Functional Systems and Modular Evolution: The Relationship between Fixation of New Domain Copies and the Present Structure of the Connections in the Functional System

The distribution of paralogous domains in relation to the structure of functional systems (FSs) is examined. It was found that the frequencies of particular domain types in genes for the hemostasis and complement FSs by far exceeded the frequencies expected on assumption of their random distribution in the genome, i.e., the domains were not randomly distributed in relation to the FS boundaries. For instance, it was shown that approximately 50% of the total mRNA of genes for the hemostasis and complement FSs encodes 20 domain types repeated on average 16 (from 2 to 115) times. Thus, the present structure of the FS connections plays a key role in the formation of new connections in the system evolution. Possible causes and mechanisms of the accumulation of paralogous genes and domains in these systems are discussed. The distribution asymmetry may be explained by the systemic character of the organization (system connectivity). Since any structural innovation must be included in the scheme of the present connections, the new protein must contain at least one functional site complementary to sites of the molecules already functioning in the system. The mechanism of preference of own domains probably consists in fixation via selection of the shortest among many alternative possible formation pathways of the new functional structure. This mechanism must promote the accumulation in the FS of copies of already functioning structures (genes, domains) that can relatively rapidly adapt for performing the new function.     

Bf and C3 complement types in rheumatoid arthritis

Bf and C3 complement types were studied in 100 male and 100 females patients from northern Sweden with erosive rheumatoid arthritis (RA) and compared with population controls. A significantly decreased frequency of the Bf FS phenotype was found particularly in males and in patients with a family history of polyarthritis. Significant Bf associations were also found with a more severe form of RA (functional classes III and IV) and with high titers of the rheumatoid factor. No significant difference with respect to C3 phenotype and gene frequencies was found between RA patients and controls. Thus, the association between RA and C3F found in some previous investigations was not confirmed.     

The investigation of allele and genotype frequencies of human C3 (rs2230199) in south Iranian population

The complement system is an important mediator of natural and acquired immunity. The complement system genes coding complement proteins have polymorphisms. Hereditary deficiencies of this system predispose to autoimmune conditions such as age-dependent macular degeneration or impairment of immunity against microorganisms. When different populations are compared, the frequency of complement polymorphism shows a very marked geographical distribution. The frequency of the functional polymorphism rs2230199 (Arg80Gly; C > G) in the C3 gene was determined in population from south of Iran (n = 200), using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). One hundred thirty-eight persons (69 %) were homozygous for C allele (CC or SS); fifty-six person (28 %) heterozygote GC (FS) and six people were homozygous for G allele (GG or FF) (3 %). The allele frequency was 82 % for C3S and 18 % for C3F. A distribution of C3C allele frequency in our population is different from the reports of Asians (100 %); Indians (90-98 %); African-American (93 %); Africans (99 %) and south Brazilian (97 %). However, this finding is similar with the findings Caucasian (80-82 %) ( http://www.ncbi.nlm.nih.gov/SNP ); Americans (80 %); Pushtoon, Hazaras, Osbek and Tajik ethnic groups in Afghanistan (88-90 %) and Tunisian population (84 %). Our study confirmed significant inter-ethnic differences in C3 (rs2230199) frequencies between south Iranians and other ethnic groups. The analysis of genetic variation in complement genes is a tool to provide new insights into the evolution of the human immune system.     

Functional gene expression domains: defining the functional unit of eukaryotic gene regulation

The term functional domain is often used to describe the region containing the cis acting sequences that regulate a gene locus. "Strong" domain models propose that the domain is a spatially isolated entity consisting of a region of extended accessible chromatin bordered by insulators that have evolved to act as functional boundaries. However, the observation that independently regulated loci can overlap partially or completely raises questions about functional requirements for physically isolated domain structures. An alternative model, the "weak" domain model, proposes that domain structure is determined by the distribution of binding sites for positively acting factors, without a requirement for functional boundaries. The domain would effectively be the region that contains these factor-binding sites. Specificity of promoter-enhancer interactions would play a major role in maintaining the functional autonomy of adjacent genes. Sequences that interfere with these interactions (frequently characterised as insulators) would be selected against if they occurred within the domain but not at the edges, or in the interdomain regions. As a result, insulators would often be found near the borders of domains without necessarily being selected to act as boundaries.     

Complement and the multifaceted functions of VWA and integrin I domains

The recent crystal structure of complement protein component C2a reveals an interface between its VWA and serine protease domains that could not exist in the zymogen C2. The implied change in VWA domain conformation between C2 and C2a differs from that described for other VWA domains, including the I domains in integrins. Here, the remarkable diversity in both conformational regulation and ligand binding among VWA domains that function in complement, hemostasis, cell adhesion, anthrax toxin binding, vesicle transport, DNA break repair, and RNA quality control is reviewed. Finally, implications for metastability of complement convertases are discussed.     

Conserved boundary elements from the Hox complex of mosquito,Anopheles gambiae

The conservation of hox genes as well as their genomic organization across the phyla suggests that this system of anterior–posterior axis formation arose early during evolution and has come under strong selection pressure. Studies in the split Hox cluster of Drosophila have shown that proper expression of hox genes is dependent on chromatin domain boundaries that prevent inappropriate interactions among different types of cis-regulatory elements. To investigate whether boundary function and their role in regulation of hox genes is conserved in insects with intact Hox clusters, we used an algorithm to locate potential boundary elements in the Hox complex of mosquito, Anopheles gambiae. Several potential boundary elements were identified that could be tested for their functional conservation. Comparative analysis revealed that like Drosophila, the bithorax region in A. gambiae contains an extensive array of boundaries and enhancers organized into domains. We analysed a subset of candidate boundary elements and show that they function as enhancer blockers in Drosophila. The functional conservation of boundary elements from mosquito in fly suggests that regulation of hox genes involving chromatin domain boundaries is an evolutionary conserved mechanism and points to an important role of such elements in key developmentally regulated loci.     

The Escherichia coli ATP-binding cassette (ABC) proteins

The recent completion of the Escherichia coli genome sequence ( Blattner et al ., 1997 ) has permitted an analysis of the complement of genomically encoded ATP-binding cassette (ABC) proteins. A total of 79 ABC proteins makes this the largest paralogous family of proteins in E . coli . These 79 proteins include 97 ABC domains (as some proteins include more than one ABC domain) and are components of 69 independent functional systems (as many systems involve more than one ABC domain). The ABC domains are often, but not exclusively, the energy-generating domains of multicomponent membrane-bound transporters. Thus, 57 of the 69 systems are ABC transporters, of which 44 are periplasmic-binding protein-dependent uptake systems and 13 are presumed exporters. The genes encoding these ABC transporters occupy almost 5% of the genome. Of the 12 systems that are not obviously transport related, the function of only one, the excision repair protein UvrA, is known. A phylogenetic analysis suggests that the majority of ABC proteins can be assigned to 10 subfamilies. Together with statistical and, importantly, biological evidence, this analysis provides insight into the evolution and function of the ABC proteins.     

Interaction between X-Delta-2 and Hox genes regulates segmentation and patterning of the anteroposterior axis

In vertebrates, the paraxial mesoderm already exhibits a complex Hox gene pattern by the time that segmentation occurs and somites are formed. The anterior boundaries of the Hox genes are always maintained at the same somite number, suggesting coordination between somite formation and Hox expression. To study this interaction, we used morpholinos to knockdown either the somitogenesis gene X-Delta-2 or the complete Hox paralogous group 1 (PG1) in Xenopus laevis. When X-Delta-2 is knocked down, Hox genes from different paralogous groups are downregulated from the beginning of their expression at gastrula stages. This effect is not via the canonical Notch pathway, as it is independent of the Notch effector Su(H). We also reveal for the first time a clear role for Hox genes in somitogenesis, as loss of PG1 gene function results in the perturbation of somite formation and downregulation of the X-Delta-2 expression in the PSM. This effect on X-Delta-2 expression is also observed during neurula stages, before the somites are formed. These results show that somitogenesis and patterning of the anteroposterior axis are closely linked via a feedback loop involving Hox genes and X-Delta-2, suggesting the existence of a coordination mechanism between somite formation and anteroposterior patterning. Such a mechanism is likely to be functional during gastrulation, before the formation of the first pair of somites, as suggested by the early X-Delta-2 regulation of the Hox genes.     

Regulatory systems of genome domains with vague boundaries

The specific features of genome domains lacking distinct boundaries are considered. These domains cannot be mapped by testing extended genome regions for nuclease sensitivity and thereby differ from structural domains determined at the level of DNA folding in chromatin. Yet they possess the properties of typical functional domains, containing a gene or several coordinated genes along with a complex of cis-regulatory elements, which control these genes. Domains with vague boundaries may be mapped with certain structural tests, e.g., by assessing histone acetylation or the distribution of tissue-specific DNase I-hypersensitive sites through extended genome regions. The mechanisms are described in detail that regulate the function of genes in domains with vague boundaries, including overlapping domains with genes differing in tissue specificity of expression.     

Diversification of olfactory receptor genes in the Japanese medaka fish, Oryzias latipes

Vertebrate olfactory receptors (OR) exists as the largest multigene family, scattered throughout the genome in clusters. Studies have shown that different animals possess remarkably diverse set of OR genes to recognize diverse odor molecules. In order to examine the evolutionary process of OR diversification, we examined three OR gene subfamilies from Japanese medaka fish (seven lines sampled from four populations). For each subfamily, the sequences of ancestral genes were inferred based on distance method. Examination of d(N)/d(S) ratios for each branch of phylogenetic trees suggested that purifying selection is the major force of evolution in medaka OR genes. However, for the mfOR1 and mfOR2 paralogous gene pairs, a nonrandom distribution of fixed amino acid changes and the d(N)>d(S) in a branch suggested that diversifying selection occurred after gene duplication. The fixed amino acid changes were observed in the third, fifth and sixth transmembrane domains, which has been predicted to serve as a ligand-binding pocket in a structural model. Compatibility test suggested that interlocus recombinations involving the fourth transmembrane domain occurred between the mfOR1 and mfOR2 gene pairs. The pattern of nucleotide substitutions in other OR genes agrees with the hypothesis that a limited number of amino acid residues are involved in odorant binding. Such comparative analyses of paralogous OR genes should provide bases for understanding the evolution, the structure, and the functional specificity of OR genes.     

Regulatory Systems of Genome Domains with Vague Boundaries

The specific features of genome domains lacking distinct boundaries are considered. These domains cannot be mapped by testing extended genome regions for nuclease sensitivity and thereby differ from structural domains determined at the level of DNA folding in chromatin. Yet they possess the properties of typical functional domains, containing a gene or several coordinated genes along with a complex of cis-regulatory elements, which control these genes. Domains with vague boundaries may be mapped with certain structural tests, e.g., by assessing histone acetylation or the distribution of tissue-specific DNase I-hypersensitive sites through extended genome regions. The mechanisms are described in detail that regulate the function of genes in domains with vague boundaries, including overlapping domains with genes differing in tissue specificity of expression.     

Characterization of AmphiF-spondin reveals the modular evolution of chordate F-spondin genes

The F-spondin genes are a family of extracellular matrix molecules united by two conserved domains, FS1 and FS2, at the amino terminus plus a variable number of thrombospondin repeats at the carboxy terminus. Currently, characterized members include a single gene in Drosophila and multiple genes in vertebrates. The vertebrate genes are expressed in the midline of the developing embryo, primarily in the floor plate of the neural tube. To investigate the evolution of chordate F-spondin genes, I have used the basal position in chordate phylogeny of the acraniate amphioxus. A single F-spondin-related gene, named AmphiF-spondin, was isolated from amphioxus. Based on molecular phylogenetics, AmphiF-spondin is closely related to a particular subgroup of vertebrate F-spondin genes that encode six thrombospondin repeats. However, unlike these genes, expression of AmphiF-spondin is not confined to the midline but is found through most of the central nervous system. Additionally, AmphiF-spondin has lost three thrombospondin repeats and gained two fibronectin type III repeats, one of which has strong identity to a fibronectin type III repeat from Deleted in Colorectal Cancer (DCC). Taken together, these results suggest a complex evolutionary history for chordate F-spondin genes that includes (1) domain loss, (2) domain gain by tandem duplication and divergence of existing domains, and (3) gain of heterologous domains by exon shuffling.      

Invariant TAD Boundaries Constrain Cell-Type-Specific Looping Interactions between Promoters and Distal Elements around the CFTR Locus

Three-dimensional genome structure plays an important role in gene regulation. Globally, chromosomes are organized into active and inactive compartments while, at the gene level, looping interactions connect promoters to regulatory elements. Topologically associating domains (TADs), typically several hundred kilobases in size, form an intermediate level of organization. Major questions include how TADs are formed and how they are related to looping interactions between genes and regulatory elements. Here we performed a focused 5C analysis of a 2.8 Mb chromosome 7 region surrounding CFTR in a panel of cell types. We find that the same TAD boundaries are present in all cell types, indicating that TADs represent a universal chromosome architecture. Furthermore, we find that these TAD boundaries are present irrespective of the expression and looping of genes located between them. In contrast, looping interactions between promoters and regulatory elements are cell-type specific and occur mostly within TADs. This is exemplified by the CFTR promoter that in different cell types interacts with distinct sets of distal cell-type-specific regulatory elements that are all located within the same TAD. Finally, we find that long-range associations between loci located in different TADs are also detected, but these display much lower interaction frequencies than looping interactions within TADs. Interestingly, interactions between TADs are also highly cell-type-specific and often involve loci clustered around TAD boundaries. These data point to key roles of invariant TAD boundaries in constraining as well as mediating cell-type-specific long-range interactions and gene regulation.     

Improving wastewater effluent filtration by changing flow regimes—Investigations in two cold climate pilot scale systems

Two pilot-scale experimental filter systems (FSs) filled with light-weight aggregates (LWA) or crushed limestone (CL) and operated under different flow regimes were established during the summer of 2005. The main objective of the study was to compare continuous-flow hybrid CL and LWA filters and batch-operated LWA filters in order to determine optimal loading, design parameters, management schemes and operational regimes for LWA-based FSs in cold climate conditions for the secondary treatment of domestic wastewater. With higher re-circulation rates of treated water, purification efficiency increased in terms of most water quality indicators in both types of filter systems. In the batch-operated FS the highest purification efficiencies of 96% and 51% for BOD₇ and N_tot, respectively, were achieved when the re-circulation rate of 200% was applied. In the continuous-flow FS the highest purification efficiencies of 99% and 81% for BOD₇ and N_tot, respectively, were achieved when the re-circulation rate up to 300% was applied. In order to achieve effective organic matter removal, nitrification/denitrification and TSS removal, the re-circulation rate must be from 100% to 300% of the inflowing wastewater. Both FS designs, the continuous-flow hybrid systems and the batch-operated filters, are suitable for the secondary treatment of domestic wastewater in cold climate conditions.     

The role of vitronectin as multifunctional regulator in the hemostatic and immune systems

Vitronectin (= complement S-protein) belongs to the group of structurally and functionally homologous adhesive proteins (fibrinogen, fibronectin, von Willebrand factor) which are essential in the procoagulant phase of the hemostatic system, interacting with platelets and the vessel wall. In addition to a structural motif in vitronectin responsible for this interaction (cell attachment domain) other functional domains in the protein molecule exist that contribute to its multifunctional role as regulator in the immune system (complement) as well as in fibrinolysis. These various activities and the ubiquitous distribution of vitronectin in the organism are discussed with regard to structure-function relationships of the protein molecule. Vitronectin may thus provide a conceptual molecular link between cell adhesion, humoral immune response and the hemostatic system, particularly at the blood-vessel wall interphase.     

Higher-order chromatin domains link eQTLs with the expression of far-away genes

Distal expression quantitative trait loci (distal eQTLs) are genetic mutations that affect the expression of genes genomically far away. However, the mechanisms that cause a distal eQTL to modulate gene expression are not yet clear. Recent high-resolution chromosome conformation capture experiments along with a growing database of eQTLs provide an opportunity to understand the spatial mechanisms influencing distal eQTL associations on a genome-wide scale. We test the hypothesis that spatial proximity contributes to eQTL-gene regulation in the context of the higher-order domain structure of chromatin as determined from recent Hi-C chromosome conformation experiments. This analysis suggests that the large-scale topology of chromatin is coupled with eQTL associations by providing evidence that eQTLs are in general spatially close to their target genes, occur often around topological domain boundaries and preferentially associate with genes across domains. We also find that within-domain eQTLs that overlap with regulatory elements such as promoters and enhancers are spatially more close than the overall set of within-domain eQTLs, suggesting that spatial proximity derived from the domain structure in chromatin plays an important role in the regulation of gene expression.     

MrpL36p, a highly diverged L31 ribosomal protein homolog with additional functional domains in Saccharomyces cerevisiae mitochondria

Translation in mitochondria utilizes a large complement of ribosomal proteins. Many mitochondrial ribosomal components are clearly homologous to eubacterial ribosomal proteins, but others appear unique to the mitochondrial system. A handful of mitochondrial ribosomal proteins appear to be eubacterial in origin but to have evolved additional functional domains. MrpL36p is an essential mitochondrial ribosomal large-subunit component in Saccharomyces cerevisiae. Increased dosage of MRPL36 also has been shown to suppress certain types of translation defects encoded within the mitochondrial COX2 mRNA. A central domain of MrpL36p that is similar to eubacterial ribosomal large-subunit protein L31 is sufficient for general mitochondrial translation but not suppression, and proteins bearing this domain sediment with the ribosomal large subunit in sucrose gradients. In contrast, proteins lacking the L31 domain, but retaining a novel N-terminal sequence and a C-terminal sequence with weak similarity to the Escherichia coli signal recognition particle component Ffh, are sufficient for dosage suppression and do not sediment with the large subunit of the ribosome. Interestingly, the activity of MrpL36p as a dosage suppressor exhibits gene and allele specificity. We propose that MrpL36p represents a highly diverged L31 homolog with derived domains functioning in mRNA selection in yeast mitochondria.