Misacylation and editing by Escherichia coli valyl-tRNA synthetase: evidence for two tRNA binding sites.

Valyl-tRNA synthetase (ValRS) has difficulty discriminating between its cognate amino acid, valine, and structurally similar amino acids. To minimize translational errors, the enzyme catalyzes a tRNA-dependent editing reaction that prevents accumulation of misacylated tRNA(Val). Editing occurs with threonine, alanine, serine, and cysteine, as well as with several nonprotein amino acids. The 3'-end of tRNA plays a vital role in promoting the tRNA-dependent editing reaction. Valine tRNA having the universally conserved 3'-terminal adenosine replaced by any other nucleoside does not stimulate the editing activity of ValRS. As a result 3'-end tRNA(Val) mutants, particularly those with 3'-terminal pyrimidines, are stably misacylated with threonine, alanine, serine, and cysteine. Valyl-tRNA synthetase is unable to hydrolytically deacylate misacylated tRNA(Val) terminating in 3'-pyrimidines but does deacylate mischarged tRNA(Val) terminating in adenosine or guanosine. Evidently, a purine at position 76 of tRNA(Val) is essential for translational editing by ValRS. We also observe misacylation of wild-type and 3'-end mutants of tRNA(Val) with isoleucine. Valyl-tRNA synthetase does not edit wild-type tRNA(Val)(A76) mischarged with isoleucine, presumably because isoleucine is only poorly accommodated at the editing site of the enzyme. Misacylated mutant tRNAs as well as 3'-end-truncated tRNA(Val) are mixed noncompetitive inhibitors of the aminoacylation reaction, suggesting that ValRS, a monomeric enzyme, may bind more than one tRNA(Val) molecule. Gel-mobility-shift experiments to characterize the interaction of tRNA(Val) with the enzyme provide evidence for two tRNA binding sites on ValRS.     

The sequence of the gorilla fetal globin genes: evidence for multiple gene conversions in human evolution

Two fetal globin genes (G gamma and A gamma) from one chromosome of a lowland gorilla (Gorilla gorilla gorilla) have been sequenced and compared to three human loci (a G gamma-gene and two A gamma-alleles). A comparison of regions of local homology among these five sequences indicates that long after the duplication that produced the two nonallelic gamma-globin loci of catarrhine primates, about 35 million years (Myr) ago, at least one gene conversion event occurred between these loci. This conversion occurred not long before the ancestral divergence (about 6 Myr ago) of Homo and Gorilla. After this ancestral divergence, a minimum of three more gene conversion events occurred in the human lineage. Each human A gamma-allele shares specific sequence features with the gorilla A gamma-gene; one such distinctive allelic feature involves the simple repeated sequence in IVS 2. This suggests that early in the human lineage the A gamma-genes may have undergone a crossing-over event mediated by this simple repeated sequence. The DNA sequences from coding regions of both G gamma- and A gamma-loci, a comparison of 292 codons in the corresponding gorilla and human genes, show an unusually low evolutionary rate, with only two nonsilent differences and, surprisingly, not even one silent substitution. The two nonsynonymous substitutions observed predict a glycine at codon 73 and an arginine at codon 104 in the gorilla A gamma-sequence rather than aspartic acid and lysine, respectively, in human A gamma. Because only arginine has been found at position 104 in gamma-chains of Old World monkeys, it may represent the ancestral residue lost in gorilla and human G gamma-chains and in the human A gamma-chain. Possibly the arginine codon (AGG) was replaced by the lysine codon (AAG) in the G gamma-gene of a common ancestor of Homo and Gorilla and then was transferred to the A gamma-gene by subsequent conversions in the human lineage. DNA sequence conversions, similar to that attributed to the fetal gamma-globin genes, appear to be relatively frequent phenomena and, if widespread throughout the genome, may have profound evolutionary consequences.      

Amplification on the Amycolatopsis (Nocardia) mediterranei plasmid pMEA100: sequence similarities to actinomycete att sites

An amplification of a 2.0-kb fragment was found on the plasmid pMEA100 isolated from a subculture of the wild-type strain LBG A3136 of Amycolatopsis (Nocardia) mediterranei. Plasmid preparations contained a mixture of molecules with copy numbers of the amplified unit in the range of 2 to 10. The amplification on pMEA100 was stable; propagation of cells for many generations did not change the pattern of the amplified DNA. Fragments of the plasmids containing the amplifiable unit of DNA (AUD) and the amplified DNA sequence (ADS) were subcloned and characterized. Sequencing of the AUD terminal regions and the junction between ADS units showed that the amplifiable unit of DNA was flanked by 12-bp direct repeats. The DNA segments adjacent to the 12-bp sequence common to the left and right AUD terminal regions also showed significant similarities. In addition, the left AUD terminal region flanking the 12-bp repeat exhibited considerable sequence similarity to actinomycete plasmid attachment sites, particularly to the pMEA 100 att site.     

The first report of RNA U to C or G editing in the mitochondrial NADH dehydrogenase subunit 5 (Nad5) transcript of wild barley

Molecular Biology Reports - Nad dehydrogenase complex in mtDNA has a significant role in cellular respiration. One of the largest subunits in the complex is subunit 5 (Nad5). Four cDNAs of the...     

C to U editing stimulates A to I editing in the anticodon loop of a cytoplasmic threonyl tRNA in Trypanosoma brucei

Editing of tRNAs is widespread in nature and either changes the decoding properties or restores the folding of a tRNA. Unlike the phylogenetically disperse adenosine (A) to inosine (I) editing, cytosine (C) to uridine (U) editing has only been previously described in organellar tRNAs. We have shown that cytoplasmic tRNA(Thr)(AGU) undergoes two distinct editing events in the anticodon loop: C to U and A to I. In vivo, every inosine-containing tRNA(Thr) is also C to U edited at position 32. In vitro, C to U editing stimulates conversion of A to I at the wobble base. Although the in vivo and in vitro requirements differ, in both cases, the C to U change plays a key role in A to I editing. Due to an unusual abundance of A34-containing tRNAs, our results also suggest that the unedited and edited tRNAs are functional, each dedicated to decoding a specific threonine codon. C to U editing of cytoplasmic tRNA expands the editing repertoire in eukaryotic cells, and when coupled to A to I changes, leads to an interrelation between editing sites.     

Primate repetitive DNAs: evidence for new satellite DNAs and similarities in non-satellite repetitive DNA sequence properties

Repetitious DNA sequences have been isolated from a number of the primates in both Suborders Anthropoidea and Prosimii by hydroxyapatite chromatography at a C₀t of 10. In addition to finding previously unreported possible AT-rich satellite DNAs in Orangutan, Gibbon, Rhesus and Slow Loris a clear similarity to human DNA was found in the non-satellite repetitious DNA sequence properties of the primates in the Suborder Anthropoidea. This is based on the presence of the hydroxyapatite isolated 1.703 and 1.714 g/cm³ DNA families in CsCl gradients in the analytical ultracentrifuge following renaturation and extensive DNA hyperpolymer network formation. Within the superfamily Hominoidea the amount of the 1.714 g/cm³ DNA family was greater than that of the 1.703 g/cm³ DNA family while the reverse situation was true within the Superfamily Cercopithecoidea. The orangutan 1.703 and 1.714 g/cm³ DNA families were shown to exhibit the same differential reassociation behavior demonstrated previously in human DNA (Marx et al., 1976a). These data are interpreted as preliminary evidence for a similar sequence organization in the Order Primates Suborder Anthropoidea.     

The ovaries of Mecoptera: basic similarities and one exception to the rule

Two entirely different types of ovaries (ovarioles) have been described in mecopterans. In the representatives of Meropeidae, Bittacidae, Panorpodidae and Panorpidae the ovarioles are of the polytrophic-meroistic type. Four regions: a terminal filament, germarium, vitellarium and ovariole stalk can be distinguished in the ovarioles. The germaria house numerous germ cell clusters. Each cluster arises as a result of 2 consecutive mitoses of a cystoblast and consists of 4 sibling cells. The oocyte always differentiates from one of the central cells of the cluster, whereas the remaining 3 cells develop into large, polyploid nurse cells. The vitellaria contain 7-12 growing egg chambers (= oocyte-nurse cell complexes). In contrast, the ovaries of the snow flea, Boreus hyemalis, are devoid of nurse cells and therefore panoistic (secondary panoistic). The ovarioles are composed of terminal filaments, vitellaria and ovariole stalks only; in adult females functional germaria are absent. Histochemical tests suggest that amplification of rDNA takes place in the oocyte nuclei. Resulting dense nucleolar masses undergo fragmentation into multiple polymorphic nucleoli. The classification of extant mecopterans as well as the phylogenetic relationships between Mecoptera and Siphonaptera are discussed in the context of presented data.     

Stabilities of consecutive A.C, C.C, G.G, U.C, and U.U mismatches in RNA internal loops: Evidence for stable hydrogen-bonded U.U and C.C.+ pairs.

The stability and structure of RNA duplexes with consecutive A.C, C.A, C.C, G.G, U.C, C.U, and U.U mismatches were studied by UV melting, CD, and NMR. The results are compared to previous results for GA and AA internal loops [SantaLucia, J., Kierzek, R., & Turner, D. H. (1990) Biochemistry 29, 8813-8819; Peritz, A., Kierzek, R., & Turner, D.H. (1991) Biochemistry 30, 6428-6436)]. The observed order for stability increments of internal loop formation at pH 7 is AG = GA approximately UU greater than GG greater than or equal to CA greater than or equal to AA = CU = UC greater than or equal to CC greater than or equal to AC. The results suggest two classes for internal loops with consecutive mismatches: (1) loops that stabilize duplexes and have strong hydrogen bonding and (2) loops that destabilize duplexes and may not have strong hydrogen bonding. Surprisingly, rCGCUUGCG forms a very stable duplex at pH 7 in 1 M NaCl with a TM of 44.8 degrees C at 1 x 10(-4) M and a delta G degrees 37 of -7.2 kcal/mol. NOE studies of the imino protons indicate hydrogen bonding within the U.U mismatches in a wobble-type structure. Resonances corresponding to the hydrogen-bonded uridines are located at 11.3 and 10.4 ppm. At neutral pH, rCGCCCGCG is one of the least stable duplexes with a TM of 33.2 degrees C and delta G degrees 37 of -5.1 kcal/mol. Upon lowering the pH to 5.5, however, the TM increases by 12 degrees C, and delta G degrees 37 becomes more favorable by 2.5 kcal/mol. The pH dependence of rCGCCCGCG may be due to protonation of the internal loop C's, since no changes in thermodynamic parameters are observed for rCGCUUGCG between pH 7 and 5.5. Furthermore, two broad imino proton resonances are observed at 10.85 and 10.05 ppm for rCGCCCGCG at pH 5.3, but not at pH 6.5. This is also consistent with C.C+ base pairs forming at pH 5.5. rCGCCAGCG and rGGCACGCC have a small pH dependence, with TM increases of 5 and 3 degrees C, respectively, upon lowering the pH from 7 to 5.5. rCGCCUGCG and rCGCUCGCG also show little pH dependence, with TM increases of 0.8 and 1.4 degrees C, respectively, upon lowering the pH to 5.5.(ABSTRACT TRUNCATED AT 400 WORDS)     

Palaeolimnological evidence for recent chemical and biological changes in U.K. Acid Waters Monitoring Network sites

1. Palaeolimnological evidence is presented for the long-term (post-1850) and recent (post-1970) trends in acidity of eleven sites in the U.K. Acid Waters Monitoring Network.
2. Sites are located throughout the U.K. in areas sensitive to acidification, and results show that all have been acidified since pre-industrial times. Although there is considerable variation in the timing and magnitude of these changes the results are consistent with other evidence of the widespread and severe acidification of sensitive U.K. freshwaters as a result of acidic deposition.
3. The most severely acidified sites generally have the highest critical load exceedance, although there is a only a poor relationship between exceedance and post-1850 pH change ( r = 0.58, P = 0.06) or diatom floristic change ( r = 0.52, P = 0.1). These results highlight the difficulty of inferring biological change or 'damage' in freshwater ecosystems from current national maps of critical load exceedances.
4. Evidence of chemical and biological response to the post-1970 reduction in U.K. S emissions is variable: seven lakes show continued acidification in the 1970s and early 1980s while four appear to have been in steady state. One afforested site shows continued acidification until at least 1990, the year of coring, suggesting that at this site increased scavenging of acid anions following canopy closure and/or increased nitrate leaching have offset the benefits of reduced S deposition.
5. Five sites appear to have been in steady state since the early to mid-1980s, and two show unambiguous evidence for a recent increase in pH and a reversal in the diatom assemblages to that of earlier levels. The results support and extend the findings of other studies and show that biological recovery is occurring in at least two chronically acidified areas of the U.K. (Galloway, SW Scotland, and north Wales), and that natural recovery can lead to the re-establishment of biota previously present at the site.     

Palaeolimnological evidence for recent chemical and biological changes in U.K. Acid Waters Monitoring Network sites

1. Palaeolimnological evidence is presented for the long-term (post-1850) and recent (post-1970) trends in acidity of eleven sites in the U.K. Acid Waters Monitoring Network.
2. Sites are located throughout the U.K. in areas sensitive to acidification, and results show that all have been acidified since pre-industrial times. Although there is considerable variation in the timing and magnitude of these changes the results are consistent with other evidence of the widespread and severe acidification of sensitive U.K. freshwaters as a result of acidic deposition.
3. The most severely acidified sites generally have the highest critical load exceedance, although there is a only a poor relationship between exceedance and post-1850 pH change ( r = 0.58, P = 0.06) or diatom floristic change ( r = 0.52, P = 0.1). These results highlight the difficulty of inferring biological change or 'damage' in freshwater ecosystems from current national maps of critical load exceedances.
4. Evidence of chemical and biological response to the post-1970 reduction in U.K. S emissions is variable: seven lakes show continued acidification in the 1970s and early 1980s while four appear to have been in steady state. One afforested site shows continued acidification until at least 1990, the year of coring, suggesting that at this site increased scavenging of acid anions following canopy closure and/or increased nitrate leaching have offset the benefits of reduced S deposition.
5. Five sites appear to have been in steady state since the early to mid-1980s, and two show unambiguous evidence for a recent increase in pH and a reversal in the diatom assemblages to that of earlier levels. The results support and extend the findings of other studies and show that biological recovery is occurring in at least two chronically acidified areas of the U.K. (Galloway, SW Scotland, and north Wales), and that natural recovery can lead to the re-establishment of biota previously present at the site.     

Lipoprotein lipase: evidence for high- and low-affinity enzyme sites.

The kinetic constants for membrane-supported lipoprotein lipase have been determined for the enzyme active in lipoprotein triglyceride catabolism in perfused heart and adipose tissues, using a nonrecirculating system. Heart endothelial lipoprotein lipase reacted as a single population of high-affinity substrate binding sites (Km' 0.07 mM triglyceride). Km' (apparent Michaelis constant for the supported enzyme species) was independent of flow rate and the enzyme was rapidly released by heparin, suggestive of a superficial membrane binding site. Lipoprotein lipase active in perfused adipose tissue had significantly different kinetic properties, including a low substrate affinity (Km' 0.70 mM triglyceride), diffusion dependence of Km' at low flow rates, and slow release of enzyme by heparin. Adipose tissue may contain a small proportion of high affinity sites. While only a small proportion of total heart tissue lipoprotein lipase was directly active in triglyceride hydrolysis, this study suggests that the major part of lipoprotein lipase in adipose tissue may be involved in the hydrolysis of circulating lipoprotein triglyceride.