Autonomous replication sequences in an extrachromosomal element of a pathogenic Entamoeba histolytica.

Entamoeba histolytica possesses a 24.5 kilobase plasmid-like molecule which encodes for the organism's ribosomal RNAs. Sequence analysis of this extrachromosomal element revealed the presence of AT rich sequences which show homology to the origin of replication of other lower eucaryotes. An 802 bp fragment containing these sequences was cloned into a yeast shuttle vector lacking the origin of replication and the construct tested for its ability to replicate autonomously in yeast. Mitotic stability tests as well as evidence for plasmid maintenance indicate that the transformed cells contained self-replicating episomes and not stably integrated molecules. The nucleotide sequence of this ARS-containing fragment is presented.     

A Proposed Genus Boundary for the Prokaryotes Based on Genomic Insights

Genomic information has already been applied to prokaryotic species definition and classification. However, the contribution of the genome sequence to prokaryotic genus delimitation has been less studied. To gain insights into genus definition for the prokaryotes, we attempted to reveal the genus-level genomic differences in the current prokaryotic classification system and to delineate the boundary of a genus on the basis of genomic information. The average nucleotide sequence identity between two genomes can be used for prokaryotic species delineation, but it is not suitable for genus demarcation. We used the percentage of conserved proteins (POCP) between two strains to estimate their evolutionary and phenotypic distance. A comprehensive genomic survey indicated that the POCP can serve as a robust genomic index for establishing the genus boundary for prokaryotic groups. Basically, two species belonging to the same genus would share at least half of their proteins. In a specific lineage, the genus and family/order ranks showed slight or no overlap in terms of POCP values. A prokaryotic genus can be defined as a group of species with all pairwise POCP values higher than 50%. Integration of whole-genome data into the current taxonomy system can provide comprehensive information for prokaryotic genus definition and delimitation.     

Nud1p, the yeast homolog of Centriolin, regulates spindle pole body inheritance in meiosis

Nud1p, a protein homologous to the mammalian centrosome and midbody component Centriolin, is a component of the budding yeast spindle pole body (SPB), with roles in anchorage of microtubules and regulation of the mitotic exit network during vegetative growth. Here we analyze the function of Nud1p during yeast meiosis. We find that a nud1-2 temperature-sensitive mutant has two meiosis-related defects that reflect genetically distinct functions of Nud1p. First, the mutation affects spore formation due to its late function during spore maturation. Second, and most important, the mutant loses its ability to distinguish between the ages of the four spindle pole bodies, which normally determine which SPB would be preferentially included in the mature spores. This affects the regulation of genome inheritance in starved meiotic cells and leads to the formation of random dyads instead of non-sister dyads under these conditions. Both functions of Nud1p are connected to the ability of Spc72p to bind to the outer plaque and half-bridge (via Kar1p) of the SPB.     

Microbial life at high salt concentrations: phylogenetic and metabolic diversity

Halophiles are found in all three domains of life. Within the Bacteria we know halophiles within the phyla Cyanobacteria, Proteobacteria, Firmicutes, Actinobacteria, Spirochaetes, and Bacteroidetes. Within the Archaea the most salt-requiring microorganisms are found in the class Halobacteria. Halobacterium and most of its relatives require over 100–150 g/l salt for growth and structural stability. Also within the order Methanococci we encounter halophilic species. Halophiles and non-halophilic relatives are often found together in the phylogenetic tree, and many genera, families and orders have representatives with greatly different salt requirement and tolerance. A few phylogenetically coherent groups consist of halophiles only: the order Halobacteriales, family Halobacteriaceae (Euryarchaeota) and the anaerobic fermentative bacteria of the order Halanaerobiales (Firmicutes). The family Halomonadaceae (Gammaproteobacteria) almost exclusively contains halophiles. Halophilic microorganisms use two strategies to balance their cytoplasm osmotically with their medium. The first involves accumulation of molar concentrations of KCl. This strategy requires adaptation of the intracellular enzymatic machinery, as proteins should maintain their proper conformation and activity at near-saturating salt concentrations. The proteome of such organisms is highly acidic, and most proteins denature when suspended in low salt. Such microorganisms generally cannot survive in low salt media. The second strategy is to exclude salt from the cytoplasm and to synthesize and/or accumulate organic 'compatible' solutes that do not interfere with enzymatic activity. Few adaptations of the cells' proteome are needed, and organisms using the 'organic-solutes-in strategy' often adapt to a surprisingly broad salt concentration range. Most halophilic Bacteria, but also the halophilic methanogenic Archaea use such organic solutes. A variety of such solutes are known, including glycine betaine, ectoine and other amino acid derivatives, sugars and sugar alcohols. The 'high-salt-in strategy' is not limited to the Halobacteriaceae. The Halanaerobiales (Firmicutes) also accumulate salt rather than organic solutes. A third, phylogenetically unrelated organism accumulates KCl: the red extremely halophilic Salinibacter (Bacteroidetes), recently isolated from saltern crystallizer brines. Analysis of its genome showed many points of resemblance with the Halobacteriaceae, probably resulting from extensive horizontal gene transfer. The case of Salinibacter shows that more unusual types of halophiles may be waiting to be discovered.     

Analysis of the gene coding for the murine cellular tumour antigen p53

A genomic clone containing the functional gene for the murine p53 cellular tumour antigen was isolated and structurally characterised. The gene contains at least 11 exons and 10 introns, the first intron possessing a length of 6.1 kb. Attempts to determine the exact 5' end of p53 mRNA were inconclusive, probably due to the presence of a remarkable stem and loop structure (delta G degrees approximately equal to -56 kcal/mol) in the 5' region of the gene. Suggestive similarities were found to exist between p53 and the protein product of the myc oncogene.     

Crystal structure of the collagen alpha1(VIII) NC1 trimer.

Collagen VIII is a major component of Descemet's membrane and is also found in vascular subendothelial matrices. The C-terminal non-collagenous domain (NC1) domain of collagen VIII, which is a member of the C1q-like protein family, forms a stable trimer and is thought to direct the assembly of the collagen triple helix, as well as polygonal supramolecular structures. We have solved the crystal structure of the mouse alpha1(VIII)(3) NC1 domain trimer at 1.9 A resolution. Each subunit of the intimate NC1 trimer consists of a ten-stranded beta-sandwich. The surface of the collagen VIII NC1 trimer presents three strips of partially exposed aromatic residues shown to interact with the non-ionic detergent CHAPS, which are likely to be involved in supramolecular assemblies. Equivalent strips exist in the NC1 domain of the closely related collagen X, suggesting a conserved assembly mechanism. Surprisingly, the collagen VIII NC1 trimer lacks the buried calcium cluster of the collagen X NC1 trimer. The mouse alpha1(VIII) and alpha2(VIII) NC1 domains are 71.5% identical in sequence, with the differences being concentrated on the NC1 trimer surface. A few non-conservative substitutions map to the subunit interfaces near the surface, but it is not obvious from the structure to what extent they determine the preferred assembly of collagen VIII alpha1 and alpha2 chains into homotrimers.     

p53 and p21 regulate error-prone DNA repair to yield a lower mutation load

Regulation of mutation rates is critical for maintaining genome stability and controlling cancer risk. A special challenge to this regulation is the presence of multiple mutagenic DNA polymerases in mammals. These polymerases function in translesion DNA synthesis (TLS), an error-prone DNA repair process that involves DNA synthesis across DNA lesions. We found that in mammalian cells TLS is controlled by the tumor suppressor p53, and by the cell cycle inhibitor p21 via its PCNA-interacting domain, to maintain a low mutagenic load at the price of reduced repair efficiency. This regulation may be mediated by binding of p21 to PCNA and via DNA damage-induced ubiquitination of PCNA, which is stimulated by p53 and p21. Loss of this regulation by inactivation of p53 or p21 causes an out of control lesion-bypass activity, which increases the mutational load and might therefore play a role in pathogenic processes caused by genetic instability.     

p53 cellular tumor antigen: analysis of mRNA levels in normal adult tissues, embryos, and tumors.

The relative levels of mRNA specific for the mouse p53 cellular tumor antigen were determined in various normal adult tissues, embryos, and tumors. All tumors studied contained concentrations of p53 mRNA well above those present in most normal tissues. Normal spleen, however, had p53 mRNA levels comparable to those found in some tumors, despite the fact that they contained barely detectable p53 protein. This apparent discrepancy was found to be due to the extremely rapid turnover rate of p53 in the spleen (half-life, approximately equal to 6 min). In developing fetuses, a marked reduction of p53 mRNA levels was manifest from day 11 onwards, whereas the levels during organogenesis (days 9 to 11) were comparable to those found in undifferentiated embryonic stem cells and in some tumors.