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The utility of proteomics in algal taxonomy: Bostrychia radicans/B. moritziana (Rhodomelaceae, Rhodophyta) as a model study
Abstract
A comparison of the proteome of eight genetically well-characterized isolates of the Bostrychia radicans (Mont.) Mont./B. moritziana (Sond. ex Kütz.) J. Agardh species complex was undertaken to establish if genetic relationships among them can be determined using proteome data. Genetic distances were calculated on the basis of common and distinct spots in two-dimensional gel electrophoresis (2-DE). Proteomes of the male and female plants of each population were compared to analyze the range of genetic difference within an isolate. Haploid male and female plants of the same species had 3.7%–7.1% sex-specific proteins. The degree of similarity of the proteome was consistent with previous DNA sequence data and sexual compatibility studies between the isolates. Two sexually compatible isolates from Venezuela showed a pair-wise distance ranging from 0.14 to 0.21. The isolates from Mexico and Venezuela, which were partially compatible, showed a maximum pair-wise distance of 0.26. A high level of genetic difference was found among isolates that were sexually incompatible. The isolate from Brazil was reproductively isolated from the Mexico and Venezuela isolates and showed a maximum pair-wise distance of 0.65 and 0.58, respectively. Comparative proteomics may be helpful for studying genetic distances among algal samples, if intraisolate variation (gene expression) can be minimized or tested.
... Proteomics using large two-dimensional polyacrylamide gel electrophoresis (2-DE) allows multiple expressed proteins to be separated and mapped, providing a convenient and powerful method for monitoring variations at translational levels (Kim et al. 2008). Proteins identified using peptide mass fingerprinting (PMF) and tandem mass spectrometry (MS/MS) ion searches can be separated and then analyzed by MS/MS. ...
... Chan et al. (2002) first reported a method for the optimization of sample preparation for 2-DE and later conducted proteomic analysis for species recognition of several harmful bloom-related dinoflagellates (Chan et al. 2004(Chan et al. , 2006. More recent studies have shown that 2-DE results are useful for analysis of the phylogenetic relationships among closely related species of algae (Kim et al. 2008). ...
... Therefore, P. micans requires only a few additional proteins to feed on algal prey. Kim et al. (2008) reported that 3.7-7.1% of the expression proteome (33-59 protein spots) was differentially expressed between male and female plants of the red algae, Bostrychia radicans and B. moritziana. These results suggests that the changes in a small portion of the expression proteome could significantly alter the morphological and physiological state of algal cells. ...
Protein profiles of a common mixotrophic dinoflagellate, Prorocentrum micans, growing autotrophically and mixo-trophically (fed on the cryptophyte Rhodomonas salina) were compared using two-dimensional gel electrophoresis (2-DE) to determine if they vary in different trophic modes. Approximately 2.3% of the detected proteins were differentially expressed in the different trophic modes. Twelve proteins observed only in the mixotrophic condition had lower pI value (<5) than the fifteen proteins observed only in the autotrophic condition (>5). When the internal amino acid sequences of five selected proteins differentially expressed between autotrophic and mixotrophic conditions were analyzed us-ing matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry, two proteins that were specifically expressed in the autotrophic condition showed homology to glyceraldehyde-3-phosphatase dehydrogenase (GAPDH) and a bacterial catalase. Three mixotrophy-specific proteins showed homology to certain hypothetical proteins from an insect and bacteria. These results suggested the presence of certain gene groups that are switched on and off according to the trophic mode of P. micans.
... Graves and Haystead [58] provide an in-depth review of proteomics techniques. For the past decade or so, several novel marine algal proteins have been identified by twodimensional electrophoresis (DE) and mass spectrometry (MS) including proteins from the macroalga Gracilaria changii [59], and the microalgae Dunaliella bardawil [60] and Nannochloropsis oculata [61]. Nevertheless, proteomics does not end with the identification of a particular protein, it also helps to uncover the underlying function of the latter including its role in evolution as well as taxonomic studies and biochemical pathways [8,61,62]. ...
... For the past decade or so, several novel marine algal proteins have been identified by twodimensional electrophoresis (DE) and mass spectrometry (MS) including proteins from the macroalga Gracilaria changii [59], and the microalgae Dunaliella bardawil [60] and Nannochloropsis oculata [61]. Nevertheless, proteomics does not end with the identification of a particular protein, it also helps to uncover the underlying function of the latter including its role in evolution as well as taxonomic studies and biochemical pathways [8,61,62]. ...
Marine algae are of high importance in their natural habitats and even more now in the world of green technology. The sprouting interest of the scientific community and industries in these organisms is driven by the fast-growing world of modern biotech‐ nology. Genomics, transcriptomics, proteomics, metabolomics and their integration collectively termed here as 'marine algal-omics' have broadened the research horizon in view of enhancing human's life by addressing environmental problems and encouraging novelty in the field of pharmaceuticals among so many more. Their use in the human society dates back to 500 B. C. in China and later across the globe; they are still being used for similar purposes and more today. There is a hiking interest in marine algae and their derivatives—from phycoremediation, food supplements, pharmaceut‐ icals to dyes. Marine algae are currently considered as an emerging panacea for the society. They are being studied in a multitude of arenas. The multi-use of marine algae is enticing and promises to be a boon for industrial applications. Yet, most marine algae face challenges that might variably constrain their commercialisation. This chapter gives an overview of marine algae including all the 'omics' technologies involved in studying marine algae and it explores their multitude applications. It also draws the various successful industries budded around them and presents some of the challenges and opportunities along with future directions.
... Proteomics using large two-dimensional polyacrylamide gel electrophoresis (2-DE) allows multiple expressed proteins to be separated and mapped, providing a convenient and powerful method for monitoring variations at translational levels (Kim et al., 2008). Proteomic technology is useful for investigating the correlation between expressed proteins and behavioral and physiological phenotypes during biological cycles. ...
... Rapid progress has been made in 2-DE methodology for marine algae where proteomic analysis done for species recognition of several harmful bloom-related dinoflagellates (Chan et al., 2006). More recent studies have shown that 2-DE results are useful for analysis of the phylogenetic relationships among closely related species of algae (Kim et al., 2008). Protein profiles of a common mixotrophic dinoflagellate, Prorocentrum micans, growing autotrophically and mixotrophically (fed on the cryptophyte Rhodomonas salina) suggest that comparative proteomics may be a useful tool for analysis of the mixotrophism of dinoflagellates. ...
Algae are one of the most primitive life forms and most abundant on earth. They consist of one or more of eukaryotic cells containing chlorophyll. It may be a single cell, colonies, filament of cells, or as in the kelp, simple tissues. The cell doubling time is typically 1-2 days, and under optimal conditions can be as short as 6 hours for reported in Chlamydomanas species (Chen et al., 2009), as compared to bacteria (20 min), animal cells (20 hours) and plant cells (2-4 days). Algae inhabit particularly the oceans, rivers, lakes, streams, ponds and swamps, but some species can be found in the soil and survive dry conditions for a long time. Because of such diverse ecological habitats ranging from seawater, freshwater or brackish water, they are equipped to flourish in extreme temperature and pHs such as at 80°C, in and around hot springs, or in the snow and ice of Arctic and Antarctic regions (Rosenberg et al., 2008). In the tropics and subtropics, algae may be found on leaves, woods and stones, within or on plants and animals. Although for many years categorized as plants because of their photosynthetic ability, algae are now placed within the diverse kingdom Protista of eukaryotic, predominantly single-celled microscopic organisms (Hollar, 2012).
... Although these glycoproteins must be important for fertilization, which can be blocked by adding the complimentary sugars or lectins, it is still uncertain whether these glycoproteins are responsible for species recognition. Quite recently, Kim et al. (2008) undertook a comparison of the proteome among eight isolates of the Bostrychia radicans/moritziana species complex showing various reproductive reactions with each other. The male and female isolates had 3.7–7.1% sex-specific proteins, and the lack of any shared sex-specific proteins across all isolates may suggest rapid evolution of these proteins (Kim et al., 2008). ...
... Quite recently, Kim et al. (2008) undertook a comparison of the proteome among eight isolates of the Bostrychia radicans/moritziana species complex showing various reproductive reactions with each other. The male and female isolates had 3.7–7.1% sex-specific proteins, and the lack of any shared sex-specific proteins across all isolates may suggest rapid evolution of these proteins (Kim et al., 2008). Such a proteome analysis is expected to become an important cue for the elucidation of the molecular mechanisms of sex as well as species recognition in red algae. ...
Allopatric speciation is the most widely accepted model proposed for speciation: once a population is divided by extrinsic
barriers, genetic flow is interrupted between these disjunct subpopulations, and reproductive isolation is established as
a by-product of the accumulation of genetic changes in these isolated populations. According to the biological species concept
(Mayr, 1942), the evolution of reproductive isolation is a defining characteristic of speciation, and reproductive isolation
contributes to the diversification of species by creating genetically independent lineages. It had been generally thought
that isolation is more difficult in marine populations, with frequent gene flow over large distances (Hoffmann, 1987; van
den Hoek, 1987; Norton, 1992; Shanks et al., 2003), as there seem to be far fewer extrinsic barriers in marine environments
than in terrestrial ones (Palumbi, 1994). Such attributes are considered to limit the isolation of a species into allopatric
populations less frequently, making allopatric speciation rarer (Mayr, 1954). However, recent molecular analyses have revealed
great genetic divergences among/within populations in various marine organisms, including macroalgae, and thus, the generalization
that speciation must be rare in marine habitats appears to be incorrect.
... A sex-specifically expressed lectin which is involved in gamete binding was isolated from this species . Sex-associated genetic markers have been found in red algae (Martinez et al. 1999, Kim et al. 2008, Guillemin et al. 2012. These have mostly been derived from random markers (RAPDs, SCARs, AFLP). ...
Diverse sex determination mechanisms have been reported in eukaryotes, but little is known about the genetic pathways leading to sex determination in red algae. Sex‐specific genes that could be involved in sex determination and sexual differentiation were investigated in the red alga Bostrychia moritziana by analyzing the transcriptomes of various phases including males, females and tetrasporophytes. Sex‐dominantly‐expressed genes which showed >10 fold difference between sexes were isolated using comparative RNA‐seq analysis. We found 19 gene homologues, 10 from males and 9 from females, that were found only in one sex in genomic amplification using strains collected from five different localities. Most of the sex‐specific genes are involved in important cellular processes including chromosome segregation, nucleo‐cytoplasmic protein shuttling or tRNA modification. Quantitative PCR analysis showed that some sex‐specific genes were differently regulated during critical events of sexual reproduction like fertilization and carposporophyte development. We could localize the expression of a male‐specific gene in spermatia before and after gamete binding using RNA in situ hybridization. Amino acid sequence identity between male and female homologues of importin alpha gene and PreQ(0) reductase were highly divergent (75% and 74%, respectively), suggesting that these divergent homologues are on non‐recombining UV‐type chromosomes in their respective sexes. Another set of transcripts were found that were sex‐dominantly‐expressed, but not sex‐speific. 19 out of 39 sex‐dominantly‐expressed transcripts were annotated to transposable elements. Our results suggest that sexual differentiation in B. moritziana may be achieved by multi‐level regulation of cellular processes, both from genes present only in one sex and differential expression of shared genes.
... Several novel proteins were identified using 2D electrophoresis and mass spectrometry from marine algae such as Botryococcus braunii (Nguyen and Harvey 2003), Dunaliella bardawil (Katz et al. 1995). Proteomics not only handles the identification of proteins involved but it also enlightens the hidden functions including the role in the evolution along with taxonomic studies and biochemical pathways (Kim et al. 2008). ...
In recent years, algal research revolves comprehensively in tapping the prospective of various approaches viz. energy sources and nutritional supplements. Researchers worldwide are exploring various aspects of microalgae as they attract the scientific community because of their various unique properties. It has been shown that changing the parameters for algae growth can stimulate these beautiful cells to form substances which are of high value. Biosynthesis of a number of compounds is governed through several enzymatic steps which are further influenced and controlled by the type and concentration of nutrient provided or present in the natural habitat that can act as rate-limiting factor. Algal omics has turned out to be the finest option in recent years for tapping algae as biofuel resource. Genomics and transcriptomics of algae have delivered decisive information to understand lipid biosynthesis. On the other hand, proteomics and metabolomics complement algal omics by offering accurate and useful understandings into the linked physiological settings. Although genomic study reveals many important parameters for various applications using algae, which can be further enhanced when complemented well with the techniques of proteomics, transcriptomics, metabolomics and lipidomics. Combination of datasets from various lipid enhancement approaches can deliver a system-wide impression. These approaches permit closer consideration in the future with an opinion to different practical impacts that are projected in modern era.
... Two-dimensional polyacrylamide gel electrophoresis (2-DE), which can separate complex protein mixtures, was used to identify protein changes that occur normally during development and tissue differentiation, as a result of disease, drug treatment, environmental change, etc. (Celis et al. 1999a, 1999b, Spandidos and Rabbitts 2002. In phycology, this branch of "omics" has been widely applied, as evident from a large number of publications accumulating recently (e.g., Kim et al. 2008, Choi et al. 2015. Proteomic approach has been applied in Bryopsis studies by Yamagishi et al. (2004) who performed 2-DE analysis of the hybrid plants that developed from hybrid protoplasts of B. plumosa obtained by mixing protoplasm of its gametophyte and sporophyte. ...
When a multinucleate cell of Bryopsis plumosa was collapsed by a physical wounding, the extruded protoplasm ag-gregated into numerous protoplasmic masses in sea water. A polysaccharide envelope which initially covered the proto-plasmic mass was peeled off when a cell membrane developed on the surface of protoplast in 12 h after the wounding. Transmission electron microscopy showed that the protoplasmic mass began to form a continuous cell membrane at 6 h after the wounding. The newly generated cell membrane repeated collapse and rebuilding process several times until cell wall developed on the surface. Golgi bodies with numerous vesicles accumulated at the peripheral region of the rebuilding cell at 24 h after the wounding when the cell wall began to develop. Several layers of cell wall with distinctive electron density developed within 48-72 h after the wounding. Proteome profile changed dramatically at each stage of cell rebuilding process. Most proteins, which were up-regulated during the early stage of cell rebuilding disappeared or reduced significantly by 24-48 h. About 70-80% of protein spots detected at 48 h after the wounding were newly appeared ones. The expression pattern of 29 representative proteins was analyzed and the internal amino acid sequences were obtained using mass spectrometry. Our results showed that a massive shift of gene expression occur during the cell-rebuilding process of B. plumosa.
... Keeping in mind that 'the fabric of life is protein-based' (Karr 2008), one can reasonably conclude that proteomics can provide insight into the mechanisms of adaptation and speciation. Although the earliest taxonomic applications were published in the beginning of the 1980s (Aquadro & Avise 1981, Ohnishi et al. 1983, the application of proteomics has intensified only in the last decade in the fields of ecology, population biology, taxonomy, and evolutionary studies (López 2005, Biron et al. 2006, Karr 2008, Kim et al. 2008, Cash 2009, Diz et al. 2012b. In contrast to discrete molecular markers, a proteomic approach to phylogenetic reconstruction has the advantage of integral analysis considering the complex system of working molecular machinery (N avas & Albar 2004). ...
During recent years, the role of proteomics in ecology, population biology and evolutionary studies has been growing. It has been used effectively to resolve taxonomic ambiguity in closely related species and for detection of environmental effects on populations. The family Littorinidae (Gastropoda; periwinkles) includes a set of widely distributed species, which are important members of intertidal communities of the seacoasts around the world. Taxonomic relationships among several species, e.g. within the subgenus Neritrema, are still poorly understood. We explored proteomes of three closely related littorinid species (the so called “saxatilis”-group: Littorina arcana, L. compressa and L. saxatilis) from different intertidal levels of one geographic area. The proteome data were obtained using fluorescence-difference 2D electrophoresis (DIGE) followed by mass-spectrometry for protein identification. We showed that L. compressa formed well-supported clade within the "saxatilis" group, while L. arcana and L. saxatilis were similar at the proteomic level as a whole. Moreover, the variable elements in the proteomes of the two former species shifted similarly along the vertical shore gradient, in contrast to L. compressa. Such proteomic divergence suggests that closely related species may function differently at the physiological level under very similar conditions.
... These authors compared different ecotypes of Bostrychia isolated from distinct communities. A proteomic comparison of eight complexes isolated from B. radicans was also performed by [5], while [6] assessed molecular and reproductive diversity. The authors considered the phylogenetic analysis of plastids and data on the mitochondrial DNA sequences which revealed the existence of seven different evolutionary lineages among samples all around the world. ...
Little is known about the morphology and location of macromolecules, especially proteins and carbohydrates, in vegetative and reproductive structures of mangrove species, including Bostrychia radicans. Therefore, to gain a better understanding of tetrasporogenesis in B. radicans, cytochemical, structural and ultrastructual analyses were performed. Thalli were collected from mangroves in Florianopolis, Santa Catarina, Brazil. Fertile branches were fixed and processed for light microscopy (LM), confocal microcopy and transmission electron microscopy (TEM) observations. The LM sections were stained with toluidine blue, periodic acid-Schiff and Coomassie brilliant blue. Tetrasporogenesis occurs in the pericentral cells of the terminal branches. This process is initially characterized by an increase in cell volume, resulting from the proliferation of organelles. The young tetrasporangia remain connected to the basal cell by pit connections. After a considerable increase in volume, the tetrasporocytes divide tetrahedrally, giving rise to haploid spores. During this process, there is an increasing production of starch grains, causing the organelles to group. As the organelles proliferate, the plasma membrane undergoes simultaneous invaginations toward the tetrasporangium center. The most conspicuous organelle throughout tetrasporogenesis was the Golgi complex. Polysaccharidic components are predominant in the tetrasporangium cell wall throughout tetrasporogenesis. Although protein components prevail in the cytoplasm of younger tetrasporangia, there is a predominance of reserve material with maturation. In the initial phase, there is an increase in the number of chloroplasts and a significant increment of Golgi bodies which contribute to the formation of the amorphous portion of the cell wall and possibly the biosynthesis of starch grains.
... Validación taxonómica de especies. Kim et al. (2008), utilizaron los genes rbcL y cox1 en poblaciones de Gracilariopsis chorda (Holmes) Ohmi, para confirmar la posición taxonómica dentro del género Gracilariopsis, en individuos designados como Gracilaria chorda, así como también discutir la posición taxonómica de Gracilariopsis dentro del orden. Ambos genes revelaron estructura genética. ...
In this paper we analyze the usefulness of population
genetics as a tool in studies of marine macroalgae.
This type of study has been developed with various
approaches, such as taxonomic, ecological, and biogeographic,
primarily in red and brown macroalgae,
and using various techniques as Microsatellites,
RAPDs, SSCP, RFLPs, Isozymes and DNA sequencing.
In Mexico there are, to date, few published studies
that incorporate this tool, probably due to the high
costs involved in this type of studies and because th
extensive theoretical knowledge prior to should be to
integrate different lines as biogeography, molecular
phylogeny and taxonomy of the group in question.
... ity and the genetic distinctness of populations) must be one of the major concepts if we wish to understand the production of diversity and the evolution of characters under selection. What controls this isolation is unknown, but results from an early study on the proteome of B.�radicans / B.�moritziana (Kim et al. 2008) has suggested useful approaches to investigating specific proteins that vary substantially between lineages or sexual stages of known reproductively compatible and incompatible isolates. This research has been very fruitful over the years and further investigation in this genus in the coming years will greatly benefit from this accumulated knowledge. ...
Studies of the red algal genus Bostrychia over the last 15 years have made it a model system for many evolutionary processes within red algal species. The combination of newly developed, or first employed methods, in red algal species studies has made Bostrychia�a pioneer genus in intraspecific studies. Bostrychia was the first genus in which a mitochon-drial marker was used for intraspecific red algal phylogeny, and the first for which a 3-genome phylogeny was undertak-en. The genus was the first red alga used to genetically show maternal plastid and mitochondria inheritance, and also to show correlation between cryptic species (genetically divergent intraspecific lineages) and reproductive incompatibility. The chemotaxonomic use, and physiological function of osmolytes, has also been extensively studied in Bostrychia. Our continuous studies of Bostrychia also highlight important aspects in algal species studies. Our worldwide sampling, and resampling in certain areas, show that intensive sampling is needed to accurately assess the genetic diversity and there-fore phylogeographic history of algal species, with increased sampling altering evolutionary hypotheses. Our studies have also shown that long-term morphological character stability (stasis) and character convergence can only be cor-rectly assessed with wide geographic sampling of morphological species. While reproductive incompatibility of diver-gent lineages supports the biological species nature of these lineages, reproductive incompatibility is also seen between isolates with little genetic divergence. It seems that reproductive incompatibility may evolve quickly in red algae and the unique early stages of fertilization (e.g., gametes covered by walls, active movement of spermatium nuclei to the distant egg nucleus), also well investigated in Bostrychia, may be key to our understanding of this process.
... Two-dimensional gel electrophoresis (2-DE) 2-DE was performed as described previously (Kim et al. 2008;Han et al. 2012). The gametes used for 2-DE were harvest at 2 h after release. ...
Metabolic pathways of cell organelles may influence the expression of nuclear genes involved in fertilization and subsequent zygote development through a retrograde
regulation. In Scytosiphon lomentaria, inheritance of chloroplast is biparental but mitochondria are maternally inherited. Male and female gametes underwent different parthenogenetic outcomes. Most (>99 %) male gametes did not differentiate rhizoid cells or survived beyond four-cell stage, while over 95 % of female gametes grew into mature asexual plants. Proteomic analysis showed that the protein contents of male and female gametes differed by approximately 1.7 %, 12 sex-specific proteins out of 700 detected proteins. Three sex-specific proteins were isolated and identified using CAF-MALDI mass spectrometry and RACE-PCR. Among them, a male gamete-specific homoaconitate hydratase (HACN) and a female gamete-specific succinate semialdehyde dehydrogenase (SSADH) were predicted to be the genes involved in mitochondrial metabolic pathways. The expression level of both mitochondrial genes was dramatically changed at the fertilization event. During parthenogenetic development the male-specific HACN and GTP-binding protein were gradually down-regulated but SSADH stayed up-regulated up to 48 h. To observe the effect of chemicals on the expression of these genes, male and female gametes were treated with γ-aminobutyric acid (GABA), hydrogen peroxide and l-ascorbic acid. Among them GABA treatment significantly reduced SSADH gene expression in female gamete but the same treatment induced high upregulation of the gene in male gamete. GABA treatment affected the behavior of gametes and their parthenogenetic development. Both gametes showed prolonged motile stage, retarded settlement and subsequent parthenogenetic development. Our results suggest that male and female gametes regulate mitochondrial metabolic pathways differentially during fertilization, which may be the reason for their physiological and behavioral differences.
... Simpson, 1961). In the not-too-distant future proteomics (L opez, 2005;Kim et al., 2008) and metabolomics (Rodr ıguez-Fern andez et al., 2011) will probably be generalized as additional sources of data for taxonomic research as such evidence is interpreted as homologous characters. ...
Names in taxonomy have seven different and important properties, some due to their existence in the context of classifications. Names confer or facilitate individuation, information storage and retrieval, and set theories of relationships, explanatory power, testable predictions, conceptual power, and language. No other way of naming in science is so powerful. And this is possible because taxonomic naming is done with full consideration of the theoretical specification of empirical data (characters) and their correspondence among taxa via homology statements. Since Darwin and Hennig, sets of homologous characters distributed among taxa allow precise hypotheses of a genealogical relationship, and this relationship is reflected in the way naming results in a classification.
... Meanwhile it is impossible to establish the precise function of these proteins due to the absence of genetic information related directly to dinoflagellates. The plankton protists on the whole are very poorly represented in the genome and proteome databases, which is a serious problem at their study [57]. Recently, much more attention has become to pay to this problem [1], so it is hoped that soon the situation will change for the better. ...
Mixotrophy is the ability to combine autotrophic and heterotrophic modes of nutrition. It is widely spread in various microorganisms, particularly in such important plankton groups as dinoflagellates and cyanobacteria. Mixotrophy has a significant impact on our comprehension of the matter and energy flows in marine ecosystems, and therefore, it is an object of much attention for several recent decades. Nevertheless, the precise data on the balance of auto- and heterotrophy during the mixotrophic growth have been absent so far, which is due, first of all, to insufficient understanding of physiological and molecular ground of this phenomenon. In this review we discuss some ecological and cytophysiological aspects of investigation of mixotrophy in microorganisms as well as possible reasons for relatively slow progress in this area.
... No research has looked at which genes may be involved in reproductive incompatibility in any red algae, although research is beginning to identify candidate genes in Bostrychia (i.e. genes associated with reproductive mode, Kim et al. 2008). Our data suggest that reproductive incompatibility is first manifested in the uniquely complex processes (i.e. ...
SUMMARY Previous studies suggested that the biodiversity of the mangrove-associated Bostrychia radicans/Bostrychia moritziana species complex on the Pacific coast of Central America, based on genetic and reproductive data, were low compared with similar areas on the Atlantic coast. Evolutionary scenarios were proposed based on either a recent introduction to the Pacific, or a more uniform environment leading to genetically connected populations and low differentiation between populations. We sampled more extensively in southern Mexico, Guatemala and El Salvador and sequenced the samples for the RuBisCo spacer. Our results show that genetic diversity is high in these populations. Many haplotypes retrieved are also found in the Atlantic Ocean (USA, Brazil), an observation not made before. Data suggest that populations are highly differentiated with little evidence of isolation-by-distance. The population at La Puntilla, El Salvador is highly differentiated from other populations. Data also suggest that diversity is reduced in a northerly direction, with only one haplotype, unique to Pacific Central America, found north of Chiapas, Mexico. This could be due to northern expansion of this unique genotype as sea surface temperatures ameliorated following the last glacial maximum. Our data do not support the previous proposition of low diversity in the east central Pacific and suggest that much of the Pacific Central America diversity is from before the closure of the Isthmus of Panama.
... Finally, the quality of the extracted proteins is improved by using the 2-D clean- Up Kit (GE Healthcare). In another important proteomic work with macroalgae developed by Kim et al. (2008) and published contemporarily with the Contreras et al. (2008) work, using as models the red algae Bostrychia radicans and B. moritziana (Plantae, Rhodophyta), used a lysis buffer comprised principally by urea and thiourea (detailed protocol in Appendix B). Although these species belong to the same group of red algae like G. changii, the simplicity of this method utilized in comparison with the phenol one (Wong et al., 2006) is due to the morphological characteristics of this species (see image in Appendix B). ...
... Macroalgae represent most of algae aquaculture both by volume and value (about 99%), being mainly cultivated as a source of food or specific substances (like iodine, algin, carrageenan and agar) [2]. Not many proteomic studies exist in macroalgae, but there are already a few [78][79][80][81][82], mostly focusing on optimizing viable proteomic workflows for algae, since these organisms tend to contain substances which make adequate protein extraction more challenging. Interestingly, despite these technical issues, there are already a large number of proteomic studies on microalgae, on a wide variety of topics. ...
Over the last forty years global aquaculture presented a growth rate of 6.9% per annum with an amazing production of 52.5 million tonnes in 2008, and a contribution of 43% of aquatic animal food for human consumption. In order to meet the world's health requirements of fish protein, a continuous growth in production is still expected for decades to come. Aquaculture is, though, a very competitive market, and a global awareness regarding the use of scientific knowledge and emerging technologies to obtain a better farmed organism through a sustainable production has enhanced the importance of proteomics in seafood biology research. Proteomics, as a powerful comparative tool, has therefore been increasingly used over the last decade to address different questions in aquaculture, regarding welfare, nutrition, health, quality, and safety. In this paper we will give an overview of these biological questions and the role of proteomics in their investigation, outlining the advantages, disadvantages and future challenges. A brief description of the proteomics technical approaches will be presented. Special focus will be on the latest trends related to the aquaculture production of fish with defined nutritional, health or quality properties for functional foods and the integration of proteomics techniques in addressing this challenging issue.
The quantitative realtime polymerase chain reaction (RT-qPCR) is a powerful and sensitive method to measure expression of targeted gene but it highly relies on the use of suitable reference genes for data normalization. We evaluated the expressions of 8 housekeeping genes: 18S ribosomal rDNA (18S rDNA), 28S ribosomal rDNA (28S rDNA),rubisco large subunit (rbc-L), β-actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1 (EF1), β-tubulin (TubB), and P-phycoerythrin B (PEB), to select the suitable reference genes for different life-history stages (tetrasporophyte, carposporophyte, and male/female gametophyte) of Gracilaria vermiculophylla by absolute quantitative method. Softwares geNorm and BestKeeper were used to verify the results acquired from copy number analysis. Results show that the expression of identified reference genes varied in comparing groups composed of different type of life stages. It is suggested that 18S rDNA and TubB could be used for highly complex samples composed of mixed ploidy and phases. 18S rDNA and 28S rDNA were also preferred for using among the matured isomorphic samples. But for samples with different maturities, TubB and ACT were recommended for tetrasporophytes and gametophytes respectively.
Background. The Gracilariaceae is one of the most diverse and abundant families of marine red algae. Most species in this family exhibit high morphological variability and overlapping of characters. In the Yucatan Peninsula 30 species have been historically recorded, but the phylogenetic identity for many of them is still unknown.
Questions: Is the current diversity of the family in the area overestimated?
Studied species: Crassiphycus caudatus, C. corneus, C. usneoides, Gracilaria flabelliformis, G. hayi, G. cf isabellana, G. microcarpa, G. occidentalis, G. suzanneae, Gracilariopsis tenuifrons.
Study site and dates: Campeche: km 33 carretera Champotón, Bahía Tortuga, Puente Xen Kan III, Punta Xen, Sabancuy, 2017, 2018; Yucatan: Puerto Sisal, Puerto Progreso, Puerto Chicxulub, Puerto Telchac, 2018; Quintana Roo: Playa 88, Xcalacoco, Punta Esmeralda, 2019.
Methods. Phylogenetic analysis (with COI-5P and rbcL sequences), supported by DNA species delimitation methods, genetic distances and morphological comparisons, allowed us to molecularly identify the specimens collected.
Results. The phylogenetic identity of 10 species of Gracilariaceae was confirmed, two of which are new records for the Mexican Atlantic: G.
hayi and G. suzanneae.
Conclusion. The study demonstrated that the molecular assessment has proved to be very useful for the diversity evaluation, thus, the future
phylogenetic identifies for the rest of morphospecies recorded in the area will allow a better approximation of Gracilariaceae diversity species.
Key words: COI-5P, diversity, morphospecies, phylogeny, rbcL.
Background
The introduction of DNA-based molecular markers made a revolution in biological systematics. However, in cases of very recent divergence events, the neutral divergence may be too slow, and the analysis of adaptive part of the genome is more informative to reconstruct the recent evolutionary history of young species. The advantage of proteomics is its ability to reflect the biochemical machinery of life. It may help both to identify rapidly evolving genes and to interpret their functions.
Methods
Here we applied a comparative gel-based proteomic analysis to several species from the gastropod family Littorinidae. Proteomes were clustered to assess differences related to species, geographic location, sex and body part, using data on presence/absence of proteins in samples and data on protein occurrence frequency in samples of different species. Cluster support was assessed using multiscale bootstrap resampling and the stability of clustering—using cluster-wise index of cluster stability. Taxon-specific protein markers were derived using IndVal method. Proteomic trees were compared to consensus phylogenetic tree (based on neutral genetic markers) using estimates of the Robinson–Foulds distance, the Fowlkes–Mallows index and cophenetic correlation.
Results
Overall, the DNA-based phylogenetic tree and the proteomic similarity tree had consistent topologies. Further, we observed some interesting deviations of the proteomic littorinid tree from the neutral expectations. (1) There were signs of molecular parallelism in two Littoraria species that phylogenetically are quite distant, but live in similar habitats. (2) Proteome divergence was unexpectedly high between very closely related Littorina fabalis and L. obtusata , possibly reflecting their ecology-driven divergence. (3) Conservative house-keeping proteins were usually identified as markers for cryptic species groups (“saxatilis” and “obtusata” groups in the Littorina genus) and for genera ( Littoraria and Echinolittorina species pairs), while metabolic enzymes and stress-related proteins (both potentially adaptively important) were often identified as markers supporting species branches. (4) In all five Littorina species British populations were separated from the European mainland populations, possibly reflecting their recent phylogeographic history. Altogether our study shows that proteomic data, when interpreted in the context of DNA-based phylogeny, can bring additional information on the evolutionary history of species.
Gracilaria falconii from Falcón, Venezuela, is described as a new species, based mainly on phylogenetic evidence. Morphologically, G. falconii is similar to G. mammillaris, a species previously reported from the Gulf of Mexico, the Caribbean Sea, Venezuela, and Brazil. Although G. falconii is morphologically different from phylogenetically closely related species, morphological boundaries between G. falconii and other flattened Gracilaria are not so clear.
Biological response of cells to variable conditions should affect the expression level of certain genes. Quantification of these changes in target genes needs stable internal controls. Real-time quantitative polymerase chain reaction (PCR) has traditionally used reference or ‘housekeeping’ genes, that are considered to maintain equal expression in different conditions, to evaluate changes in target genes between samples and experimental conditions. Recent studies showed that some housekeeping genes may vary considerably in certain biological samples. This has not been evaluated in red algae. In order to identify the optimal internal controls for real-time PCR, we studied the expression of eleven commonly used housekeeping genes; elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase, β-actin, polyubiquitin, 30S ribosomal gene, 60S ribosomal gene, beta-tubulin, alpha-tubulin, translation initiation factor, ubiquitin-conjugating enzyme, and isocitrate dehydrogenase in different life-history stages of Bostrychia moritziana. Our results suggest that glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) and 30S ribosomal gene, have the most stable gene expression levels between the different life history stages (male, female, carposporophyte, and tetrasporophyte), while the other genes are not satisfactory as internal controls. These results suggest that the combinations of GAPDH and 30S would be useful as internal controls to assess expression level changes in genes that may control different physiological processes in this organism or that may change in different life history stages. These results may also be useful in other red algal systems.
Aquaculture has shown an amazing growth rate in fish production over the last decade, though nowadays it is a very competitive market in the food industry. Fish and shellfish demand will continue to grow, in part as a response to population growth. Provision of seafood from capture fish is declining and is partly not sustainable. Seafood from aquaculture will potentially overcome this supply issue. It can deliver a product of defined quality, composition and safety to the market in all seasons of the year enabling a greater penetration of fish products in consumer’s diet.
Species recognition in algae is often extremely difficult due to the paucity of morphological characters and high
environmental plasticity. If environment is important to morphology, then related species growing in sympatry are good
candidates to discover diagnostic characters. We studied algae collected in sympatric populations in tropical Atlantic
Mexico. Hydropuntia cornea and H. usneoides have high morphological variation and have been difficult to diagnose
morphologically. We used four molecular markers (RuBisCo spacer, cox2–3 spacer, rbcL and COI) to investigate the
genetic relationship between samples that correspond to either H. cornea or H. usneoides; in addition, we determined if
molecular-characterized groups were morphologically distinguishable. RuBisCo and cox2–3 spacers revealed low genetic
variation but showed two genetic groups: groups I and II. rbcL and COI phylogenies also showed a separation into two
groups, corresponding with cox2–3 spacer groups I and II. Group I matched sequences in GenBank of H. usneoides and
group II with H. cornea. Populations were mixed for these genetic groups, with group I prominent in Quintana Roo
populations. Morphological analysis of samples in both genetic clades showed that they are not distinguishable. As the
difference between the two groups is only genetic, they are, consequently, cryptic species. While the extremes of
morphology in allopatric populations may be distinguishable, we do not feel that in most cases these species can be
recognized. Therefore, we propose that these two ‘species’ should be designated as a species complex (the Hydropuntia
cornea/H. usneoides complex).
Species recognition in algae is often extremely difficult due to the paucity of morphological characters and high
environmental plasticity. If environment is important to morphology, then related species growing in sympatry are good candidates to discover diagnostic characters. We studied algae collected in sympatric populations in tropical Atlantic Mexico. Hydropuntia cornea and H. usneoides have high morphological variation and have been difficult to diagnose morphologically. We used four molecular markers (RuBisCo spacer, cox2–3 spacer, rbcL and COI) to investigate the genetic relationship between samples that correspond to either H. cornea or H. usneoides; in addition, we determined if molecular-characterized groups were morphologically distinguishable. RuBisCo and cox2–3 spacers revealed low genetic variation but showed two genetic groups: groups I and II. rbcL and COI phylogenies also showed a separation into two groups, corresponding with cox2–3 spacer groups I and II. Group I matched sequences in GenBank of H. usneoides and group II with H. cornea. Populations were mixed for these genetic groups, with group I prominent in Quintana Roo populations. Morphological analysis of samples in both genetic clades showed that they are not distinguishable. As the difference between the two groups is only genetic, they are, consequently, cryptic species. While the extremes of morphology in allopatric populations may be distinguishable, we do not feel that in most cases these species can be recognized. Therefore, we propose that these two ‘species’ should be designated as a species complex (the Hydropuntia
cornea/H. usneoides complex).
Biodiversity studies and conservation measures depend on good and up-to-date taxonomic data. General faunistic lists are the result of long periods of sampling. How many species on a global list are actually living in an area can only be answered by updating inventories. The progressive disappearance of taxonomic specialists and the undervaluation of their work is not only unjustified, but could lead to specialists from other disciplines working with meaningless data.
This study describes the isolation and structural determination of two amides, isolated for the first time: N,4-dihydroxy-N-(2'-hydroxyethyl)-benzamide (0.019%) and N,4-dihydroxy-N-(2'-hydroxyethyl)-benzeneacetamide (0.023%). These amides, produced by the red macroalgae Bostrychia radicans, had their structures assigned by NMR spectral data and MS analyses. In addition, this chemical study led to the isolation of cholesterol, heptadecane, squalene, trans-phytol, neophytadiene, tetradecanoic and hexadecanoic acids, methyl hexadecanoate and methyl 9-octadecenoate, 4-(methoxymethyl)-phenol, 4-hydroxybenzaldehyde, methyl 4-hydroxybenzeneacetate, methyl 2-hydroxy-3-(4-hydroxyphenyl)-propanoate, hydroquinone, methyl 4-hydroxymandelate, methyl 4-hydroxybenzoate, 4-hydroxybenzeneacetic acid and (4-hydroxyphenyl)-oxo-acetaldehyde. This is the first report concerning these compounds in B. radicans, contributing by illustrating the chemical diversity within the Rhodomelaceae family.
The application of proteomics to marine sciences has increased in recent years because the proteome represents the interface between genotypic and phenotypic variability and, thus, corresponds to the broadest possible biomarker for eco-physiological responses and adaptations. Likewise, proteomics can provide important functional information regarding biosynthetic pathways, as well as insights into mechanism of action, of novel marine natural products. The goal of this review is to (1) explore the application of proteomics methodologies to marine systems, (2) assess the technical approaches that have been used, and (3) evaluate the pros and cons of this proteomic research, with the intent of providing a critical analysis of its future roles in marine sciences. To date, proteomics techniques have been utilized to investigate marine microbe, plant, invertebrate, and vertebrate physiology, developmental biology, seafood safety, susceptibility to disease, and responses to environmental change. However, marine proteomics studies often suffer from poor experimental design, sample processing/optimization difficulties, and data analysis/interpretation issues. Moreover, a major limitation is the lack of available annotated genomes and proteomes for most marine organisms, including several "model species". Even with these challenges in mind, there is no doubt that marine proteomics is a rapidly expanding and powerful integrative molecular research tool from which our knowledge of the marine environment, and the natural products from this resource, will be significantly expanded.
A gamete recognition mechanism in Antithamnion sparsum Tokida is proposed based on experiments using various lectins and carbohydrates. Spermatial binding to trichogynes is inhibited
by pre-incubation of spermatia with concanavalin A (ConA) and/or L-fucose, while trichogyne receptors are blocked by the complementary
carbohydrate α-methyl D-mannose and/or the lectin Ulex europaeus agglutinin (UeA1). Binding inhibition (40–50%) was observed with 10–50 mM carbohydrates and 25–50 μg ml-1 lectins. The inhibitory effects of ConA and UeA1 is partially reversed (to 80–90% of controls) by addition of α-methyl D-mannose
and L-fucose, respectively. Lectin binding to spermatial surfaces was visualized by Fluorescein isothiocyanate (FITC) conjugated
ConA, whereas carbohydrate receptors along the trichogyne and spermatium were localized with α-mannosylated-FITC-albumin and
L-fucosylated-FITC-albumin, respectively. These results suggest that gamete recognition in Antithamnion sparsum is mediated by a double-docking recognition system consisting of spermatia possessing surface L-fucose receptors and α-methyl
D-mannose moiety, and trichogynes possessing the complementary receptors.
Two-dimensional gel electrophoresis (2-DE) is a unique method of large-scale protein characterisation, and is a powerful approach in the study of protein expression. In the present work, the experimental conditions for 2-DE of foot proteins from the mussel Mytilus galloprovincialis Lmk were established; the technique was performed with intertidal and cultured mussels using Melanie 3 software for data analysis. This powerful technique enabled the visualisation of a total of 750 protein spots consistently expressed in the foot. The intensity of 92 selected spots was compared between intertidal and cultured mussels, and statistically significant differences were detected in the expression of 45 (48.9 %) of the 92 proteins analysed. In 31 of these proteins, intensity was higher in the cultured stock than in the intertidal mussels, while in 14 proteins spot intensity was higher in the latter. Using mass spectrometry (MS) combined with sequence database searching, 6 of the most prominent differentially expressed proteins were analysed. Of these, 1 was identified as being Heat-shock Protein 70, and 2 were shown to be cytoskeleton-associated proteins, myosin and actin. Heat-shock Protein 70, which is known to be involved in cellular transport and chaperoning and associated with stress situations, was more highly expressed in intertidal mussels living in littoral areas than in cultured mussels. These findings are discussed in connection with the molecular changes involved in the adaptation of mussels to different ecological conditions.
The subfamily Bostrychioideae (Rhodomelaceae, Ceramiales) presently consists of two genera and about 17 species. Generic distinction within the subfamily is based on the number of tier cells (the results of transversely dividing pericentral cells) per axial cell, with two tier cells designating the genus Bostrychia and more than two characterising Stictosiphonia. Phylogenetic analysis of all but two species of this subfamily based on DNA sequences from the nuclear-encoded 26S ribosomal RNA gene and plastid-encoded rbcL gene shows that this generic split is not supported. Our data suggest that the subfamily may be split into two sections based on one or another type of attachment structure, as first proposed by Erika Post, but we propose that all species be returned to the genus Bostrychia due to as yet not fully resolved or supported phylogeny. We also recommend that Bostrychia tenella ssp. flagellifera be returned to B. flagellifera because it does not group with B. tenella samples and shows a diagnostic synapomorphy (branching interval). Our data additionally show that many currently circumscribed species are not monophyletic. Even when B. tenuissima is regarded as synonymous with B. simpliciuscula, due to sequence identity, the resulting amalgamated species is still polyphyletic when populations from broad geographic regions are analysed. Another possible polyphyletic species is B. calliptera. The clade containing cladohaptera (specialized attachment organs) is taxonomically unresolved but contains a heavily corticated species (B. pilulifera), a parasite (Bostrychiocolax australis), plus several lineages in the Bostrychia radicans/B. moritziana species complex that are morphologically very similar.
Bostrychia moritziana is a widespread species commonly associated with mangroves. The ribulose-1-5-bispbosphate carboxylase/oxygenase spacer region (RUBISCO spacer) was used to score haplotypes using single-stranded conformational polymorphisms (SSCP) in over 140 individual isolates. Sequence data showed no variation in isolates with identical SSCP banding patterns. Phylogenetic relationships revealed five separate lineages. Lineage 1 consisted of plants found over a large geographic area (Australia, New Zealand, New Caledonia, Fiji,South Africa, Japan) and showed little variation in RUBISCO spacer sequence. This lineage also contained the holotype of Bostrychia bispora, and other asexually reproducing isolates; thus, the data did not support the recognition of the species B. bispora. Internal transcribed spacer 1 (ITS1)-RFLP data of lineage I isolates showed some biogeographic clustering, based on proximity, although all sexual isolates within this Lineage were interfertile. Other lineages had higher sequence variation and, for the most part, consisted of isolates with compound monosiphonous branch apices. This morphological character seemed to be useful in distinguishing between B. moritziana and the closely allied species B, radicans, but not all isolates fit this pattern.
Hybridization of isolates of Bostrychia radicans (Montagne) Montagne and B. moritziana (Sonder ex Kutzing) J. Agardh from various geographical locations was performed in laboratory culture. Positive crosses (successful formation of carposporophytes and release of carpospores) were observed between single isolates of B. moritziana from Australia and South Africa. but this species did not hybridize with B. radicans isolates. Hybridization occurred between isolates of B. radicans from Venezuela and Pacific Mexico and between Peruvian and some Brazilian isolates. Not all Brazilian isolates were able to hybridize with each other. DNA sequences of the intergenic spacer of the plastid-encoded large- and small-subunit genes of ribulose bisphosphate carboxylase/oxygenase (RUBISCO) were used to determine relationships between and within these two species. Bostrychia radicans formed a clade distinct from B. moritziana. All phylogenetic analyses resulted in two major clades within B. radicans that did not completely correspond to geographical proximity of the isolates. The phylogenetic relationships generally correlated with the hybridization data, although isolates that were not able to hybridize were sometimes closely related, based on DNA comparisons. Incongruence between DNA sequence phylogeny and proposed relationships based on crossing results with one B. radicans isolate can be interpreted as a case of plastid introgression. Tests of alternative hypotheses, grouping isolates based on geographic proximity, ocean basin distribution, northern and southern hemisphere distribution, or parasite susceptibility/resistance were tested and were not supported by the DNA sequence data. A biogeographical history for the present distribution of the species must consider both vicariant and dispersal events.
Field and culture samples of Bostrychia and Stictosiphonia species were analyzed for D-dulcitol and D-sorbitol that function as osmoregulatory solutes and photoassimilation products. Bostrychia bispora West et Zuccarello, B. calliptera (Montagne) Montagne, B. montagnei Harvey, B. moritziana (Sonder ex Kutzing) J. Agardh, B. pinnata J. Tanaka et Chihara, B. radicans (Montagne) Montagne, B. simpliciuscula Harvey ex J. Agardh and B. tenella (Lamouroux) J. Agardh contain both polyols, except for one isolate of B. radicans (2879/80/81) that contains only D-sorbitol. Bostrychia tenuissima King et Puttock and Stictosiphonia hookeri (Harvey) Hooker et Harvey and S. kelanensis (Grunow ex Post) King et Puttock contain only D-sorbitol. Polyol accumulation is greater in nutrient-stressed culture of B. moritziana than in nonstressed culture. In Stictosiphonia hookeri D-sorbitol accumulation is lower at 15-degrees-C than at 25-degrees-C. Generally, in all species, field samples have much lower polyol concentrations than culture samples.
Bostrychia moritziana (Sonder ex Kützing) J. Agardh is recorded from many regions around the world. Our laboratory culture investigations have verified a sexual life cycle in isolates from Australia, Venezuela, Colombia, South Africa, Fiji, New Zealand and Indonesia. By contrast, asexual isolates producing successive generations of tetrasporophytes in laboratory culture and, presumably, in the field, are known from Australia. New Caledonia and Japan. In Australia, asexual reproduction is absent only in Victoria. In Western Australia, Northern Territory and Queensland, 99% of the isolates have asexual reproduction. In New South Wales (NSW), asexual and sexual populations are often intermixed. Of the 176 worldwide field collections, 58% were vegetative, 39% were tetrasporic, 2% were female and 1% were male. After several years of observations on the asexual isolates in culture, at least 30 successive asexual tetrasporophytic generations have developed. Only two asexual isolates (3558 and 3575) from NSW have formed a single male and female gametophyte in culture. In a self-cross of 3568, the carpospores developed into tetrasporophytes that recycled asexually. All outcrosses done with normal sexual isolates produced normal carposporophytes and the carpospores developed into tetrasporophytes that also recycled asexually. Asexual populations may arise repeatedly by loss of meiosis in tetrasporangia of sexual populations. Asexual reproduction apparently does not diminish the overall dispersal and abundance in the field. Our present bio-geographic data show that sexually reproducing popuiations of B. moritziana occur worldwide, while asexuaily reproducing populations are confined to the western Pacific. Bostrychia bispora West et Zuccarello, initially described on the basis of its asexual reproduction to distinguish it from B, moritziana, is now reduced to synonymy with B. moritziana.
Red algae of the Bostrychia radicans/B. moritziana complex are common in warm temperate areas of North America. Phylogenetic analysis of both plastid and mitochondrial DNA sequence data revealed seven distinct evolutionary lineages among worldwide samples. Although only two haplotypes (plastid and mitochondrial) were found in Pacific Mexico, four plastid and 11 mitochondrial haplotypes were found in a similar latitudinal spread along the Atlantic coast of the United States. On the U.S. Atlantic coast only one plastid haplotype was found in northern samples (Connecticut to North Carolina), whereas further south several plastid haplotypes were found. Phylogenetic analyses suggested that this single plastid haplotype found among northern samples could be the result of a northward range expansion possibly since the last glacial maximum. Crossing data of samples within the same evolutionary lineage showed that samples with the same plastid haplotypes were generally sexually compatible; samples with different plastid haplotypes were reproductively isolated. Samples from Pacific Mexico were partially reproductively compatible with some samples from the Atlantic USA (plastid haplotype C) and were more closely related to these samples than these U.S. samples were to other U.S. Atlantic samples. Compatible solute types mirrored the plastid haplotype, with plastid haplotype B having only sorbitol, whereas all other haplotypes also contained dulcitol. Samples from Atlantic USA, with different plastid haplotypes (e.g. B vs. C), but within the same evolutionary lineage, were reproductively isolated from each other. Data indicate that reproductive isolation occurs between and within supported evolutionary lineages and that the number of cryptic species is high.
Using two-dimensional polyacrylamide gel electrophoresis, the genetic variation of proteins was examined in three organs (needle, bud, and pollen) from 18 trees of maritime pine. Three types of variation were noted: presence/absence, staining intensity, and position variation of the spots. Of the 902 polypeptides scored in the three organs, 245 (27.2%) were polymorphic. Moreover, among these variable spots, 117 were found in a single organ, demonstrating an increased polymorphism of the organ-specific polypeptides (56.0% vs 18.4% for the organ-unspecific polypeptides). Finally, a positive correlation was found between variability level and subunit molecular weight for spots showing position variation but not for spots showing presence/absence or staining intensity variations. Possible explanations for this observation are discussed.
The binding of fluorescein isothiocyanate (FITC) conjugated lectins to gametes of Aglaothamnion oosumiense Itono during fertilization was studied by the use of confocal microscopy. The physiological effects of lectins and carbohydrates on gamete binding were also examined. Four different lectins, concanavalin A (ConA), soybean agglutinin (SBA), Dolichos biflorus agglutinin (DBA) and wheat germ agglutinin (WGA) bound to the surface of spermatia, but each lectin labelled a different region of the spermatium. SBA and DBA bound only to the spermatial appendages but ConA bound to all the spermatial surface except the spermatial appendages. WGA labelled a narrow region that connects the spermatial body and appendages. During fertilization, the ConA and WGA specific substances on the spermatial surface moved towards the area contacting the trichogyne and accumulated on the surface of the fertilization canal. Spermatial binding to trichogynes was inhibited by pre-incubation of spermat ia with SBA or ConA, while trichogyne receptors were blocked by the complementary carbohydrates, D-glucose or N-acetyl-galactosamine, respectively. WGA and DBA as well as their complementary carbohydrates had little effect on gamete binding. The inhibitory effects of ConA and SBA were increased when the two lectins were applied simultaneously. The inhibitory effects of both lectins were partially reversed (to 80–90% of controls) by addition of complementary carbohydrates at the same time. The results suggested that SBA and ConA receptors on the spermatial surface are involved in gamete recognition in Aglaothamnion oosumiense.
In this study, a proteomic approach was applied for the generation of reference maps and subsequently to detect, quantify and compare the global protein expression between two related species of marine mussels, Mytilus
edulis and Mytilus
galloprovincialis, growing in their own geographical habitats. A comparative study of the protein profiles generated from analytical two-dimensional electrophoresis gels was performed, and changes in protein expression were analysed quantitatively by computer analysis. An average of 1,278 spots per gel was detected in 16 individuals (8 M. edulis and 8 M. galloprovincialis); however, not all spots were included in the study. Expression of 420 spots was compared, and significant differences in the intensity levels were detected in 37 protein spots (8.8%). Fifteen proteins showed higher expression in M. edulis, and 22 proteins, in M. galloprovincialis. The technique of peptide mass fingerprinting using MALDI-TOF (matrix-assisted laser desorption ionisation/time-of-flight) and/or nanoelectrospray double subfragmentation mass spectrometry enabled the unambiguous identification of 15 of these 37 differentially expressed proteins. Most of the identified proteins can be grouped basically into four broad functional classes: cytoskeletal and myofibrillar proteins, proteins associated with stress response, proteins associated with the storage or production of energy, and proteins related to rearrangement in the synthesis of native structures. These results expand our understanding of the molecular differentiation of the two mussel taxa and serve as a useful base for future ecological, physiological and genetic studies.
A technique has been developed for the separation of proteins by two-dimensional polyacrylamide gel electrophoresis. Due to its resolution and sensitivity, this technique is a powerful tool for the analysis and detection of proteins from complex biological sources. Proteins are separated according to isoelectric point by isoelectric focusing in the first dimension, and according to molecular weight by sodium dodecyl sulfate electrophoresis in the second dimension. Since these two parameters are unrelated, it is possible to obtain an almost uniform distribution of protein spots across a two-diminsional gel. This technique has resolved 1100 different components from Escherichia coli and should be capable of resolving a maximum of 5000 proteins. A protein containing as little as one disintegration per min of either 14C or 35S can be detected by autoradiography. A protein which constitutes 10 minus 4 to 10 minus 5% of the total protein can be detected and quantified by autoradiography. The reproducibility of the separation is sufficient to permit each spot on one separation to be matched with a spot on a different separation. This technique provides a method for estimation (at the described sensitivities) of the number of proteins made by any biological system. This system can resolve proteins differing in a single charge and consequently can be used in the analysis of in vivo modifications resulting in a change in charge. Proteins whose charge is changed by missense mutations can be identified. A detailed description of the methods as well as the characteristics of this system are presented.
We describe here a computer system for the analysis of high-resolution two-dimensional gel-electrophoresis patterns, with some initial applications. The system (called TYCHO) comprises programs for image acquisition, background subtraction and smoothing, spot detection, gaussian spot modeling, and pattern matching and comparison. It is based on a conventional minicomputer, but makes extensive use of a high-speed array processor in the image-processing and -modeling steps. Used in concert with the ISO-DALT two-dimensional electrophoresis system (Anal. Biochem. 85:331-354, 1978), TYCHO allows quantitative measurement of hundreds of proteins in complex biological samples, and constitutes the initial data-reduction system required for work towards a Human Protein Index.
A protein map of the smallest known self-replicating organism, Mycoplasma genitalium (Class: Mollicutes), revealed a high proportion of acidic proteins. Amino acid composition was used to putatively identify, or provide unique parameters, for 50 gene products separated by two-dimensional gel electrophoresis. A further 19 proteins were subjected to peptide-mass fingerprinting using matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry and 4 were subjected to N-terminal Edman degradation. The majority of M. genitalium proteins remain uncharacterised. However, the combined approach of amino acid analysis and peptide-mass fingerprinting allowed gene products to be linked to homologous genes in a variety of organisms. This has allowed proteins to be identified prior to detection of their respective genes via the M. genitalium sequencing initiative. The principle of 'hierarchical' analysis for the mass screening of proteins and the analysis of microbial genomes via their protein complement or 'proteome' is detailed. Here, characterisation of gene products depends upon the quickest and most economical technologies being employed initially, so as to determine if a large number of proteins are already present in both homologous and heterologous species databases. Initial screening, which lends itself to automation and robotics, can then be followed by more time and cost intensive procedures, when necessary.
In the search for new markers of human endometrial hyperplasia and adenocarcinoma the method of quantitative two-dimensional gel electrophoresis was applied to study the protein expression profiles of metabolically [(35)S]-methionine-labelled proteins of endometrial explants. Approximately 1700 protein spots were resolved by the two-dimensional gel electrophoresis, and the expression pattern of each of these proteins was assessed for increased expression during hyperplasia or adenocarcinoma. In total, six protein spots showed increased expression in hyperplasia, 19 in carcinoma, and eight in both hyperplasia and carcinoma. Twelve of these 33 differentially expressed proteins were identified by peptide mass mapping combined with sequence database searching. Among the identified proteins were proteins involved in cellular transport and chaperoning, i.e. heat shock protein 27 kDa protein, heat shock 70 kDa protein, heat shock cognate 71 kDa protein, and serotransferrin. Other identified proteins were: regulatory chain protein of cAMP-dependent protein kinase, prohibitin, and heterogeneous nuclear ribonucleoprotein A2/B1. Finally we identified proteins associated with the cytoskeleton, vimentin and tropomyosin isoform 3, and the glycolytic pathway, alpha enolase, and phosphoglycerate kinase. The remaining unidentified proteins were either not contained in the database and must be assumed to be novel proteins, or were present in too low amounts to allow characterization.
The characteristics of protein detection and quantitation with SYPRO Ruby protein gel stain in one‐ and two‐dimensional polyacrylamide gels were evaluated. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) analyses of three different purified recombinant proteins showed that the limits of detection were comparable to the limits of detection with ammoniacal silver staining and were protein‐specific, ranging from 0.5 to 5 ng. The linearity of the relationship between protein level and SYPRO Ruby staining intensity also depended on the individual protein, with observed linear dynamic ranges of 200‐, 500‐, and, 1000‐fold for proteins analyzed by SDS‐PAGE. SYPRO Ruby protein gel stain was also evaluated in two‐dimensional electrophoretic (2‐DE) analysis of Escherichia coli proteins. The experiment involved analysis of replicates of the same sample as well as dilution of the sample from 0.5 to 50 νg total protein across gels. In addition to validating the 2‐DE system itself, the experiment was used to evaluate three different image analysis programs: Z3 (Compugen), Progenesis (Nonlinear Dynamics), and PDQuest (Bio‐Rad). In each program, we analyzed the 2‐DE images with respect to sensitivity and reproducibility of overall protein spot detection, as well as linearity of response for 20 representative proteins of different molecular weights and pI. Across all three programs, coefficients of variation (CV) in total number of spots detected among replicate gels ranged from 4 to 11%. For the 20 representative proteins, spot quantitation was also comparable with CVs for gel‐to‐gel reproducibility ranging from 3 to 33%. Using Progenesis and PDQuest, a 1000‐fold linear dynamic range of SYPRO Ruby was demonstrated with a single known protein. These two programs were more suitable than Z3 for examining individual protein spot quantity across a series of gels and gave comparable results.
The characteristics of protein detection and quantitation with SYPRO Ruby protein gel stain in one- and two-dimensional polyacrylamide gels were evaluated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of three different purified recombinant proteins showed that the limits of detection were comparable to the limits of detection with ammoniacal silver staining and were protein-specific, ranging from 0.5 to 5 ng. The linearity of the relationship between protein level and SYPRO Ruby staining intensity also depended on the individual protein, with observed linear dynamic ranges of 200-, 500-, and, 1000-fold for proteins analyzed by SDS-PAGE. SYPRO Ruby protein gel stain was also evaluated in two-dimensional electrophoretic (2-DE) analysis of Escherichia coli proteins. The experiment involved analysis of replicates of the same sample as well as dilution of the sample from 0.5 to 50 νg total protein across gels. In addition to validating the 2-DE system itself, the experiment was used to evaluate three different image analysis programs: Z3 (Compugen), Progenesis (Nonlinear Dynamics), and PDQuest (Bio-Rad). In each program, we analyzed the 2-DE images with respect to sensitivity and reproducibility of overall protein spot detection, as well as linearity of response for 20 representative proteins of different molecular weights and pI. Across all three programs, coefficients of variation (CV) in total number of spots detected among replicate gels ranged from 4 to 11%. For the 20 representative proteins, spot quantitation was also comparable with CVs for gel-to-gel reproducibility ranging from 3 to 33%. Using Progenesis and PDQuest, a 1000-fold linear dynamic range of SYPRO Ruby was demonstrated with a single known protein. These two programs were more suitable than Z3 for examining individual protein spot quantity across a series of gels and gave comparable results.
Use of isozymes (including allozymes) in studies of population genetics and systematics of seaweeds has increased sufficiently in the last decade to allow some generalization. Only a single locus has been observed for about half the enzymes analysed in seaweeds, compared with 29% in vascular plants. Compared with higher plants, macroalgal species generally have low amounts of electrophoretically detectable genetic variation; the lowest levels of genetic variation found in natural populations are those reported for seaweeds. Nonetheless, seaweeds show an association between levels of genetic diversity as revealed by isozymes and species-specific attributes, such as mating system and predominance of asexual versus sexual reproduction. In systematic studies, isozymes have revealed cryptic species and identified pairs of: sibling taxa. The quaternary structure of enzymes appears to be conserved at the phylum level. With the current availability of improved techniques for enzyme electrophoresis and for data interpretation, we expect future studies utilizing isozyme electrophoresis to provide further insight into population and evolutionary processes in seaweeds.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
In the search for new markers of human endometrial hyperplasia and adenocarcinoma the method of quantitative two-dimensional gel electrophoresis was applied to study the protein expression profiles of metabolically [ 35 S]-methionine-labelled proteins of endometrial explants. Approximately 1700 protein spots were resolved by the two-dimensional gel electrophoresis, and the expression pattern of each of these proteins was assessed for increased expression during hyperplasia or adenocarcinoma. In total, six protein spots showed increased expression in hyperplasia, 19 in carcinoma, and eight in both hyperplasia and carcinoma. Twelve of these 33 differentially expressed proteins were identified by peptide mass mapping combined with sequence database searching. Among the identified proteins were proteins involved in cellular transport and chaperoning, i.e. heat shock protein 27 kDa protein, heat shock 70 kDa protein, heat shock cognate 71 kDa protein, and serotransferrin. Other identified proteins were: regulatory chain protein of cAMPdependent protein kinase, prohibitin, and heterogeneous nuclear ribonucleoprotein A2/B1. Finally we identified proteins associated with the cytoskeleton, vimentin and tropomyosin isoform 3, and the glycolytic pathway, a enolase, and phosphoglycerate kinase. The remaining unidentified proteins were either not contained in the database and must be assumed to be novel proteins, or were present in too low amounts to allow characterization.
We have studied allelic variation at gene loci coding for 23 proteins in nine species and subspecies of hominoids. The organisms studied comprise the three families and the six genera of extant hominoids as follows. Family Hominidae: man (Homo sapiens). Family Pongidae: chimpanzee (two species, Pan troglodytes and P. paniscus), gorilla (Gorilla gorilla), and orangutan (two subspecies, Pongo pygmaeus pygmaeus and P. p. abelii). Family Hylobatidae: gibbon (two species, Hylobates lar and H concolor) and siamang (Symphalangus syndactylus). The degree of genetic variation observed in the nonhuman hominoids is low; the expected heterozygosity is 2.3%, compared with a 6.7% for humans. Although the number of individuals studied is very small (59 if humans are excluded), it seems safe to conclude that at least the large apes are genetically less polymorphic than humans. The phylogeny inferred from the electrophoretic data shows man, the chimpanzee, the gorilla, and the orangutan about equally divergent from each other, but considerably separated from the siamang and the gibbon. Thus, our data are not consistent with phylogenies proposed by some molecular biologists showing the orangutan as being as different from man and the African apes as the gibbon and the siamang are. On the other hand, our data are not incompatible with phylogenies showing the orangutan as slightly more different from man and the African apes than these are from each other-a phylogeny which in our view is favored by the available information when all sources of evidence are taken into account. Concerning the phylogenetic relationships between humans, chimpanzees, and gorillas, the electrophoretic data agree with other molecular data indicating that these three genera are about equally related to each other This is inconsistent with phylogenies proposed by paleontologists and comparative anatomists showing the gorilla and the chimpanzee as more closely related to each other than either one is to man. The degree of genetic differentiation among man, chimpanzee, gorilla, and orangutan is similar to that observed among congeneric species in other groups of organisms, although they involve comparisons between genera (chimpanzee-gorilla-orangutan) or between families (man versus any of the great apes). The genetic distance between the Hylobatidae and the Pongidae (great apes) or Hominidae (man) is slightly higher than the overall average distance between congeneric species, but lower than the typical distance observed between confamilial genera, in other groups of organisms.
The concentration of protein actually solubilized in sample buffer in preparation for analysis by two-dimensional polyacrylamide gel electrophoresis cannot be directly determined by the Lowry, Biuret, or Bradford protein methods due to interference by the combinational effect of presence of urea, detergents, carrier ampholytes, and thiol compounds in sample solubilization buffers. Determinations of the actual amount of protein solubilized and applied to gels is required to accurately quantitatively and qualitatively evaluate second-dimension polypeptide maps. It was found that when sample buffer consisting of 9 M urea, 4 % Nonidet P-40, 2 % Ampholines, and 2 % 2-mercaptoethanol containing solubilized sample(s) was acidified prior to dilution, protein concentrations over a range of 0.5 to 50 μg could be reproducibly determined utilizing a modified Bradford assay. The modified assay generates two near-linear segments, one over the range < 0.5 to 5 μg total protein that permits the application of Beer's law and a second linear response encompassing 5 to 50 μg total protein. The assay did not tolerate presence of greater than 0.1 % sodium dodecyl sulfate but addition of sodium chloride and protamine sulfate did not adversely affect protein quantitation. The modified assay allows direct quantitation of protein solubilized in sample buffers containing urea, carrier ampholytes, nonionic detergents, and thiol compounds.
Bostrychia radicans(Montagne) Montagne is a pantropical/temperate red alga associated with mangroves and saltmarsh plants. Collections were made from a similar north-south geographic distribution along both the Pacific and Atlantic coasts of North America. Hybridization studies were performed with cultured isolates to assess the extent of interfertility and reproductive isolation along these two coastlines. All male and female gametophytes derived from single tetrasporophytes were intercompatible. Almost all isolates extending over 1500 km of coast line from northern Pacific Mexico are compatible, forming cystocarps that released viable carpospores. Even isolates which morphologically would be placed in two species [B. radicans and B. moritziana(Sender ex Kützing) J. Agardh], based on the presence or absence of monosiphonous branches, were capable of hybridizing. Crosses of isolates from the Atlantic USA showed a greater amount of incompatibility. Certain isolates were not compatible with any other isolates including isolates collected in close proximity (North Carolina isolates), while other isolates from the same locality were compatible (South Carolina). An isolate from South Carolina formed tetrasporophytes with isolates from Pacific Mexico but tetraspores were not viable. Certain incompatible crosses formed ‘pseudocystocarps’ but viable carposporophytes did not develop. Generalizations about reproductive isolation within a species must also consider differences between populations from different biogeographic regions that may reflect different paleoclimatological histories, founder effects and unique dispersal events.
A cDNA library from a mixed population of female and male gametophytes of Griffithsia japonica Okamura was constructed, and a cDNA clone, designated GjFP-1 (G. japonica female predominant-1), was isolated by differential screening of the cDNA library. The transcript corresponding to GjFP-1 was abundant in female gametophytes, but only basal levels of the transcript were detected in male and tetrasporangial thalli. Determination of the nucleotide sequence of GjFP-1 identified a putative open reading frame encoding 313 amino acids and a 3′ untranslated region of 116 nucleotides. The deduced amino acid sequence of GjFP-1 for the putative open reading frame shared high homology with the previously reported amino acid sequences of heat shock protein 90 (hsp90). RNA blot hybridization analysis for the transcripts of heat-shocked G. japonica and DNA hybridization analysis for the genomic DNA of G. japonica with GjFP-1 as a probe suggested that GjFP-1 is a cDNA clone for the hsp90 gene in G. japonica.In situ hybridization for the GjFP-1 transcript again showed a differential specificity of the transcript in female gametophytes. The data reported here suggest a possibility of hsp90 function linked to the development of the female gametophytes in G. japonica.
The application of proteomics in alga research is still quite limited. The present report describes the establishment of the proteome of a red alga of eco-nomic importance, Gracilaria changii (Xia et Abb-ott) Abbott, Zhang et Xia. Initially, four protein extraction methods including direct precipitation by trichloroacetic acid/acetone, direct lysis using urea buffer, Tris buffer, and phenol/chloroform ex-traction were compared for their suitability to gen-erate G. changii proteins for two-dimensional gel electrophoresis (2-DE). The phenol/chloroform pro-tein extraction method gave the best 2-DE resolu-tion of the proteins. Using these 2-DE gels and mass spectrometry, several proteins including pigment proteins, metabolic enzymes, and ion transporters were identified. These findings highlight the poten-tial of using proteomic approaches for the investi-gation of G. changii protein function.
When the coenocytic green alga Bryopsis plumosa (Huds.) Ag. was cut open and the cell contents were expelled, the cell organelles agglutinated rapidly in seawater to form protoplasts. Aggregation of cell organelles in seawater was mediated by a lectin–carbohydrate complementary system. Two sugars, N-acetyl-d-glucosamine and N-acetyl-d-galactosamine inhibited aggregation of cell organelles. The presence of these sugars on the surface of chloroplasts was verified with their complementary fluorescein isothiacyanate-labeled lectins. An agglutination assay using human erythrocytes showed the presence of lectins specific for N-acetyl-d-galactosamine and N-acetyl-d-glucosamine in the crude extract. One-step column purification using N-acetyl-d-glucosamine-agarose affinity chromatography yielded a homogeneous protein. The protein agglutinated the cell organelles of B. plumosa, and its agglutinating activity was inhibited by the above sugars. Sodium dodecyl sulfate polyacrylamide gel electrophoresis results showed that this protein might be composed of two identical subunits cross-linked by two disulfide bridges. Enzyme and chemical deglycosylation experiments showed that this protein is deficient in glycosylation. The molecular weight was determined as 53.8 kDa by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The N-terminal 15 amino acid sequence of the lectin was Ser–Asp–Leu–Pro–Thr–X–Asp–Phe–Phe–His–Ile–Pro–Glu–Arg–Tyr, and showed no sequence homology to those of other reported proteins. These results suggest that this lectin belongs to a new class of lectins. We named this novel lectin from B. plumosa“bryohealin.”
Zuccarello and West (2003) reported on the phylogenetic diversity of algae identified as Bostrychia radicans (Montagne) Montagne and B. moritziana (Sonder ex Kützing) J. Agardh from around the world. They showed that the species complex consisted of seven distinct lineages, of which two lineages were common on the East Coast of the USA and eastern Gulf of Mexico. The distribution of haplotypes within these lineages on the East Coast of the USA showed a general north–south distribution. One haplotype of lineage 5 (B) was mostly collected in northern areas, while the other common haplotype (C) was more southerly in distribution. Samples in lineage 6 (haplotype D) were not found north of Sapelo Island, Georgia. Increased sampling from the eastern USA over 5 years later has revealed an altered pattern. Haplotype D is distributed in North Carolina and is common in some populations. Haplotype C is rare or absent in many sampled populations. Haplotype B is only observed in the northern sampled sites on both sides of the Florida peninsula. This disjunct distribution agrees with geological scenarios for a strait between the western Gulf of Mexico and southern Georgia in the Miocene/Pliocene, which closed in the late Pliocene. This paper highlights the importance of increased sampling to determine phylogeographic patterns and hypotheses of dispersal scenarios in algae.
Field and culture samples of the red algal genera Bostrychia and Stictosiphonia from all around the world were analyzed for the polyols D-sorbitol and dulcitol, that function as osmolytes, as well as for the heteroside digeneaside by using 13C-NMR spectroscopy and HPLC. While all plants exhibited D-sorbitol, the occurrence of dulcitol and digeneaside was highly variable. Therefore, different types of low molecular weight carbohydrate distribution patterns were found in Bostrychia and Stictosiphonia. The presence of D-sorbitol seems to be a reliable chemosystematic character for both genera, because no other red alga is known to contain this compound. The lack of dulcitol may be correlated with the geographical origin of the Bostrychia and Stictosiphonia samples: while all tropical isolates exhibited both sugar alcohols, in cold-temperate plants only D-sorbitol was determined. In warm-temperate species, however, both polyol distribution types may occur. These data are discussed in terms of possible temperature sensitivity of the dulcitol pathway. However, the biological function of digeneaside (the main photo-assimilated compound in members of the order Ceramiales) is still obscure.
Two-dimensional gel electrophoresis of seedling proteins has been undertaken on diploid representatives of the A, S, and DTriticum genomes. The 457 reproducible spots of the hexaploid Chinese Spring were taken into account in the comparisons. More than 82% of the hexaploid spots were found among the representatives of each of the three genomes. Thus, it was proposed that the homoeologous structural loci, on the three homoeologous genomes, can be present in the same allelic forms and that from 72% to 86% of the spots of the hexaploid pattern are in two or three doses. The dendrogram, deduced from the similarity indices computed between the 16 diploid accessions, was very consistent with our present knowledge of the genetic relationships in theTriticum genus. This result, in addition to previous ones, shows that the comparison of two-dimensional protein patterns represents a novel experimental approach for studying the phylogenetic relationships in theTriticinae.
Two-dimensional gel electrophoresis (2-DE), a method which can be used to analyze the expression of many proteins, is a promising and powerful approach which we have begun to use in the characterization of the complex pathologic processes in Alzheimer’s disease (AD). In the present study, a reliable 2-DE database of human brain proteins was created by improving the reproducibility of 2-DE images using an immobilized pH gradient (IPG) for the first dimension gel electrophoresis and Melanie II as the program for data analysis. The brain samples were taken from the temporal cortex of brains at autopsy from 15 AD patients and 15 age-matched controls with non-neurological disorders. About 700 spots were located as consistently expressed proteins in the human brain, all of which were expressed also in AD brains. Comparing the density of spots between AD and normal control, we found that five protein spots were significantly increased, 28 spots were significantly decreased and nine spots were detected only in AD. Two spots among those significantly increased and one spot among those significantly decreased were identified as glial fibrillary acidic proteins. The database of brain proteins in AD constructed for the present study, including the statistical data of density changes in AD, should be a useful beginning for a comprehensive human 2-DE database available via the Internet, which will facilitate further investigation of pathogenic protein alterations in AD.
In this article, the growth, mortality, pathological conditions and protein expression of hatchery obtained mussels from pure and hybrid crosses between individuals from three genetically divergent European populations were evaluated in the Rı́a de Arousa (NW Spain) under raft-suspended cultivation conditions. Progenitors for the crosses were obtained from a Mytilus edulis population from The Netherlands and from two M. galloprovincialis populations located on each of the two sides of a major genetic break, associated with the Almerı́a–Oran oceanographic front in the Iberian Peninsula. Results indicated that mussels from crosses between individuals of M. galloprovincialis populations have a significantly higher biomass production than those from hybrid crosses between individuals of M. galloprovincialis and M. edulis populations. This different performance was not due to differences in growth rate but rather to the considerably higher mortality, during the warmer season, of the mussels from hybrid crosses. This strong viability selection operating against hybrid mussels with respect to pure M. galloprovincialis crosses under raft cultivation conditions in the Rı́a de Arousa was very similar to that operating in natural populations of the M. edulis/M. galloprovincialis hybrid zone in SW England, reported in previous studies. Our results also show that the lower viability of hybrid mussels is clearly associated with both higher parasitization by the protistan Marteilia refringens and lower levels of the stress proteins calreticulin and heat shock protein 70. Among the mussels from the different M. galloprovincialis crosses, those from crosses between males and females of autochthonous (Galician) origin are the ones which show a better performance.
Use of isozymes (including allozymes) in studies of population genetics and systematics of seaweeds has increased sufficiently in the last decade to allow some generalization. Only a single locus has been observed for about half the enzymes analysed in seaweeds, compared with 29% in vascular plants. Compared with higher plants, macroalgal species generally have low amounts of electrophoretically detectable genetic variation; the lowest levels of genetic variation found in natural populations are those reported for seaweeds. Nonetheless, seaweeds show an association between levels of genetic diversity as revealed by isozymes and species-specific attributes, such as mating system and predominance of asexual versus sexual reproduction. In systematic studies, isozymes have revealed cryptic species and identified pairs of sibling taxa. The quaternary structure of enzymes appears to be conserved at the phylum level. With the current availability of improved techniques for enzyme electrophoresis and for data interpretation, we expect future studies utilizing isozyme electrophoresis to provide further insight into population and evolutionary processes in seaweeds.
The proteins extracted from etiolated seedlings of 10 accessions, representatives of the species of the Sitopsis section of Triticum were analyzed by two-dimensional (2D) gel electrophoresis. The 2D patterns were compared and similarity indices were computed between all accessions. From the similarity matrix a dendrogram was deduced, that gave interesting information concerning the genetic relationships between the species of the Sitopsis section. The 2D pattern obtained from the Chinese Spring variety of Triticum aestivum was then included in the analysis. From the dendrogram obtained and also from the number of species-specific proteins shared between Chinese Spring and each of the Sitopsis species, it is proposed that the B genome donor of the cultivated wheats originated from a species closer to the T. speltoides-T. aucheri group than to any other diploid Sitopsis species.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Samples from pre- and post-rigor cod mince, surimi (a concentrate of fish myofibrillar proteins obtained after washing and dewatering the fish mince) and water from the first wash in the surimi manufacture, processed with and without the addition of 7.5 mM CaCl2 and 15 mM MgCl2, were analyzed by two-dimensional electrophoresis. The results showed that the main myofibrillar proteins, including myosin, actin and tropomyosin, remained in the surimi. Several other proteins were selectively removed during the washing procedure. Some additional major spots were detected in the two-dimensional gels containing samples of the wash water and surimi processed with the addition of Ca2+ and Mg2+ salts. These spots were either absent or present in minor amounts in the samples of post-rigor cod mince, wash water and surimi processed without Ca2+ and Mg2+ salts and in all the pre-rigor samples. This induced us to suggest that the new additional spots may constitute fragments of proteins originated by increased proteolytic activity during the surimi manufacture upon the addition of the Ca2+ and Mg2+ salts. Two-dimensional electrophoresis has proved to be a valuable tool to quickly and easily assess the effect of different processing conditions on the protein content of the products.
The construction and analysis of protein databases using the QUEST system is described, and the REF52 protein database is presented. A protein database provides the means to store and compare quantitative and descriptive data for up to 2000 proteins from many experiments that employ computer-analyzed two-dimensional gel electrophoresis. The QUEST system provides the tools to manage, analyze, and communicate these data. The REF52 database contains experiments with normal and transformed rat cell lines. In this report, many of the proteins on the REF52 map are identified by name, by subcellular localization, and by mode of post-translational modification. The quantitative experiments analyzed and compared here include 1) a study of the quantitative reproducibility of the analysis system, 2) a study of the clonal reproducibility of REF52 cells, 3) a study of growth-related changes in REF52 cells, and 4) a study of the effects of labeling cells for varying lengths of time. Of the proteins analyzed from REF52 cells, 10% are nuclear, 6% are phosphoproteins, and 4% are mannose-labeled glycoproteins. The mannose-labeled proteins are more prominent in patterns from quiescent cells, while the synthesis of cytoskeletal proteins is generally repressed at quiescence. A small set of proteins, selected for elevated rates of synthesis is generally repressed at quiescence. A small set of proteins, selected for elevated rates of synthesis in quiescent versus proliferating cells includes one of the tropomyosin isoforms, a myosin light chain isoform, and several prominent glycoproteins. These proteins are thought to be characteristic of the differentiated state of untransformed REF52 cells. Proteins induced early versus late after refeeding quiescent cells show very different patterns of growth regulation. These studies lay the foundations of the REF52 database and provide information needed to interpret the experiments with transformed REF52 cells, which are reported in the accompanying paper (Garrels, J., and Franza, B. R., Jr. (1989) J. Biol. Chem. 264, 5299-5312).
A powerful data processing methodology for analysis and classification of two-dimensional gels is introduced. The approach is based on correspondence analysis (CA) and ascendant hierarchical classification (AHC), and significantly differs from the more classical principal-component decomposition. Starting with a series of gels, each having a large number of spots, CA allows their representation in a factorial space of reduced dimension; classification into meaningful groups is then performed using AHC. Simultaneous representation of both spots and gels in the same space can be done. This precisely indicates the key spots pertinent for the classification, and therefore the characteristic proteins representative of a particular class of gels (i.e. of a particular disease or biological status). In addition, knowledge of these characteristic spots greatly simplifies the screening of future gels. After a brief overview of the Mélanie system for analyzing 2D gels, the theory of correspondence analysis and ascendant hierarchical classification is summarized. Equations are given that are easily ammenable to computation. How classification of two-dimensional gel electrophoretograms is accomplished is then detailed. Experimental results support the power of this approach.
We have developed and refined a system for quantitative computer analysis of two-dimensional polyacrylamide gel electrophoretograms. The system, named Elsie 4, is based on one described by Vo et al. (Anal. Biochem. 112, 258 (1981]. It is highly automated. Elsie 4 can find, and measure the intensity of, almost any spot resolvable on two-dimensional gels, including spots visible only as shoulders off larger spots and spots so close together that there is no "valley" between them. It can automatically match the spot patterns of different gels, potentially without the need for a user to provide landmark matches. The matches between paired gels let us follow the synthesis of any spot through a set of gels. Information about a group of matched spots can be obtained by referring to any spot in the group. There is generally no need for a standard or reference gel. Data for two experiments can be combined and compared by matching any gel in one experiment with any gel in the other. There are ways to automatically find possible mismatches in sets of gels. Scans and the results of the analysis can be shown on an image displayer. The programs use function libraries; this helps ensure consistency and increase portability. The programs and functions can be linked together in many ways; this lets users build custom programs for analysis of specific experiments.
Peripheral blood lymphocytes from 40 children have been examined for genetic variation in their protein constituents by two-dimensional polyacrylamide gel electrophoresis. One hundred six polypeptides chosen without respect to genetic variability were scored in gels from the 40 children. For each child, gels from both parents were also examined to substantiate the genetic basis of variants observed. Of the total of 4,240 polypeptides, 23 could not be scored unambiguously. Fourteen of the polypeptides showed genetic variants in one or more of the children. One hundred twenty-nine of 4,217 polypeptides scored exhibited the combination of a normal and a variant polypeptide. All variants were present in at least one of the parents of the subject. The index of heterozygosity observed (3.05% +/- .23%) indicates substantial genetic variation in cellular protein constituents.
cornerstone of the theory of evolution by gradual change is that the rate of A evolution is absolutely limited by the amount of genetic variation in the evolv- ing population. FISHER'S "Fundamental Theorem of Natural Selection" ( 1930) is a mathematical statement of this generalization, but even without mathematics it is clear that genetic change caused by natural selection presupposes genetic differences already existing, on which natural selection can operate. In a sense, a description of the genetic variation in a population is the fundamental datum of evolutionary studies; and it is necessary to explain the origin and maintenance of this variation and to predict its evolutionary consequences. It is not surprising, then, that a major effort of genetics in the last 50 years has been to characterize the amounts and kinds of genetic variation existing in natural or laboratory populations of various organisms. The results so far have told us a great deal about cytological variation such as polymorphisms for inversions and translocations, about frequencies of rare visible mutations at many loci, and about frequencies of chromosomes that are deleterious when homozygous together with the degree of that deleterious effect. In addition, we know of some striking singlelocus polymorphisms. These results are familiar to all students of population genetics and evolution, and have been well reviewed by DOBZHANSKY (195 1 ) and more recently by MAYR (1 963).
Two-dimensional gel electrophoresis of shoot proteins was used to study the relationships between Triticum, Secale, and Hordeum. A high level of polymorphism was found among the 1275 spots scored: only 198 spots were found common to all. But, under the hypothesis that only allelic variations were observed, the mean number of alleles per locus was only two. Phenograms were built from different distance indices. All of them showed Triticum genomes A and D close to each other, Hordeum far from the Triticum cluster, and Secale at an intermediate position. A discussion on the use of various distance indices is presented.
Proteins of two plants, Arabidopsis thaliana and rice (Oryza sativa) were subjected to two-dimensional electrophoresis analysis with two modifications: (i) comigration of external standard marker proteins with resultant horizontal and vertical stripes in the gel, and (ii) deblocking with a vapor of aqueous heptafluorobutyric acid for N-acetylserine. Approximately 5000 protein spots were separated from both the five tissues of Arabidopsis and the nine tissues of rice. Over one hundred spots were electroblotted for N-terminal sequencing. Among the newly sequenced proteins, 62 were from Arabidopsis and 51 from rice.
Biological speciation ultimately results in prezygotic isolation-the inability of incipient species to mate with one another-but little is understood about the selection pressures and genetic changes that generate this outcome. The genus Chlamydomonas comprises numerous species of unicellular green algae, including numerous geographic isolates of the species C. reinhardtii. This diverse collection has allowed us to analyze the evolution of two sex-related genes: the mid gene of C. reinhardtii, which determines whether a gamete is mating-type plus or minus, and the fus1 gene, which dictates a cell surface glycoprotein utilized by C. reinhardtii plus gametes to recognize minus gametes. Low stringency Southern analyses failed to detect any fus1 homologs in other Chlamydomonas species and detected only one mid homolog, documenting that both genes have diverged extensively during the evolution of the lineage. The one mid homolog was found in C. incerta, the species in culture that is most closely related to C. reinhardtii. Its mid gene carries numerous nonsynonymous and synonymous codon changes compared with the C. reinhardtii mid gene. In contrast, very high sequence conservation of both the mid and fus1 sequences is found in natural isolates of C. reinhardtii, indicating that the genes are not free to drift within a species but do diverge dramatically between species. Striking divergence of sex determination and mate recognition genes also has been encountered in a number of other eukaryotic phyla, suggesting that unique, and as yet unidentified, selection pressures act on these classes of genes during the speciation process.
A methodological overview of proteome analysis is provided along with details of efforts to achieve high-throughput screening (HTS) of protein samples derived from two-dimensional electrophoresis gels. For both previously sequenced organisms and those lacking significant DNA sequence information, mass spectrometry has a key role to play in achieving HTS. Prototype robotics designed to conduct appropriate chemistries and deliver 700-1000 protein (genes) per day to batteries of mass spectrometers or liquid chromatography (LC)-based analyses are well advanced, as are efforts to produce high density gridded arrays containing > 1000 proteins on a single matrix assisted laser desorption ionisation/time-of-flight (MALDI-TOF) sample stage. High sensitivity HTS of proteins is proposed by employing principally mass spectrometry in an hierarchical manner: (i) MALDI-TOF-mass spectrometry (MS) on at least 1000 proteins per day; (ii) electrospray ionisation (ESI)/MS/MS for analysis of peptides with respect to predicted fragmentation patterns or by sequence tagging; and (iii) ESI/MS/MS for peptide sequencing. Genomic sequences when complemented with information derived from hybridisation assays and proteome analysis may herald in a new era of holistic cellular biology. The current preoccupation with the absolute quantity of gene-product (RNA and/or protein) should move backstage with respect to more molecularly relevant parameters, such as: molecular half-life; synthesis rate; functional competence (presence or absence of mutations); reaction kinetics; the influence of individual gene-products on biochemical flux; the influence of the environment, cell-cycle, stress and disease on gene-products; and the collective roles of multigenic and epigenetic phenomena governing cellular processes. Proteome analysis is demonstrated as being capable of proceeding independently of DNA sequence information and aiding in genomic annotation. Its ability to confirm the existence of gene-products predicted from DNA sequence is a major contribution to genomic science. The workings of software engines necessary to achieve large-scale proteome analysis are outlined, along with trends towards miniaturisation, analyte concentration and protein detection independent of staining technologies. A challenge for proteome analysis into the future will be to reduce its dependence on two-dimensional (2-D) gel electrophoresis as the preferred method of separating complex mixtures of cellular proteins. Nonetheless, proteome analysis already represents a means of efficiently complementing differential display, high density expression arrays, expressed sequence tags, direct or subtractive hybridisation, chromosomal linkage studies and nucleic acid sequencing as a problem solving tool in molecular biology.
This review describes briefly proteome science. It explains why proteome science or proteomics emerged only recently and why a shift from genomics to proteomics is occurring. This review further illustrates that proteomics can unravel new domains in nature's complexity. Finally, it demonstrates that proteomics is offering new tools for the study of complex biological or medical problems.