Small-molecule metabolism: an enzyme mosaic

Escherichia coli has been a popular organism for studying metabolic pathways. In an attempt to find out more about how these pathways are constructed, the enzymes were analysed by defining their protein domains. Structural assignments and sequence comparisons were used to show that 213 domain families constitute 90% of the enzymes in the small-molecule metabolic pathways. Catalytic or cofactor-binding properties between family members are often conserved, while recognition of the main substrate with change in catalytic mechanism is only observed in a few cases of consecutive enzymes in a pathway. Recruitment of domains across pathways is very common, but there is little regularity in the pattern of domains in metabolic pathways. This is analogous to a mosaic in which a stone of a certain colour is selected to fill a position in the picture.     

Translesion DNA polymerases in eukaryotes: what makes them tick?

Life as we know it, simply would not exist without DNA replication. All living organisms utilize a complex machinery to duplicate their genomes and the central role in this machinery belongs to replicative DNA polymerases, enzymes that are specifically designed to copy DNA. “Hassle-free” DNA duplication exists only in an ideal world, while in real life, it is constantly threatened by a myriad of diverse challenges. Among the most pressing obstacles that replicative polymerases often cannot overcome by themselves are lesions that distort the structure of DNA. Despite elaborate systems that cells utilize to cleanse their genomes of damaged DNA, repair is often incomplete. The persistence of DNA lesions obstructing the cellular replicases can have deleterious consequences. One of the mechanisms allowing cells to complete replication is “Translesion DNA Synthesis (TLS)”. TLS is intrinsically error-prone, but apparently, the potential downside of increased mutagenesis is a healthier outcome for the cell than incomplete replication. Although most of the currently identified eukaryotic DNA polymerases have been implicated in TLS, the best characterized are those belonging to the “Y-family” of DNA polymerases (pols η, ι, κ and Rev1), which are thought to play major roles in the TLS of persisting DNA lesions in coordination with the B-family polymerase, pol ζ. In this review, we summarize the unique features of these DNA polymerases by mainly focusing on their biochemical and structural characteristics, as well as potential protein–protein interactions with other critical factors affecting TLS regulation.     

Xylanases, xylanase families and extremophilic xylanases

Xylanases are hydrolytic enzymes which randomly cleave the beta 1,4 backbone of the complex plant cell wall polysaccharide xylan. Diverse forms of these enzymes exist, displaying varying folds, mechanisms of action, substrate specificities, hydrolytic activities (yields, rates and products) and physicochemical characteristics. Research has mainly focused on only two of the xylanase containing glycoside hydrolase families, namely families 10 and 11, yet enzymes with xylanase activity belonging to families 5, 7, 8 and 43 have also been identified and studied, albeit to a lesser extent. Driven by industrial demands for enzymes that can operate under process conditions, a number of extremophilic xylanases have been isolated, in particular those from thermophiles, alkaliphiles and acidiphiles, while little attention has been paid to cold-adapted xylanases. Here, the diverse physicochemical and functional characteristics, as well as the folds and mechanisms of action of all six xylanase containing families will be discussed. The adaptation strategies of the extremophilic xylanases isolated to date and the potential industrial applications of these enzymes will also be presented.     

The evolutionary genetics of egg size plasticity in a butterfly

Abstract The evolution of phenotypic plasticity requires that it is adaptive, genetically determined, and exhibits sufficient genetic variation. For the tropical butterfly Bicyclus anynana there is evidence that temperature-mediated plasticity in egg size is an adaptation to predictable seasonal change. Here we set out to investigate heritability in egg size and genetic variation in the plastic response to temperature in this species, using a half-sib breeding design. Egg size of individual females was first measured at a high temperature 4 days after eclosion. Females were then transferred to a low temperature and egg size was measured after acclimation periods of 6 and 12 days respectively. Overall, additive genetic variance explained only 3-11% of the total phenotypic variance, whereas maternal effects were more pronounced. Genotype-environment interactions and cross-environmental correlations of less than unity suggest that there is potential for short-term evolutionary change. Our findings strengthen the support for the adaptive nature of temperature-mediated plasticity in egg size.     

The structure and mechanism of action of cellulolytic enzymes

The modern structural classification of polysaccharases comprising cellulase–hemicellulase enzyme systems is dis cussed. Their catalytic domains are currently grouped into 15 of more than 80 known glycosyl hydrolase families, whereas substrate binding domains fall into 13 families. The structures of catalytic and substrate binding domains, as well as linker sequences, are briefly considered. A hypothetical mechanism of concerted action of catalytic and substrate binding domains of cellobiohydrolases on the surface of highly ordered cellulose is suggested.     

Radiation hybrid mapping of the canine type I and type IV collagen gene subfamilies

We are interested in the collagen gene superfamily and its involvement in hereditary diseases of the human and domestic dog. Presented here is radiation hybrid mapping of the type I and type IV collagen gene subfamilies on the most recent version of the canine map. The col1A1 gene was mapped to chromosome 9, col1A2 was mapped to chromosome 14, col4A1 and col4A2 were mapped to chromosome 22 and col4A3 and col4A4 were mapped to chromosome 25. The col4A5 and col4A6 genes, while linked to one another, are not linked in the present version of the canine map but likely are present on the X chromosome. These data provide an insight into the molecular evolution of these subfamilies and increase the number of mapped genes in discrete regions of the canine genome. J.K. Lowe and R. Guyon contributed equally to this work Sequences determined during the course of this work have been deposited in GenBank. Accession numbers are AF291995 (col1A1) and AF291996 (col1A2)     

Epigenetic silencing may aid evolution by gene duplication

Gene duplication is commonly regarded as the main evolutionary path toward the gain of a new function. However, even with gene duplication, there is a loss-versus-gain dilemma: most newly born duplicates degrade to pseudogenes, since degenerative mutations are much more frequent than advantageous ones. Thus, something additional seems to be needed to shift the loss versus gain equilibrium toward functional divergence. We suggest that epigenetic silencing of duplicates might play this role in evolution. This study began when we noticed in a previous publication (Lynch M, Conery JS [2000] Science 291:1151–1155) that the frequency of functional young gene duplicates is higher in organisms that have cytosine methylation (H. sapiens, M. musculus, and A. thaliana) than in organisms that do not have methylated genomes (S. cerevisiae, D. melanogaster, and C. elegans). We find that genome data analysis confirms the likelihood of much more efficient functional divergence of gene duplicates in mammals and plants than in yeast, nematode, and fly. We have also extended the classic model of gene duplication, in which newly duplicated genes have exactly the same expression pattern, to the case when they are epigenetically silenced in a tissue- and/or developmental stage-complementary manner. This exposes each of the duplicates to negative selection, thus protecting from pseudogenization. Our analysis indicates that this kind of silencing (i) enhances evolution of duplicated genes to new functions, particularly in small populations, (ii) is quite consistent with the subfunctionalization model when degenerative but complementary mutations affect different subfunctions of the gene, and (iii) furthermore, may actually cooperate with the DDC (duplication– degeneration–complementation) process. Dedicated to the memory of Susumu Ohno     

A Survey of the Molecular Evolutionary Dynamics of Twenty-Five Multigene Families from Four Grass Taxa

We surveyed the molecular evolutionary characteristics of 25 plant gene families, with the goal of better understanding general processes in plant gene family evolution. The survey was based on 247 GenBank sequences representing four grass species (maize, rice, wheat, and barley). For each gene family, orthology and paralogy relationships were uncertain. Recognizing this uncertainty, we characterized the molecular evolution of each gene family in four ways. First, we calculated the ratio of nonsynonymous to synonymous substitutions (d _N/d _S) both on branches of gene phylogenies and across codons. Our results indicated that the d _N/d _S ratio was statistically heterogeneous across branches in 17 of 25 (68%) gene families. The vast majority of d _N/d _S estimates were <<1.0, suggestive of selective constraint on amino acid replacements, and no estimates were >1.0, either across phylogenetic lineages or across codons. Second, we tested separately for nonsynonymous and synonymous molecular clocks. Sixty-eight percent of gene families rejected a nonsynonymous molecular clock, and 52% of gene families rejected a synonymous molecular clock. Thus, most gene families in this study deviated from clock-like evolution at either synonymous or nonsynonymous sites. Third, we calculated the effective number of codons and the proportion of G+C synonymous sites for each sequence in each gene family. One or both quantities vary significantly within 18 of 25 gene families. Finally, we tested for gene conversion, and only six gene families provided evidence of gene conversion events. Altogether, evolution for these 25 gene families is marked by selective constraint that varies among gene family members, a lack of molecular clock at both synonymous and nonsynonymous sites, and substantial variation in codon usage. Received: 25 May 2000 / Accepted: 16 October 2000     

Gene Conversion Drives Within Genic Sequences: Concerted Evolution of Ribosomal RNA Genes in Bacteria and Archaea

Multiple copies of a given ribosomal RNA gene family undergo concerted evolution such that sequences of all gene copies are virtually identical within a species although they diverge normally between species. In eukaryotes, gene conversion and unequal crossing over are the proposed mechanisms for concerted evolution of tandemly repeated sequences, whereas dispersed genes are homogenized by gene conversion. However, the homogenization mechanisms for multiple-copy, normally dispersed, prokaryotic rRNA genes are not well understood. Here we compared the sequences of multiple paralogous rRNA genes within a genome in 12 prokaryotic organisms that have multiple copies of the rRNA genes. Within a genome, putative sequence conversion tracts were found throughout the entire length of each individual rRNA genes and their immediate flanks. Individual conversion events convert only a short sequence tract, and the conversion partners can be any paralogous genes within the genome. Interestingly, the genic sequences undergo much slower divergence than their flanking sequences. Moreover, genomic context and operon organization do not affect rRNA gene homogenization. Thus, gene conversion underlies concerted evolution of bacterial rRNA genes, which normally occurs within genic sequences, and homogenization of flanking regions may result from co-conversion with the genic sequence. Received: 31 March 2000 / Accepted: 15 June 2000     

Methodologies for target selection in structural genomics

As the number of complete genomes that have been sequenced keeps growing, unknown areas of the protein space are revealed and new horizons open up. Most of this information will be fully appreciated only when the structural information about the encoded proteins becomes available. The goal of structural genomics is to direct large-scale efforts of protein structure determination, so as to increase the impact of these efforts. This review focuses on current approaches in structural genomics aimed at selecting representative proteins as targets for structure determination. We will discuss the concept of representative structures/folds, the current methodologies for identifying those proteins, and computational techniques for identifying proteins which are expected to adopt new structural folds.