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Studies of chromosome and genome biology often focus on condensed chromatin in the form of chromosomes and neglect the non-dividing cells. Even when interphase nuclei are considered, they are often then treated as interchangeable round objects. However, different cell types can have very different nuclear shapes, and these shapes have impacts on cellular function; indeed, many pathologies are linked with alterations to nuclear shape. In this review, we describe some of the nuclear morphologies beyond the spherical and ovoid. Many of the leukocytes of the immune system have lobed nuclei, which aid their flexibility and migration; smooth muscle cells have a spindle shaped nucleus, which must deform during muscle contractions; spermatozoa have highly condensed nuclei which adopt varied shapes, potentially associated with swimming efficiency. Nuclei are not passive passengers within the cell. There are clear effects of nuclear shape on the transcriptional activity of the cell. Recent work has shown that regulation of gene expression can be influenced by nuclear morphology, and that cells can drastically remodel their chromatin during differentiation. The link between the nucleoskeleton and the cytoskeleton at the nuclear envelope provides a mechanism for transmission of mechanical forces into the nucleus, directly affecting chromatin compaction and organisation.

Mitochondrial flashes (mitoflashes) are recently discovered mitochondrial activity which reflects chemical and electrical excitation of the organelle. Emerging evidence indicates that mitoflashes represent highly regulated, elementary signaling events that play important roles in physiological and pathophysiological processes in eukaryotes. Furthermore, they are regulated by mitochondrial ROS, Ca 2+ , and protons, and are intertwined with mitochondrial metabolic processes. As such, targeting mitoflash activity may provide a novel means for the control of mitochondrial metabolism and signaling in health and disease. In this brief review, we summarize salient features and mechanisms of biogenesis of mitoflashes, and synthesize data on mitoflash biology in the context of metabolism, cell differentiation, stress response, disease, and ageing.

The evolution of life from the simplest, original form to complex, intelligent animal life occurred through a number of key innovations. Here we present a new tool to analyze these key innovations by proposing that the process of evolutionary innovation may follow one of three underlying processes, namely a Random Walk, a Critical Path, or a Many Paths process, and in some instances may also constitute a "Pull-up the Ladder" event. Our analysis is based on the occurrence of function in modern biology, rather than specific structure or mechanism. A function in modern biology may be classified in this way either on the basis of its evolution or the basis of its modern mechanism. Characterizing key innovations in this way helps identify the likelihood that an innovation could arise. In this paper, we describe the classification, and methods to classify functional features of modern organisms into these three classes based on the analysis of how a function is implemented in modern biology. We present the application of our categorization to the evolution of eukaryotic gene control. We use this approach to support the argument that there are few, and possibly no basic chemical differences between the functional constituents of the machinery of gene control between eukaryotes, bacteria and archaea. This suggests that the difference between eukaryotes and prokaryotes that allows the former to develop the complex genetic architecture seen in animals and plants is something other than their chemistry. We tentatively identify the difference as a difference in control logic, that prokaryotic genes are by default 'on' and eukaryotic genes are by default 'off.' The Many Paths evolutionary process suggests that, from a 'default off' starting point, the evolution of the genetic complexity of higher eukaryotes is a high probability event.

The processing of particles, cells, and droplets for reactions, analyses, labeling, and coating is an important aspect of many microfluidic workflows. However, performing multi-step processes is typically a laborious and time-consuming endeavor. By exploiting the laminar nature of flow within microchannels, such procedures can benefit in terms of both speed and simplicity. This can be achieved either by manipulating the flow streams around the objects of interest, particularly for the localized perfusion of cells, or by manipulating the objects themselves within the streams via a range of forces. Here, we review the variety of methods that have been employed for performing such "multilaminar flow" procedures on particles, cells, and droplets.

Actin-like proteins comprise a large group of polymorphic proteins that readily form filaments engaged in cytoskeletal functions. Various members have been identified in prokaryotic and eukaryotic cells, e.g. MreB, ParM and Ta0583, and actin and the actin-related proteins, ARPs, respectively. Therefore, it is assumed that an ancestor of actin/MreB/ParM already existed in the last common progenitor of all cells. In eubacteria and archaea, actin-like proteins are either membrane-associated or freely soluble, and their activities are related to motility, cell shape maintenance, subcellular organization and cell cycle progression. In eukaryotes, all these functions are executed by actin in various isoforms. Additional functions have been described for actin and ARPs in the nucleus of the eukaryotic cell, and some of those were also discovered in prokaryotes. In the current essay, we compare structures and selected functions of prokaryotic and eukaryotic actins and discuss various aspects on how actins may have found their way into bacteria, into the eukaryotic cytoplasm and into the nuclear compartment.

A new scenario of the origin of eukaryotic cell and multicellularity is presented. A concentric pH-gradient has been shown to exist in the cytosol of eukaryotic cells. The most probable source of such gradient is its self-formation in gradient of electric field between center and periphery of a cell. Theoretical analysis has shown that, for example, a cell of Saccharomyces cerevisiae has enough energy to continuously sustain such gradient of strength about 1.5 kV/cm, the value sufficient for effective isoelectric focusing of cytoplasmic proteins. Focusing of enzymes could highly increase the effectiveness of an otherwise diffusion-limited metabolism of large cells by concentrating enzymes into small and distinct parts of a cytoplasm. By taking away an important physical constraint to the volume of cytoplasm, the intracellular isoelectric focusing enabled evolution of cells 3-4 order of magnitude larger than typical prokaryotic cells. This opened the way for the origin of phagocytosis and lately for the development of different forms of endosymbiosis, some of them resulting in an endosymbiotic origin of mitochondria and plastids. The large volume of a cell-enabled separation of nuclear and cytoplasmic compartments which was a precondition for separation of transcription and translation processes and therefore also for the origin of various RNA-preprocessing mechanisms. The possibility to regulate gene expression by postprocessing RNA and to regulate metabolism by an electrophoretic translocation enzymes between different parts of cytoplasm by changing their isoelectric points opened the way for cell and tissue differentiation and therefore for the origin of complex multicellular organisms.

We investigated the genetic diversity of eukaryotic microorganisms (0.8-20 microm) by sequencing cloned 18S rRNA genes in six genetic libraries constructed from six locations in Lake Taihu, a large shallow subtropical lake in China. Genetic libraries of eukaryotic ribosomal RNA were screened by restriction fragment length polymorphism (RFLP) analysis, and one clone representative of each RFLP pattern was partially sequenced. A total of 528 clones were clustered into 165 RFLP patterns and finally into 131 operational taxonomic unit (OTUs). Phylogenetic analysis revealed that each library included many unique OTUs, as well as members of distantly related phylogenetic groups. A majority of the clones were from alveolates, stramenopiles, cercozoa, cryptophytes, chlorophytes, and fungi, with members of choanoflagellida, euglenida, centroheliozoa, ancyromonadidae, ichthyosporea, and kathablepharid representing a minor fraction of the library. Six OTUs (15 clones) were not related to any known eukaryotic group. Canonical correspondence analysis suggested that the differences in eukaryotic microorganism community composition of in the six regions were partially related to trophic status, sediment resuspension, and top-down regulation by metazooplankton.

An in situ colonization assay was performed to study the early stages of biofilm formation in Rio Tinto (SW, Spain), an extremely acidic environment (pH ca. 2). Eukaryotic assemblages were monitored at monthly intervals for 1 year. Diversity, colonization rates, and seasonal variations were analyzed. Structural features of naturally grown biofilms were explored by light and scanning electron microscopy in backscattered electron mode. A total of 14 taxa were recognized as constituents of the eukaryotic assemblages. The eukaryotic communities were dissimilar at the different sampling sites. The lowest diversity was found at the most extreme locations, in terms of pH and heavy metal concentrations. The biofilms were mainly formed by species from the genera Dunaliella and Cyanidium. Two genera of filamentous algae, Zygnemopsis and Klebsormidium, were principally responsible for the variability in the cell number throughout the year. These species appear in June to decrease almost completely between October and November. In contrast, the number of heterotrophic flagellates and ciliates remained constant throughout the year. The microcolonization sequence showed an initial accumulation of amorphous particles composed of bacteria and inorganic grains of minerals. By the end of the second month, the organic matrix was also populated by fungi, bacteria, and a few eukaryotic heterotrophs such as amoebae and small flagellates. Diatoms only showed significant colonization in regions where mycelial matrices were first established. Flagellated green algae such as Dunaliella or Chlamydomonas as well as Euglena were also present at the very beginning of the biofilm development, although in low numbers (<100 cells cm(-2)). After the flagellated cells, sessile species of algae such Chlorella or Cyanidium appeared. Filamentous algae were the last species to colonize the biofilms. Most of the naturally grown biofilms were found to be structures composed of different species organized in different layers separated, probably by extracellular polymeric substances, although more analysis should be done in this regard. The possible implications of the biofilm structure in the adaptation to this extreme habitat are discussed.

Sequence directed mutagenesis is a mechanism by which imperfect repeats "repair" each other to become perfect, generating mutations. This process is known to be prevalent in prokaryotes and it has been implicated in several human genetic diseases. Here we test whether sequence directed mutagenesis occurs in the protein coding sequences of eukaryotes using extensive DNA sequence data from humans, mice, Drosophila, nematodes, yeast, and Arabidopsis. Using two tests we find little evidence of sequence directed mutagenesis. We conclude that sequence directed mutagenesis is not prevalent in eukaryotes and that the examples of human diseases, apparently caused by sequence directed mutagenesis, are probably coincidental.

Balamuthia mandrillaris is a recently identified free-living protozoan pathogen that can cause fatal granulomatous encephalitis in humans. Recent studies have shown that B. mandrillaris consumes eukaryotic cells such as mammalian cell cultures as food source. Here, we studied B. mandrillaris interactions with various eukaryotic cells including, monkey kidney fibroblast-like cells (COS-7), human brain microvascular endothelial cells (HBMEC) and Acanthamoeba (an opportunistic protozoan pathogen) as well as prokaryotes, Escherichia coli. B. mandrillaris exhibited optimal growth on HBMEC compared with Cos-7 cells. In contrast, B. mandrillaris did not grow on bacteria but remained in the trophozoite stage. When incubated with Acanthamoeba trophozoites, B. mandrillaris produced partial Acanthamoeba damage and the remaining Acanthamoeba trophozoites underwent encystment. However, B. mandrillaris were unable to consume Acanthamoeba cysts. Next, we observed that B. mandrillaris-mediated Acanthamoeba encystment is a contact-dependent process that requires viable B. mandrillaris. In support, conditioned medium of B. mandrillaris did not stimulate Acanthamoeba encystment nor did lysates of B. mandrillaris. Overall, these studies suggest that B. mandrillaris target Acanthamoeba in the trophozoite stage; however, Acanthamoeba possess the ability to defend themselves by forming cysts, which are resistant to B. mandrillaris. Further studies will examine the mechanisms associated with food selectivity in B. mandrillaris.

The relationship among the three cellular domains Archaea, Bacteria, and Eukarya has become a central problem in unraveling the tree of life. This relationship can now be studied as the completely sequenced genomes of representatives of these cellular domains become available. We performed a bioinformatic investigation of the Encephalitozoon cuniculi proteome. E. cuniculi has the smallest sequenced eukaryotic genome, 2.9 megabases coding for 1997 proteins. The proteins of E. cuniculi were compared with a previously characterized set of eukaryotic signature proteins (ESPs). ESPs are found in a eukaryotic cell, whether from an animal, a plant, a fungus, or a protozoan, but are not found in the Archaea and the Bacteria. We demonstrated that 85% of the ESPs have significant sequence similarity to proteins in E. cuniculi. Hence, E. cuniculi, a minimal eukaryotic cell that has removed all inessential proteins, still preserves most of the ESPs that make it a member of the Eukarya. The locations and functions of these ESPs point to the earliest history of eukaryotes.

Attempts were made to define the relationship among the three domains (eukaryotes, archaea, and eubacteria) using phylogenetic tree analyses of 16S rRNA sequences as well as of other protein sequences. Since the results are inconsistent, it is implied that the eukaryotic genome has a chimeric structure. In our previous studies, the origin of eukaryotes to be the symbiosis of archaea into eubacteria using the whole open reading frames (ORF) of many genomes was suggested. In these studies, the species participating in the symbiosis were not clarified, and the effect of gene duplication after speciation (in-paralog) was not addressed. To avoid the influence of the in-paralog, we developed a new method to calculate orthologous ORFs. Furthermore, we separated eukaryotic in-paralogs into three groups by sequence similarity to archaea, eubacteria (other than alpha-proteobacteria), and alpha-proteobacteria and treated them as individual organisms. The relationship between the three ORF groups and the functional classification was clarified by this analysis. The introduction of this new method into the phylogenetic tree analysis of 66 organisms (4 eukaryotes, 13 archaea, and 49 eubacteria) based on gene content suggests the symbiosis of pyrococcus into gamma-proteobacteria as the origin of eukaryotes.

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.

The negative correlation between the frequencies of usage of amino acids and their biosynthetic cost suggests that organisms minimize costs of protein biosynthesis. Empirical results support that: (1) free-living organisms (Archaea, Bacteria, and Eucaryota) minimize the usage of heavy amino acids more than intracellular organisms (viruses, chloroplasts, and mitochondria), a result confirmed by comparing intracellular Bacteria with other Bacteria; (2) avoidance of amino acids with low impact on protein structure (Chou-Fasman indices) is greater than for those with equal molecular weight but greater structural impact: constraints on protein function limit cost-minimization; (3) amino acid weight minimization (WM) for a protein correlates positively with the protein's expression level and with its size; (4) preliminary results suggest that for different proteins, the evolutionary rate of amino acid replacements correlates negatively with WM in these proteins; (5) results suggest that WM decreases with genome-size; and (6) developmental rates correlate positively with WM (within primates and rodents), even after confounding factors were accounted for. Effects of biosynthetic cost-minimization at whole-organism levels vary with metabolic and ecological strategies. Biosynthetic cost-minimization is an adaptive hypothesis that yields a semi-mechanistic explanation for small differences in allele fitness.

There is accumulating evidence that the general shape of the ribosomal DNA-based phylogeny of Eukaryotes is strongly biased by the long-branch attraction phenomenon, leading to an artifactual basal clustering of groups that are probably highly derived. Among these groups, Foraminifera are of particular interest, because their deep phylogenetic position in ribosomal trees contrasts with their Cambrian appearance in the fossil record. A recent actin-based phylogeny of Eukaryotes has proposed that Foraminifera might be closely related to Cercozoa and, thus, branch among the so-called crown of Eukaryotes. Here, we reanalyze the small-subunit ribosomal RNA gene (SSU rDNA) phylogeny by removing all long-branching lineages that could artifactually attract foraminiferan sequences to the base of the tree. Our analyses reveal that Foraminifera branch together with the marine testate filosean Gromia oviformis as a sister group to Cercozoa, in agreement with actin phylogeny. Our study confirms the utility of SSU rDNA as a phylogenetic marker of megaevolutionary history, provided that the artifacts due to the heterogeneity of substitution rates in ribosomal genes are circumvented.

The relative rates of change for eight sets of ubiquitous proteins were determined by a test in which anciently duplicated paralogs are used to root the universal tree and distances are calculated between each taxonomic group and the last common ancestor. The sets included ATPase subunits, elongation factors, signal recognition particle and its receptor, three sets of tRNA synthetases, transcarbamoylases, and an internal duplication in carbamoyl phosphate synthase. In each case phylogenetic trees were constructed and the distances determined for all pairs. Taken over the period of time since their last common ancestor, average evolutionary rates are remarkably similar for Bacteria and Eukarya, but Archaea exhibit a significantly slower average rate.

One of the most important omissions in recent evolutionary theory concerns how eukaryotes could emerge and evolve. According to the currently accepted views, the first eukaryotic cell possessed a nucleus, an endomembrane system, and a cytoskeleton but had an inefficient prokaryotic-like metabolism. In contrast, one of the most ancient eukaryotes, the metamonada Giardia lamblia, was found to have formerly possessed mitochondria. In sharp contrast with the traditional views, this paper suggests, based on the energetic aspect of genome organization, that the emergence of eukaryotes was promoted by the establishment of an efficient energy-converting organelle, such as the mitochondrion. Mitochondria were acquired by the endosymbiosis of ancient alpha-purple photosynthetic Gram-negative eubacteria that reorganized the prokaryotic metabolism of the archaebacterial-like ancestral host cells. The presence of an ATP pool in the cytoplasm provided by this cell organelle allowed a major increase in genome size. This evolutionary change, the remarkable increase both in genome size and complexity, explains the origin of the eukaryotic cell itself. The loss of cell wall and the appearance of multicellularity can also be explained by the acquisition of mitochondria. All bacteria use chemiosmotic mechanisms to harness energy; therefore the periplasm bounded by the cell wall is an essential part of prokaryotic cells. Following the establishment of mitochondria, the original plasma membrane-bound metabolism of prokaryotes, as well as the funcion of the periplasm providing a compartment for the formation of different ion gradients, has been transferred into the inner mitochondrial membrane and intermembrane space. After the loss of the essential function of periplasm, the bacterial cell wall could also be lost, which enabled the naked cells to establish direct connections among themselves. The relatively late emergence of mitochondria may be the reason why multicellularity evolved so slowly.

We describe a sequential (step by step) Darwinian model for the evolution of life from the late stages of the RNA world through to the emergence of eukaryotes and prokaryotes. The starting point is our model, derived from current RNA activity, of the RNA world just prior to the advent of genetically-encoded protein synthesis. By focusing on the function of the protoribosome we develop a plausible model for the evolution of a protein-synthesizing ribosome from a high-fidelity RNA polymerase that incorporated triplets of oligonucleotides. With the standard assumption that during the evolution of enzymatic activity, catalysis is transferred from RNA --> RNP --> protein, the first proteins in the "breakthrough organism" (the first to have encoded protein synthesis) would be nonspecific chaperone-like proteins rather than catalytic. Moreover, because some RNA molecules that pre-date protein synthesis under this model now occur as introns in some of the very earliest proteins, the model predicts these particular introns are older than the exons surrounding them, the "introns-first" theory. Many features of the model for the genome organization in the final RNA world ribo-organism are more prevalent in the eukaryotic genome and we suggest that the prokaryotic genome organization (a single, circular genome with one center of replication) was derived from a "eukaryotic-like" genome organization (a fragmented linear genome with multiple centers of replication). The steps from the proposed ribo-organism RNA genome --> eukaryotic-like DNA genome --> prokaryotic-like DNA genome are all relatively straightforward, whereas the transition prokaryotic-like genome --> eukaryotic-like genome appears impossible under a Darwinian mechanism of evolution, given the assumption of the transition RNA --> RNP --> protein. A likely molecular mechanism, "plasmid transfer," is available for the origin of prokaryotic-type genomes from an eukaryotic-like architecture. Under this model prokaryotes are considered specialized and derived with reduced dependence on ssRNA biochemistry. A functional explanation is that prokaryote ancestors underwent selection for thermophily (high temperature) and/or for rapid reproduction (r selection) at least once in their history.

Taken together, the available data appear to be consistent with a model in which Myc proteins function downstream of D-type cyclins and synergize with E2F proteins in the activation of the cyclin E/cdk2 kinase. This view of Myc proteins appears strikingly similar to established models for the E2F/DP family of proteins. However, it should be noted that there are clear differences and several predictions of such a model that have been critically tested for E2F proteins are still untested for Myc in this model. First, it appears that at least some target genes of Myc implicated in this process are still unknown; second, clear data from knockout cells that link p107 to Myc function are missing; and third, we are not aware of studies of tumour samples that clarify whether mutations in myc genes relieve the requirement for mutations in the cyclin D/p16 pathway.

In this study we constructed a bootstrapped distance tree of 500 small subunit ribosomal RNA sequences from organisms belonging to the so-called crown of eukaryote evolution. Taking into account the substitution rate of the individual nucleotides of the rRNA sequence alignment, our results suggest that (1) animals, true fungi, and choanoflagellates share a common origin: The branch joining these taxa is highly supported by bootstrap analysis (bootstrap support [BS] > 90%), (2) stramenopiles and alveolates are sister groups (BS = 75%), (3) within the alveolates, dinoflagellates and apicomplexans share a common ancestor BS > 95%), while in turn they both share a common origin with the ciliates (BS > 80%), and (4) within the stramenopiles, heterokont algae, hyphochytriomycetes, and oomycetes form a monophyletic grouping well supported by bootstrap analysis (BS > 85%), preceded by the well-supported successive divergence of labyrinthulomycetes and bicosoecids. On the other hand, many evolutionary relationships between crown taxa are still obscure on the basis of 18S rRNA. The branching order between the animal-fungal-choanoflagellates clade and the chlorobionts, the alveolates and stramenopiles, red algae, and several smaller groups of organisms remains largely unresolved.When among-site rate variation is not considered, the inferred tree topologies are inferior to those where the substitution rate spectrum for the 18S rRNA is taken into account. This is primarily indicated by the erroneous branching of fast-evolving sequences. Moreover, when different substitution rates among sites are not considered, the animals no longer appear as a monophyletic grouping in most distance trees.

The AAA proteins (ATPases Associated with a variety of cellular Activities) are found in eubacterial, archaebacterial, and eukaryotic species and participate in a large number of cellular processes, including protein degradation, vesicle fusion, cell cycle control, and cellular secretory processes. The AAA proteins are characterized by the presence of a 230 to 250-amino acid ATPase domain referred to as the Conserved ATPase Domain or CAD. Phylogenetic analysis of 133 CAD sequences from 38 species reveal that AAA CADs are organized into discrete groups that are related not only in structure but in cellular function. Evolutionary analyses also indicate that the CAD was present in the last common ancestor of eubacteria, archaebacteria, and eukaryotes. The eubacterial CADs are found in metalloproteases, while CAD-containing proteins in the archaebacterial and eukaryotic lineages appear to have diversified by a series of gene duplication events that lead to the establishment of different functional AAA proteins, including proteasomal regulatory, NSF/Sec, and Pas proteins. The phylogeny of the CADs provides the basis for establishing the patterns of evolutionary change that characterize the AAA proteins.

Eukaryotic ribosomal RNA (rRNA) contains numerous modified nucleotides: about 115 methyl groups and some 95 pseudouridines in vertebrates; about 65 methyl groups and some 45 pseudouridines in Saccharomyces cerevisiae. All but about ten of the methyl groups are ribose methylations. The remaining ten are on heterocyclic bases. The ribose methylations occur very rapidly upon the primary rRNA transcript in the nucleolus, probably on nascent chains, and they appear to play an important role in ribosome maturation, at least in vertebrates. All of the methyl groups occur in the conserved core of rRNA. However, there is no consensus feature of sequence or secondary structure for the methylation sites; thus the nature of the signal(s) for site-specific methylations had until recently remained a mystery. The situation changed dramatically with the discovery that many of the ribose methylation sites are in regions that are precisely complementary to small nucleolar RNA (snoRNA) species. Experimental evidence indicates that structural motifs within the snoRNA species do indeed pinpoint the precise nucleotides to be methylated by the putative 2'-O-methyl transferase(s). Regarding base methylations, the gene DIM1, responsible for modification of the conserved dimethyladenosines near the 3' end of 18S rRNA, has been shown to be essential for viability in S. cerevisiae and is suggested to play a role in the nucleocytoplasmic transport of the small ribosomal subunit. Recently nearly all of the pseudouridines have also been mapped in the rRNA of several eukaryotic species. As is the case for ribose methylations, most pseudouridine modifications occur rapidly upon precursor rRNA, within core sequences, and in a variety of local primary and secondary structure environments. In contrast to ribose methylation, no potentially unifying process has yet been identified for the enzymic recognition of the many pseudouridine modification sites. However, the new data afford the basis for a search for any potential involvement of snoRNAs in the recognition process.

Available sequences that correspond to the E. coli ribosomal proteins L11, L1, L10, and L12 from eubacteria, archaebacteria, and eukaryotes have been aligned. The alignments were analyzed qualitatively for shared structural features and for conservation of deletions or insertions. The alignments were further subjected to quantitative phylogenetic analysis, and the amino acid identity between selected pairs of sequences was calculated. In general, eubacteria, archaebacteria, and eukaryotes each form coherent and well-resolved nonoverlapping phylogenetic domains. The degree of diversity of the four proteins between the three groups is not uniform. For L11, the eubacterial and archaebacterial proteins are very similar whereas the eukaryotic L11 is clearly less similar. In contrast, in the case of the L12 proteins and to a lesser extent the L10 proteins, the archaebacterial and eukaryotic proteins are similar whereas the eubacterial proteins are different. The eukaryotic L1 equivalent protein has yet to be identified. If the root of the universal tree is near or within the eubacterial domain, our ribosomal protein-based phylogenies indicate that archaebacteria are monophyletic. The eukaryotic lineage appears to originate either near or within the archaebacterial domain.

This report summarizes data on the induction, repair and biological relevance of five types of radiation-induced DNA lesions for which repair kinetic studies have been performed in eukaryotic cells by various laboratories. These lesions are: DNA-protein crosslinks, base damage, single-strand breaks, double-strand breaks and bulky lesions (clustered base damage in the nm-range). The influence of various factors, such as oxia/anoxia, linear energy transfer of the radiation used, incubation medium, cell cycle stage, thiol content, hyperthermia, on the induction and repair of these lesions is described. Radiation-sensitive cell lines are also included.

We present theoretical considerations that suggest that synonymous-codon usage might be expected to be close to an equilibrium distribution given a very homogeneous process of silent substitution. By homogeneous we mean that substitution depends only on the two bases involved, so that 12 base-substitution rates completely describe the silent substitution process. We have developed a method of statistically testing for such homogeneous equilibrium and applied it to reported data on the codon usages of different classes of organisms. Weakly expressed bacterial sequences and both mammalian and nonmammalian eukaryotic sequences deviate significantly from a random pattern of codon usage, in the direction of homogeneous equilibrium. On the other hand, highly expressed bacterial sequences do not exhibit homogeneous equilibrium, which may be correlated with recent experimental results showing that they are optimized to accept the most abundant tRNAs. To examine the effect of amino acid replacements on the homogeneous model of silent substitution, we divided the amino acids with degenerate codes into two classes, those with high mutabilities and those with low, and performed the same analysis on bacterial and eukaryotic data sets. The codon sets of the highly mutable class of amino acids are not further from homogeneous equilibrium than are the codon sets of the class with low mutabilities. We also found for the eukaryotic data that these independent classes of codon sets show very similar equilibrium patterns. The various results suggest a high level of uniformity in the process of silent fixation in the different synonymous-codon sets, especially in eukaryotes.

The published translation termination sequences have been compiled and analysed to aid the interpretation of experiments on termination codon usage in the Xenopus oocyte (Bienz et al. 1981). There are significant differences between prokaryotes and eukaryotes concerning the usage of the three termination codons and of tandem stops. In addition viruses show termination strategies that differ from those of their hosts. Preferred context sequences flanking termination codons are described. Contexts vary within the last codon according to the nature of the termination codon, but are uniform within the first triplet following the terminators.

The properties of heterologous in vitro transcription systems utilizing components from mammalian and yeast cells have been investigated. Purified yeast RNA polymerase II, when supplemented with a full complement of mammalian transcription factors, does not promote specific transcription initiation on cloned mammalian class II genes. Similarly, a complete mammalian transcription system does not initiate specific transcription on cloned yeast class II genes. These results indicate evolutionary divergence in function of yeast and mammalian class II genes and the associated transcription apparatus. The functional differences observed in this study are corroborated by previously reported structural differences between yeast and mammalian RNA polymerase II and class II genes. In contrast, the mechanism of eukaryotic class III gene transcription appears to be evolutionarily conserved. Thus, a mammalian transcription extract specifically transcribes the cloned yeast 5S rRNA gene. This system synthesizes without processing the 130 base 5S rRNA precursor, and this primary transcript may be processed to the mature 120 base RNA using a partially purified yeast processing activity. An homologous yeast class III transcription system has also been developed. This yeast system contains all components necessary for proper synthesis, processing and splicing of yeast tRNA precursors. Using the homologous yeast transcription system, a template in which the 5' flanking region and first 5 base pairs of the 5S rRNA gene had been deleted is utilized to synthesize a 120 base transcript, but the efficiency of transcription is reduced to about 10% that of the wild type gene. Thus, the yeast 5S rRNA gene has an internal transcription control region, but the immediate 5' flanking sequence have effects on the level of transcription.

Experimental work published elsewhere has shown that the Archaebacteria encompass several distinct subgroups including methanogens, extreme halophiles, and various thermoacidophiles. The common characteristics of Archaebacteria known to date are these: (1) the presence of characteristic tRNAs and ribosomal RNAs; (2) the absence of peptidoglycan cell walls, with in many cases, replacement by a largely proteinaceous coat; (3) the occurrence of ether linked lipids built from phytanyl chains and (4) in all cases known so far, their occurrence only in unusual habitats. These organisms contain a number of 'eucaryotic features' in addition to their many bacterial attributes. This is interpreted as a strong indication that the Archaebacteria, while not actually eucaryotic, do indeed represents a third separate, line of descent as originally proposed.

We study the equilibrium in the use of synonymous codons by eukaryotic organisms and find five equations involving substitution rates that we believe embody the important implications of equilibrium for the process of silent substitution. We then combine these five equations with additional criteria to determine sets of substitution rates applicable to eukaryotic organisms. One method employs the equilibrium equations and a principle of maximum entropy to find the most uniform set of rates consistent with equilibrium. In a second method we combine the equilibrium equations with data on the man-mouse divergence to determine that set of rates that is most neutral yet consistent with both types of data (i.e., equilibrium and divergence data). Simulations show this second method to be quite reliable in spite of significant saturation in the substitution process. We find that when divergence data are included in the calculation of rates, even though these rates are chosen to be as neutral as possible, the strength of selection inferred from the nonuniformity of the rates is approximately doubled. Both sets of rates are applied to estimate the human-mouse divergence time based on several independent subsets of the divergence data consisting of the quartet, C- or T-ending duet, and A- or G-ending duet codon sets. Both rate sets produce patterns of divergence times that are shortest for the quartet data, intermediate for the CT-ending duets, and longest for the AG-ending duets. This indicates that rates of transitions in the duet-codon sets are significantly higher than those in the quartet-codon sets; this effect is especially marked for A----G, the rate of which in duets must be about double that in quartets.

We examined the codon usages in well-conserved and less-well-conserved regions of vertebrate protein genes and found them to be similar. Despite this similarity, there is a statistically significant decrease in codon bias in the less-well-conserved regions. Our analysis suggests that although those codon changes initially fixed under amino acid replacements tend to follow the overall codon usage pattern, they also reduce the bias in codon usage. This decrease in codon bias leads one to predict that the rate of change of synonymous codons should be greater in those regions that are less well conserved at the amino acid level than in the better-conserved regions. Our analysis supports this prediction. Furthermore, we demonstrate a significantly elevated rate of change of synonymous codons among the adjacent codons 5' to amino acid replacement positions. This provides further support for the idea that there are contextual constraints on the choice of synonymous codons in eukaryotes.