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Regulatory gene adaptation: an evolutionary model.

There is increasing support for the notion that changes in regulatory loci have played a major role in eukaryotic evolution. Assuming a model of genetic regulation based upon presently accepted views of the organisation of the eukaryotic genome and basic models of evolutionary genetics, one can provide a context for speculation about the advantages and disadvantages of adaptation via regulatory genes. As additional information is obtained on particular regulatory systems, the evolutionary genetic model can be modified and expanded.

Adaptation, Biological↗

Networks of gene regulation, neural development and the evolution of general capabilities, such as human empathy.

A network of gene regulation organized in a hierarchical and combinatorial manner is crucially involved in the development of the neural network, and has to be considered one of the main substrates of genetic change in its evolution. Though qualitative features may emerge by way of the accumulation of rather unspecific quantitative changes, it is reasonable to assume that at least in some cases specific combinations of regulatory parts of the genome initiated new directions of evolution, leading to novel capabilities of the brain. These notions are applied, in this paper, to the evolution of the capability of cognition-based human empathy. It is suggested that it has evolved as a secondary effect of the evolution of strategic thought. Development of strategies depends on abstract representations of one's own possible future states in one's own brain to allow assessment of their emotional desirability, but also on the representation and emotional evaluation of possible states of others, allowing anticipation of their behaviour. This is best achieved if representations of others are connected to one's own emotional centres in a manner similar to self-representations. For this reason, the evolution of the human brain is assumed to have established representations with such linkages. No group selection is involved, because the quality of strategic thought affects the fitness of the individual. A secondary effect of this linkage is that both the actual states and the future perspectives of others elicit vicarious emotions, which may contribute to the motivations of altruistic behaviour.

Biological Evolution↗

Expression divergence between duplicate genes.

A general picture of the role of expression divergence in the evolution of duplicate genes is emerging, thanks to the availability of completely sequenced genomes and functional genomic data, such as microarray data. It is now clear that expression divergence, regulatory-motif divergence and coding-sequence divergence all increase with the age of duplicate genes, although their exact interrelationships remain to be determined. It is also clear that gene duplication increases expression diversity and enables tissue or developmental specialization to evolve. However, the relative roles of subfunctionalization and neofunctionalization in the retention of duplicate genes remain to be clarified, especially for higher eukaryotes. In addition, the relationship between gene duplication and evolution of transcriptional regulatory networks is largely unexplored.

Evolution, Molecular↗

The origin of vertebrate limbs.

The earliest tetrapod limbs are polydactylous, morphologically varied and do not conform to an archetypal pattern. These discoveries, combined with the unravelling of limb developmental morphogenetic and regulatory mechanisms, have prompted a re-examination of vertebrate limb evolution. The rich fossil record of vertebrate fins/limbs, although restricted to skeletal tissues, exceeds the morphological diversity of the extant biota, and a systematic approach to limb evolution produces an informative picture of evolutionary change. A composite framework of several phylogenetic hypotheses is presented incorporating living and fossil taxa, including the first report of an acanthodian metapterygium and a new reconstruction of the axial skeleton and caudal fin of Acanthostega gunnari. Although significant nodes in vertebrate phylogeny remain poorly resolved, clear patterns of morphogenetic evolution emerge: median fin origination and elaboration initially precedes that of paired fins; pectoral fins initially precede pelvic fin development; evolving patterns of fin distribution, skeletal tissue diversity and structural complexity become decoupled with increased taxonomic divergence. Transformational sequences apparent from the fish-tetrapod transition are reiterated among extant lungfishes, indicating further directions for comparative experimental research. The evolutionary diversification of vertebrate fin and limb patterns challenges a simple linkage between Hox gene conservation, expression and morphology. A phylogenetic framework is necessary in order to distinguish shared from derived characters in experimental model regulatory systems. Hox and related genomic evolution may include convergent patterns underlying functional and morphological diversification. Brachydanio is suggested as an example where tail-drive patterning demands may have converged with the regulation of highly differentiated limbs in tetrapods.

Animals↗

A genomewide survey of developmentally relevant genes in Ciona intestinalis. VIII. Genes for PI3K signaling and cell cycle.

Cell growth and cell divisions are two fundamental biological processes for cells in multi-cellular organisms. The molecules involved in these biological processes are highly conserved within eukaryotes, including plants and unicellular organisms such as yeast. However, some regulatory molecules seem to be innovated during animal evolution. Therefore, to understand how the ubiquitous systems have evolved or have been conserved, we examined genes for the phosphoinositide 3-kinase (PI3K) pathway that is important for cell growth, and genes for cell cycle regulation in the genome of Ciona intestinalis. It was found that the Ciona intestinalis genome contains all the essential constituents of the PI3K pathway. In addition, the class IB PI3K catalytic and regulatory subunits, which had not previously been known in animals other than mammals, were found in the Ciona genome. Similarly, all essential cyclins and CDKs were found in the Ciona genome, while cyclin G and cyclin L were likely to be independently lost in the ascidian lineage, which may be dispensable for the cell cycle. Cyclin F, which was previously known only in vertebrates, was not found in the Ciona genome. Therefore, this gene was probably innovated during the evolution of vertebrates to be involved in vertebrate-specific cell cycle regulation. Since Ciona is regarded as one of the most primitive extant chordates, the present analysis gives us an insight into how these fundamental biological genes are evolved or are conserved during chordate evolution.

Animals↗

Endogenous retroviruses provide the primary polyadenylation signal for two new human genes (HHLA2 and HHLA3).

By screening the expressed sequence tag (EST) database, we identified transcripts of two new human genes that are polyadenylated within a long terminal repeat (LTR) of the HERV-H endogenous retrovirus family. The first gene, termed HHLA2, is represented by two EST clones and one cDNA clone, all of which have a polyadenylated LTR as their 3' end. The gene has an open reading frame (ORF) of 414 amino acids with three immunoglobulin-like domains and is expressed primarily in intestinal tissues, kidney, and lung. Seven small EST clones from several different tissues were found for the second gene, termed HHLA3. As with HHLA2, all HHLA3 ESTs utilized a HERV-H LTR as the polyadenylation signal. Three types of alternatively spliced HHLA3 transcripts that could encode proteins of 76, 121, or 153 amino acids were detected. Interestingly, the ORF for two of these transcripts continues into the LTR. For both HHLA2 and 3, no major human transcripts that utilized a non-LTR polyadenylation signal were detected. Analysis of RNA from baboon, which lacks the LTRs at these genomic loci, showed that the baboon HHLA2 and 3 genes use other polyadenylation signals. This study demonstrates that ancient retroviral insertions have assumed gene regulatory functions during the course of human evolution.

Amino Acid Sequence↗

Myb p75 oncoprotein is expressed in developing otic and epibranchial placodes.

The c-myb proto-oncogene encodes a transcriptional regulatory protein which is highly conserved throughout evolution. Myb has been considered to be normally restricted to hematopoietic tissues, but there are indications that this might not always be the case. The present work shows the expression of a p75 Myb oncoprotein in the otic vesicle and epibranchial placodes of the early chick embryo. Expression was sequential and followed the same time course as the formation of placode-derived cranial ganglia. The results suggest a potential role for c-myb in regulation of placode development and neurogenesis.

Animals↗

Inhibition by opioids of phagocytosis in peritoneal macrophages.

We have tested the effect of prototypic opioid agonists on phagocytosis of sheep erythrocytes by mouse peritoneal macrophages. It was found that morphine and all the opioid peptides tested inhibited phagocytosis by a biphasic, naloxone-reversible mechanism. Delta agonists were the most effective inhibitors, suggesting that the response is mediated by a delta receptor. Chronic exposure to morphine apparently results in the development of tolerance since under these conditions the inhibitory effect of the opiate is abolished. These results are similar to previously reported effects of opioids on endocytosis in other systems, which suggests that this inhibition is part of a basic regulatory mechanism that has been conserved in evolution.

Animals↗

Structural characterization of the gene encoding the large zinc finger protein ZAS3: implication to the origin of multiple promoters in eukaryotic genes.

ZAS3 is a large zinc finger protein that regulates kappaB-mediated transcription and TNF-driven signal transduction pathway. Herein, we have characterized the mouse ZAS3 gene that spans 400 kb and splits into 16 exons. Four ZAS3 exons, ranging from 676 to 3956 nucleotides, are significantly larger than the average size of mammalian internal exons. Intron 10, when retained in mRNAs, encodes N-terminal DNA binding domain, called ZASN. As predicted from cDNAs, 5' untranslated region composed of the 2317 nucleotides is extremely long and contains upstream open reading frames, suggesting that translation initiation of ZAS3 transcripts by conventional cap-dependent ribosome scanning mechanism may be inefficient. Additionally, cDNA data analysis followed by reporter gene assays shows that the ZAS3 locus harbors two promoters that are 80 kb apart. The data suggest that the expression of ZAS3 is controlled by a combination of differential promoter usage, alternative splicing, and possible intergenic splicing. The distribution and degree of conservation of exons within the ZAS3 locus, together with the complex alternative splicing events and upstream open reading frame in 5' untranslated exons, lead us to speculate that multiple promoters of an eukaryotic gene might be residual traces of regulatory regions of other genes lost in evolution.

5' Untranslated Regions↗

Channel catfish hepcidin expression in infection and anemia.

Hepcidin, originally identified as a 25 amino acid antimicrobial peptide made in the liver, is a key regulator of iron balance and recycling in humans and mice. Closely related hepcidin genes and peptides have also been identified in a number of fish species and in teleosts are thought to function as endogenous antibiotics involved in host defense against infection. Here we report the transcriptional regulation of hepcidin expression by infection and anemia in the channel catfish. Changes in hepcidin expression in catfish challenged with Edwardsiella ictaluri and in fish affected by channel catfish anemia (CCA) were measured by real time quantitative PCR. Hepcidin transcript levels in the livers were increased 4, 19, and 22-fold at 4, 24, and 48h following bacterial challenge, respectively. However, augmented hepcidin expression in the intestine and olfactory sac was detected only at 48h post-infection. Hepcidin transcript levels in the livers of catfish affected by CCA were less than 14% of that present in healthy counterparts. Hepatic hepcidin transcript levels correlated significantly with serum iron concentrations (r=0.54, p<0.05) and with the percent saturation of transferrin (r=0.63, p<0.05). Similar to mammalian hepcidins, channel catfish hepcidin is an iron-responsive gene and may also play important roles in innate host defense to infection and in iron homeostasis. Mammalian hepcidins may have evolved from an antimicrobial peptide and its structure and transcriptional regulatory mechanisms have been conserved throughout vertebrate evolution.

Amino Acid Sequence↗

Cloning of the mRNA of overexpression in colon carcinoma-1: a sequence overexpressed in a subset of colon carcinomas.

We have identified a novel human cDNA overexpressed in a colon carcinoma cell line, TC7, established from a tumor with a normal karyotype arising in a patient with a hereditary nonpolyposis colorectal carcinoma. The OCC-1 (overexpressed in colon carcinoma-1) gene is composed of six exons and located in the q24.1 region of chromosome 12. It is transcribed as two mRNA species that differ in their 5'- and 3'-terminal ends. Abundant accumulation of both transcripts was found in placenta, skeletal muscle, kidney, and pancreas tissues. Absent or very faint expression was observed in heart, brain, lung and liver tissues. Overexpressed in colon carcinoma-1 cDNA direct in vitro translation of several polypeptides whose size is shorter than 9 kDa. Attempts to produce antibodies against these synthesized polypeptides in Escherichia coli failed. The absence of sequences at the mRNA and DNA levels hybridizing with mouse sequences together with the absence of a large open reading frame raise the possibility that OCC-1 sequences appeared recently in evolution and are transcribed as two noncoding regulatory RNA. Elevated levels of OCC-1 mRNA were observed in three of eight colon carcinomas as compared to normal mucosa of the same patient. Since these tumors shared the same characteristics of having a near diploid karyotype, OCC-1 overexpression may be a hallmark of this subset of colon carcinomas.

Adult↗

Otx genes in brain morphogenesis.

Most of the gene candidates for the control of developmental programmes that underlie brain morphogenesis in vertebrates are the homologues of Drosophila genes coding for signalling molecules or transcription factors. Among these, the orthodenticle group includes the Drosophila orthodenticle (otd) and the vertebrate Otx1 and Otx2 genes, which are mostly involved in fundamental processes of anterior neural patterning. These genes encode transcription factors that recognise specific target sequences through the DNA binding properties of the homeodomain. In Drosophila, mutations of otd cause the loss of the anteriormost head neuromere where the gene is transcribed, suggesting that it may act as a segmentation "gap" gene. In mouse embryos, the expression patterns of Otx1 and Otx2 have shown a remarkable similarity with the Drosophila counterpart. This suggested that they could be part of a conserved control system operating in the brain and different from that coded by the HOX complexes controlling the hindbrain and spinal cord. To verify this hypothesis a series of mouse models have been generated in which the functions of the murine genes were: (i) fully inactivated, (ii) replaced with each others, (iii) replaced with the Drosophila otd gene. Otx1-/- mutants suffer from epilepsy and are affected by neurological, hormonal, and sense organ defects. Otx2-/- mice are embryonically lethal, they show gastrulation impairments and fail in specifying anterior neural plate. Analysis of the Otx1-/-; Otx2+/- double mutants has shown that a minimal threshold level of the proteins they encode is required for the correct positioning of the midbrain-hindbrain boundary (MHB). In vivo otd/Otx reciprocal gene replacement experiments have provided evidence of a general functional equivalence among otd, Otx1 and Otx2 in fly and mouse. Altogether these data highlight a crucial role for the Otx genes in specification, regionalization and terminal differentiation of rostral central nervous system (CNS) and lead to hypothesize that modification of their regulatory control may have influenced morphogenesis and evolution of the brain.

Animals↗

Search for the mechanism of toxicity of dioxins. A lesson in toxicology.

The development of modern toxicology was strongly influenced by parallel evolutions of sister disciplines as well as regulatory requirements. This led to an overemphasis of some areas of toxicology while other aspects of it experienced at least a temporary benign neglect. The author of this paper believes that the few and far between successes in elucidating the mechanism of toxicity of chemicals are related to this uneven approach to a very complex problem. Based on one of the most important and controversial contemporary issues in toxicology, viz. that of dioxins, it is argued that by a systematic step-wise use of methodologies borrowed from sister disciplines, our capabilities of elucidating the mechanism of action of toxicants can be considerably improved.

Animals↗

Screening of antifeedant activity in brain extracts led to the identification of sulfakinin as a satiety promoter in the German cockroach. Are arthropod sulfakinins homologous to vertebrate gastrins-cholecystokinins?

The feeding cycle of the adult female cockroach Blattella germanica parallels vitellogenesis. The study of the mechanisms that regulate this cycle led us to look for food-intake inhibitors in brain extracts. The antifeedant activity of brain extracts was tested in vivo by injecting the extract and measuring the carotenoids contained in the gut from carrot ingested after the treatment. By HPLC fractionation and tracking the biological activity with the carrot test, we isolated the sulfakinin EQFDDY(SO3H) GHMRFamide (Pea-SK). A synthetic version of the peptide inhibited food intake when injected at doses of 1 microg (50% inhibition) and 10 microg (60% inhibition). The sulfate group was required for food-intake inhibition. These biological and structural features are similar to those of the gastrin-cholecystokinin (gastrin-CCK) family of vertebrate peptides. However, heterologous feeding assays (human CCK-8 tested on B. germanica, and Pea-SK tested on the goldfish Carassius auratus) were negative. In spite of this, alignment and cluster analysis of these and other structurally similar peptide families suggest that sulfakinins and gastrin-CCKs are homologous, and that mechanisms of feeding regulation involving these regulatory peptides may have been conserved during evolution between insects and vertebrates.

Amino Acid Sequence↗

Mechanisms of glycosaminoglycan activation of the serpins in hemostasis.

Serpins are the predominant protease inhibitors in the higher organisms and are responsible, in humans, for the control of many highly regulated processes including blood coagulation and fibrinolysis. The serpin inhibitory mechanism has recently been revealed by the solution of a crystallographic structure of the final serpin-protease complex. The serpin mechanism, in contrast to the classical lock-and-key mechanism, involves dramatic conformational change in both the inhibitor and the inhibited protein. The final result is a stable covalent complex in which the properties of each component are altered so as to allow clearance from the circulation. Several serpins are involved in hemostasis: antithrombin (AT) inhibits many coagulation proteases, most importantly factor Xa and thrombin; heparin cofactor II (HCII) inhibits thrombin; protein C inhibitor (PCI) inhibits activated protein C and thrombin bound to thrombomodulin; plasminogen activator inhibitor 1 inhibits tissue plasminogen activator; and alpha2-antiplasmin inhibits plasmin. Nearly all of these reactions are accelerated through interactions with glycosaminoglycans (GAGs) such as heparin or heparan sulfate. Recent structures of AT, HCII and PCI have revealed how in each case the serpin mechanism has been fine-tuned by evolution to bring about high levels of regulatory control, and how seemingly disparate mechanisms of GAG binding and activation can share critical elements. By considering the serpins involved in hemostasis together it is possible to develop a deeper understanding of their complex individual roles.

Animals↗

Premature arrest of the male flower meristem precedes sexual dimorphism in the dioecious plant Silene latifolia.

Most dioecious plant species are believed to derive from hermaphrodite ancestors. The regulatory pathways that have been modified during evolution of the hermaphrodite ancestors and led to the emergence of dioecious species still remain unknown. Silene latifolia is a dioecious plant species harboring XY sex chromosomes. To identify the molecular mechanisms involved in female organ suppression in male flowers of S. latifolia, we looked for genes potentially involved in the establishment of floral organ and whorl boundaries. We identified homologs of Arabidopsis thaliana SHOOTMERISTEMLESS (STM) and CUP SHAPED COTYLEDON (CUC) 1 and CUC2 genes in S. latifolia. Our phylogenetic analyses suggest that we identified true orthologs for both types of genes. Detailed expression analyses showed a conserved expression pattern for these genes between S. latifolia and A. thaliana, suggesting a conserved function of the corresponding proteins. Comparative in situ hybridization experiments between male, female, and hermaphrodite individuals reveal that these genes show a male-specific pattern of expression before any morphological difference become apparent. Our results make SlSTM and SlCUC strong candidates for being involved in sex determination in S. latifolia.

Flowers↗

Enhancer-dependent expression of human kappa immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation.

We have developed a general method for introducing cloned genes into mammalian cells that affords substantial benefits over current technology. It is simple, rapid, and applicable to many (perhaps all) cell types, including those that are refractory to traditional transfection procedures. The method involves exposure of a suspension of cells and cloned DNA to a high-voltage electric discharge. In a model application of this transfection procedure, we have studied the expression of cloned human and mouse Ig kappa genes stably introduced into mouse pre-B cells and fibroblasts. We find that there is a B-cell-specific enhancer-activator region in the J-C intron of the human kappa gene that is necessary for efficient transcription of the cloned gene in mouse pre-B lymphocytes. This suggests that both the DNA element and the proteins required for its regulatory activity have been highly conserved in evolution and that these elements operate at the pre-B-cell stage of immunocyte development, a stage that precedes productive kappa gene rearrangement.

Animals↗

A primate-dominant third glycosylation site of the beta2-adrenergic receptor routes receptors to degradation during agonist regulation.

beta(2)-adrenergic receptors (beta(2)AR) of all species are N-linked glycosylated at amino terminus residues approximately 6 and approximately 15. However, the human beta(2)AR has a potential third N-glycosylation site at ECL2 residue 187. To determine whether this residue is glycosylated and to ascertain function, all possible single/multiple Asn --> Gln mutations were made in the human beta(2) AR at positions 6, 15, and 187 and were expressed in Chinese hamster fibroblast cells. Substitution of Asn-187 alone or with Asn-6 or Asn-15 decreased the apparent molecular mass of the receptor on SDS-PAGE in a manner consistent with Asn-187 glycosylation. All receptors bound the agonist isoproterenol and functionally coupled to adenylyl cyclase. However, receptors without 187 glycosylation failed to display long term agonist-promoted down-regulation. In contrast, loss of Asn-6/Asn-15 glycosylation did not alter down-regulation. Cell surface distribution and agonist-promoted internalization of receptors and recruitment of beta-arrestin 2 were unaffected by the loss of 187 glycosylation. Furthermore, acutely internalized wild-type and Gln-187 receptors were both localized by confocal microscopy to early endosomes. During prolonged agonist exposure, wild-type beta(2)AR co-localized with lysosomes, consistent with trafficking to a degradation compartment. However, Gln-187 beta(2)AR failed to co-localize with lysosomes despite agonist treatments up to 18 h. Phylogenetic analysis revealed that this third glycosylation site is found in humans and other higher order primates but not in lower order primates such as the monkey. Nor is this third site found in rodents, which are frequently utilized as animal models. These data thus reveal a previously unrecognized beta(2)AR regulatory motif that appeared late in primate evolution and serves to direct internalized receptors to lysosomal degradation during long term agonist exposure.

Adenylyl Cyclases↗