmitic acid inhibited (?)-(R)-fenoprofen–CoA formation in the same extent in all animal species. The stereoselectivity of the thioesterification was also species dependent. © 1995 Wiley-Liss, Inc.     

The human cornea has a high incidence of acquired chromosome abnormalities

Structurally and functionally, the human cornea is a highly specialized tissue. The corneal stromal collagen matrix is uniquely transparent and yet maintains a mechanically tough and chemically impermeable barrier between the eye and environment. We report for the first time that stromal keratocytes of the human cornea show cytogenetic abnormalities with a frequency that is unprecedented among normal tissues. The abnormalities are acquired, clonal and nonclonal, primarily aneuploid in nature, and present in normal as well as diseased corneas. Received: 10 February 1997 / Accepted: 21 May 1997     

In vitro recombination of non-homologous genes can result in gene fusions that confer a switching phenotype to cells

Regulation of protein activity is central to the complexity of life. The ability to regulate protein activity through exogenously added molecules has biotechnological/biomedical applications and offers tools for basic science. Such regulation can be achieved by establishing a means to modulate the specific activity of the protein (i.e. allostery). An alternative strategy for intracellular regulation of protein activity is to control the amount of protein through effects on its production, accumulation, and degradation. We have previously demonstrated that the non-homologous recombination of the genes encoding maltose binding protein (MBP) and TEM1 β-lactamase (BLA) can result in fusion proteins in which β-lactamase enzyme activity is allosterically regulated by maltose. Here, through use of a two-tiered genetic selection scheme, we demonstrate that such recombination can result in genes that confer maltose-dependent resistance to β-lactam even though they do not encode allosteric enzymes. These 'phenotypic switch' genes encode fusion proteins whose accumulation is a result of a specific interaction with maltose. Phenotypic switches represent an important class of proteins for basic science and biotechnological applications in vivo.     

The vitamin K-dependent carboxylase has been acquired by Leptospira pathogens and shows altered activity that suggests a role other than protein carboxylation

Leptospirosis is an emerging infectious disease whose pathology includes a hemorrhagic response, and sequencing of the Leptospira interrogans genome revealed an ortholog of the vitamin K-dependent (VKD) carboxylase as one of several hemostatic proteins present in the bacterium. Until now, the VKD carboxylase was known to be present only in the animal kingdom (i.e. metazoans that include mammals, fish, snails, and insects), and this restricted distribution and high sequence similarity between metazoan and Leptospira orthologs strongly suggests that Leptospira acquired the VKD carboxylase by horizontal gene transfer. In metazoans, the VKD carboxylase is bifunctional, acting as an epoxidase that oxygenates vitamin K to a strong base and a carboxylase that uses the base to carboxylate Glu residues in VKD proteins, rendering them active in hemostasis and other physiologies. In contrast, the Leptospira ortholog showed epoxidase but not detectable carboxylase activity and divergence in a region of identity in all known metazoan VKD carboxylases that is important to Glu interaction. Furthermore, although the mammalian carboxylase is regulated so that vitamin K epoxidation does not occur unless Glu substrate is present, the Leptospira VKD epoxidase showed unfettered epoxidation in the absence of Glu substrate. Finally, human VKD protein orthologs were not detected in the L. interrogans genome. The combined data, then, suggest that Leptospira exapted the metazoan VKD carboxylase for some use other than VKD protein carboxylation, such as using the strong vitamin K base to drive a new reaction or to promote oxidative damage or depleting vitamin K to indirectly inhibit host VKD protein carboxylation.     

Phytophthora infestans has a plethora of phospholipase D enzymes including a subclass that has extracellular activity

In eukaryotes phospholipase D (PLD) is involved in many cellular processes. Currently little is known about PLDs in oomycetes. Here we report that the oomycete plant pathogen Phytophthora infestans has a large repertoire of PLDs divided over six subfamilies: PXPH-PLD, PXTM-PLD, TM-PLD, PLD-likes, and type A and B sPLD-likes. Since the latter have signal peptides we developed a method using metabolically labelled phospholipids to monitor if P. infestans secretes PLD. In extracellular medium of ten P. infestans strains PLD activity was detected as demonstrated by the production of phosphatidic acid and the PLD specific marker phosphatidylalcohol.     

The class 3 outer membrane protein (PorB) of Neisseria meningitidis: gene sequence and homology to the gonococcal porin PIA

The class 3 protein (PorB) is an important component of the meningococcal outer membrane. The structural gene (porB) encoding the class 3 protein has been cloned using primers suitable for the amplification of the corresponding chromosomal fragment by the polymerase chain reaction (PCR). The complete nucleotide sequence was determined and predicts a mature protein of 310 amino acids, preceded by a signal peptide of 19 residues. The predicted protein sequence of the class 3 protein exhibits essential structural homology to the gonococcal porin PIA. The class 3 protein encoding gene was expressed in Escherichia coli under the control of an inducible promoter.     

Transposon-encoded site-specific recombination: nature of the Tn3 DNA sequences which constitute the recombination site res.

The tnpR gene of transposon Tn3 encodes a site-specific recombination enzyme that acts at res, a DNA region adjacent to tnpR, to convert co-integrate intermediates of interreplicon transposition to the normal transposition end-products. We have used two complementary approaches to study the nature of the Tn3 recombination region, res. Firstly, the DNA-binding sites for tnpR protein were determined in DNase I protection experiments. These identified a 120-bp region between the tnpA and tnpR genes that can be subdivided into three separate protein-binding sites. Genetic dissection experiments indicate that few, if any, other sequences in addition to this 120-bp region are required for res function. Moreover, we have shown that the two directly repeated res regions within a molecule are unequal partners in the recombination reaction: a truncated res region, which is unable to recombine with a second identical res region, can recombine efficiently with an intact res region. This demonstration, along with the observation that tnpR/res recombination acts efficiently on directly repeated res regions within a molecule but inefficiently both on inverted res regions in the same molecule and in the fusion reaction between res regions in different molecules, leads us to propose that one-dimensional diffusion (tracking) of tnpR protein along DNA is used to locate an initial res region, and then to bring a second directly repeated res region into a position that allows recombination between the res regions.     

Carbonic anhydrase: Enzyme that has transformed the biosphere

The bases of modern type biosphere were laid down about two billion years ago during the predominance of prokaryotes on the Earth. Cyanobacteria changed radically the composition of the Proterozoic atmosphere by saturating it with photosynthetic oxygen. At the same time, large quantities of atmospheric CO₂ became sequestered in carbonates owing to mineralization of ancient cyano-bacterial communities; the latter have reached us in the form of laminated limestone deposits, termed stromatolites. The mechanism of carbonate depositing by cyanobacteria is still poorly understood. It is not yet clear whether physiological processes are involved in cell mineralization or if the outer membranes of cyanobacteria serve as a kind of crystallization center and arrange the structure for natural accumulation of sediments. We proposed that a key role in the mechanism of biomineralization belongs to the enzyme carbonic anhydrase (CA), which regulates the equilibrium between the inorganic carbon forms (C_i), including bicarbonate that participates in natural sedimentation of calcium. Since the deposition of calcium carbonate by prokaryotes occurs in the pericellular space and this deposition is controlled by pH, it seems likely that CA, localized on the periphery of cyanobacterial cells, is involved in stabilizing the external pH and in promoting cell mineralization. This review summarizes information concerning possible mechanisms of biogenic calcification (CaCO₃ deposition). The function of CA in the living cell and the role of this enzyme in biological processes are considered, and the data on localization of CA in cyano-bacterial cells are presented. Based on available evidence, a scheme is suggested to describe the role of extracellular CA in photosynthetic carbon assimilation and to relate this process with CaCO₃ deposition during mineralization of cyanobacteria.     

In vivo genetic engineering: homologous recombination as a tool for plasmid construction

This paper describes a novel method for creating exact DNA fusions between any two points in a plasmid carried in Bacillus subtilis. It exploits the homologous in vivo recombination between directly repeated sequences that can be established by insertion of a synthetic oligodeoxyribonucleotide. The method was used to enhance the productivity in B. subtilis of a cloned alpha-amylase (Amy)-encoding gene originating from Bacillus stearothermophilus. Thus, an exact fusion between nucleotide sequences encoding the expression signals, including the signal peptide, of a Bacillus licheniformis Amy-encoding gene and the mature Amy of B. stearothermophilus, was created. The resulting hybrid translational product was processed correctly in B. subtilis during secretion, giving rise to an Amy identical to the mature Amy secreted by B. stearothermophilus.     

DNA shuffling: Modifying the hand that nature dealt

Summary DNA shuffling is a technique being utilized for in vitro recombination of a single gene or pools of homologous genes. The genes are fragmented into randomly sized pieces, and polymerase chain reaction (PCR) reassembly of full-length genes from the fragments, via self-priming, yields recombination due to PCR template switching. After these PCR products are screened and the interesting products sequenced, improved clones are reshuffled to recombine useful mutations in additive or synergistic ways, in effect mimicking the process of natural sexual recombination. Proteins can be ‘bred’ with the appropriate individual properties and then their ‘progeny’ screened for the desired combination of traits. DNA shuffling is a powerful tool enabling rapid and directed evolution of new genes, operons and whole viral genomes.