5-Aminolevulinate synthase and the first step of heme biosynthesis

5-Aminolevulinate synthase catalyzes the condensation of glycine and succinyl-CoA to yield 5-aminolevulinate. In animals, fungi, and some bacteria, 5-aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway. Mutations on the human erythroid 5-aminolevulinate synthase, which is localized on the X-chromosome, have been associated with X-linked sideroblastic anemia. Recent biochemical and molecular biological developments provide important insights into the structure and function of this enzyme. In animals, two aminolevulinate synthase genes, one housekeeping and one erythroid-specific, have been identified. In addition, the isolation of 5-aminolevulinate synthase genomic and cDNA clones have permitted the development of expression systems, which have tremendously increased the yields of purified enzyme, facilitating structural and functional studies. A lysine residue has been identified as the residue involved in the Schiff base linkage of the pyridoxal 5-phosphate cofactor, and the catalytic domain has been assigned to the C-terminus of the enzyme. A conserved glycine-rich motif, common to all aminolevulinate synthases, has been proposed to be at the pyridoxal 5phosphate-binding site. A heme-regulatory motif, present in the presequences of 5-aminolevulinate synthase precursors, has been shown to mediate the inhibition of the mitochondrial import of the precursor proteins in the presence of heme. Finally, the regulatory mechanisms, exerted by an iron-responsive element binding protein, during the translation of erythroid 5-aminolevulinate synthase mRNA, are discussed in relation to heme biosynthesis.     

Molecular evolution of duplicate copies of genes encoding cytosolic glutamine synthetase in Pisum sativum

Here, we describe two nearly identical expressed genes for cytosolic glutamine synthetase (GS3A and GS3B) in Pisum sativum L. RFLP mapping data indicates that the GS3A and GS3B genes are separate loci located on different chromosomes. DNA sequencing of the GS3A and GS3B genes revealed that the coding regions are 99% identical with only simple nucleotide substitutions resulting in three amino acid differences. Surprisingly, the non-coding regions (5 non-coding leader, the 11 introns, and 3 non-coding tail) all showed a high degree of identity (96%). In these non-coding regions, 25% of the observed differences between the GS3A and GS3B genes were deletions or duplications. The single difference in the 3 non-coding regions of the GS3A and GS3B genes was a 25 bp duplication of an AU-rich element in the GS3B gene. As the GS3B mRNA accumulates to lower levels than the GS3A gene, we tested whether this sequence which resembles an mRNA instability determinant functioned as such in the context of the GS mRNA. Using the GS3B 3 tail as part of a chimeric gene in transgenic plants, we showed that this AU-rich sequence has little effect on transgene mRNA levels. To determine whether the GS3A/GS3B genes represent a recent duplication, we examined GS3-like genes in genomic DNA of ancient relatives of P. sativum. We observed that several members of the Viceae each contain two genomic DNA fragments homologous to the GS3B gene, suggesting that this is an ancient duplication event. Gene conversion has been invoked as a possible mechanism for maintaining the high level of nucleotide similarity found between the GS3A and GS3B genes. Possible evolutionary reasons for the maintenance of these twin GS genes in pea, and the general duplication of genes for cytosolic GS in all plant species are discussed.     

The response of nodulated alfalfa to water supply, temperature and elevated CO2: photosynthetic downregulation

Plants grown in an environment of elevated CO₂ and temperature often show reduced CO₂ assimilation capacity, providing evidence of photosynthetic downregulation. The aim of this study was to analyse the downregulation of photosynthesis in elevated CO₂ (700 µmol mol⁻¹) in nodulated alfalfa plants grown at different temperatures (ambient and ambient + 4°C) and water availability regimes in temperature gradient tunnels. When the measurements were taken in growth conditions, a combination of elevated CO₂ and temperature enhanced the photosynthetic rate; however, when they were carried out at the same CO₂ concentration (350 and 700 µmol mol⁻¹), elevated CO₂ induced photosynthetic downregulation, regardless of temperature and drought. Intercellular CO₂ concentration measurements revealed that photosynthetic acclimation could not be accounted for by stomatal limitations. Downregulation of plants grown in elevated CO₂ was a consequence of decreased carboxylation efficiency as a result of reduced rubisco activity and protein content; in plants grown at ambient temperature, downregulation was also induced by decreased quantum efficiency. The decrease in rubisco activity was associated with carbohydrate accumulation and depleted nitrogen availability. The root nodules were not sufficiently effective to balance the source–sink relation in elevated CO₂ treatments and to provide the required nitrogen to counteract photosynthetic acclimation.     

Relationship between changes in ploidy and stable cellular resistance to hydrogen peroxide

Stable hydrogen peroxide (H₂O₂)-resistant variants of the Chinese hamster ovary HA-1 line have been isolated by culturing cells in progressively increasing concentrations of H₂O₂ (>200 days, in 50–800 μM H₂O₂). Increases in catalase activity in these variant cell lines were shown to correlate with increased H₂O₂ resistance. Stable (>240 days) H₂O₂-resistant cell lines, seven quasidiploid (21–22 chromosomes/cell) and six quasitetraploid (40–44 chromosomes/cell) were clonally isolated from the 800 μM adapted H₂O₂-resistant variants which were heterogeneous with respect to ploidy. The H₂O₂ dose-modifying factors (DMFs) were 3, 5, 8, 13, 15, 26, and 27 for the seven quasidiploid cell lines, and 21, 32, 38, 40, 42, and 49 for the six quasitetraploid cell lines. The mean DMF was 14±10 for the former and 37±10 for the latter. Our data show that on the average the quasitetraploid cell lines were significantly more resistant to H₂O₂-mediated cell killing than the quasidiploid cell lines derived from the same mixed population of 800 μM H₂O₂-adapted cells. When catalase activities (k units/cell) of the HA-1 cells and three of the clonally derived cell lines (two quasidiploid and one quasitetraploid) were determined and plotted vs. H₂O₂–DMF, a positive linear correlation was obtained (correlation coefficient = 0.99). This result was further confirmed when immunoreactive catalase protein/cell was detected by Western blots. Our data show that chronic exposure of cells to H₂O₂ stress (800 μM) was accompanied by increases in quasitetraploid cells within the population. Quasitetraploid cell lines derived from this population demonstrated increased stable H₂O₂-resistance which may be related to stable increases in the expression of catalase.     

Replication and Control of Circular Bacterial Plasmids

An essential feature of bacterial plasmids is their ability to replicate as autonomous genetic elements in a controlled way within the host. Therefore, they can be used to explore the mechanisms involved in DNA replication and to analyze the different strategies that couple DNA replication to other critical events in the cell cycle. In this review, we focus on replication and its control in circular plasmids. Plasmid replication can be conveniently divided into three stages: initiation, elongation, and termination. The inability of DNA polymerases to initiate de novo replication makes necessary the independent generation of a primer. This is solved, in circular plasmids, by two main strategies: (i) opening of the strands followed by RNA priming (theta and strand displacement replication) or (ii) cleavage of one of the DNA strands to generate a 3′-OH end (rolling-circle replication). Initiation is catalyzed most frequently by one or a few plasmid-encoded initiation proteins that recognize plasmid-specific DNA sequences and determine the point from which replication starts (the origin of replication). In some cases, these proteins also participate directly in the generation of the primer. These initiators can also play the role of pilot proteins that guide the assembly of the host replisome at the plasmid origin. Elongation of plasmid replication is carried out basically by DNA polymerase III holoenzyme (and, in some cases, by DNA polymerase I at an early stage), with the participation of other host proteins that form the replisome. Termination of replication has specific requirements and implications for reinitiation, studies of which have started. The initiation stage plays an additional role: it is the stage at which mechanisms controlling replication operate. The objective of this control is to maintain a fixed concentration of plasmid molecules in a growing bacterial population (duplication of the plasmid pool paced with duplication of the bacterial population). The molecules involved directly in this control can be (i) RNA (antisense RNA), (ii) DNA sequences (iterons), or (iii) antisense RNA and proteins acting in concert. The control elements maintain an average frequency of one plasmid replication per plasmid copy per cell cycle and can “sense” and correct deviations from this average. Most of the current knowledge on plasmid replication and its control is based on the results of analyses performed with pure cultures under steady-state growth conditions. This knowledge sets important parameters needed to understand the maintenance of these genetic elements in mixed populations and under environmental conditions.     

Synthesis of ribosomal proteins during growth of Streptomyces coelicolor

Changes in expression of ribosomal protein genes during growth and stationary phase of Streptomyces coelicolor A3(2) in liquid medium were studied. Proteins being synthesized were pulse-labelled with [³⁵ S]-methionine, separated by two-dimensional poly-acrylamide gel electrophoresis, and quantified using the Bioimage computer software. Most of the ribosomal proteins were synthesized throughout the life cycle. Exceptions were two proteins whose synthesis drastically decreased at the approach of stationary phase. These two proteins were identified in purified ribosomes as homologues of Escherichia coli ribosomal proteins L10 and L7/L12, using antibodies raised against fusion proteins between these ribosomal proteins and Escherichia coliβ-galactosldase. The genes (rplJ and rplL) encoding the L10 and L7/L12 proteins were contained in a 1.2 kb BamHl fragment that was cloned and sequenced. The linkage and order of the genes coincide with other L10-L7/L12 operons. However, L11 and L1 genes were not present immediately upstream of the L10 gene, as is the case for E. coli and other bacteria. Instead, two open reading frames of unknown function were found immediately upstream of the L10 gene, in an adjacent 1.9 kb BamHl fragment.     

Human aldehyde dehydrogenase: chromosomal assignment of the gene for the isozyme that metabolizes γ-aminobutyraldehyde

A cDNA clone of the E3 isozyme of human liver aldehyde dehydrogenase consisting of a 1320-base pair (bp) coding region and a 180-bp non-coding region at the 3 end was used for chromosomal localization of the E3 gene. Using a panel of human/hamster somatic cell hybrids we have localized, the gene coding for the E3 isozyme to human chromosome 1.     

Evading pre-existing anti-hinge antibody binding by hinge engineering

Antigen-binding fragments (Fab) and F(ab′)₂ antibodies serve as alternative formats to full-length anti-bodies in therapeutic and immune assays. They provide the advantage of small size, short serum half-life, and lack of effector function. Several proteases associated with invasive diseases are known to cleave antibodies in the hinge-region, and this results in anti-hinge antibodies (AHA) toward the neoepitopes. The AHA can act as surrogate Fc and reintroduce the properties of the Fc that are otherwise lacking in antibody fragments. While this response is desired during the natural process of fighting disease, it is commonly unwanted for therapeutic antibody fragments. In our study, we identify a truncation in the lower hinge region of the antibody that maintains efficient proteolytic cleavage by IdeS protease. The resulting neoepitope at the F(ab′)₂ C-terminus does not have detectable binding of pre-existing AHA, providing a practical route to produce F(ab′)₂ in vitro by proteolytic digestion when the binding of pre-existing AHA is undesired. We extend our studies to the upper hinge region of the antibody and provide a detailed analysis of the contribution of C-terminal residues of the upper hinge of human IgG1, IgG2 and IgG4 to pre-existing AHA reactivity in human serum. While no pre-existing antibodies are observed toward the Fab of IgG2 and IgG4 isotype, a significant response is observed toward most residues of the upper hinge of human IgG1. We identify a T₂₂₅L variant and the natural C-terminal D₂₂₁ as solutions with minimal serum reactivity. Our work now enables the production of Fab and F(ab′)₂ for therapeutic and diagnostic immune assays that have minimal reactivity toward pre-existing AHA.