Processivity of translation in the eukaryote cell: Role of aminoacyl-tRNA synthetases

Several lines of evidence led to the conclusion that mammalian ribosomal protein synthesis is a highly organized biological process in vivo. A wealth of data support the concept according to which tRNA aminoacylation, formation of the ternary complex on EF1A and delivery of aminoacyl-tRNA to the ribosome is a processive mechanism where tRNA is vectorially transferred from one component to another. Polypeptide extensions, referred to as tRBDs (tRNA binding domains), are appended to mammalian and yeast aminoacyl-tRNA synthetases. The involvement of these domains in the capture of deacylated tRNA and in the sequestration of aminoacylated tRNA, suggests that cycling of tRNA in translation is mediated by the processivity of the consecutive steps. The possible origin of the tRBDs is discussed.     

Bacterial, archaeal, and eukaryal diversity in the intestines of Korean people

The bacterial, archaeal, and eukaryal diversity in fecal samples from ten Koreans were analyzed and compared by using the PCR-fingerprinting method, denaturing gradient gel electrophoresis (DGGE). The bacteria all belonged to the Firmicutes and Bacteroidetes phyla, which were known to be the dominant bacterial species in the human intestine. Most of the archaeal sequences belonged to the methane-producing archaea but several halophilic archarea-related sequences were also detected unexpectedly. While a small number of eukaryal sequences were also detected upon DGGE analysis, these sequences were related to fungi and stramenopiles (Blastocystis hominis). With regard to the bacterial and archaeal DGGE analysis, all ten samples had one and two prominent bands, respectively, but many individual-specific bands were also observed. However, only five of the ten samples had small eukaryal DGGE bands and none of these bands was observed in all five samples. Unweighted pair group method and arithmetic averages clustering algorithm (UPGMA) clustering analysis revealed that the archaeal and bacterial communities in the ten samples had relatively higher relatedness (the average Dice coefficient values were 68.9 and 59.2% for archaea and bacteria, respectively) but the eukaryal community showed low relatedness (39.6%).     

Assembly of the human origin recognition complex occurs through independent nuclear localization of its components

Initiation of eukaryotic genome duplication begins when a six-subunit origin recognition complex (ORC) binds to DNA. However, the mechanism by which this occurs in vivo and the roles played by individual subunits appear to differ significantly among organisms. Previous studies identified a soluble human ORC(2-5) complex in the nucleus, an ORC(1-5) complex bound to chromatin, and an Orc6 protein that binds weakly, if at all, to other ORC subunits. Here we show that stable ORC(1-6) complexes also can be purified from human cell extracts and that Orc6 and Orc1 each contain a single nuclear localization signal that is essential for nuclear localization but not for ORC assembly. The Orc6 nuclear localization signal, which is essential for Orc6 function, is facilitated by phosphorylation at its cyclin-dependent kinase consensus site and by association with Kpna6/1, nuclear transport proteins that did not co-purify with other ORC subunits. These and other results support a model in which Orc6, Orc1, and ORC(2-5) are transported independently to the nucleus where they can either assemble into ORC(1-6) or function individually.     

Genomic BLAST: custom-defined virtual databases for complete and unfinished genomes

BLAST (Basic Local Alignment Search Tool) searches against DNA and protein sequence databases have become an indispensable tool for biomedical research. The proliferation of the genome sequencing projects is steadily increasing the fraction of genome-derived sequences in the public databases and their importance as a public resource. We report here the availability of Genomic BLAST, a novel graphical tool for simplifying BLAST searches against complete and unfinished genome sequences. This tool allows the user to compare the query sequence against a virtual database of DNA and/or protein sequences from a selected group of organisms with finished or unfinished genomes. The organisms for such a database can be selected using either a graphic taxonomy-based tree or an alphabetical list of organism-specific sequences. The first option is designed to help explore the evolutionary relationships among organisms within a certain taxonomy group when performing BLAST searches. The use of an alphabetical list allows the user to perform a more elaborate set of selections, assembling any given number of organism-specific databases from unfinished or complete genomes. This tool, available at the NCBI web site http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/genom_table_cgi, currently provides access to over 170 bacterial and archaeal genomes and over 40 eukaryotic genomes.     

Prospects of electron cryotomography to visualize macromolecular complexes inside cellular compartments: implications of crowding

Electron cryotomography has unique potential for three-dimensional visualization of macromolecular complexes at work in their natural environment. This approach is based on reconstructing three-dimensional volumes from tilt series of electron micrographs of cells preserved in their native states by vitrification. Resolutions of 5–8 nm have already been achieved and the prospects for further improvement are good. Since many intracellular activities are conducted by complexes in the megadalton range with dimensions of 20–50 nm, current resolutions should suffice to identify many of them in tomograms. However, residual noise and the dense packing of cellular constituents hamper interpretation. Recently, tomographic data have been collected on vitrified eukaryotic cells (Medalia et al., Science (2002) in press). Their cytoplasm was found to be markedly less crowded than in the prokaryotes previously studied, in accord with differences in crowding between prokaryotic and eukaryotic cells documented by other (indirect) biophysical methods. The implications of this observation are twofold. First, complexes should be more easily identifiable in tomograms of eukaryotic cytoplasm. This applies both to recognizing known complexes and characterizing novel complexes. An example of the latter—a 5-fold symmetric particle is—given. Second, electron cryotomography offers an incisive probe to examine crowding in different cellular compartments.     

Evidence in a nematode for regulation of transposon excision by tissue-specific factors

Summary The transposable element Tc1 in Caenorhabditis elegans undergoes an excision reaction, which can be detected in a Southern hybridization as the appearance of empty chromosomal insertion sites. This excision reaction is under tissue-specific regulation in that it occurs at much higher frequency in somatic cells than in the germ line. We show here that this regulation is likely to be due to the action of tissue-specific factors that either promote excision in somatic tissues or repress it in the germ line. The rate of excision of elements at five distinct chromosomal sites has been measured by a method that avoids ambiguities due to cell division. All these elements are found to undergo excision at closely similar rates during the L1 larval stage. No distinct difference exists among the elements at different sites that would suggest regulation by flanking sequences.     

Early evolutionary origin of the planktic foraminifera inferred from small subunit rDNA sequence comparisons

Phylogenetic analysis of five partial planktic foraminiferal small subunit (SSU) ribosomal (r) DNA sequences with representatives of a diverse range of eukaryote, archaebacterial, and eubacterial taxa has revealed that the evolutionary origin of the foraminiferal lineage precedes the rapid eukaryote diversification represented by the crown of the eukaryotic tree and probably represents one of the earliest splits among extant free-living aerobic eukaryotes. The foraminiferal rDNA sequences could be clearly separated from known symbionts, commensals, and food organisms. All five species formed a single monophyletic group distinguished from the crown group by unique foraminiferal specific insertions as well as considerable nucleotide distance in aligned regions.     

The First Sexual Lineage and the Relevance of Facultative Sex

Models for the origin of the sex incorporate either obligate or facultative sexual cycles. The relevance of each assumption to the ancestral sexual population can be examined by surveying the sexual cycles of eukaryotes, and by determining the first lineage to diverge after sexuality evolved. Two protistan groups, the parabasalids and the oxymonads, have been suggested to be early-branching sexual lineages. A maximum-likelihood analysis of elongation factor-1α sequences shows that the parabasalids diverged prior to the oxymonads and thus represent the earliest sexual lineage of eukaryotes. Since both of these protist lineages and most other eukaryotes are facultatively sexual, it is likely that the common ancestor of all known eukaryotes was facultatively sexual as well. This finding has important implications for the ``Best-Man hypothesis' and other models for the origin of sex. Received: 21 August 1998 / Accepted: 26 December 1998