An RNA Phage Lab: MS2 in Walter Fiers’ Laboratory of Molecular Biology in Ghent, from Genetic Code to Gene and Genome, 1963–1976

The importance of viruses as model organisms is well-established in molecular biology and Max Delbrück’s phage group set standards in the DNA phage field. In this paper, I argue that RNA phages, discovered in the 1960s, were also instrumental in the making of molecular biology. As part of experimental systems, RNA phages stood for messenger RNA (mRNA), genes and genome. RNA was thought to mediate information transfers between DNA and proteins. Furthermore, RNA was more manageable at the bench than DNA due to the availability of specific RNases, enzymes used as chemical tools to analyse RNA. Finally, RNA phages provided scientists with a pure source of mRNA to investigate the genetic code, genes and even a genome sequence. This paper focuses on Walter Fiers’ laboratory at Ghent University (Belgium) and their work on the RNA phage MS2. When setting up his Laboratory of Molecular Biology, Fiers planned a comprehensive study of the virus with a strong emphasis on the issue of structure. In his lab, RNA sequencing, now a little-known technique, evolved gradually from a means to solve the genetic code, to a tool for completing the first genome sequence. Thus, I follow the research pathway of Fiers and his ‘RNA phage lab’ with their evolving experimental system from 1960 to the late 1970s. This study illuminates two decisive shifts in post-war biology: the emergence of molecular biology as a discipline in the 1960s in Europe and of genomics in the 1990s.     

Contrasting Approaches to a Biological Problem: Paul Boyer, Peter Mitchell and the Mechanism of the ATP Synthase, 1961–1985

Attempts to solve the puzzling problem of oxidative phosphorylation led to four very different hypotheses each of which suggested a different view of the ATP synthase, the phosphorylating enzyme. During the 1960s and 1970s evidence began to accumulate which rendered Peter Mitchell’s chemiosmotic hypothesis, the novel part of which was the proton translocating ATP synthase (ATPase), a plausible explanation. The conformational hypothesis of Paul Boyer implied an enzyme where ATP synthesis was driven by the energy of conformational changes in the respiratory proteins. This was finally abandoned as an explanation of the overall process. Nevertheless the conformational understanding of the enzyme became an acceptable proposal during the early 1970s and eventually led Boyer to a view of the enzyme that incorporated both hypotheses. The correspondence between Mitchell and Boyer, both Nobel laureates, exposes their different approaches to both this enzyme and to the hypotheses of oxidative phosphorylation and illuminates a key step in the development of bioenergetics. In particular Boyer was suspicious of proton gradients, because he could not envisage a chemical mechanism for the synthesis of ATP, while Mitchell distrusted conformational arguments because he believed the proton must act vectorially at the active site of the enzyme. This resulted in two different views of the mechanisms operating in this enzyme. Ultimately while Boyer was able to marry the two approaches, Mitchell retained his insistence on the role of the proton at the active site and was thus unable to give significance to Boyer’s conformational ideas. The underlying issues in this debate are discussed particularly with reference to the differing styles of Boyer and Mitchell and the influence of molecular biology, especially the development of protein technology.     

Molecular Characteristics and Clinical Outcomes of EGFR Exon 19 C-Helix Deletion in Non–Small Cell Lung Cancer and Response to EGFR TKIs

Epidermal growth factor receptor (EGFR) exon 19 deletion (E19del) is the most common activating mutation in advanced non–small cell lung cancer (NSCLC) and associates with the sensitivity of EGFR tyrosine kinase inhibitors (TKIs) treatment. However, not all mutant patterns of E19del have been well studied for the limited coverage of regular EGFR mutation testing. Here, we performed a retrospective cohort study of the C-helix E19del in advanced NSCLC patients based on the screening data by the next-generation sequencing (NGS) platform. From May 2012 to December 2019, clinical information and specimen from 7544 consecutive advanced (IIIB/IV) NSCLC patients were collected and screened for EGFR gene mutations by NGS from multicenters in China. The molecular characteristics and responsiveness to first-line EGFR TKIs therapy in NSCLC patients with C-helix E19del were analyzed. The clinical characteristics were also compared between patients with classical E19del and C-helix E19del. Thirty-eight (2.6%) patients with C-helix E19del and 1400 (97.4%) patients with classical E19dels were identified from 1438 patients with E19del. No significant difference in clinical characteristics was observed between the C-helix E19del and classical E19del groups (P > .05), except for histology (P < .001). All 22 patients with C-helix E19del as p.S752_I759del, p.A750_E758del, p.A750_E758delinsP, p.T751_A755delinsNY, p.T751_I759delinsG, p.T751_I759delinsLD, p.T751_I759delinsN, p.T751_L760delinsNL, and p.T751_D761delinsLY reached the best response as partial response rate (72.7%), and the progression-free survival (PFS) was 12.0 months. The PFS after EGFR TKIs in patients with C-helix E19del tended to be longer than patients with classical E19del but has no statistical significance (12.0 months vs 8.5 months, P = .06). The C-helix E19del could be a positive biomarker for predicting response to EGFR TKIs in advanced NSCLC patients. NGS should be the appropriate platform to identify this rare population, especially when patients harbor no actionable driver mutation initially and are reluctant to accept chemotherapy as first-line therapy.     

Between (Racial) Groups and a Hard Place: An Exploration of Social Science Approaches to Race and Genetics, 2000–2014

As the social sciences expand their involvement in genetic and genomic research, more information is needed to understand how theoretical concepts are applied to genetic data found in social surveys. Given the layers of complexity of studying race in relation to genetics and genomics, it is important to identify the varying approaches used to discuss and operationalize race and identity by social scientists. The present study explores how social scientists have used race, ethnicity, and ancestry in studies published in four social science journals from 2000 to 2014. We identify not only how race, ethnicity, and ancestry are classified and conceptualized in this growing area of research, but also how these concepts are incorporated into the methodology and presentation of results, all of which structure the discussion of race, identity, and inequality. This research indicates the slippage between concepts, classifications, and their use by social scientists in their genetics-related research. The current study can assist social scientists with clarifying their use and interpretations of race and ethnicity with the incorporation of genetic data, while limiting possible misinterpretations of the complexities of the connection between genetics and the social world.     

Left–Right Determination: Involvement of Molecular Motor KIF3, Cilia,and Nodal Flow

Mammalian left–right determination is a good example for how multiple cell biological processes coordinate in the formation of a basic body plan. The leftward movement of fluid at the ventral node, called nodal flow, is the central process in symmetry breaking on the left–right axis. Nodal flow is autonomously generated by the rotation of posteriorly tilted cilia that are built by transport via KIF3 motor on cells of the ventral node. How nodal flow is interpreted to create left–right asymmetry has been a matter of debate. Recent evidence suggests that the leftward movement of sheathed lipidic particles, called nodal vesicular parcels (NVPs), may result in the activation of the noncanonical hedgehog signaling pathway, an asymmetric elevation in intracellular Ca²⁺ and changes in gene expression.Although the human body is apparently bilaterally symmetrical on the surface, the visceral organs are arranged asymmetrically in a stereotyped manner. The heart, spleen, and pancreas reside on the left side of the body, whereas the gall bladder and most of the liver are on the right side (Fig. 1A). Because the human body is formed from a spherically symmetrical egg (oocyte), symmetry breakdown is one of the fundamental processes of development.Open in a separate windowFigure 1.(A) Left–right asymmetric arrangements of internal organs in the human body. (Left) Normal arrangement (situs solitus). Most humans (>99%) have the heart on the left side and the liver on the right side. (Right) Mirrored arrangement (situs inversus). Half of patients with Kartagener''s syndrome have this arrangement, whereas the remaining patients are normal. Therefore, the left–right bilateral symmetry is randomly broken in this disease. (B–E) Scanning electron micrographs of wild-type (B, D) and Kif3b^−/− (C, E) mouse embryos. (B, C) Full-length images. Wild-type embryos at this stage have already turned with a right-sided tail (B), whereas Kif3b^−/− embryos remain unturned (C). In panel C, the dilated pericardial sac has been removed, and the heart loop is inverted (arrow). (D, E) Higher-magnification images and schematic representations of the heart loops showing a normal loop in the wild-type embryo (D) and an inverted loop in the mutant embryo (E). (F–I) Scanning electron micrographs of a mouse node. (F) Low-magnification view of a mouse embryo at 7.5 days postcoitum. Reichert''s membrane is removed, and the embryo is observed from the ventral side. The node is indicated by a black rectangle. The orientation is indicated in the panel as anterior (A), posterior (P), left (L), and right (R). Scale bar = 100 µm. (G) Higher-magnification view of the mouse node. The orientation is the same as in panel A. Scale bar = 20 µm. (H) Higher-magnification view of the nodal cilia (arrows) and nodal pit cells. Scale bar = 5 µm. (I) Nodal pit cells of Kif3b^−/− embryos. Nodal cilia are absent in these genetically manipulated embryos. (J) Intraflagellar transport. Protein components in the cilia and flagella are transported by KIF3A/B complexes (light and dark blue) along the doublet microtubules of the axoneme. (Panels A and J were reproduced with permission from JT Biohistory Research Hall/TokyoCinema. B–I were modified from Nonaka et al. 1998, Okada et al. 2005, and Hirokawa et al. 2006, with permission.)In many lower vertebrates and invertebrates, eggs are asymmetrical even before fertilization (as in Drosophila [Gilbert 2003]). In some organisms, such as fish and frog, the dorsoventral (DV) and anteroposterior (AP) axes are determined at fertilization by the distribution of the yolk and the entry position of the sperm (Gilbert 2003). In human, mouse, and other mammals, the embryo is initially cylindrically symmetrical when it implants itself into the wall of the uterus. The DV axis is first to be specified as the proximal–distal axis from the implantation site. Subsequently, the AP axis is arbitrarily determined in the plane perpendicular to the DV axis (Alarcon and Marikawa 2003; Beddington and Robertson 1999). In either case, L/R is thus the last axis to be determined, and needs to be consistent with the preceding DV and AP axes. Because the chirality of the body is predetermined by chiral molecules, such as amino acids and nucleic acids, the laterality or orientation of the L/R axis is established theoretically or potentially once the AP and DV axes are determined. The problem here is how this potentially established laterality is materialized through developmental events. This mechanism is still totally unknown for the invertebrates. However, recent studies of mouse embryo clarified the L/R determination mechanism in mammalian embryos (Hirokawa et al. 2006).Until recently, little was known about the mechanism for breaking L/R symmetry. The initial clue to tackling this question was a human genetic disease called Kartagener''s syndrome. Approximately half of these patients have their organs in the reversed orientation (situs inversus). Thus, the L/R determination is randomized in this syndrome. Patients with Kartagener''s syndrome also suffer from sinusitis and bronchiectasis (Kartagener 1933). A number of male patients with Kartagener''s syndrome are sterile. Affected individuals have immotile sperm and defective cilia in their airway (Afzelius 1976). Airway cilia and sperm from these patients have abnormal ultrastructures. Specifically, the axonemes, the interior supramolecular complexes that produce the movement of cilia and flagella, lack dynein arms, the molecular motors required for cilia and flagella motility (Afzelius 1976). However, the relationships between these phenotypes and the causes of situs inversus remained unknown for more than 20 years.Approximately 10 years ago, molecular biological studies identified several genes that are asymmetrically expressed in the L/R orientation before L/R asymmetric morphogenesis of the embryo. Morphological L/R asymmetry first becomes apparent with the orientation of the heart-tube loop (Fig. 1B,D) (Kaufman 1992), but initial L/R asymmetric gene expression precedes the morphological changes. In early embryos (7.5 days for mice) at the stage of somatogenesis, genes such as Lefty-2 (Ebaf), Nodal, and Pitx2 are expressed in the left lateral plate mesoderm, a structure located on the side of the embryos (Capdevila et al. 2000; Hamada 2002; Harvey 1998; Levin 2005; Yost 1999). However, the upstream phenomena that cause asymmetrical expression of these genes remain enigmatic.Many studies have suggested that the so-called node, a concave triangular region transiently formed during gastrulation at the ventral midline surface of early embryos, is important for L/R determination (Harvey 1998). When viewed from above the ventral side, the node of mouse embryos appears as a roughly triangular depression with the apex pointed toward the anterior (Fig. 1F,G), and it is 50–100 µm in width and 10–20 µm in depth. This nodal pit is covered by Reichert''s membrane, and the cavity is filled with extraembryonic fluid. The ventral embryonic surface of the nodal pit consists of an epithelial sheet of a few hundred monociliated cells (nodal pit cells).Nodal pit cells have one or sometimes two cilia that appear as rodlike protrusions approximately 5 µm in length and 0.3 µm in diameter (Fig. 1G,H). Because Kartagener''s syndrome suggests a potential link between cilia motility and L/R determination, the cilia in the node have been postulated to be motile and responsible for L/R determination.However, the ultrastructure of these nodal cilia is similar to that of immotile primary cilia. In motile cilia and flagella, nine pairs of doublet microtubules are arranged longitudinally along the axes (Fig. 2A) (Satir and Christensen 2007). Adjacent pairs of doublet microtubules are connected by dynein arms, which generate the motility of cilia and flagella. In addition, there are two microtubules in the center of cilia and flagella, referred to as the central pair, which define the direction of the beating plane. This essential central pair of microtubules is missing in primary cilia. Similar to other immotile primary cilia, the monocilia of nodal pit cells lack the central pair of microtubules and thus have a 9 + 0 microtubule arrangement. Therefore, based on their ultrastructure and initial video-microscopic observations, nodal monocilia were originally considered immotile (Bellomo et al. 1996).Open in a separate windowFigure 2.(A) Ultrastructures of normal cilia and primary cilia. (Left) Normal cilia and flagella have nine pairs of doublet microtubules (yellow) and two central microtubules (yellow). Adjacent doublet microtubules are connected with dynein motors (blue and green). The orientation of the central pair of microtubules is considered to determine the beating plane (purple). (Right) The central pair of microtubules is missing in immotile primary cilia and nodal cilia. In nodal cilia, the dynein motors remain in a chiral arrangement and produce a rotation-like movement (purple). (B–E) Rotation of nodal cilia and leftward nodal flow. The images are views from the ventral side. The orientation is indicated in the panels as anterior (A), posterior (P), left (L), and right (R). (B) Trajectory of a fluorescent bead attached to the tip of a nodal cilium (Movies 1 and 2). Three consecutive video frames with 33-ms exposures at 16-ms intervals (interlaced scan) are shown. The moving bead produces arc-shaped images (traced by green arrows). The beads rotate clockwise when viewed above the node. (C) Trajectories of the tips of nodal cilia traced from a high-speed video sequence (500 frames/s) (Movie 3). The red circles show the positions of the ends of the cilia at 10-ms time intervals, and the yellow circles show the positions of the roots of the cilia. The white ellipses show the trajectories of the tips. Scale bar = 5 µm. (D) The positions of beads that entered the node from the right edge traced for 4 seconds at 0.33-second intervals. Different symbols indicate different beads. Most beads go straight to the left edge of the node. Scale bar = 20 µm. (E) The trajectories of four beads selected to illustrate the streamline of the nodal flow. The flow is mostly laminar and straight in the middle of the node, but often makes small vortices near the left edge (arrowheads). (Panel A was reproduced with permission from JT Biohistory Research Hall/TokyoCinema. B–E were modified from Okada et al. [1999, 2005] with permission.)

Movie 1

Download video file.^{(3.2M, mov)}Leftward nodal flow. Nodal flow was visualized by adding fluorescent beads to the medium surrounding the ventral node of a mouse embryo at the early somite stage. 4× time lapse.

Movie 2

Open in a separate windowClick here to view.^{(104K, gif)}Rotatory movement of nodal cilia.

Movie 3

Download video file.^{(39M, mov)}Posteriorly-tilted rotation of nodal cilia. Nodal cilia were observed by high-speed video microscopy (500 frames/s). The focus was adjusted to approximately 3 µm above the surface of the ventral node. The images of the cilia thus blur when they are near the floor and become clear when they come up to the focal plane. Note that the particle (highlighted by a red circle) does not go down to the surface but eventually goes toward the left side of the node due to the movement of the cilia. Reproduced from Okada et al. (2005) with permission.Through studies of the flow of materials within cells, we serendipitously found that nodal cilia are actually motile and vigorously rotating. This rotation generates the leftward flow of extraembryonic fluid in the nodal pit. The directionality of this flow, termed nodal flow, determines laterality. Thus, quite unexpectedly, a physical process, fluid flow, was identified as the initial L/R symmetry-breaking event. In this review, we first summarize the discovery of nodal flow and then discuss how this leftward linear flow is generated in a fluid dynamic manner by the rotational movement of cilia. We further discuss the mechanisms by which L/R asymmetry is determined by this nodal flow.     

The Prion Challenge to the `Central Dogma' of Molecular Biology, 1965–1991: Part I: Prelude to Prions

Since the 1930s, scientists studying the neurological disease scrapie had assumed that the infectious agent was a virus. By the mid 1960s, however, several unconventional properties had arisen that were difficult to reconcile with the standard viral model. Evidence for nucleic acid within the pathogen was lacking, and some researchers considered the possibility that the infectious agent consisted solely of protein. In 1982, Stanley Prusiner coined the term `prion' to emphasize the agent's proteinaceous nature. This infectious protein hypothesis was denounced by many scientists as `heretical'.This essay asks why the concept of an infectious protein was considered controversial. Some biologists justified their evaluation of this hypothesis on the grounds that an infectious protein contradicted the `central dogma of molecular biology'. Others referred to vague theoretical constraints such as molecular biology's `theoretical structure' or `framework'. Examination of the objections raised by researchers reveals exactly what generalizations were being challenged by a protein model of infection.This two-part survey of scrapie and prion research reaches several conclusions: (1) A theoretical framework is present in molecular biology, exerting its influence in hypothesis formation and evaluation; (2) This framework consists of several related, yet separable, generalizations or `elements', including Francis Crick's Central Dogma and Sequence Hypothesis, plus notions concerning infection, replication, protein synthesis, and protein folding; (3) The term `central dogma' has stretched beyond Crick's original 1958 definition to encompass at least two other `framework elements': replication and protein synthesis; and (4) From the study of scrapie and related diseases, biological information has been delineated into at least two classes: sequential and what I call `conformational'.In Part I of this essay, a brief review of the central dogma, as outlined by both Francis Crick and James Watson, will be given. The developments in scrapie research from 1965 to 1972 will then be traced. This section will summarize many of the puzzling, non-viral-like properties of the scrapie agent. Alternative hypotheses to the viral explanation will also be presented, including early versions of a protein-only hypothesis. Part II of this essay will follow the developments in scrapie and prion research from the mid 1970s through 1991. The growing prominence of a protein-only model of infection will be balanced by continued objections from many researchers to a pathogen devoid of nucleic acid. These objections will help illuminate those generalizations in molecular biology that were indeed challenged by a protein-only model of infection.     

Preferential Energy- and Potential-Dependent Accumulation of Cisplatin–Gutathione Complexes in Human Cancer Cell Lines (GLC4 and K562): A Likely Role of Mitochondria

cis-Diamminedichloroplatinum(II) (CDDP) is an important chemotherapeutic agent used in the treatment of a wide variety of solid tumors. We have recently shown that aquated forms of cisplatin (aqua-Pt) rapidly accumulate in K562 and GLC4 cultured cells, in comparison to CDDP. Thus, when cells are incubated with aquated forms of cisplatin a gradient of concentration is observed after a short time, approximately 40 min, with an intracellular concentration of aqua-Pt of 20–30 times higher than that of extracellular aqua-Pt. The same gradient of concentration is observed when cells are incubated with CDDP but it takes a longer time, i.e., about 24 h. Therefore, the question arises as to the identity of the intracellular sites of accumulation of aqua-Pt. Using several agents to modulate membrane potential, acidic compartment pH and/or ATP level, we obtained evidence that aqua-Pt may accumulate rapidly inside mitochondria as this accumulation is energy- and membrane-potential-dependent. However, aqua-Pt complexes are not characterized by a delocalized charge and a lipophilic character that would permit their movement through the inner membrane. Therefore, it is suggested that intracellular aqua-Pt reacts rapidly with glutathione with the resultant complex being transported inside the mitochondria via one of the known glutathione transporters, i.e., dicarboxylate and/or 2-oxoglutarate transporters present in the inner membrane.     

Changing Patterns of Disease and Mortality at the Children’s Hospital,Accra: Are Infections Rising?

BackgroundThe Millennium Development Goals (MDGs) have led to reductions in child mortality world-wide. This has, invariably, led to the changes in the epidemiology of diseases associated with child mortality. Although facility based data do not capture all deaths, they provide an opportunity to confirm diagnoses and insight into these changes which are relevant for further disease control.ObjectiveTo identify changes in the disease pattern of children who died at the Princess Marie Louise Children’s Hospital (PML) in Ghana from 2003–2013.MethodsA cross sectional review of mortality data was carried out at PML. The age, sex, duration of admission and diagnosis of consecutive patients who died at the hospital between 2003 and 2013 were reviewed. This information was entered into an Access database and analysed using Stata 11.0 software.ResultsAltogether, 1314 deaths (3.6%) occurred out of a total of 37,012 admissions. The majority of the deaths, 1187 (90.3%), occurred in children under the age of 5 years. While deaths caused by malaria, malnutrition, HIV infection and diarrhoea decreased, deaths caused by pneumonia were rising. Suspected septicaemia and meningitis showed a fluctuating trend with only a modest decrease between 2012 and 2013. The ten leading causes of mortality among under-fives were malnutrition, 363 (30.6%); septicaemia, 301 (25.4%); pneumonia, 218 (18.4%); HIV infection, 183 (15.4%); malaria, 155 (13.1%); anaemia, 135 (11.4%); gastroenteritis/dehydration, 110 (9.3%); meningitis, 58 (4.9%); tuberculosis, 34 (2.9%) and hypoglycaemia, 27 (2.3%). For children aged 5–9 years, the leading causes of mortality were malaria, 42 (42.9%); HIV infection, 27 (27.6%); anaemia, 14 (14.3%); septicaemia, 12 (12.2%); meningitis, 10 (10.2%); malnutrition, 9 (9.2%); tuberculosis, 5 (5.1%); pneumonia, 4 (4.1%); encephalopathy, 3 (3.1%); typhoid fever, 3 (3.1%) and lymphoma, 3 (3.1%). In the adolescent age group, malaria, 8 (27.6%); anaemia, 6 (20.7%); HIV infection, 5 (17.2%); sickle cell disease, 3 (10.3%) and meningitis, 3 (10.3%) were most common.ConclusionThere has been a decline in the under-five mortality at PML over the years; however, deaths caused by pneumonia appear to be rising. This highlights the need for better diagnostic services, wider HIV screening and clinical audits to improve outcomes in order to achieve further reductions in child mortality and maintain the gains.     

Of Fighting Flies,Mice, and Men: Are Some of the Molecular and Neuronal Mechanisms of Aggression Universal in the Animal Kingdom?

Aggressive behavior is widespread in the animal kingdom, but the degree of molecular conservation between distantly related species is still unclear. Recent reports suggest that at least some of the molecular mechanisms underlying this complex behavior in flies show remarkable similarities with such mechanisms in mice and even humans. Surprisingly, some aspects of neuronal control of aggression also show remarkable similarity between these distantly related species. We will review these recent findings, address the evolutionary implications, and discuss the potential impact for our understanding of human diseases characterized by excessive aggression.     

11th European Cell Cycle Conference: April 23–26, 1997, Gardone Riviera (Italy) and Symposium on Cell Cycle Regulation (17th International Congress of Biochemistry and Molecular Biology): August 24–29, 1997, San Francisco,CA

In the following pages I have summarized some of the findings presented at two recent `cell cycle gatherings'. I have focused on those topics which in my opinion represent a substantial advancement in our understanding of the cell cycle regulatory pathways.     

Combined Proteomic and Molecular Approaches for Cloning and Characterization of Copper–Zinc Superoxide dismutase (Cu, Zn-SOD2) from Garlic (Allium sativum)

Superoxide dismutases (SODs; EC 1.15.1.1) are key enzymes in the cells protection against oxidant agents. Thus, SODs play a major role in the protection of aerobic organisms against oxygen-mediated damages. Three SOD isoforms were previously identified by zymogram staining from Allium sativum bulbs. The purified Cu, Zn-SOD2 shows an antagonist effect to an anticancer drug and alleviate cytotoxicity inside tumor cells lines B16F0 (mouse melanoma cells) and PAE (porcine aortic endothelial cells). To extend the characterization of Allium SODs and their corresponding genes, a proteomic approach was applied involving two-dimensional gel electrophoresis and LC-MS/MS analyses. From peptide sequence data obtained by mass spectrometry and sequences homologies, primers were defined and a cDNA fragment of 456?bp was amplified by RT-PCR. The cDNA nucleotide sequence analysis revealed an open reading frame coding for 152 residues. The deduced amino acid sequence showed high identity (82-87%) with sequences of Cu, Zn-SODs from other plant species. Molecular analysis was achieved by a protein 3D structural model.