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PubMed1999

Vesicular stomatitis.

Vesicular stomatitis is a disease of livestock caused by some members of the Vesiculovirus genus (Family Rhabdoviridae), two of which are called 'vesicular stomatitis virus'. Clinical disease presents as severe vesiculation and/or ulceration of the tongue, oral tissues, feet, and teats, and results in substantial loss of productivity. Except for its appearance in horses, it is clinically indistinguishable from foot-and-mouth disease. Unlike foot-and-mouth disease, it is very infectious for man and can cause a temporarily debilitating disease. Vesicular stomatitis occurs seasonally every year in the southeastern USA, southern Mexico, throughout Central America and in northern South America, and emerges from tropical areas to cause sporadic epidemics in cooler climates during the summer months. Other Vesiculoviruses are endemic in India and Africa. Vesiculoviruses are arthropod-borne and it is possible they are actually well adapted insect viruses that incidentally infect mammals. Vesiculoviruses are relatively simple, having a linear, single stranded, negative sense RNA genome encased in a bullet-shaped virion made from only five proteins. Upon infection of cultured cells, viral products turn off cellular gene expression and seize the entire metabolic potential of the cell. They also depolymerize the cytoskeleton to cause rapid tissue destruction. Virus infection in animals provokes interferon and nitric oxide responses, which quickly control viral replication, and an antibody response that prevents further viral replication. Vesiculovirus genome replication is error-prone, resulting in viral progeny containing many variants. This allows rapid adaptation. Nevertheless, vesicular stomatitis virus genomic sequences appear relatively stable within single endemic areas, and vary progressively on a North-South axis in the Western Hemisphere. Numerous important fundamental discoveries in immunology and virology have come from recent studies of vesicular stomatitis virus. However, these discoveries have not led to a safe and fully effective vaccine for man or beast. In the absence of a vaccine, the continual increase in rapid intercontinental travel, the increase in numbers and concentration of susceptible animals, the plasticity of the viral genome, and the underappreciation of vesiculoviruses as veterinary and zoonotic pathogens by regulators and biomedical researchers, are combining with potentially explosive consequences.

Animals↗
PubMed1987

Tubulinlike protein from Spirochaeta bajacaliforniensis.

Tubulin proteins are the fundamental subunits of all polymeric microtubule-based eukaryotic structures. Long, hollow structures each composed of 13 protofilaments as revealed by electron microscopy, microtubules (240 angstroms in diameter) are nearly ubiquitous in eukaryotes. These proteins have been the subject of intense biochemical and biophyiscal interest since the early 1970s and are of evolutionary interest as well. If tubulin-based structures (i.e., neurotubules, mitotic spindle tubules, centrioles, kinetosomes, axonemes, etc.) evolved from spirochetes by way of motility symbioses, tubulin homologies with spirochete proteins should be detectable. Tubulin proteins are widely thought to be limited to eukaryotes. Yet both azotobacters and spirochetes have shown immunological cross-reactivity with antitubulin antibodies. In neither of these studies was tubulin isolated nor any specific antigen identified as responsible for the immunoreactivity. Furthermore, although far less uniform in structure than eukaryotic microtubules, various cytoplasmic fibers and tubules (as seen by electron microscopy) have been reported in several types of prokaryotes (e.g., Spirochaeta; large termite spirochetes; treponemes; cyanobacteria; and Azotobacter. This work forms a part of our long-range study of the possible prokaryotic origin of tubulin and microtubules. Spirochetes are helically shaped gram-negative motile prokaryotes. They differ from all other bacterial in that the position of their flagella is periplasmic: their flagella lie between the inner and outer membranes of the gram-negative cell wall. Some of the largest spirochetes have longitudinally aligned 240 angstrom microtubules. Unfortunately, in spite of many attempts, all of the larger spirochetes (family Pillotaceae) with well-defined cytoplasmic tubules and antitubulin immunoreactivity are not cultivable. However, a newly described spirochete species (Spirochaeta bajacaliforniensis) possessing cytoplasmic fibers displays antitubulin immunoreactivity in whole-cell preparations. Since preliminary observations suggested that Spirochaeta bajacaliforniensis proteins may be related to eukaryotic tubulins, their characterization was undertaken. Brain tubulin can be purified by utilizing its ability to polymerize at warm temperatures and to depolymerize in the cold. After several cycles of sedimentation and redissolution the microtubule fraction is comprised of 75% tubulin and 20% high molecular mass microtubule-associated proteins (MAPs). In this paper we report that components of cell lysates, prepared from a spirochete that contains cytoplasmic fibers (Spirochaeta bajacaliforniensis), also exhibit the property of temperature-dependent cyclical sedimentation. Additionally we report the identification and characterization of the polypeptide responsible for cross-reactivity with antitubulin antiserum.

Animals↗
PubMed1993

RNA based evolutionary optimization.

The notion of an RNA world has been introduced for a prebiotic scenario that is dominated by RNA molecules and their properties, in particular their capabilities to act as templates for reproduction and as catalysts for several cleavage and ligation reactions of polynucleotides and polypeptides. This notion is used here also for simple experimental assays which are well suited to study evolution in the test tube. In molecular evolution experiments fitness is determined in essence by the molecular structures of RNA molecules. Evidence is presented for adaptation to environment in cell-free media. RNA based molecular evolution experiments have led to interesting spin-offs in biotechnology, commonly called 'applied molecular evolution', which make use of Darwinian trial-and-error strategies in order to synthesize new pharmacological compounds and other advanced materials on a biological basis. Error-propagation in RNA replication leads to formation of mutant spectra called 'quasispecies'. An increase in the error rate broadens the mutant spectrum. There exists a sharply defined threshold beyond which heredity breaks down and evolutionary adaptation becomes impossible. Almost all RNA viruses studied so far operate at conditions close to this error threshold. Quasispecies and error thresholds are important for an understanding of RNA virus evolution, and they may help to develop novel antiviral strategies. Evolution of RNA molecules can be studied and interpreted by considering secondary structures. The notion of sequence space introduces a distance between pairs of RNA sequences which is tantamount to counting the minimal number of point mutations required to convert the sequences into each other. The mean sensitivity of RNA secondary structures to mutation depends strongly on the base pairing alphabet: structures from sequences which contain only one base pair (GC or AU are much less stable against mutation than those derived from the natural (AUGC) sequences. Evolutionary optimization of two-letter sequences in thus more difficult than optimization in the world of natural RNA sequences with four bases. This fact might explain the usage of four bases in the genetic language of nature. Finally we study the mapping from RNA sequences into secondary structures and explore the topology of RNA shape space. We find that 'neutral paths' connecting neighbouring sequences with identical structures go very frequently through entire sequence space. Sequences folding into common structures are found everywhere in sequence space.(ABSTRACT TRUNCATED AT 400 WORDS)

Adaptation, Physiological↗
PubMed2000

Signature-based analysis of MET proto-oncogene mutations using DHPLC.

Research tools which improve mutation detection, SNP discovery, and allele characterization will facilitate studies of cancer, inherited disease, and genomic evolution. Denaturing High-Performance Liquid Chromatography (DHPLC) is a recently developed methodology for detection of heteroduplexes formed in DNA samples containing mismatches between wild type and mutant strands. In an effort to develop a rapid, sensitive mutation detection method for studies of families with inherited kidney cancer, we evaluated DHPLC for detection and analysis of MET proto-oncogene mutations in papillary renal carcinomas (PRC). We found DHPLC to be 100% accurate in detecting 15 known disease-associated MET mutations. Significantly, each MET mutation and two novel SNPs generated a characteristic chromatographic profile or signature with reproducible distinguishing features. Standardization of DHPLC reagents and improved methods design were critical to the reliability and accuracy of mutation prediction. Improvements included addition of a 75% acetonitrile wash followed by a rejuvenating gradient, and detailed analysis of signature shape, retention time (RT), RT differences (DeltaRT), and temperature-dependent (melt) profiling. We used signatures to predict mutations in new PRC samples, mutation carriers in asymptomatic hereditary PRC family members, and in a blind study of previously characterized DNAs. Application to SNP discovery is discussed. Wiley-Liss, Inc.

Carcinoma, Renal Cell↗
PubMed1976

Comparative ultrastructural investigations of the pineal organ of the blind cave fish, Anoptichthys jordani, and its ancestor, the eyed river fish, Astyanax mexicanus.

A comparative ultrastructural study has been made of the pineal organ in specimens of two closely related populations of the characid fish, Astyanaz mexicanus. The specimens of one population are living in the river, under natural light conditions. The specimens of the other population, originally described as Anoptichthys jordani, are living in a completely dark cave. In specimens of both populations the pineal organ consists of a spindle shaped end-vesicle, connected to the diencephalic roof by a slender stalk. The pineal tissue is compact and consists predominantly of glia-like supporting cells and sensory cells resembling the photoreceptor cells of the lateral vertebrate eye. Phagocytotic microglia-like cells can be found in close contact with the outer segments of the sensory cells. Nerve cells are located in the neighbourhood of neuropil formations, in which synaptic contacts are established between sensory cells and nerve cells. From these nerve cells fibers are emerging, forming the pineal tract that runs down the pineal stalk towards the diencephalon. On the basis of the ultrastructure described by other authors it is concluded that the pineal organ in specimens of the river population of Astyanax mexicanus resembles the pineal organ of other fish species. In specimens of the river population, reared under normal light-dark conditions for 3, 9 or 18 months, conspicuous morphological changes have not been detected in the presumably light-sensitive outer segments of the sensory cells or in other parts of the pineal tissue. In specimens of the cave populations, reared under identical conditions, an age-dependent, gradual regression of the regular outer segment organization of the pineal sensory cells takes place. In other parts of the pineal tissue, only small morphological changes can be observed. In specimens of the cave population, reared in constant darkness, the regression of the pineal outer segment organization begins earlier and is obvious. It is postulated that the gradual age-dependent regression of the regular organization of the outer segments in the pineal organ of cave specimens of Astyanax mexicanus is genetically determined and indicates a regressive evolution of the pineal light sensitivity. The expression of the regressive traits is dependent on the environmental light conditions.

Age Factors↗
PubMed1979

Ultrastructure of graniferous tracheary elements in the haustorium of Exocarpus bidwillii, a root hemi-parasite of the Santalaceae.

The xylem in the body of the haustorium of E. bidwillii has the shape of an inverted conical flask with the expanded portion being known as the vascular core. The tracheary elements of the vascular core are notable for the occurrence of numerous granules within their lumina and the presence of mostly imperforate walls. Elsewhere in the haustorium graniferous tracheary elements are absent and the cells are usually ordinary vessel elements. Thin sections for transmission electron microscopy, post-stained in potassium permanganate, show that the secondary wall thickenings of the graniferous tracheary elements consist of eccentric layers in which the microfibrils of each successive layer run alternately longitudinally and transversely. The granules of the tracheary elements average 2 micrometer in diameter and consist of a homogeneous matrix which shows a fine fibrillar structure on high resolution. The granules are naked and mostly remain as separate structures within the lumen of the cell, but occasionally they fuse into small groups or irregular masses. In some cells the granules become transformed into fibrillar material that disperses throughout the lumen. This dispersed material may accumulate in vessels of the interrupted zone proximal to the vascular core. Occasionally, the granules also change into compacted amorphous masses that adhere to the walls of the cell. Ultrastructural cytochemistry confirms that the granules are protein and not starch as was originally believed for the Santalaceae. The function of the vascular core and its graniferous tracheary elements is discussed and we suggest that it might help regulate the pressure and flow of xylem sap entering the parasite from the host. Graniferous tracheary elements in the Santalaceae and in root parasites of the Serophulariaceae are compared and it is concluded that they represent examples of convergent evolution.

Histocytochemistry↗
PubMed1992

Primary structure of the Thermoplasma proteasome and its implications for the structure, function, and evolution of the multicatalytic proteinase.

The proteasome or multicatalytic proteinase is a high molecular mass multisubunit complex ubiquitous in eukaryotes but also found in the archaebacterial proteasome is made of two different subunits only, and yet the complexes are almost identical in size and shape. Cloning and sequencing the gene encoding the small (beta) subunit of the T. acidophilum complex completes the primary structure of the archaebacterial proteasome. The similarity of the derived amino acid sequences of 233 (alpha) and 211 (beta) residues, respectively, indicates that they arose from a common ancestral gene. All the sequences of proteasomal subunits from eukaryotes available to date can be related to either the alpha-subunit or beta-subunit of the T. acidophilum "Urproteasome", and they can be distinguished by means of a highly conserved N-terminal extension, which is characteristic for alpha-type subunits. On the basis of circumstantial evidence we suggest that the alpha-subunits have regulatory and targeting functions, while the beta-subunits carry the active sites.

Amino Acid Sequence↗
PubMed1978

Chemical composition of the peptidoglycan-free cell walls of methanogenic bacteria.

Cell walls were prepared from freeze-dried samples of 7 strains of Methanobacterium by mechanical disintegration of the cells followed by incubation with trypsin. Electron microscopy revealed the presence of sacculi exhibiting the shape of the original cells, on which no surface structure could be detected. Ultrathin sections of the isolated sacculi showed a homogenously electron dense layer of about 10--15 nm in width. The ash content varied between 8 and 18% of dry weight. The sacculi of all the strains contained Lys: Ala:Glu:GlcNAc or GalNAc in a molar ratio of about 1:1.2:2:1. In one strain (M. ruminantium M1) alanine is replaced by threonine, however, Neutral sugars and--in some strains--additional amounts of the amino sugars were present in variable amounts, and could be removed by formamide extraction or HF treatment without destroying the sacculi. No muramic acid or D-amino acids typical of peptidoglycan were found. Therefore, the sacculi of the methanobacteria consist of a different polymer containing a set of three L-amino acids and one N-acetylated amino sugar. From cells of Methanospirillum hungatii no sacculi, but tube-like sheaths could be isolated, which tend to fracture perpendicularly to the long axis of the sheath along the fibrills seen on the surface. The sheaths consist of protein containing 18 amino acids and small amounts of neutral sugars. They are resistent to the proteinases tested and are not disintegrated by boiling in 2% sodium dodecylsulfate for 30 min. The three Gram-negative strains Black Sea isolate JR-1, Cariaco isolate JR-1 and Methanobacterium mobile do not contain a rigid sacculus, but merely a SDS-sensitive surface layer composed of regularly arranged protein subunits. This evidence indicates that, within the methanogens, different cell wall polymers characteristic of particular groups of organisms may have evolved during evolution, and supports the hypothesis that the evolution of the methanogens was separated from that of the peptidoglycan-containing procaryotic organisms at a very early stage.

Acetates↗
PubMed2004

Multiple developmental pathways leading to a single morph: monosulcate pollen (examples from the Asparagales).

BACKGROUND AND AIMS: Early developmental events in microsporogenesis are known to play a role in pollen morphology: variation in cytokinesis type, cell wall formation, tetrad shape and aperture polarity are responsible for pollen aperture patterning. Despite the existence of other morphologies, monosulcate pollen is one of the most common aperture types in monocots, and is also considered as the ancestral condition in this group. It is known to occur from either a successive or a simultaneous cytokinesis. In the present study, the developmental sequence of microsporogenesis is investigated in several species of Asparagales that produce such monosulcate pollen, representing most families of this important monocot clade. METHODS: The developmental pathway of microsporogenesis was investigated using light transmission and epifluorescence microscopy for all species studied. Confocal microscopy was used to confirm centripetal cell plate formation. KEY RESULTS: Microsporogenesis is diverse in Asparagales, and most variation is generally found between families. It is confirmed that the whole higher Asparagales clade has a very conserved microsporogenesis, with a successive cytokinesis and centrifugal cell plate formation. Centripetal cell wall formation is described in Tecophilaeaceae and Iridaceae, a feature that had so far only been reported for eudicots. CONCLUSIONS: Monosulcate pollen can be obtained from several developmental pathways, leading thus to homoplasy in the monosulcate character state. Monosulcate pollen should not therefore be considered as the ancestral state unless it is produced through the ancestral developmental pathway. The question about the ancestral developmental pathway leading to monosulcy remains open.

Biological Evolution↗
PubMed1999

Analysis of heregulin symmetry by weighted evolutionary tracing.

Heregulins are members of the protein family of EGF-like growth and differentiation factors. The primary cell-surface targets of heregulins are heterodimers of the EGF-receptor homolog HER2 with either HER3 or HER4. We used a weighted evolutionary trace analysis to identify structural features that distinguish the EGF-like domain (hrg) of heregulins from other members of the EGF family. In this analysis, each amino acid sequence is weighted according to its uniqueness and the variability in each position is assigned by an amino acid substitution matrix. Conserved residues in heregulin that are variable in other EGF-like domains are considered possible specificity-conferring residues. This analysis identifies two clusters of residues at the foot of the boot-shaped hrg domain. The residues in one cluster are recruited from the N-terminus; those in the other are from the ohm-loop region and show a weak sequence similarity to the N-terminal residues at the opposite side of the boot. The remaining residues with high conservation scores distribute themselves into these two distinct surfaces on hrg. This pseudo-twofold symmetry and the presence of two distinct interfaces may reflect the preference of hrg for heterodimeric versus homodimeric HER complexes.

Amino Acid Sequence↗
PubMed2003

Differentiation of cartilaginous anlagen in entire embryonic mouse limbs cultured in a rotating bioreactor.

Mechanisms involved in development of the embryonic limb have remained the same throughout eons of genetic and environmental evolution under Earth gravity (1 g). During the spaceflight era it has been of interest to explore the ancient theory that form of the skeleton develops in response to gravity, and that changes in gravitational forces can change the developmental pattern of the limb. This has been shown in vivo and in vitro, allowing the hypergravity of centrifugation and microgravity of space to be used as tools to increase our knowledge of limb development. In recapitulations of spaceflight experiments, premetatarsals were cultured in suspension in a bioreactor, and found to be shorter and less differentiated than those cultured in standard culture dishes. This study only measured length of the metatarsals, and did not account for possible changes due to the skeletal elements having a more in vivo 3D shape while in suspension vs. flattened tissues compressed by their own weight. A culture system with an outcome closer to in vivo and that supports growth of younger limb buds than traditional systems will allow studies of early Hox gene expression, and contribute to the understanding of very early stages of development. The purpose of the current experiment was to determine if entire limb buds could be cultured in the bioreactor, and to compare the growth and differentiation with that of culturing in a culture dish system. Fore and hind limbs from E11-E13 ICR mouse embryos were cultured for six days, either in the bioreactor or in center-well organ culture dishes, fixed, and embedded for histology. E13 specimens grown in culture dishes were flat, while bioreactor culture specimens had a more in vivo-like 3D limb shape. Sections showed excellent cartilage differentiation in both culture systems, with more cell maturation, and hypertrophy in the specimens cultured in the bioreactor. Younger limb buds fused together during culture, so an additional set of E11.5 limb buds was cultured with and without encapsulation in alginate prior to culturing in the bioreactor. Encapsulated limbs grown in the bioreactor did not fuse together, but developed only the more proximal elements while limbs grown in culture dishes formed proximal and distal elements. Alginate encapsulation may have reduced oxygenation to the progress zone of the developing limb bud resulting in lack of development of the more distal elements. These results show that the bioreactor supports growth and differentiation of skeletal elements in entire E13 limb buds, and that a method to culture younger limb buds without fusing together needs to be developed if any morphometric analysis is to be performed.

Alginates↗
PubMed1993

Molecular analysis of chicken embryo SPARC (osteonectin).

SPARC is a secreted glycoprotein that modulates cell shape and cell-matrix interactions. Levels of SPARC are increased at sites of somitogenesis, osteogenesis, and angiogenesis in the embryo and during wound repair in the adult. We have cloned and characterized SPARC from chicken embryo. A 2.2-kbp cDNA, obtained by a novel use of the polymerase chain reaction, was determined to encode a 298-residue protein that is 85% identical to human SPARC. Antigenic sites in particular appear to be highly conserved, as antibodies against C-terminal sequences of murine and bovine SPARC reacted with a 41-43 kDa protein in chicken embryo extracts. Chicken SPARC can be defined by four sequence signatures: (a) a conserved spacing of 11 cysteine residues in domain II, (b) the pentapeptide KKGHK in domain II, which is contained within a larger region of 31 identical residues, (c) a 100% conserved region of 10 residues in domain III, and (d) a C-terminal, calcium-binding EF-hand motif. SPARC mRNAs in the 10-day-old chicken embryo are represented by three sizes of 1.8, 2.2 and 3.0 kb. The relative steady-state levels for the 2.2-kb mRNA were determined as aorta > or = skeletal muscle > calvarium > vertebra > anterior limb > kidney > heart > brain > skin and lung >> liver. The relative abundance of the 1.8-kb and 2.2-kb mRNAs varied among tissues and indicated that differential processing of SPARC mRNAs might occur. All three RNA species were detected by a cDNA probe for the N-terminal part of the coding region. Thus, the three mRNA species appear to arise from differential 3' splicing and/or polyadenylation. Collective evidence demonstrates that SPARC has been well-conserved during vertebrate evolution, a finding that indicates a fundamental role for this protein in development.

Amino Acid Sequence↗
PubMed2006

Protamines and male infertility.

Protamines are the major nuclear sperm proteins. The human sperm nucleus contains two types of protamine: protamine 1 (P1) encoded by a single-copy gene and the family of protamine 2 (P2) proteins (P2, P3 and P4), all also encoded by a single gene that is transcribed and translated into a precursor protein. The protamines were discovered more than a century ago, but their function is not yet fully understood. In fact, different hypotheses have been proposed: condensation of the sperm nucleus into a compact hydrodynamic shape, protection of the genetic message delivered by the spermatozoa, involvement in the processes maintaining the integrity and repair of DNA during or after the nucleohistone-nucleoprotamine transition and involvement in the epigenetic imprinting of the spermatozoa. Protamines are also one of the most variable proteins found in nature, with data supporting a positive Darwinian selection. Changes in the expression of P1 and P2 protamines have been found to be associated with infertility in man. Mutations in the protamine genes have also been found in some infertile patients. Transgenic mice defective in the expression of protamines also present several structural defects in the sperm nucleus and have variable degrees of infertility. There is also evidence that altered levels of protamines may result in an increased susceptibility to injury in the spermatozoan DNA causing infertility or poor outcomes in assisted reproduction. The present work reviews the articles published to date on the relationship between protamines and infertility.

Amino Acid Sequence↗
PubMed1999

Comparative biochemistry of CO2 fixation and the evolution of autotrophy.

Carbon dioxide fixation is a polyphyletic trait that has evolved in widely separated prokaryotic branches. The three principal CO2-assimilation pathways are (i) the reductive pentose-phosphate cycle, i.e. the Calvin-Benson cycle; (ii) the reductive citric acid (or Arnon) cycle; and (iii) the net synthesis of acetyl-CoA from CO/CO2, or Wood pathway. Sequence analysis and the comparative biochemistry of these routes suggest that all of them were shaped to a considerable extent by the evolutionary recruitment of enzymes. Molecular phylogenetic trees show that the Calvin-Benson cycle was a relatively late development in the (eu)bacterial branch, suggesting that some form(s) of carbon assimilation may have been operative before chlorophyll-based photosynthesis. On the other hand, the ample phylogenetic distribution of both the Arnon and the Wood pathways does not allow us to infer which one of them is older. However, different lines of evidence, including experimental reports on the NiS/FeS-mediated C-C bond formation from CO and CH3SH are used here to argue that the first CO2-fixation route may have been a semi-enzymatic Wood-like pathway.

Acetyl Coenzyme A↗
PubMed1973

The phylogenetic relationships of the members of the Drosophila robusta group.

The phylogenetic relationships among the species of the D. robusta group were investigated by the analysis of chromosomal differences. Six of the ten known members of the D. robusta group were available for the study: D. colorata and D. robusta from the United States, and D. sordidula, D. pseudosordidula, D. lacertosa, and D. moriwakii from Japan. Analysis of the metaphase chromosomes from larval ganglion cells suggests that D. moriwakii and D. colorata, with rod-shaped X-chromosomes, are the more ancestral species, while D. sordidula, D. pseudosordidula, D. robusta, and D. lacertosa, with V-shaped X-chromosomes, are derived. The ancestral position of D. colorata and D. moriwakii is further strengthened by the fact that these are the two species in the D. robusta group that are cytologically closest to D. nigromelanica of the related D. melanica group. Of the four derived species, D. sordidula was found to be the closest to the ancestral species. The phylogeny based on the analysis of the gene sequences in the homologous chromosomes agreed with that indicated by the metaphase chromosomes. Since all attempts to obtain hybrids were unsuccessful except for the cross involving D. moriwakii females and D. colorata males, photographic maps of the salivary chromosomes were used to determine homology between the chromosomes of the different species. Evidence is presented to indicate that the D. robusta group originated in Asia (Japan), and that there were two migrations to the New World, the first leading to D. robusta, and the second to D. colorata. It is suggested that the route of migrations was across the Bering Land Bridge, and further, that the migrations occurred during the period from late Oligocene to middle Miocene, 20-25 million years ago.

Animals↗
PubMed2001

The deprivation syndrome is the driving force of phylogeny, ontogeny and oncogeny.

Energy is the motor of life. Energy ensures the organism's survival and competitive advantage for reproductive success. For almost 3 billion years, unicellular organisms were the only life form on earth. Competition for limited energy resources and raw materials exerted an incessant selective pressure on organisms. In the adverse environment and due to their 'feast and famine' life style, hardiness to a variety of stressors, particularly to nutrient deprivation, was the selection principle. Both resistance and mutagenic adaptation to stressors were established as survival strategies by means of context-specific processes creating stability or variability of DNA sequence. The conservation of transduction pathways and functional homology of effector molecules clearly bear witness that the principles of life established during prokaryotic and eukaryotic unicellular evolution, although later diversified, have been unshakably cast to persist during metazoan phylogenesis. A wealth of evidence suggests that unicellular organisms evolved the phenomena of differentiation and apoptosis, sexual reproduction, and even aging, as responses to environmental challenges. These evolutionary accomplishments were elaborated from the dichotomous resistance/mutagenesis response and sophisticated the capacity of cells to tune their genetic information to changing environmental conditions. Notably, the social deprivation responses, differentiation and apoptosis, evolved as intercellularly coordinated events: a multitude of differentiation processes were elaborated from sporulation, the prototypic stress resistance response, while apoptosis, contrary to current concepts, is no altruistic cell suicide but was programmed as a mutagenic survival response; this response, however, is socially thwarted leading into mutagenic error catastrophe. In the hybrid differentiation-apoptosis process, cytocide and cannibalism of apoptotic cells thus serve the purpose of fueling the survival of the selfish genes in the differentiating cells. However, successful mutagenesis, although repressed, persisted in the asocial stress response of carcinogenesis as a regression to primitive unicellular behavior following failure of intercellular communication. While somatic mutagenesis was largely prevented, Metazoa elaborated germ cell mutagenesis as an evolutionary vehicle. Genetic competence, a primitive, stress-induced mating behavior, evolved into sexual reproduction which harnessed mutagenesis by subjecting highly mutable germ cells to a rigid viability selection. These processes were programmatically fixed as life- and cell-cycle events but retained their deprivation response phenotypes. Thus, the differentiation-apoptosis tandem evolved as the 'clay' to mold the specialized structures and functions of a multicellular organism while sexual reproduction elaborated the principle of quality-checked mutagenesis to create the immense diversity of Metazoa following the Cambrian explosion. Throughout these events, reactive oxygen and nitrogen species, which are regulated by energy homeostasis, shape the genetic information in a regulated but random, uncoded process providing the fitness-related feedback of phenotype to genotype. The interplay of genes and environment establishes a dynamic stimulus-response feedback cycle which, in animate nature, may be the organizing principle to contrive the reciprocal duality of energy and matter.

Aging↗
PubMed2004

Retroposon mapping in molecular systematics.

Advances in genome sciences are demonstrating the dynamic nature of noncoding DNA regions, which are comprised largely of repetitive elements with no apparent function. Retroposons are one class of mobile genetic elements that amplify and move about the genome via a copy-and-paste mechanism that employs an RNA intermediate. Short and long interspersed elements (SINEs and LINEs, respectively) are types of retroposons of particular interest because of their active role in shaping the architecture of genomes and their diagnostic value as evolutionary markers for studies of phylogeny and population biology. Although the use of SINEs and LINEs for molecular systematic studies is proliferating, a comprehensive laboratory protocol that explicitly outlines how to isolate and characterize retroposons for systematic studies in a detailed, step-by-step fashion has been lacking. The present chapter addresses this gap in the literature by focusing on the strategy for isolating new SINEs from a genomic library, the screening process, the sequencing and characterization of clones into subfamilies, quantification of copy number in host taxa, and the critical diagnosis of phylogenetically informative SINE and LINE insertion patterns. Practical limits to the method are discussed in relation to sampling design, systematic character theory, and the empirical distribution of elements observed in eukaryotic lineages. Major steps in the experimental process are illustrated with case examples from a diversity of taxonomic groups and by published results in the molecular biology and systematics literature.

Animals↗
PubMed2002

A strong signature of balancing selection in the 5' cis-regulatory region of CCR5.

CCR5 encodes a cell surface chemokine receptor molecule that serves as the principal coreceptor, with CD4, for HIV-type 1 (HIV-1). Varied HIV-1 susceptibility and time to progression to AIDS have been associated with polymorphisms in CCR5. Many of these polymorphisms are located in the 5' cis-regulatory region of CCR5, suggesting that it may have been a target of natural selection. We characterized CCR5 sequence variation in this region in 400 chromosomes from worldwide populations and compared it to a genome-wide analysis of 100 Alu polymorphisms typed in the same populations. Variation was substantially higher than expected and characterized by an excess of intermediate-frequency alleles. A genealogy of CCR5 haplotypes had deep branch lengths despite markedly little differentiation among populations. This finding suggested a deviation from neutrality not accounted for by population structure, which was confirmed by tests for natural selection. These results are strong evidence that balancing selection has shaped the pattern of variation in CCR5 and suggest that HIV-1 resistance afforded by CCR5 5' cis-regulatory region haplotypes may be the consequence of adaptive changes to older pathogens.

Alleles↗