The primer tRNA for Moloney murine leukemia virus DNA synthesis. Nucleotide sequence and aminoacylation of tRNAPro.

The nucleotide sequence of a tRNA primer molecule for initiation of Moloney murine leukemia virus DNA synthesis has been determined. The sequence is heterogeneous in two positions but both forms, when drawn in a cloverleaf structure, have anticodon specificities for proline. We have termed these isoacceptors tRNA1Pro and tRNA2Pro. Aminoacylation studies confirmed the specificity for proline. The two forms occur in approximately equal amounts in uninfected mouse and chicken cells and in Moloney leukemia virus particles.     

Exploring the pharmacokinetic properties of phosphorus-containing selective HDAC 1 and 2 inhibitors (SHI-1:2)

We report the preparation and structure–activity relationships of phosphorus-containing histone deacetylase inhibitors. A strong trend between decreasing phosphorus functional group size and superior mouse pharmacokinetic properties was identified. In addition, optimized candidates showed tumor growth inhibition in xenograft studies.     

Genetic and structural analysis of base substitutions in the central pseudoknot of Thermus thermophilus 16S ribosomal RNA

Characterization of base substitutions in rRNAs has provided important insights into the mechanism of protein synthesis. Knowledge of the structural effects of such alterations is limited, and could be greatly expanded with the development of a genetic system based on an organism amenable to both genetics and structural biology. Here, we describe the genetic analysis of base substitutions in 16S ribosomal RNA of the extreme thermophile Thermus thermophilus, and an analysis of the conformational effects of these substitutions by structure probing with base-specific modifying agents. Gene replacement methods were used to construct a derivative of strain HB8 carrying a single 16S rRNA gene, allowing the isolation of spontaneous streptomycin-resistant mutants and subsequent genetic mapping of mutations by recombination. The residues altered to give streptomycin resistance reside within the central pseudoknot structure of 16S rRNA comprised of helices 1 and 27, and participate in the U13–U20–A915 base triple, the G21–A914 type II sheared G–A base pair, or the G885–C912 Watson–Crick base pair closing helix 27. Substitutions at any of the three residues engaged in the base triple were found to confer resistance. Results from structure probing of the pseudoknot are consistent with perturbation of RNA conformation by these substitutions, potentially explaining their streptomycin-resistance phenotypes.     

Wound healing and arm regeneration in Ophioderma longicaudum and Amphiura filiformis (Ophiuroidea, Echinodermata): comparative morphogenesis and histogenesis

All species of the Ophiuroidea have exceptional regenerative capabilities; in particular, they can replace arms lost following traumatic or self-induced amputation. In order to reconstruct this complex phenomenon, we studied arm regeneration in two different ophiuroids, Ophioderma longicaudum (Retzius, 1805) and Amphiura filiformis O. F. Müller, 1776, which are quite distantly related. These species present contrasting regeneration and differentiation rates and differ in several ecological traits. The aim of this paper is to interpret the primary sequence of morphogenetic and histogenetic events leading to the complete reconstruction of a new arm, comparing the arm regenerative processes of these two ophiuroid species with those described in crinoids. Arm regeneration in ophiuroids is considered an epimorphic process in which new structures develop from a typical blastema formed from an accumulation of presumptive undifferentiated cells. Our results showed that although very different in some respects such as, for instance, the regeneration rate (0.17 mm/week for O. longicaudum and 0.99 mm/week for A. filiformis), morphogenetic and histogenetic aspects are surprisingly similar in both species. The regenerative process presents similar characteristics and follows a developmental scheme which can be subdivided into four phases: a repair phase, an early regenerative phase, an intermediate regenerative phase and an advanced regenerative phase. In terms of histogenesis, the regenerative events involve the development of new structures from migratory pluripotent cells, which proliferate actively, in addition in both cases there is a significant contribution from dedifferentiated cells, in particular dedifferentiating myocytes, although to varying extents. This evidence confirms the plasticity of the regenerative phenomenon in echinoderms, which can apparently follow different pathways in terms of growth and morphogenesis, but nevertheless involve both epimorphic and morphallactic contributions at the cellular level.     

Structural analysis of kasugamycin inhibition of translation

The prokaryotic ribosome is an important target of antibiotic action. We determined the X-ray structure of the aminoglycoside kasugamycin (Ksg) in complex with the Escherichia coli 70S ribosome at 3.5-A resolution. The structure reveals that the drug binds within the messenger RNA channel of the 30S subunit between the universally conserved G926 and A794 nucleotides in 16S ribosomal RNA, which are sites of Ksg resistance. To our surprise, Ksg resistance mutations do not inhibit binding of the drug to the ribosome. The present structural and biochemical results indicate that inhibition by Ksg and Ksg resistance are closely linked to the structure of the mRNA at the junction of the peptidyl-tRNA and exit-tRNA sites (P and E sites).     

Amelioration of the cost of conjugative plasmid carriage in Eschericha coli K12

Although plasmids can provide beneficial functions to their host bacteria, they might confer a physiological or energetic cost. This study examines how natural selection may reduce the cost of carrying conjugative plasmids with drug-resistance markers in the absence of antibiotic selection. We studied two plasmids, R1 and RP4, both of which carry multiple drug resistance genes and were shown to impose an initial fitness cost on Escherichia coli. To determine if and how the cost could be reduced, we subjected plasmid-containing bacteria to 1100 generations of evolution in batch cultures. Analysis of the evolved populations revealed that plasmid loss never occurred, but that the cost was reduced through genetic changes in both the plasmids and the bacteria. Changes in the plasmids were inferred by the demonstration that evolved plasmids no longer imposed a cost on their hosts when transferred to a plasmid-free clone of the ancestral E. coli. Changes in the bacteria were shown by the lowered cost when the ancestral plasmids were introduced into evolved bacteria that had been cured of their (evolved) plasmids. Additionally, changes in the bacteria were inferred because conjugative transfer rates of evolved R1 plasmids were lower in the evolved host than in the ancestral host. Our results suggest that once a conjugative bacterial plasmid has invaded a bacterial population it will remain even if the original selection is discontinued.     

Coordinated nuclear export of 60S ribosomal subunits and NMD3 in vertebrates

60S and 40S ribosomal subunits are assembled in the nucleolus and exported from the nucleus to the cytoplasm independently of each other. We show that in vertebrate cells, transport of both subunits requires the export receptor CRM1 and Ran.GTP. Export of 60S subunits is coupled with that of the nucleo- cytoplasmic shuttling protein NMD3. Human NMD3 (hNMD3) contains a CRM-1-dependent leucine-rich nuclear export signal (NES) and a complex, dispersed nuclear localization signal (NLS), the basic region of which is also required for nucleolar accumulation. When present in Xenopus oocytes, both wild-type and export-defective mutant hNMD3 proteins bind to newly made nuclear 60S pre-export particles at a late step of subunit maturation. The export-defective hNMD3, but not the wild-type protein, inhibits export of 60S subunits from oocyte nuclei. These results indicate that the NES mutant protein competes with endogenous wild-type frog NMD3 for binding to nascent 60S subunits, thereby preventing their export. We propose that NMD3 acts as an adaptor for CRM1-Ran.GTP-mediated 60S subunit export, by a mechanism that is conserved from vertebrates to yeast.     

Compensating effects on the cytotoxicity of ribonuclease A variants

Ribonuclease (RNase) A can be endowed with cytotoxic activity by enabling it to evade the cytosolic ribonuclease inhibitor protein (RI). Enhancing its conformational stability can increase further its cytotoxicity. Herein, the A4C/K41R/G88R/V118C variant of RNase A was created to integrate four individual changes that greatly decrease RI affinity (K41R/G88R) and increase conformational stability (A4C/V118C). Yet, the variant suffers a decrease in ribonucleolytic activity and is only as potent a cytotoxin as its precursors. Thus, individual changes that increase cytotoxicity can have offsetting consequences. Overall, cytotoxicity correlates well with the maintenance of ribonucleolytic activity in the presence of RI. The parameter (k(cat)/K(m))(cyto), which reports on the ability of a ribonuclease to manifest its ribonucleolytic activity in the cytosol, is especially useful in predicting the cytotoxicity of an RNase A variant.     

Biosynthesis and degradation of storage protein in spores of the fungus Botryodiplodia theobromae

Muiridin, a spore-specific protein of the fungus Botryodiplodia theobromae, comprises about 25% of the mature pycnidiospore protein. It has an apparent molecular weight of 16,000 to 17,000 and is rich in glutamine, asparagine, and arginine. Muiridin is synthesized in developing spores via a precursor with an apparent molecular weight of 24,000. Two other polypeptides present in young developing spores with apparent molecular weights of 18,000 and 15,000 are immunologically related to muiridin. We propose a pathway for muiridin synthesis. Muiridin is actively degraded during the germination of spores from 30-day-old cultures. This degradation is independent of exogenous amino acids in the germination medium. In contrast, glutamine and, to a lesser extent, asparagine partially inhibit the degradation of muiridin during germination of spores from 7-day-old cultures.